The Pennsylvania State University
The Graduate School
College of Medicine
INSIGHTS INTO THE MECHANISM OF ACTION OF 13-CIS RETINOIC ACID
IN SUPPRESSING SEBACEOUS GLAND FUNCTION
A Thesis in
Molecular Medicine
by
Amanda Marie Nelson
© 2007 Amanda Marie Nelson
Submitted in Partial Fulfillment
of the Requirements
for the Degree of
Doctor of Philosophy
May 2007
The thesis of Amanda Marie Nelson was reviewed and approved* by the following:
Diane M. Thiboutot
Professor of Dermatology
Thesis Advisor
Chair of Committee
Gary A. Clawson
Professor of Pathology, Biochemistry and Molecular Biology
Mark Kester
Distinguished Professor of Pharmacology
Jeffrey M. Peters
Associate Professor of Molecular Toxicology
Jong K. Yun
Assistant Professor of Pharmacology
Craig Meyers
Professor of Microbiology and Immunology
Co-chair, Molecular Medicine Graduate Degree Program
*Signatures are on file in the Graduate School
iii
ABSTRACT
Nearly 40-50 million people of all races and ages in the United States have acne,
making it the most common skin disease. Although acne is not a serious health threat, severe
acne can lead to disfiguring, permanent scarring; increased anxiety; and depression. Isotretinoin
(13-cis Retinoic Acid) is the most potent agent that affects each of the pathogenic features of
acne: 1) follicular hyperkeratinization, 2) the activity of Propionibacterium acnes, 3) inflammation
and 4) increased sebum production.
Isotretinoin has been on the market since 1982 and even though it has been prescribed
for 25 years, extensive studies into its molecular mechanism of action in human skin and
sebaceous glands have not been done. Since isotretinoin is a teratogen, there is a clear need
for safe and effective alternative therapeutic agents. The studies undertaken in this thesis were
designed to increase our understanding of the effects of 13-cis RA on the sebaceous gland and
its mechanism of action in sebum suppression.
It is well established that isotretinoin drastically reduces the size and lipid secretion of
sebaceous glands. We hypothesized that isotretinoin decreases the size of the sebaceous
gland by inducing cell cycle arrest and/or apoptosis and that sebum suppression is most likely
an indirect result of the reduced size of the sebaceous gland.
Our studies show that 13-cis RA, unlike 9-cis RA or ATRA, induces cell cycle arrest and
apoptosis in SEB-1 sebocytes. Its ability to induce apoptosis is not inhibited in the presence of
functional retinoic acid receptor (RAR) pan antagonist AGN 193198, suggesting an RARindependent mechanism of apoptosis. Gene expression analysis was performed in cultured
SEB-1 sebocytes that were treated with 13-cis RA and in biopsies of skin taken from patients
that were treated for one week with isotretinoin. These data indicate that 13-cis RA increases
expression of neutrophil gelatinase associated lipocalin (NGAL) and Tumor Necrosis Factor
related apoptosis inducing ligand (TRAIL). In turn, we report that both NGAL and TRAIL induce
apoptosis within SEB-1 sebocytes and, as such, are potential mediators of 13-cis RA induced
apoptosis in human sebocytes.
These studies into the mechanism of action of 13-cis RA in sebaceous glands suggest
that 13-cis RA mediates its sebosuppressive effect through preferential induction of apoptosis in
sebaceous glands. Furthermore, these data provide a rationale for drug discovery of alternative
agents that are capable of selectively inducing apoptosis in sebaceous glands as a treatment for
severe acne.
iv
TABLE OF CONTENTS
LIST OF FIGURES ....................................................................................................ix
LIST OF TABLES.......................................................................................................xvi
LIST OF ABBREVIATIONS .......................................................................................xvii
ACKNOWLEDGEMENTS ..........................................................................................xx
Chapter 1 Literature Review .....................................................................................1
1.1 Introduction ...................................................................................................1
1.2 Sebaceous gland anatomy and physiology ..................................................1
1.2.1 Skin.....................................................................................................2
1.2.2 Anatomy of the Sebaceous Gland ......................................................3
1.2.2.1 Histology ...................................................................................3
1.2.2.2 Location ....................................................................................4
1.2.2.3 Embryogenesis and Morphogenesis ........................................5
1.2.2.4 Physiology of the Sebaceous Gland: Holocrine Secretion .......6
1.2.2.5 Lipid Composition of Sebum.....................................................7
1.2.2.6 Function of Sebum....................................................................7
1.2.3 Regulation of sebaceous gland size and sebum production...............8
1.3 Acne..............................................................................................................10
1.3.1 Epidemiology ......................................................................................10
1.3.2 Pathophysiology .................................................................................11
1.3.3 Classifications of acne lesions ............................................................13
1.3.4 Model systems for acne research-animal ...........................................14
1.3.4.1 Rat preputial gland....................................................................14
1.3.4.2 Hamster flank organ and ear ....................................................15
1.3.4.3 Rhino mouse.............................................................................16
1.3.5 Models for acne research: isolated human sebaceous gland organ
culture. ..................................................................................................17
1.3.6 Model systems for acne research: sebocyte cell culture ....................18
1.3.7 Current Treatments for Acne ..............................................................19
1.3.7.1 Cleansers: follicular hyperkeratinization ...................................19
1.3.7.2 Antibiotics: P. acnes and inflammation .....................................20
1.3.7.3 Hormonal therapy: sebum suppression ....................................20
1.3.7.4 Topical Retinoids: inflammation, follicular hyperkeratinization..22
1.3.7.5 Oral Retinoid: Isotretinoin (Accutane®, 13-cis Retinoic Acid)....22
1.4 Retinoids .......................................................................................................24
1.4.1 Retinoid Biology ..................................................................................25
1.4.1.1 What are retinoids?...................................................................25
1.4.1.2 Retinoid receptors.....................................................................26
1.4.1.3 Retinoid binding proteins and retinoid metabolizing enzymes ..28
1.4.1.4 Retinoid Function......................................................................30
1.4.1.4.1 Proliferation.....................................................................30
1.4.1.4.2 Differentiation..................................................................32
1.4.1.4.3 Apoptosis ........................................................................33
1.5 Retinoids in dermatology ..............................................................................36
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1.5.1 Significance of research project..........................................................36
Chapter 2 13-cis Retinoic Acid Induces Apoptosis and Cell Cycle Arrest in Human SEB1 Sebocytes ........................................................................................................37
2.1 Chapter Abstract ...........................................................................................37
2.2 Introduction ...................................................................................................38
2.3 Results ..........................................................................................................39
2.3.1 13-cis RA exhibits a more rapid onset of growth inhibition of SEB-1
sebocytes compared to 9-cis RA and ATRA. .......................................39
2.3.2 13-cis RA significantly inhibits DNA synthesis in SEB-1 sebocytes....41
2.3.3 13-cis RA, but not 9-cis RA or ATRA, increases p21 levels in SEB-1
sebocytes..............................................................................................43
2.3.4 13-cis RA, but not 9-cis RA or ATRA, decreases cyclin D1 protein in SEB1 sebocytes...........................................................................................45
2.3.5 13-cis RA induces apoptosis in SEB-1 sebocytes but not in HaCaT
keratinocytes or NHEK. ........................................................................45
2.3.6 13-cis RA specifically increases levels of cleaved caspase 3 in SEB-1
sebocytes..............................................................................................47
2.3.7 13-cis RA, but not 9-cis RA or ATRA, increases TUNEL staining in SEB-1
sebocytes..............................................................................................49
2.3.8 Apoptosis induction by 13-cis RA in SEB-1 sebocytes is not blocked by
RAR antagonist AGN 193109...............................................................52
2.3.9 13-cis RA is isomerized to ATRA over time in SEB-1 sebocytes........52
2.4 Discussion.....................................................................................................55
2.5 Materials and Methods..................................................................................59
2.5.1 Cell Culture .........................................................................................59
2.5.2 Effects of retinoids on SEB-1 proliferation ..........................................60
2.5.3 3H thymidine incorporation assay .......................................................60
2.5.4 Western blot analysis for p21, cyclin D1 and cleaved caspase 3 .......61
2.5.5 Annexin V-FITC/Propidium Iodide FACS Apoptosis Assay ................62
2.5.6 TdT-Mediated dUTP Nick End Labeling (TUNEL) Staining ................63
2.5.7 Quantitative Polymerase Chain Reaction (QPCR) .............................64
2.5.8 HPLC ..................................................................................................64
Chapter 3 Array profiling of skin from patients on isotretinoin provides insights into
potential mediators of its apoptotic effect on sebaceous glands.........................66
3.1 Chapter Abstract ...........................................................................................66
3.2 Introduction ...................................................................................................67
3.3 Results ..........................................................................................................68
3.3.1 Patient selection and procedures........................................................68
3.3.2 Histology reveals statistically significant decrease in sebaceous gland
size after 8 weeks of treatment.............................................................69
3.3.3 Significant decreases in genes that regulate lipid metabolism were noted
in the gene array expression analysis of skin biopsies taken from patients
at 8 weeks into isotretinoin therapy. .....................................................71
3.3.4 Gene expression analysis of skin from patients treated with 13-cis RA for
one-week revealed significant increases in genes encoding calcium binding
vi
proteins, retinoid signaling molecules, solute carriers and serine
proteases……………………………………………………………………..73
3.3.5 Gene expression analysis in SEB-1 sebocytes and HaCaT keratinocytes
with 72 hour 13-cis RA treatment. ........................................................75
3.3.6 QPCR verification of select genes from array analyses......................78
3.3.7 Cluster Analysis ..................................................................................80
3.3.8 Functional categorization of significantly changed genes...................82
3.3.9 Promoter analysis of genes ................................................................84
3.3.10 Comparison of gene changes at one-week and 8-week revealed only 3
common genes. ....................................................................................85
3.3.11 Comparisons between one-week, SEB-1 sebocytes and HaCaT
keratinocytes array data revealed only one gene in common between all
three arrays...........................................................................................85
3.3.12 Immunohistochemistry and western analysis showed increased NGAL
expression after 13-cis RA treatment in patient skin and our cell lines,
respectively...........................................................................................87
3.3.13 Isotretinoin increased apoptosis in one-week patient sections. ........89
3.3.14 Purified NGAL protein induces apoptosis in SEB-1 sebocytes but not in
HaCaT or NHEK keratinocytes. ............................................................90
3.3.15 Apoptosis induced by NGAL is mediated by specific NGAL receptor
isoforms. ...............................................................................................91
3.4 Discussion.....................................................................................................92
3.5 Materials and Methods..................................................................................98
3.5.1 Patient selection and tissue biopsies ..................................................98
3.5.2 Image analysis of sebaceous gland size ............................................99
3.5.3 Cell Culture .........................................................................................99
3.5.4 Gene expression microarray analysis.................................................100
3.5.5 Quantitative real-time polymerase chain reaction (QPCR) .................100
3.5.6 Cluster Analysis ..................................................................................101
3.5.7 Database promoter analysis of genes whose expression was significantly
changed by 13-cis RA...........................................................................101
3.5.8 Comparisons of gene expression arrays ............................................102
3.5.9 NGAL immunohistochemistry .............................................................102
3.5.10 Western blotting ................................................................................103
3.5.11 TdT-mediated dUTP Nick End Labeling (TUNEL) staining...............103
Chapter 4 Mechanisms involved in induction of apoptosis in SEB-1 sebocytes:
Activation of the extrinsic death receptor pathway by Tumor Necrosis Factor related
apoptosis inducing ligand (TRAIL) ......................................................................105
4.1 Chapter Abstract ...........................................................................................105
4.2 Introduction ...................................................................................................106
4.3 Results ..........................................................................................................107
4.3.1 13-cis RA up-regulates genes involved in apoptosis in SEB-1 sebocytes.
…………………………………………………………………………………107
4.3.2 13-cis RA increases cleaved caspase 8 to a greater extent than 9-cis RA
or ATRA. ...............................................................................................108
4.3.3 13-cis RA increases TRAIL expression in SEB-1 sebocytes ..............109
4.3.4 TRAIL increases levels of cleaved caspase 3 in SEB-1 sebocytes ....111
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4.3.5 siRNA knockdown of TRAIL inhibits activation of caspase 3 by 13-cis RA
……………………………………………………………………………...…112
4.4 Discussion.....................................................................................................116
4.5 Materials and Methods..................................................................................120
4.5.1 Reagents ............................................................................................120
4.5.2 Quantitative Polymerase Chain Reaction (QPCR) .............................120
4.5.3 siRNA knockdown of TRAIL by nucleofection.....................................121
4.5.4 Western blot analysis for TRAIL and cleaved caspase 3....................122
Chapter 5 Development and Characterization of a Temperature Sensitive Sebocyte Cell
Line (TSS-1)........................................................................................................123
5.1 Chapter Abstract ...........................................................................................123
5.2 Introduction ...................................................................................................124
5.3 Results ..........................................................................................................124
5.3.1 Temperature Sensitive Sebocytes (TSS) persist in culture ................124
5.3.2 TSS sebocytes display a differentiated phenotype: Increased intracellular
lipid and expression of the androgen receptor and the melanocortin 5
receptor.................................................................................................127
5.3.3 TSS sebocytes enter senescence following prolonged incubation at
‘restrictive’ temperatures.......................................................................129
5.3.4 Total lipogenesis is increased at elevated temperatures in TSS
sebocytes..............................................................................................131
5.3.5 Synthetic androgen R1881 increases and 13-cis RA decreases lipids in
TSS-1 sebocytes ..................................................................................134
5.3.6 13-cis RA decreases TSS-1 proliferation............................................135
5.3.7 13-cis RA induces apoptosis in TSS-1 sebocytes at the restrictive
temperature. .........................................................................................136
5.4 Discussion....................................................................................................138
5.5 Materials and Methods..................................................................................142
5.5.1 Sebocyte Culture ................................................................................142
5.5.2 Establishment of TSS Sebocytes and Individual Clonal Cell Lines ....142
5.5.3 Cell Growth and Viability.....................................................................143
5.5.4 Immunohistochemistry and Oil Red O Staining ..................................144
5.5.5 Western Analysis ................................................................................144
5.5.6 β-galactosidase Senescence Assay ...................................................145
5.5.7 Lipogenesis Assay: 14C-actetate incorporation into neutral lipids .......145
5.5.8 TdT-Mediated dUTP Nick End Labeling (TUNEL) Staining ................146
Chapter 6 Discussion and Future Directions ............................................................147
6.1 Introduction ...................................................................................................147
6.2 Rationale, hypothesis, and results of this work.............................................147
6.3 Explanation of model ....................................................................................148
6.4 Future Directions of this project ....................................................................150
6.4.1 Why is 13-cis RA superior to 9-cis RA or ATRA in the treatment of
acne?... .................................................................................................150
6.4.2 What RAR-independent events can lead to 13-cis RA-induced
apoptosis?..................................................................................... ……152
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6.4.3 Lipogenesis vs. Apoptosis: Does 13-cis RA preferentially affect one of
these processes?..................................................................................153
6.4.4 Does TRAIL mediate isotretinoin-induced apoptosis within the sebaceous
gland? ...................................................................................................156
6.4.5 Why are the apoptotic effects of 13-cis RA limited to sebocytes and do not
occur within keratinocytes?...................................................................158
6.5 Conclusion ....................................................................................................160
Appendix A Supplemental gene expression array tables .........................................161
A.1 All significantly changed genes after 8 weeks isotretinoin therapy...............161
A.2 All significantly changed gene in SEB-1 sebocytes after 72 hours 13-cis RA
treatment......................................................................................................178
A.3 All significantly changed genes in HaCaT keratinocytes after 72 hour 13-cis RA
treatment......................................................................................................180
References…………………………………………………………………………………..182
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LIST OF FIGURES
Figure 1: Cross-section of skin. Image obtained from www.visualinfo.com. Copyright
2005-2006 – Bernard Déry. ................................................................................3
Figure 2: Hematoxylin and eosin stained longitudinal section of human sebaceous
gland showing its multi-lobular structure. .....................................................4
Figure 3: Signaling pathways and transcription factors involved in cell lineage
determinations. As daughter cells migrate from the bulge region, changes in the
expression patterns of numerous transcription factors determine their final cell
lineage. Data is far from complete in this area; it is very likely that other pathways
and transcription factors play a significant role in determining each cell
lineage.......... ......................................................................................................6
Figure 4: Development of acne lesions Follicular hyperkeratinization, increased
sebum production, active P. acnes bacteria, and inflammation all contribute to the
development of acne. Diagram taken from “Fast Facts-Acne” 2004 Health Press
Limited. ...............................................................................................................12
Figure 5: Examples of the chemical structure of retinoids. Figure modified from
Roos, T.C., Jugert, F. K., Merk, H. F., Bickers, D. R. Retinoid Metabolism in Skin.
(1998) Pharmacolgical Reveiws 50(2): 315-333.................................................26
Figure 6: Functions involving retinoids Figure taken from Napoli, JL. Biochemical
pathways of retinoid transport, metabolism, and signal transduction. (1996) Clinical
Immunology and Immunopathology. 80(3):S52-62.............................................30
Figure 7: Simplified view of cell cycle control with focus on G1 and S phase of the
cell cycle. Diagram from
http://www.scielo.br/img/fbpe/rimtsp/v44n1/a07fig03.gif.....................................31
Figure 8: Diagram of extrinsic and intrinsic apoptosis pathways. Type 1: extrinsic
(death receptor) pathway. Type II: intrinsic (mitochondrial) pathway. Figure modified
from I. Petak, J.A. Houghton. Pathology Oncology Research, Vol 7(2), 95-106,
2001. ...................................................................................................................34
Figure 9: 13-cis RA, 9-cis RA and ATRA differentially inhibit SEB-1 sebocyte
proliferation. (a-c) Time-dependent inhibition of SEB-1 sebocyte proliferation with
individual retinoid compounds. SEB-1 cells were cultured in the presence of ethanol
vehicle alone (0.01% or less; control), 0.1 µM, 0.5 µM or 1 µM concentrations of 13cis RA, 9-cis RA, ATRA for 24, 48 or 72 hours. Attached cells were collected,
stained with trypan blue, and counted manually by hemacytometer. Data represent
mean ± SEM, n = 12. Statistical analysis was performed by ANOVA Two-Factor with
Replication. * p < 0.05, **p < 0.01, *** p < 0.0001. .............................................40
Figure 10: 13-cis RA inhibits DNA synthesis to a greater extent than 9-cis RA or
ATRA. (a-c) SEB-1 sebocytes were treated with ethanol vehicle (0.01% or less;
control) or 0.1, 0.5, 1 µM concentrations of 13-cis RA, 9-cis RA or ATRA for 24, 48
or 72 hours. 1µCi 3H thymidine was added to each sample 8 hours prior to
collection. Cells were washed and collected for liquid scintillation counting. Data
represent mean ± SEM, n ≥12. Statistical analysis was performed by ANOVA TwoFactor with Replication. *p < 0.005 and **p < 0.01. ............................................42
Figure 11: 13-cis RA increases p21 and decreases cyclin D1 proteins. (a) SEB-1
cells were treated with 0.1 µM, 1µM, 10 µM 13-cis RA or vehicle. (b-c) Parallel
experiments were performed with 0.1 µM 0.5 µM, or 1 µM concentrations of 9-cis
RA and ATRA. Blots were incubated with primary antibodies to p21 and β-actin for
loading control normalization and analyzed by densitometry. (d) SEB-1 cells were
treated with 0.1 µM, 1µM, 10 µM 13-cis RA or vehicle and blots were incubated with
primary antibodies to cyclin D1 and β-actin. Magic Mark XP (MM) indicates band
size. Blots are representative of a minimum of three western blots. Graphs
represent normalized values relative to vehicle (control) expression of a minimum of
three independent western blots. Mean ± SEM. * p < 0.05 ** p = 0.01..............44
Figure 12: 13-cis RA induces late apoptosis in SEB-1 sebocytes but not in HaCaT
keratinocytes or NHEK. (a) SEB-1 cells were treated with vehicle (negative
control), 13-cis RA (0.1 µM or 1 µM), or staurosporine (S) (positive control) for
indicated times. (b) HaCaT cells were treated with vehicle, 13-cis RA (0.1 µM or 1
µM), or staurosporine (S) for the indicated times. (c) NHEK cells were treated with
vehicle, 13-cis RA (0.1 µM or 1 µM), or staurosporine (S) for indicated times. In all
experiments, cells were prepared according to manufacturer’s protocol for Annexin
V-FITC / PI staining. (BD ApoAlert, BD Biosciences) Data was analyzed with Cell
Quest Software and represent mean ± SEM, n ≥ 12. Statistical analysis was
performed with ANOVA Two Factor with Replication. *p<0.01, **p<0.00001. ....46
Figure 13: 13-cis RA induces cleaved caspase 3 expression in SEB-1 sebocytes.
(a) SEB-1 sebocytes were treated with vehicle, 0.1 µM, 1 µM, or 10 µM 13-cis RA.
(b-c) Parallel experiments were performed with 0.1 µM, 0.5 µM, or 1 µM
concentrations of 9-cis RA or ATRA. Blots were incubated with primary antibodies to
cleaved caspase 3 (1:1000) and actin (1:1000) for loading control normalization and
analyzed by densitometry. p17 and p19 are cleaved caspase 3 active fragments.
Blots are representative of a minimum of 4 independent experiments. Graph
represents normalized values relative to vehicle (control) expression for 4
independent western blots. Data represent mean ± SEM * p < 0.01. ...............48
Figure 14: Cleaved caspase 3 is not detected in NHEK treated with 13-cis RA.
NHEK were treated with vehicle, 0.1 µM, 0.5 µM, or 1 µM concentrations of 13-cis
RA or 1 µM staurosporine (S; positive control). Blots were incubated with primary
antibodies to cleaved caspase 3 (1:1000) and β-actin (1:1000) for loading control
normalization and analyzed by densitometry. Representative blot is shown......49
Figure 15: The increase in TUNEL staining with 13-cis RA is not inhibited in the
presence of RAR pan antagonist AGN 193109. (a) Representative images of
control, 0.1 µM, 1 µM, and 10 µM 13-cis RA, 9-cis RA, ATRA, and fenretinide
treatments at 72 hours. (48 hour data not shown) (b) Quantification of the
percentage of TUNEL positive stained cells per treatment at 48 and 72 hours. (9-cis
RA not shown) Data represent mean + SEM, n = 6-12. Statistical analyses were
performed with ANOVA Two Factor with Replication. * p < 0.01 ** p < 0.001 (c)
x
xi
Representative images of negative control, 1 µM 13-cis RA, AGN 193109, and 13cis RA combined with 10 µM AGN 193109 at 72 hours. (48 hour data not shown) (d)
Quantification of the percentage of TUNEL positive cells at 72 hours. Data represent
mean + SEM, n = 12. Statistical analyses were performed with ANOVA Two Factor
with Replication. * p < 0.05 when compared to control; + not statistically different.
(e) QPCR verification of RAR antagonist AGN 193109 activity in SEB-1 sebocytes.
Bars represent the efficiency corrected normalized average fold change of TIG1
under the experimental conditions as determined by REST-XL software. n = 4
...............................................................................................................................51
Figure 16: 13-cis RA is isomerized to ATRA within SEB-1 sebocytes. HPLC analysis
of (a) SEB-1 medium alone, (b) medium removed from SEB-1 sebocyte-containing
plates and (c) SEB-1 sebocytes after 5 µM 13-cis RA treatment for the indicated
times. Points are the average of duplicate samples. ..........................................54
Figure 17: 13-cis RA decreases sebaceous gland volume. (a) Hematoxylin and eosin
sections of back skin from patients before and after 8 weeks of treatment reveals a
significant decrease in sebaceous gland volume. (b) Variable changes in sebaceous
gland size were noted after one week of treatment compared to baseline biopsies
(c) Area of sebaceous glands. Statistical significance was determined by paired ttest. Representative images are shown at a total magnification of 100X.
Magnification bars = 250 µm...............................................................................70
Figure 18: QPCR verification of gene array gene changes. (a) One-week (b) 8-week.
Data represent the mean ± SEM of the fold change in gene expression as
determined by REST-XL (QPCR) in 4-5 subjects compared to 6-8 subjects as
determined by gene array. (c) SEB-1 (d) HaCaT (e) NHEK. Data represent the
mean ± SEM of the fold change in gene expression as determined by REST-XL
(QPCR) in 3 samples compared to 3 samples as determined by gene array. ....79
Figure 19: Hierarchical clustering diagram of one-week isotretinoin patient
samples. Hierarchical clustering was used to compute a dendrogram that
assembled all genes and samples into a single tree. Patient samples included skin
biopsies taken prior to treatment and at one-week of treatment. Normalized array
data was imported into dChip software version 1.3. The information files for the
Affymetrix human genome HG-U133A 2.0 array was obtained from www.dChip.org
(8-week data not shown). Each row represents a single gene and each column
represents a patient sample. (B=baseline and A=after treatment). The color reflects
the level of expression when compared to the mean level of expression for the
entire biopsy set. Red indicates expression higher than the mean and blue indicates
lower expression than the mean. ........................................................................81
Figure 20: NGAL increased after one-week isotretinoin treatment.
Immunohistochemistry for NGAL on sections on back skin taken before and at oneweek treatment reveals notable increase in NGAL expression in sebaceous gland
and hair follicle. Sections were incubated overnight with a 1:50 dilution of mouse
monoclonal lipocalin 2/NGAL antibody. Negative control sections omitted primary
antibody. All sections were counterstained with hematoxylin. NC=negative control;
pre=before treatment; post=after treatment; SG=sebaceous gland; and Fol=follicle.
Representative images are shown. Total magnification: 400X ...........................87
xii
Figure 21: 13-cis RA increases NGAL protein expression. SEB-1 sebocytes, HaCaT
keratinocytes and NHEK keratinocytes were treated with vehicle control or 13-cis
RA (0.1 or 1 µM). Protein expression was verified by western blot. Blots were
incubated with primary antibody to lipocalin 2 and β-actin for loading control
normalization followed by densitometry. Graph represents normalized fold-change
values relative to control expression for a minimum of six independent blots. Mean ±
SEM. ...................................................................................................................88
Figure 22: TUNEL staining in sebaceous glands increased in patient skin after oneweek isotretinoin treatment. Two representative “after isotretinoin” images are
shown. Skin sections were obtained from paraffin blocks of patients 9-15 and were
subjected to TUNEL-peroxidase assay, according to manufacturer’s instructions.
Assay controls included DNase I treated positive and negative controls with primary
antibody omitted in negative control. At least 2 sections from every patient (before
and after) were analyzed. Sections were counter-stained with hematoxylin. Data
represent mean ± SD, n=6 patients; paired t-test was used for statistical analysis.
Total magnification 400X. ...................................................................................89
Figure 23: NGAL increases TUNEL staining in SEB-1 sebocytes SEB-1 sebocytes,
HaCaT and NHEK keratinocytes were treated in duplicate with vehicle control,
1pg/mL, 10pg/mL, 1ng/mL and 10ng/mL purified recombinant human NGAL protein
(R&D Systems) for 24 hours. (a) Representative images of SEB-1 sebocytes are
shown. Total magnification 200X. (b) Quantification of the percentage of TUNEL
positive stained cells per treatment at 24 hours. Data represent mean + SEM, n = 46. Statistical analyses were performed with ANOVA Two Factor with Replication. *
p< 0.05, ** p < 0.01, *** p < 0.0001.....................................................................90
Figure 24: Cell-specific expression of 24p3R/NGAL-R isoforms is influenced by 13cis RA. (a) Protein lysates (vehicle and 1µM 13-cis RA 48 hours) were
immunoblotted with affinity purified 24p3 receptor antibody. Positive (+) and
negative (-) control protein lysates obtained from Dr. Michael Green. Variable
expression of receptor isoforms (short, long, and high molecular weight forms) are
noted across cell lines and in response to 13-cis RA. (b) Relative quantification of
isoforms. Blots were incubated with β-actin for loading control normalization
followed by densitometry. Graph represents normalized fold-change values relative
to control expression for three independent blots. Mean ± SD. ..........................92
Figure 25: Increased active caspase 8 with 13-cis RA treatment. Protein lysates from
SEB-1 sebocytes treated with 1 µM concentrations of 13-cis RA, 9-cis RA and
ATRA or vehicle control (0.01% ethanol) and 1 µM) for 48 hours were
immunoblotted with mouse Caspase 8 antibody (Cell Signaling Technology). A
representative blot is shown. Graph represents mean ± SD fold-change values
normalized to control for 2 independent blots (13-cis RA) and 4 independent blots
(9-cis RA and ATRA) ..........................................................................................109
Figure 26: TRAIL expression is increased by 13-cis RA treatment in SEB-1
sebocytes. (a) QPCR was performed for TRAIL at 24, 48 and 72 hours. Bars
represent mean fold change of 3 independent samples as determined by REST-XL
software. (b) TRAIL protein expression at 72 hours. Preliminary western blot
xiii
shown. Graph shows relative level of TRAIL protein for one experiment to
date......................................................................................................................111
Figure 27: TRAIL increases expression of cleaved caspase 3 protein. SEB-1
sebocytes were treated with increasing concentrations of purified recombinant
human TRAIL (rhTRAIL) protein for 48 hours. (a) Representative blot is shown. (b)
Graph represents normalized values relative to control expression of three
independent western blots. Mean ± SD. * p < 0.05............................................112
Figure 28: QPCR shows siRNA knockdown of TRAIL mRNA siCONTROL and TRAIL
siRNA (2 concentrations) were nucleofected into SEB-1 sebocytes using Amaxa
Nucleofection Kit T and nucleofector device. Twenty-four hours later, 0.1 µM 13-cis
RA was added to induce TRAIL expression. Total RNA was isolated at 24, 48 and
72 hours of 13-cis RA treatment. Graph represents fold-change in level of TRAIL
mRNA for one sample.........................................................................................113
Figure 29: siRNA knockdown of TRAIL inhibits active caspase 3 protein
expression. siCONTROL and TRAIL siRNA (2 concentrations) were nucleofected
into SEB-1 sebocytes using Amaxa Nucleofection Kit T and nucleofector device.
Twenty-four hours later, 0.1 µM 13-cis RA was added to induce TRAIL expression.
Protein was isolated at 24, 48 and 72 hours of 13-cis RA treatment and subjected to
immunoblotting with TRAIL (1:500) and cleaved caspase 3 (1:800) antibodies.
Graphs represent normalized relative levels (compared to control) of TRAIL or
cleaved caspase 3 protein of one sample...........................................................115
Figure 30: TSS sebocytes express SV40 large T antigen. TSS sebocytes (33ºC),
SEB-1 sebocytes (37ºC) and HaCaT Keratinocytes (37ºC) were incubated with
primary antibody to large T antigen and detected by anti-mouse, FITC secondary
antibody. Cells were analyzed by fluorescence microscopy. Representative images
are shown. ..........................................................................................................125
Figure 31: SV40 large T antigen expression declines with increasing temperature
within TSS sebocytes. TSS sebocytes were grown at 33ºC. Sebocytes then
remained at 33ºC or were shifted to 37ºC or 41ºC for 72 hours to “shut-off” large T
antigen protein. As a control, SEB-1 sebocytes were also grown under all three
temperatures. Cells were incubated with primary antibody to large T antigen and
detected by anti-mouse, FITC secondary antibody. Representative images are
shown..................................................................................................................126
Figure 32: TSS sebocyte growth declines with increasing temperature. TSS-1
sebocytes were plated and placed at 33ºC, 37ºC, 39ºC or 41ºC. Manual cells counts
were performed every three days. Data points represent average of three
independent samples at each time point. ...........................................................127
Figure 33: Oil Red O staining in TSS sebocytes increased with elevating
temperatures. TSS and SEB-1 sebocytes were cultured at 33ºC, 37ºC and 41ºC
temperatures f6r 6 days followed by O Red O staining to detect intracellular neutral
lipids. Cells were counterstained with hematoxylin. Representative images are
shown. Magnification: 400X. ...............................................................................128
xiv
Figure 34: Androgen receptor and melanocortin 5 receptor were expressed in
differentiated TSS sebocyte cell lines. a) Total protein lysates from TSS-1, TSS2, TSS-3 and TSS-4 cell lines cultured at 33ºC, 37ºC and 39ºC for three days were
analyzed for expression of androgen receptor (110kD) via western blotting.
Representative blots are shown. b) Relative levels of androgen receptor
expression. Mean ± SE, n= 3. c) Total protein lysates from TSS-1, TSS-2, TSS-3
and TSS-4 cell lines at cultured 33ºC, 37ºC and 39ºC were analyzed for expression
of the melanocortin 5 receptor (32kD) via western blotting. Controls included lysates
from SEB-1, rat preputial cells (Rat P.C.) and human placenta (Plac.) Magic
Markers XP (M.M.) were used as size indicators................................................129
Figure 35: TSS-1 sebocytes entered senescence after prolonged incubation at 39C.
TSS-1 sebocytes were cultured at 33ºC, 37ºC and 39ºC for 3, 5 or 7 days followed
by β-galactosidase assay procedures. Representative images are shown.
Magnification: 400X ............................................................................................130
Figure 36: TSS-1 sebocyte culture and treatment model. This diagram outlines the
timing and temperatures involved in using TSS-1 sebocytes. Experiments analyzing
basal conditions are conducted on Day 5. Depending on the treatment length,
assays are conducted on Days 6-9. In most cases, parallel plates at 33ºC and 39ºC
are examined. .....................................................................................................131
Figure 37: TSS sebocyte lipogenesis was greatest at 37ºC incubation with little to
no difference between 33 and 39ºC temperatures. TSS sebocytes were cultured
and total lipogenesis was performed at 3 days (one-week data not shown) after
temperature switch. Mean ± SEM; n = 6 samples. Statistical significance was
determined by ANOVA Two Factor with Replication and considered significant if *p
< 0.05..................................................................................................................132
Figure 38: Incorporation of 14C acetate into lipids was greatest at 37ºC in TSS-1.
TSS-1 sebocytes were cultured and lipogenesis assays were performed at 3 days
(a) or one week (b) after temperature switch. Mean ± SD; n = 4 samples.
C=cholesterol, FOH=fatty alcohol, OA=oleic acid, TAG=triglycerides, WE=wax
esters, CO=cholesterol oleate, and SQ=squalene. Statistical significance was
determined by paired t-test and considered significant if *p < 0.05. ›: All
temperatures statistically different from each other. ...........................................133
Figure 39: Synthetic androgen R1881 increased total lipogenesis in TSS-1
sebocytes. TSS-1 sebocytes were cultured as illustrated in Figure 36 and treated
with R1881 (1 X 10-8 M) or vehicle alone (control) for 24 hours prior to lipogenesis
assay. Assay was repeated three independent times. Mean ± SEM; n = 9. Statistical
significance was determined by ANOVA Two Factor with Replication and
considered significant if p < 0.05. .......................................................................134
Figure 40: 13-cis RA decreased total lipogenesis in TSS-1 sebocytes. TSS-1
sebocytes were cultured as illustrated in Figure 36 and treated with 13-cis RA (0.1
µM) or vehicle alone for 24 hours prior to lipogenesis assay. Assay was repeated
three independent times. Mean ± SEM; n = 9. Statistical significance was
determined by ANOVA Two Factor with Replication and considered significant if p <
0.05. ....................................................................................................................135
xv
Figure 41: 13-cis RA causes growth inhibition in TSS-1 sebocytes. (a) TSS-1 (33ºC)
(b) TSS-1 (39ºC). Time-dependent inhibition of TSS-1 sebocyte proliferation. TSS-1
cells were cultured in the presence of ethanol vehicle alone (0.01% or less; control),
0.1 µM, 1 µM or 10 µM concentrations of 13-cis RA 24, 48 or 72 hours. Attached
cells were collected, stained with trypan blue, and counted manually. Data represent
mean ± SEM, n = 9. Statistical analysis was performed by ANOVA Two-Factor with
Replication. * p < 0.05, **p < 0.01.......................................................................136
Figure 42: TSS-1 sebocytes undergo apoptosis with 13-cis RA treatment: TUNEL
Staining. Representative images of control, 0.1 µM, 1µM, and 10 µM 13-cis RA
treatment at 48 and 72 hours at 39ºC. (b) Quantification of the percentage of
TUNEL positive stained cells per treatment at 48 and 72 hours. Data represent
mean + SEM, n = 6. Statistical analyses were performed with ANOVA Two Factor
with Replication. * p < 0.05 ** p < 0.001 ..........................................................137
Figure 43: Model of 13-cis RA induces apoptosis and cell cycle arrest in SEB-1
sebocytes and human sebaceous glands. .....................................................149
xvi
LIST OF TABLES
Table 1: Comparison of animal models for studying sebaceous glands and acne.…17
Table 2: Comparisons of sebocyte cell culture models .......................................19
Table 3: Retinoid binding proteins with ligand identification. Table taken from
Napoli, JL. Biochemical pathways of retinoid transport, metabolism, and signal
transduction. (1996) Clinical Immunology and Immunopathology. 80(3):S5262………………………………………………………………………………………..29
Table 4: Retinoid induced apoptotic mechanisms................................................35
Table 5: Isotretinoin patient demographics ...........................................................69
Table 6: Selected significantly changed genes after 8 weeks isotretinoin therapy…72
Table 7: Significantly changed genes after 1 week isotretinoin therapy ...........74
Table 8: Selected significantly changed gene in SEB-1 sebocytes after 13-cis RA
treatment............................................................................................................76
Table 9: Selected significantly changed genes in HaCaT keratinocytes after 13-cis
RA treatment .....................................................................................................77
Table 10: Functional categorization of significantly changed one-week genes.….82
Table 11: Protein domains enriched within genes significantly changed at 8
weeks.. ...............................................................................................................83
Table 12: Down-regulated pathways enriched within genes significantly changed
at 8 weeks ..........................................................................................................84
Table 13: Common significantly changed genes within one-week isotretinoin, SEB1 sebocyte and HaCaT keratinocyte gene arrays. .........................................86
Table 14: Genes involved in apoptosis whose expression is significantly changed
by 13-cis RA in SEB-1.......................................................................................107
xvii
LIST OF ABBREVIATIONS
13-cis RA
17β-HSD
3T3
9-cis RA
ABC
ACTH
AEC
AGN 193109
ANOVA
APAF
AR
Asebia
ATRA
Bcl
CD 3254
CDK
CDKI
cpm
CRABP
CYP
DD
DED
DHEAS
DHT
DMEM
DMSO
DR
EDTA
EGF
ER
ERK
FACS
FADD
FasL
FBS
FDA
FDR
FGFR
FITC
Gap1
HaCaT
H&E
HPLC
JNK
13-cis retinoic acid
17β hydroxysteroid dehydrogenase
albino Swiss mouse embryo fibroblast cell line
9-cis retinoic acid
avidin:biotinylated enzyme complex
adrenocorticotropic hormone
3-amino-9-ethylcarbazole
RAR antagonist
analysis of variance
apoptotic protease activating factor
androgen receptor
sebaceous gland deficient mouse
all-trans retinoic acid
B cell lymphoma
RXR pan-agonist
cyclin-dependent kinase
cyclin-dependent kinase inhibitor
counts per minute
cellular retinoic acid binding protein
cytochrome P450
death domain
death effector domain
dehydroepiandrosterone sulfate
dihydrotestosterone
Dulbecco's Modified Eagle Medium
dimethyl sulfoxide
direct repeat
ethylenediamine tetraacetic acid
epidermal growth factor
estrogen receptor
extracellular signal-related-kinase
fluorescent activated cell sorter
Fas-associated death domain
Fas Ligand
fetal bovine serum
Food and Drug Administration
false discovery rate
fibroblast growth factor receptor
fluorescein isothiocyanate
growth phase 1
human keratinocyte cell line
hematoxylin & eosin
high performance liquid chromatography
c-Jun N terminal kinase
xviii
KGM
MAPK
mm
MM or M.M.
MSH
NGAL
NHEK
P. acnes
PBS
P.C.
PI
PKC
Plac.
PPAR
QPCR
RA
RALDH
RAR
RARE
Rat P.C.
Rb
RBP
REST
rhTRAIL
RNA
RNAi
ROR
RSV
RT-PCR
RXR
RXRE
S
SAPK
SEB-1
SEM
SERPINS
SG
Shh
siRNA
SV40
SZ95
TBP
Tcf3
TESS
TIG1
TLR
TNF
TNF-R
keratinocyte growth medium
mitogen-activated protein kinase
millimeter
Magic Mark XP
melanocyte stimulating hormone
neutrophil gelatinase associated lipocalin
normal human epidermal keratinocytes
Propionibacterium acnes
phosphate buffered saline
preputial cells
propidium iodide
protein kinase C
placenta
peroxisome proliferator activated receptor
quantitative real time polymerase chain reaction
retinoic acid
retinaldehyde dehydrogenase
retinoic acid receptor
retinoic acid receptor response element
rat preputial cells
retinoblastoma tumor suppressor
retinol binding protein
Relative Expression Software Tool
recombinant human tumor necrosis factor related apoptosis
inducing ligand
ribonucleic acid
RNA interference
RAR-related orphan receptor
Rous Sarcoma Virus
reverse transcription-polymerase chain reaction
rexinoid receptor, retinoid X receptor
rexinoid receptor response element
staurosporine
stress activated protein kinase
SV-40 large T antigen immortalized sebocyte cell line
standard error of mean
serine protease inhibitors
sebaceous gland
Sonic Hedgehog
small interfering RNA
simian virus 40
SV-40 large T antigen immortalized sebocyte cell line
TATA binding protein
transcription factor 3
Transcription Element Search System
tazarotene-induced gene 1
Toll-like receptor
tumor necrosis factor
tumor necrosis factor receptor
xix
TR
TRADD
TRAIL
TSS
TUNEL
VDR
thyroid receptor
TNF-R1 associated death domain protein
tumor necrosis factor related apoptosis inducing ligand
temperature sensitive sebocyte cell line
TdT-Mediated dUTP Nick End Labeling
vitamin D receptor
xx
ACKNOWLEDGEMENTS
Words cannot express my gratitude for my thesis advisor and mentor, Dr. Diane
Thiboutot M.D. I will forever by grateful to Diane for allowing me the freedom to explore and
develop this project from the ground up. Throughout the entire thesis process, Diane was
always supportive, encouraging, patient and positive. With her guidance, I was able to
effectively learn from my mistakes, becoming more confident and trusting in my abilities as a
budding scientist with each passing year. In addition to helping me succeed in science, she has
shown me that balancing family and career is possible and just takes organization and flexibility.
For all your support, I say ‘THANK YOU FOR EVERYTHING’. I look forward to our interactions
in the years to come.
The work presented in this thesis could not have been accomplished without the daily
support of my co-workers and dear friends: Kathryn Gilliland, Zhaoyuan Cong, Kimberly Smith
Dr. Heidi Devlin, and past members, Chelsea Billingsley and Dr. Terry Smith. I was warmly
welcomed into the Thiboutot Lab by Kathy and Zhaoyuan, each of whom with smiles and lots of
patience helped me get my research project started on the right foot. My daily conversations
and interactions with all these wonderful people have positively influenced and challenged me to
become the best scientist and person that I can be.
For excellent scientific and technical advice, I must thank my thesis committee: Dr. Gary
Clawson, Dr. Mark Kester, Dr. Jeffrey Peters and Dr. Jong Yun. These individuals always
provided good advice and challenged me to think critically at every step of this project.
I have truly enjoyed my time in graduate school at Penn State University College of
Medicine. My graduate school classmates provided a listening ear and also the much needed,
humor, to survive graduate school. In addition to classmates, I must thank Kathy Simon and
Kathy Shuey, two of the best administrative professionals in the Graduate School. Both Kathy
Simon and Kathy Shuey helped me tremendously with all the necessary paperwork from thesis
committee appointments to graduation deadlines in addition to being available to help with any
problem that arose during graduate school. Without them, I would still be lost.
Finally, I would like to acknowledge the continual support from my family. To say thank
you to my parents, Ken and Theresa Nelson, does not even begin to cover all they have done
for me. Without a doubt, I would not be the person I am today without them. Brad, my loving
husband, deserves a medal for his support throughout my graduate school career. He was
xxi
always there to encourage me and give me strength throughout these last 5 ½ years. I am so
grateful to have him in my life.
Chapter 1
Literature Review
1.1 Introduction
Acne is one of the most common skin conditions encountered by dermatologists. Most
acne occurs during adolescence, an already socially and psychologically challenging period in
an individual’s life; although, it can persist into adulthood. There is no cure for acne, but
successful treatments are available.
The most effective drug for severe acne is isotretinoin. However, isotretinoin is a potent
teratogen and as such, use of this drug is closely monitored through an FDA mandated registry
program: iPLEDGE. Although isotretinoin has been prescribed for over 20 years, extensive
studies into its molecular mechanism of action(s) in human skin and especially the sebaceous
glands have not been done. By understanding the sebaceous gland and, specifically, the
sebocyte response to isotretinoin in terms of changes in gene expression or cellular pathways, it
may be possible for the development of alternative therapies for acne without the teratogenic
side-effects.
The first section of this chapter will review sebaceous gland anatomy and physiology.
The second section will review epidemiological data on acne, the causes of acne, sebaceous
gland model systems and current treatments of acne. Retinoid biology and functions are
discussed within the final section of this chapter.
1.2 Sebaceous gland anatomy and physiology
Acne is a disease of the sebaceous gland. In order to understand the pathophysiology of
this disease, it is important to understand the “normal” condition of the sebaceous gland.
2
1.2.1 Skin
The skin is the largest organ in the human body with an average area of 2 meters2 in
adults. Its thickness ranges from 0.5mm to 4mm depending on body location with the thickest
skin on the soles of the feet. Skin provides a physical and physiological barrier between the
external environment and internal environment of the body. Skin has numerous functions
including physical protection, wound healing, immune defense, sensory awareness,
thermoregulation, secretion and permeability (Chuong et al, 2002). The skin is divided into two
major components: epidermis and dermis.
The epidermis is the most superficial layer of the skin and is composed of stratified
squamous epithelium. Epidermal cells are called keratinocytes. The epidermis can be
subdivided into 4-5 distinct layers. The deepest layer, stratum germinativium or stratum basale,
is a single layer of proliferative cells that divide when necessary and also keeps the epidermis
strongly attached to the underlying dermis. As cells migrate upwards into the stratum spinosum,
stratum granulosum, and stratum lucidum layers, the cells are undergoing the differentiation
process and acquiring new cytoskeletal framework, cell-cell connections, lipids and keratin
proteins. Finally, fully differentiated cells reach the outermost layer of the skin, stratum corneum.
This layer consists of numerous layers of flattened keratinocytes joined tightly together to form
an impermeable layer. This top layer is constantly shed and under normal conditions,
keratinocytes can make the journey from cell division to desquamation within a month
(Blumenberg and Tomic-Canic, 1997). In addition to keratinocytes, the epidermis is composed
of melanocytes, melanin producing cells responsible for our skin color; Langerhans cells,
resident immune cells; and Merkel cells, which help with sensory perception (Kerr, 1999).
The dermis is the deepest layer of our skin. It is composed of fibroblasts, abundant
collagen and lesser amounts of elastic and reticular fibers. The dermis is strongly connected to
the epidermis through hemidesmosomes and is the support system of the skin. The dermis
contains blood and lymph vessels, glands (sweat and sebaceous), nerves (free and
encapsulated nerve endings) and hair follicles (Figure 1). Blood vessels provide nutritional
support as well as thermoregulation. Although located in the dermis, both sweat glands and
sebaceous glands are derivatives of the epidermis and extend through the epidermis to the
skin’s surface. Nerve endings receive and transmit information regarding temperature, pain,
pressure and vibration (Kerr, 1999).
3
Figure 1: Cross-section of skin. Image obtained from www.visualinfo.com. Copyright 20052006 – Bernard Déry.
Beneath the dermis is a layer of subcutaneous tissue that is comprised of a thick layer of
connective tissue and adipose tissue.
1.2.2 Anatomy of the Sebaceous Gland
1.2.2.1 Histology
Sebaceous glands, located in the dermis, are uni-lobular or multi-lobular structures that
consist of acini connected to a common excretory duct composed of stratified squamous
epithelium and are usually associated with a hair follicle. The glands are composed of lipidproducing sebocytes and keratinocytes that line the sebaceous ducts. Just inside the basement
membrane at the periphery of the sebaceous gland is a basal cell layer composed of small,
cuboidal, nucleated, highly mitotic sebocytes. Cells progress toward the middle of the gland and
accumulate lipid droplets as they terminally differentiate. These fully differentiated sebocytes are
4
filled with lipid and lack all other cellular organelles. Surrounding the glands are connective
tissue capsules composed of collagen fibers that provide physical support (Downie et al, 2004)
(Figure 2).
Figure 2: Hematoxylin and eosin stained longitudinal section of human sebaceous
gland showing its multi-lobular structure.
1.2.2.2 Location
Sebaceous glands are associated with hair follicles all over the body. A sebaceous gland
associated with a hair follicle is termed a pilosebaceous unit. The glands may also be found in
certain non-hairy sites including the eyelids (Meibomian glands), the nipples (Montgomery
glands) and around the genitals (Tyson glands). Only the palms and soles, which have no hair
follicles, are totally devoid of sebaceous glands. Sebaceous glands vary considerably in size,
even in the same individual and in the same anatomic area. Most sebaceous glands are only a
fraction of a millimeter in size. The largest glands and the greatest density of glands (up to 400900 glands per square centimeter) are found on the face and scalp (Downie, et al., 2004;
Montagna and Parakkal, 1974; Strauss and Pochi, 1963).
In the oral epithelium, sebaceous glands known as Fordyce spots are sometimes
present. Fordyce spots are visible to the unaided eye because of their large size (up to 2 to 3
mm) and the transparency of the oral epithelium (Dreher and Grevers, 1995). In this location,
the sebaceous ducts open directly to the surface.
5
1.2.2.3 Embryogenesis and Morphogenesis
In the human fetus, sebaceous glands develop in the 13th to 16th week of gestation from
bulges (epithelial placodes) on the developing hair follicles (Muller et al, 1991; Williams et al,
1988). The bulge region of the follicle contains the epidermal stem cells that generate multiple
cell lineages including epidermal and follicular keratinocytes as well as sebaceous glands. As
daughter cells migrate from the bulge region, changes in the expression patterns of numerous
transcription factors determine their final cell lineage. Multipotent progenitors of the bulge region
express Tcf3 (transcription factor 3) and this expression represses terminally differentiated
states including hair follicles and sebaceous glands (Nguyen et al, 2006). Wnt/wingless (Wnt)
and Sonic Hedgehog (Shh) signaling pathways are intricately involved in embryonic patterning
and cell fate decisions. Cells destined to become sebocytes have increased Shh and Myc
signaling and decreased Wnt signaling (Figure 3). In transgenic mouse models, intact Wnt
signaling promotes hair follicle differentiation, whereas inhibition of Wnt signaling by preventing
Lef1/β-catenin interaction leads to sebocyte differentiation (Merrill et al, 2001). Loss-of-function
and gain-of-function transgenic mouse models demonstrated that blocking Shh signaling
inhibited normal sebocyte differentiation and constitutively activating Shh signaling resulted in
increased number and size of sebaceous glands in skin (Allen et al, 2003). When fully formed,
the glands remain attached to the hair follicles by a duct through which sebum flows into the
follicular canal and eventually to the skin surface.
6
Figure 3: Signaling pathways and transcription factors involved in cell lineage
determinations. As daughter cells migrate from the bulge region, changes in the
expression patterns of numerous transcription factors determine their final cell lineage.
Data is far from complete in this area; it is very likely that other pathways and
transcription factors play a significant role in determining each cell lineage.
1.2.2.4 Physiology of the Sebaceous Gland: Holocrine Secretion
The sebaceous glands release lipids by disintegration of entire cells, a process known
as holocrine secretion. The life span of a sebocyte from cell division to holocrine secretion is
approximately 21-25 days (Plewig and Christophers, 1974; Plewig et al, 1971). Because of the
constant state of renewal and secretion of the sebaceous gland, individual cells within the same
gland are engaged in different metabolic activities dependent upon their differentiation state
(Potter et al, 1979). The stages of this process are evident in the histology of the gland (Ito,
1984). The outermost cells, basal cell layer membrane, are small, nucleated, and devoid of lipid
droplets. This layer contains the dividing cells that replenish the gland as cells are lost in the
process of lipid excretion. As cells are displaced into the center of the gland, they begin to
produce lipid, which accumulates in droplets. Eventually the cells become greatly distended with
7
lipid droplets and the nuclei and other subcellular structures disappear. As the cells approach
the sebaceous duct, they disintegrate and release their contents. Less polar and more neutral
lipids reach the skin surface (Nicolaides et al, 1970). Proteins, nucleic acids, and the
membrane phospholipids are digested and apparently recycled during the disintegration of the
cells.
1.2.2.5 Lipid Composition of Sebum
Human sebum contains cholesterol, cholesterol esters, squalene, wax esters, di- and triglycerides and fatty acids (Stewart and Downing, 1991). Squalene and wax esters are
presumed to be unique to human sebum and distinguish sebum from the lipids of human
internal organs, which contain minimal squalene and no wax esters. The particular fatty acid unsaturation patterns of the fatty acids in the triglycerides, wax esters, and cholesterol esters also
distinguish human sebum from the lipids of other organs. The general desaturation pathway
involves inserting a double bond between the ninth and tenth carbon of stearic acid (18:0) to
produce oleic acid (18:1∆9). However, in human sebaceous glands, the predominant pattern is
the insertion of a ∆6 double bond into palmitic acid (16:0) by delta-6 desaturase (fatty acid
desaturase 2), which is the dominate fatty acid desaturase in sebaceous glands (Ge et al,
2003). The resulting product, sapienic acid (16:1∆6) is the major fatty acid of adult human
sebum (Perisho et al, 1988). Elongation of the chain by two carbons and insertion of another
double bond gives sebaleic acid (18:2∆5,8), which is unique to human sebum (Nicolaides,
1974).
1.2.2.6 Function of Sebum
The precise function of sebum in humans is unknown. It has been proposed that its
solitary role is to cause acne (Cunliffe and Shuster, 1969). It has also been suggested that
sebum reduces water loss from the skin’s surface and functions to keep skin soft and smooth,
although evidence for these claims in humans is minimal. As demonstrated in the sebaceous
gland deficient mouse (Asebia) model, glycerol derived from triglyceride hydrolysis in sebum is
8
critical for maintaining stratum corneum hydration (Flurh et al, 2003). Sebum has been shown
to have mild antibacterial action, protecting the skin from infection by bacteria and fungi,
because it contains immunoglobulin A, which is secreted from most exocrine glands (Gebhart et
al, 1989). Vitamin E delivery to the upper layers of the skin protects the skin and its surface
lipids from oxidation. Thus, sebum flow to the surface of the skin may provide the transit
mechanism necessary for vitamin E to function (Thiele et al, 1999).
1.2.3 Regulation of sebaceous gland size and sebum production
Production of sebum is continuous and is not controlled by neural mechanisms, although
substance P (neuropeptide) has been shown to increase proliferation and differentiation of
sebaceous glands (Saint-Leger and Cohen, 1985; Thiboutot, 2004; Toyoda et al, 2002). The
exact mechanisms underlying the regulation of human sebum production have not been
defined. Clearly, sebaceous glands are regulated by androgens and retinoids, but recently,
other factors, such as melanocortins, peroxisome proliferator-activated receptors (PPARs), and
fibroblast growth factor receptors (FGFRs) have been postulated to play a role as well.
It has long been recognized that sebaceous glands require androgenic stimulation to
produce significant quantities of sebum. Androgen receptors have been localized to both the
keratinocytes of the outer root sheath of hair follicles as well as the basal layer of the sebaceous
gland (Kariya et al, 2005). Individuals with a genetic deficiency of androgen receptors
(complete androgen insensitivity) have no detectable sebum secretion and do not develop acne
(Imperato-McGinley et al, 1993). Conversely, addition of testosterone and
dihydroepiandrosterone increases the size and secretion of sebaceous glands (Pochi and
Strauss, 1969). There is still a question as to which androgen is physiologically significant. The
most potent androgens are testosterone and dihydrotestosterone (DHT); however, levels of
testosterone do not parallel the patterns of sebaceous gland activity. Sebum secretion starts to
increase in children (5-6 years of age) during adrenarche although the levels of androgens are
very low at this time (Pochi et al, 1977). It is possible that the sebaceous gland is responsive to
these very low levels of androgens. In addition, testosterone levels are significantly higher in
males than in females, with no overlap between the sexes, while average rates of sebum
secretion are only slightly higher in males than in females, with considerable overlap between
9
the sexes (Harris, 1983; Thiboutot et al, 1999). The majority of females with acne have serum
androgen levels that, although higher, are within normal limits and it has been hypothesized that
locally-produced androgens within the sebaceous gland may contribute to acne (Levell, 1989;
Lookingbill et al, 1985). The weak adrenal androgen, dehydroepiandrosterone sulfate
(DHEAS), may regulate sebaceous gland activity through its conversion to testosterone and
dihydrotestosterone within the sebaceous gland. The enzymes required to convert DHEAS to
more potent androgens are present within sebaceous glands (Chen et al, 2002). The
predominant isozymes in the sebaceous gland include the type 1 3β-hydroxysteroid
dehydrogenase, the type 2 17β-hydroxysteroid dehydrogenase (17β-HSD) and the type 1 5αreductase (Fritsch et al, 2001; Thiboutot et al, 1995; Thiboutot et al, 1998). Investigations into
the influence of locally-produced androgens indicated that the activities of 5α-reductase and
17β-HSD enzymes within the sebaceous gland are not higher in male or female patients with
acne compared to patient controls with no acne. Due to the small sample size, the influence of
local androgen synthesis can not be ruled out (Thiboutot, et al., 1999). Clearly, androgens
influence sebaceous glands and sebum production, although which androgens are important
and the mechanism of their influence is not known.
Melanocortins include melanocyte-stimulating hormone (MSH) and adrenocorticotropic
hormone (ACTH). In rodents, melanocortins increase sebum production. Human primary
sebocyte cultures treated with MSH have increased numbers of cytoplasmic lipid droplets
(Zhang et al, 2003). Transgenic mice deficient in the melanocortin-5 receptor have hypoplastic
sebaceous glands and reduced sebum production (Chen et al, 1997). The melanocortin-5
receptor has been identified in human sebaceous glands where it may play a role in the
modulation of sebum production (Thiboutot et al, 2000). Further experimentation is required to
test this hypothesis.
Peroxisome proliferator activated receptors (PPARs) are orphan nuclear receptors that
are similar to retinoid receptors in many ways. Each of these receptors form heterodimers with
retinoid X receptors in order to regulate the transcription of genes involved in a variety of
processes, including lipid metabolism and cellular proliferation and differentiation (Kim et al,
2001; Rosen et al, 1999; Schoonjans et al, 1996; Spiegelman et al, 1997). Rat preputial cells
serve as a model for human sebocytes in the laboratory (Laurent et al, 1992). In rat preputial
cells, agonists of the PPARα and PPARγ receptors induced lipid droplet formation in preputial
sebocytes but not in epidermal cells while lineolic acid (PPARβ/δ agonist) induced lipid
formation in both preputial sebocytes and epidermal cells (Rosenfield et al, 1999). Based on
10
the results from their studies, Rosenfield et. al. propose that PPARα activation plays a role in
the beginning stages of lipogenesis, PPARβ/δ activation enhances the lipogenesis and PPARγ
activation controls the transition to a more differentiated state complete with more lipid droplets
within the cells, clearly identifying PPARs as a key player in sebocyte differentiation (Rosenfield,
et al., 1999). Within human sebocytes, PPAR-α, -β/δ, and -γ receptor subtypes are expressed
in basal sebocytes. PPAR-γ is also present in differentiated sebocytes (Chen et al, 2003;
Downie et al, 2004; Trivedi et al, 2006). In patients receiving fibrates (PPAR-α agonists) for
hyperliperdemia or thiazolidinediones (PPAR-γ agonists) for diabetes, sebum secretion rates
are increased (Trivedi, et al., 2006) indicating that PPARs do play a role in sebocyte
differentiation and maturation in humans.
Fibroblast growth factor receptors 1 and 2 (FGFR1, 2) are expressed in the epidermis
and skin appendages. Expression of FGFR3 and FGFR4 are localized to dermal vessels and
microvessels and are notably absent in epidermis and appendages (Hughes, 1997). FGFR2
plays an important role during embroygenesis in skin formation (Li et al, 2001). Germline
mutations in FGFR2 lead to Apert’s syndrome, which is commonly associated with acne. In
addition, somatic mutations in the same location can lead to acne, but how this receptor is
involved in sebaceous gland development and how its mutation leads to acne is unknown
(Gilaberte et al, 2003; Munro and Wilkie, 1998).
The regulation of human sebum production is complex. Advances are being made in
this area which may lead to alternative therapies for the reduction of sebum and improvement in
acne.
1.3 Acne
1.3.1 Epidemiology
Acne is one of the most prevalent skin conditions encountered by dermatologists,
affecting nearly 85% of the people between the ages of 12 and 24 years including 40-50 million
people in the United States each year (Cordrain et al, 2002; White, 1998). Although most
11
prevalent during adolescence, acne also affects infants, prepubescent children, and mature
adults (Layton et al, 2004).
Acne is not life-threatening; however, it does have a significant psychosocial impact.
Embarrassment, low self-esteem, anxiety, anger, frustration, feelings of depression and social
withdrawal may be associated with acne (Baldwin, 2002; Cunliffe, 1986; Dermatology, 2006). A
meta-analysis performed by researchers on behalf of The American Academy of Dermatology
and The Society of Investigative Dermatology demonstrated that the direct cost of acne (cystic
and vulgaris) was $2.5 billion dollars in 2004. Furthermore, adding in the loss of productivity,
unemployment and social impact of acne, the total cost of acne is estimated at $12 billion
dollars per year in the United States (Bickers et al, 2006).
1.3.2 Pathophysiology
Acne is a disease of the pilosebaceous unit. Acne is the culmination of the interaction of
4 distinct factors: 1) increased sebum production 2) increased follicular hyperkeratinization 3)
the activity of Propionibacterium acnes and 4) inflammation (Figure 4). The precise sequence
of events leading to acne is unknown, that is, which factor begins the process is a mystery.
12
Figure 4: Development of acne lesions Follicular hyperkeratinization, increased sebum
production, active P. acnes bacteria, and inflammation all contribute to the development of
acne. Diagram taken from “Fast Facts-Acne” 2004 Health Press Limited.
Very early studies demonstrated that sebum excretion correlates with the severity of
acne, in both males and females (Cotterill et al, 1971). Sebum production begins slowly with
only a few follicles exuding sebum onto the skin’s surface but, sebum production increases
significantly with age and pubertal stage. Furthermore, children who developed acne had higher
sebum productivity than those who did not develop acne (Mourelatos et al, 2007). Increased
sebaceous lipogenesis and sebum excretion does correlate with severity of acne in male
patients (Cooper et al, 1976). Sebum production is stimulated by androgens (Pochi and
Strauss, 1969) and inhibited by 13-cis retinoic acid, corresponding to improvement in acne
(Jones et al, 1980).
Follicular hyperkeratinization plays a role in the pathophysiology of acne. How
hyperkeratinization occurs and contributes to acne is not known, although studies have
investigated proliferative states of keratinocytes and adhesion of keratinocytes. Using the Ki-67
antibody as an indicator of cell proliferation, Knaggs et. al. demonstrated increased Ki-67
staining in normal follicles from acne-affected skin when compared to non-acne-affected skin.
Furthermore, cell proliferation in acne lesions (comedones) was increased compared to normal
follicles (Knaggs et al, 1994). To assess whether hyperkeratinization results from abnormal
13
cohesion between keratinocytes, the distribution of desmosomal components in normal and
acne patients was compared. No differences between these components was detected
between acne lesions (non-inflammatory vs. inflammatory) and between normal epithelium from
control or acne subjects (Knaggs et al, 1994). This study does not rule out the possibility that
other factors may play a role in increased cohesiveness of keratinocytes.
The predominant organism in the follicular flora is the gram positive, anaerobic,
pleomorphic diphtheroid Propionibacterium acnes (P. acnes) although aerobic Staphylococcus
epidermidis may also be present (Marples et al, 1974; Puhvel et al, 1975). The
microenvironment within the sebaceous gland is anaerobic; therefore, favoring the survival of P.
acnes bacteria over others. The P. acnes bacterium relies on sebaceous lipids as a nutrient
source and breaks down triglycerides into free fatty acids (Gribbon et al, 1993). Free fatty acids
within sebum can be irritating and contribute to the inflammatory response (Ro and Dawson,
2005). P. acnes plays an important role in the production of inflammatory acne by stimulating
the classical and alternative complement pathways (Webster, 1979). P. acnes releases a
variety of lytic enzymes and cytokines that are chemotactic for inflammatory cells (Webster,
1982; Webster, 1979). In addition, P. acnes lysates are capable of stimulating the production of
both proinflammatory cytokines/chemokines and anti-microbial peptides from keratinocytes and
cultured sebocytes (SZ95), indicating that keratinocytes and sebocytes, themselves, may play a
role in the pathogenesis of acne (Graham et al, 2004; Nagy et al, 2006). Toll-like receptor
(TLR) activation plays a role in innate immune response (Aderem and Ulevitch, 2000). Studies
have demonstrated increased TLR-2 and TLR-4 expression within acne lesions and have
shown that P. acnes induces TLR-2 and TLR-4 expression in cultured keratinocytes, suggesting
that these receptors are involved in acne-induced inflammation (Jugeau et al, 2005).
Many myths surround the development of acne. Acne is not caused by poor diet, bad
hygiene, or greasy cosmetics. Acne may flare around the time of menses or in times of
increased stress, and is probably the result of hormonal influences (Dermatology, 2006).
1.3.3 Classifications of acne lesions
Acne vulgaris is the most common type of acne and is characterized by both noninflamed and inflamed lesions as well as scaring. Non-inflamed lesions, also known as
14
comedones, are either closed comedones (whiteheads) or open comedones (blackheads).
Closed comedones are classified according to size: microcomedones (1-mm in diameter) or
macrocomedones (>2-mm in diameter). Open comedones are of similar size and are black in
color due to the oxidation of the skin pigment, melanin. Inflamed lesions can be papules,
pustules, or nodules and are red to yellow in color, lasting from 1-3 weeks. Most patients have a
mixture of non-inflamed and inflamed lesions (Layton, et al., 2004).
Other forms of acne include acne conglobata, which is characterized by comedones,
cysts, and abscesses, and acne fulmians, which is characterized by inflamed nodules, cysts and
plaques.
1.3.4 Model systems for acne research-animal
Acne research is limited by the lack of animal models. The only mammal that exhibits
acne is the human. Currently, no one animal model completely mimics all aspects of the human
situation; however, there are a few models that have been used to study the different aspects of
acne.
1.3.4.1 Rat preputial gland
The rat preputial gland is useful to study sebaceous gland biochemistry and regulation of
sebum production. The preputial glands are exocrine glands and pairs of glands are located
near the genitals in both male and female rats. The secretions of the preputial glands are
believed to play a role in both mating behavior (secreting pheromones) and territory marking
(Pietras, 1981; Thody and Dijkstra, 1978). The preputial gland is structured identically to human
sebaceous glands in that acini are connected to a branching duct system and more lipid-laden
cells are present in the center of the gland (Laurent, et al., 1992). Gland size and secretions are
influenced by systemic administration of hormones (androgens, progesterones, estrogens),
which is similar to human sebaceous glands (Alves et al, 1986; Ebling et al, 1971; Ebling et al,
1969; Thody and Dijkstra, 1978).
15
Single cell suspensions can be made by disruption of the rat preputial gland. These
individual cells grow as a monolayer when co-cultured with 3T3 fibroblast cells and are like
sebaceous cells in that they are slow growing and express K4, a sebaceous-specific keratin.
However, under these conditions, preputial cells did not exhibit the characteristic decrease in
proliferation when exposed to retinoic acid (Laurent, et al., 1992). Like the whole preputial
gland, hormone (estrogen) stimulation increases lipogenesis in preputial cells (Alves, et al.,
1986). The preputial cell model system has two clear disadvantages: cells are not responsive to
androgens and the percentage of each individual lipid varies dramatically from those in human
sebum (Alves et al, 1986; Rosenfield, et al., 1999; Thiboutot, 2004).
1.3.4.2 Hamster flank organ and ear
The hamster flank organ (costovertebral gland) is another model to study sebaceous
gland biochemistry and regulation of sebum production. The paired flank organs are located on
the back of golden Syrian hamsters. The organ contains sebaceous glands, hair follicles and
melanocytes which is similar to human skin (Franz et al, 1989). Because the glands are large
(roughly 6-8mm in diameter) and paired, they are extremely useful for investigations of topical
compounds, with the second gland being used as a non-treatment control. Androgen stimulation
increases the size of the flank organs while retinoid stimulation significantly decreases the size
of the flank organs; a response identical to that seen in human sebaceous glands (Ferrari et al,
1978; Gomez, 1981). Not all compounds which inhibit the flank organ have the same effect in
the human sebaceous gland and therefore the usefulness of this model may be limited (Franz,
et al., 1989).
The hamster ear model has been extensively used as a model for human sebaceous
glands. The ventral surface of the hamster ear contains multiple sebaceous glands (size of
gland varies by location within ear) (Matias and Orentreich, 1983). These sebaceous glands
are very similar to human sebaceous glands in that they have similar turn-over rates and size
(Plewig and Luderschmidt, 1977). In addition, hamster ear sebaceous glands are sensitive to
androgens and retinoids (Geiger, 1995; Matias and Orentreich, 1983). However, not all
retinoids which inhibit hamster sebaceous glands are effective in humans. For example,
16
isotretinoin and etritinate are both effective in the hamster ear but only isotretinoin is effective in
humans (Geiger, 1995).
1.3.4.3 Rhino mouse
The rhino mouse model is useful for screening anti-keratinizing agents as well as
comedolytic agents. This mouse model has a recessive mutation on chromosome 14 that
results in a mouse devoid of body hair and having wrinkled skin. The hair follicles become
detached from the underlying dermis and are no longer active, instead becoming filled with
sloughing keratinocytes to create huge numbers of hornfilled utriculi which resemble
comedones (Seiberg et al, 1997). Topical application of salicylic acid, lactic acid, and benzoyl
peroxide had partial de-scaling effects. Application of retinoic acid completely reversed the
excessive scaling and “normalized” the wrinkled phenotype (Kligman and Kligman, 1979).
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Table 1: Comparison of animal models for studying sebaceous glands and acne.
Animal Model System
Useful to Study
Rat Preputial
Glands/Cells
sebaceous gland,
sebum
Hamster Flank Organ
sebaceous gland,
sebum
Hamster Ear
sebaceous gland,
sebum
Rhino Mouse
follicular keratinization
Advantages
GLANDS: androgen
responsive, testing of
systemic compounds
CELLS: grown as
monolayer, express
sebocyte markers
similar morphology to
human sebaceous glands,
other gland can serve as
control, testing of topical
compounds, androgen
and retinoid responsive
multiple sebaceous glands
per ear, other ear can
serve as control, testing of
topical compounds,
androgen and retinoid
responsive
skin contains huge
numbers of horn-filled
utriculi that resemble
comedones, able to test
topical agents that affect
differentiation and lose of
cohesion between
keratinocytes
Disadvantages
sebum composition
is significantly
different from
human sebaceous
glands
not all effective
compounds are
effective in human
sebaceous glands
not all effective
compounds are
effective in human
sebaceous glands
?
1.3.5 Models for acne research: isolated human sebaceous gland organ culture
Human sebaceous glands can be isolated and maintained in culture for 7 days. Guy et.
al. have demonstrated that whole sebaceous glands in culture maintain cell division and
lipogenesis rates as in vivo for up to 7 days. In addition, 13-cis retinoic acid inhibits lipogenesis
as it does in vivo; however, in this system, testosterone did not increase lipogenesis as one
would predict based on the sebaceous glands’ in vivo response in previous studies (Guy et al,
1996; Pochi and Strauss, 1969). The difficulty with this model system is obtaining skin samples,
dissection of sebaceous glands, and the limited amount of time for experimentation.
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1.3.6 Model systems for acne research: sebocyte cell culture
Primary sebocytes are very difficult to grow and maintain in cell culture. In one method,
the epidermal and dermal layers of the skin are separated and the dermis is scraped to obtain
sebocytes which are co-cultured with 3T3 fibroblasts (Doran et al, 1991). In a second method,
whole sebaceous glands are isolated from the surrounding tissue and placed in culture medium.
Primary sebocytes are detected as out-growths from the sebaceous lobules (Abdel-Naser,
2004; Xia et al, 1989). These cells are not highly proliferative, grow in colonies, have limited
sub-culturing capabilities, and express proteins characteristic of differentiated sebocytes even
though they do not completely differentiate as they would in vivo (Xia, et al., 1989; Zouboulis et
al, 1994; Zouboulis et al, 1991). The sebocytes undergo holocrine rupture long before there are
sufficient numbers of cells for experimentation, which limits their usefulness. Despite the
difficulties in obtaining ample numbers of primary sebocytes, they respond to retinoids with
decreased proliferation and decreased lipogenesis (Zouboulis et al, 1991). Primary sebocyte
proliferation is stimulated by androgens and is blocked by spironolactone, an androgen receptor
blocker (Zouboulis et al, 1998).
In order to circumvent the problem of “low yield” of primary sebocytes, immortalized
human sebaceous cell lines have been created by Simian Virus 40 (SV40) large T antigen
immortalization of primary sebocytes: SZ95 and SEB-1 (Thiboutot et al, 2003; Zouboulis et al,
1999). Both of these cell lines 1) express characteristics of differentiated sebocytes; 2) produce
sebocyte specific lipids, wax esters and squalene; 3) are androgen responsive; and 4) exhibit
inhibited proliferation in the presence of retinoids (Nelson et al, 2006; Thiboutot, et al., 2003;
Zouboulis, et al., 1999). The major benefits of these cell lines are 1) enough cells can be
obtained for repeated experimentation and 2) investigators can examine sebocyte-specific
responses.
19
Table 2: Comparisons of sebocyte cell culture models
Cell Culture Model System
Primary Sebocytes
Immortalized Sebocyte Cell
Lines (SZ95, SEB-1)
Useful to
Study
sebaceous
gland
regulation,
sebum
production
sebaceous
gland
regulation,
sebum
production
Advantages
Disadvantages
maintains characteristics of
sebocytes in vivo, retinoid
and androgen responsive
low sebocyte
numbers, difficult to
obtain
sufficient numbers of cells
for experiments;
responsive to androgens,
retinoids; produce
triglycerides, wax esters
and squalene; express
sebocyte specific markers
unable to completely
terminally
differentiate; SV40
large T antigen
interferes with
"normal" growth and
differentiation
1.3.7 Current Treatments for Acne
There are numerous over-the-counter soaps, washes and preparations as well as
dermatologist-prescribed drugs available to treat mild to severe acne. For the purposes of this
thesis, I will discuss the mechanism of action of each major category in the treatment of acne.
1.3.7.1 Cleansers: follicular hyperkeratinization
Body washes and facial cleansers containing hydroxy acids and benzoyl peroxide have
anti-acne properties. These products are available as soaps, creams, gels, washes, lotions,
scrubs and peels. α-hydroxy acids, including lactic acid and glycolic acid, are water soluble and
penetrate the dermis. β-hydroxy acids, like salicylic acid, are lipid soluble and penetrate the
upper epidermis and pilosebaceous unit. Both acid forms decrease cohesion amongst
keratinocytes and cause exfoliation, helping remove the “keratinocyte plug” (Davies and Marks,
1976; Van Scott and Yu, 1984). Benzoyl peroxide works by an anti-bacterial effect in the
treatment of acne, thereby decreasing the numbers of P. acnes.
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1.3.7.2 Antibiotics: P. acnes and inflammation
Oral antibiotics function to reduce acne by inhibiting protein synthesis and proliferation of
P. acnes. Trimethoprim inhibits the enzyme dihydrofolate reductase, resulting in the inhibition of
tetrahydrofolic acid synthesis, a key precursor in DNA purine and pyrimidine synthesis. This
inhibition interferes with P. acnes proliferation. Antibiotics of the tetracycline family inhibit protein
synthesis by binding near the A-site on the 30S bacterial ribosomal subunit thus interfering with
the binding of the amino-acid charged tRNA required for mRNA translation (Spahn and
Prescott, 1996; Wirmer and Westhof, 2006). Additionally, tetracycline family members,
doxycycline and minocycline, may exhibit some anti-inflammatory properties by decreasing
each of the following: lipase production by P. acnes, cytokine production, white blood cell
chemotaxis and activity of matrix metalloproteinases (Higaki et al, 2004; Leyden, 2001; Li et al,
2006).
Topical antibiotics such as erythromycin and clindamycin are effective in reducing the
number of inflammatory lesions and inhibiting P. acnes proliferation They are available in many
formulations. Antibiotics within the macrolide family work similarly to tetracyline family members,
except that they irreversibly bind the 50S ribosomal subunit inhibiting translocation of peptidyl
tRNA and interfering with protein synthesis. Erythromycin and clindamycin may also be given
orally to treat acne.
1.3.7.3 Hormonal therapy: sebum suppression
Hormonal therapy, which functions to decrease sebum production, is available for
female patients and is particularly helpful in those with “acne flares” around the time of menses.
Anti-androgens, oral contraceptives and glucocorticoids are all types of hormonal therapy
(Thiboutot and Chen, 2003).
Anti-androgens function as androgen receptor blockers and include spironolactone,
flutamide and cyproterone acetate. Spironolactone (aldosterone antagonist) is a steroidal
androgen receptor blocker which has been used for over 20 years for the treatment of acne and
hirsutism (Shaw and White, 2002; Yemisci et al, 2005). This drug decreases sebum production
and inhibits type 2 17β-hydroxysteroid dehydrogenase, thereby inhibiting the conversion of
21
androstenedione to testosterone (Tremblay et al, 1999; Zouboulis et al, 1994). Additional
indirect mechanisms include the inhibition of 5α−reductase and elevation of steroid hormone
binding globulin (Archer and Chang, 2004). Cyproterone acetate, a progestin, acts as an
androgen receptor blocker and inhibits ovulation. Its effectiveness in the treatment of acne
results from its ability to reduce sebum production and perhaps comedogenesis (Stewart et al,
1986). Although not approved in the U.S., it is approved in Canada, Europe, and Asia for the
treatment of severe acne that is resistant to traditional therapy. Flutamide is a non-steroidal
androgen receptor blocker effective in the treatment of prostate cancer (Harper, 2006). It is
converted to a potent metabolite, 2-hydroxyflutamide, which inhibits the binding of
dihydrotestosterone (DHT) to androgen receptors (Brogden and Clissold, 1989).
Oral contraceptives are combinations of two agents: an estrogen (most commonly
ethinyl estradiol) and a progestin. Oral contraceptives also suppress ovulation, which results in
a decrease in the ovarian production of androgens and subsequent decrease sebum
production. Within the sebaceous gland, estrogen receptors (ERα and ERβ) are expressed in
both basal and differentiating sebocytes (Pelletier and Ren, 2004; Thornton et al, 2003). The
most active estrogen is estradiol, which is produced from testosterone by the action of the
enzyme aromatase. Aromatase is active in the ovary, adipose tissue and other peripheral
tissues. Estradiol can be converted to the less potent estrogen, estrone, by the action of 17β
hydroxysteroid dehydrogenase (17βHSD) enzyme. Both aromatase and 17βHSD are present in
the skin (Hay et al, 1982; Sawaya and Price, 1997). Estrogens regulate cell activities by binding
to and activating estrogen receptors.
Like estrogens, progestins act through nuclear hormone receptors triggering activation
or suppression of progesterone target genes leading to a cell-specific response. In the
sebaceous gland, progesterone receptor B is expressed in both basal and differentiating
sebocytes (Kariya, et al., 2005). First and second generation progestins can react with the
androgen receptor, thus aggravating acne, hirsutism, and androgenic alopecia (Thiboutot,
2000). Newer third generation progestins are more selective for the progesterone receptor than
the androgen receptor. In addition, progesterone has been shown to modulate pro-inflammatory
and anti-inflammatory cytokines (Davies et al, 2004). Whether progesterone receptor activation
can lead to decreased inflammation in sebaceous glands is not yet known.
Currently in the United States, there are only two oral contraceptives approved for use in
the treatment of acne: 35 µg ethinyl estradiol/norgestimate (Ortho Tri-Cyclen® Ortho, Raritan,
NJ) or 20-35 µg ethinyl estradiol/norethindrone acetate (Estrostep®, Parke Davis, Detroit, MI).
22
Glucocorticoids block androgen production by the adrenal gland and prednisone is the
preferred agent due to the increased risk of adrenal suppression with dexamethasone
(Thiboutot and Chen, 2003).
1.3.7.4 Topical Retinoids: inflammation, follicular hyperkeratinization
Topical retinoids are key in the treatment of acne due to their ability to inhibit the
formation of microcomedones. Tretinoin, tazarotene and adapalene are topical retinoids that are
currently available for the treatment of acne and are useful for inflammatory and noninflammatory acne. Tretinoin, the acid form of vitamin A (all-trans retinoic acid, (ATRA) Retin
A®), is the original topical retinoid discovered by Albert Kligman in 1967 (Kligman et al, 1969)
and is available in multiple formulations. It functions by binding retinoid nuclear receptors:
retinoic acid receptors (RAR α,β,γ) and rexinoid receptors (RXR α,β, γ) and modulating gene
expression. The mechanism of action of retinoids will be discussed in great detail in section 1.4.
Tretinoin use is associated with skin irritation including redness, burning, and excessive dryness
and peeling, which decreases with long-term use. Interestingly, tretinoin is also effective against
wrinkles.
Tazarotene (synthetic retinoid) and adapalene (naphthoic acid derivative), RARβ and
RARγ agonists, are effective against inflammatory and non-inflammatory acne similar to
tretinoin; although, the typical side effects are diminished.
1.3.7.5 Oral Retinoid: Isotretinoin (Accutane®, 13-cis Retinoic Acid)
Isotretinoin was originally prescribed for and effective in the treatment of lamellar
ichthyosis, a hereditary disorder of keratinization causing excessive scaling of the skin (Peck
and Yoder, 1976). Dr. Peck noticed a very positive side effect of this drug. In the subset of his
patients with acne who were receiving isotretinoin for ichthyosis, their acne showed dramatic
improvement (Peck, 1979). On this observation, isotretinoin was pushed into patient clinical
trials for acne treatment.
Isotretinoin (13-cis RA), marketed under the trade name of Accutane® by Hoffman-La
Roche, was approved by the U.S. Food and Drug Administration (FDA) in 1982 for the
23
treatment of recalcitrant nodular cystic acne. 13-cis RA is prescribed in cases where acne is
severe, non-responsive to other treatments, or when physical scarring is present. Patients
receive doses of 0.5mg/kg/day-2mg/kg/day; with the dose depending on age, gender and body
weight. The course of treatment lasts approximately 16-20 weeks. In rare cases, a second
course of treatment is needed. Most patients report minor side effects including chapped lips,
dry eyes, and generally dry skin.
Isotretinoin is a potent teratogen and, as such, two forms of contraception and monthly
negative pregnancy tests are required of females of child-bearing age. Children exposed to 13cis RA in utero have severe hindbrain and forebrain malformations, limb malformations,
craniofacial and cardiovascular defects. If the fetus is not spontaneously aborted, children who
survive are afflicted with serious motor and sensory delays and mental retardation (Lammer et
al, 1985; Lammer et al, 1985).
In April 2002, Hoffman-La Roche implemented the “System to Manage Accutane Related
Teratogenicity (S.M.A.R.T)” program, aimed at preventing pregnant women from receiving
isotretinoin. (Roche-Laboratories, 2001) As of March 1, 2006, isotretinoin use is restricted by
iPLEDGE, an FDA-mandated risk management program that seeks to limit fetal exposure to
isotretinoin. This program requires registration of patients, prescribing dermatologists and
pharmacists. For more information, please visit https://www.ipledge.com.
Isotretinoin belongs to the class of drugs known as retinoids, which includes all naturally
occurring and synthetic derivatives of vitamin A. In regards to acne, experimental evidence
supports the fact that 13-cis RA can influence each of the factors involved in the pathogenesis
of acne: 1) follicular hyperkeratinization, 2) bacterial colonization of the follicle, 3) inflammation
and 4) sebum production.
Isotretinoin decreases follicular keratinization by approximately 50%. (Plewig et al, 2004)
How this is accomplished is unknown; although, it is known that isotretinoin does not affect the
metabolic activity of the keratinocytes in the follicular duct epithelium or interfollicular epidermis
(Dalziel et al, 1987). Isotretinoin therapy causes a significant reduction in the gram positive,
anaerobic Propionibacterium acnes bacteria, including antibiotic resistant strains, with levels of
bacteria slowly returning to baseline after discontinuing treatment (Coates et al, 2005; Leyden et
al, 1986). It is not known how this reduction is achieved, however it may have a(n) 1) direct
killing effect on P. acnes, 2) indirect effect by decreasing sebum production, thereby removing
the food supply, or 3) increasing the host’s defense mechanisms. In support of the latter,
24
retinoic acid supplementation has been shown to “prime” the immune system to protect against
bacterial lipopolysaccharide (LPS) challenges in rats (Seguin-Devaux et al, 2005).
13-cis RA has been shown to competitively inhibit the 3α-hydroxysteriod activity of
retinol dehydrogenase, leading to decreased androgen synthesis (Karlsson et al, 2003). In
addition, it inhibits the migration of polymorphonuclear leukocytes and monocytes into the skin,
supporting its role in reducing the inflammation that is associated with acne (Norris et al, 1987;
Wozel et al, 1991). The classical and alternative complement activation pathways are
stimulated by P. acnes, possibly contributing to the inflammatory response (Webster, 1979). P.
acnes releases a variety of lytic enzymes and pro-inflammatory substances that are chemotactic
for inflammatory cells (Webster, 1979). With the reduction of P. acnes from isotretinoin
treatment, inflammation is likely to diminish.
The majority of studies have examined the sebosuppressive effect of isotretinoin. Yet,
how this sebosuppression is achieved is poorly understood. It is well established that
isotretinoin drastically reduces the size and lipid secretion of sebaceous glands in human and
animal models, in in vitro cell cultures of human sebocytes and in immortalized sebocyte cell
lines, SZ95 and SEB-1 (Goldstein et al, 1982; Gomez and Moskowitz, 1980; Landthaler et al,
1980; Nelson, et al., 2006; Strauss et al, 1980; Zouboulis, et al., 1991; Zouboulis, et al., 1999;
Zouboulis et al, 1991). Processes such as cell cycle arrest or apoptosis may explain the
histological data in human skin biopsies that demonstrate a drastic decrease in the size, shape,
and lipid content of sebaceous glands after 16 weeks of treatment with isotretinoin (Goldstein, et
al., 1982). It seems likely that the decrease in sebum production may be the net result of
sebaceous gland involution in response to isotretinoin treatment rather than its direct target.
1.4 Retinoids
There are no less than 4000 currently published review articles on retinoid biology and
retinoid therapy for various diseases. The field of retinoid research is so vast, that I will give an
overview of retinoid biology followed by a description of retinoid effects.
25
1.4.1 Retinoid Biology
1.4.1.1 What are retinoids?
In the simplest definition: retinoids are vitamin A (retinol) or natural or synthetic vitamin
A-like derivatives. Structurally, retinoid molecules consist of a cyclic end group, either
cyclohexenyl ring or aromatic ring, attached to a polyene chain, and ending in a polar group.
Examples of retinoid structures are shown in Figure 5. Derivatives of the oxidized form of retinol,
retinoic acid, have been extensively synthesized and to date, over 5000 retinoid compounds
have been produced (Dawson and Hobbs, 1994; Roos et al, 1998). Vitamin A and its retinoid
derivatives are lipid soluble molecules that can readily transverse the lipid bilayer of the plasma
membrane. The classic view of retinoid functionality is that retinoids affect cellular functions by
binding nuclear hormone receptors and affecting gene expression (Giguere et al, 1987;
Mangelsdorf et al, 1995). However, within the cancer field, more attention has been focused on
retinoid receptor-independent effects within the cell, such as modulation of signal transduction
kinase cascades including mitogen-activated protein kinase (MAPK) pathways: extracellular
signal-related-kinases (ERKs), p38MAPK, and stress activated protein kinase/c-Jun N terminal
kinase (SAPK/JNK) (Nakagawa et al, 2003; Olson and Hallahan, 2004; Pettersson et al, 2004).
26
Figure 5: Examples of the chemical structure of retinoids. Figure modified from Roos, T.C.,
Jugert, F. K., Merk, H. F., Bickers, D. R. Retinoid Metabolism in Skin. (1998) Pharmacological Reviews
50(2): 315-333.
1.4.1.2 Retinoid receptors
Retinoid receptors belong to the superfamily of nuclear hormone receptors. This
superfamily is subdivided into two groups: steroid nuclear hormone receptors, which include
androgen and estrogen receptors; or the non-steroid receptors, which include the thyroid,
vitamin D and retinoid receptors (Mangelsdorf, et al., 1995). Retinoids exert their specific
cellular effects through activation of retinoic acid or retinoid X nuclear receptors (RARs, RXRs).
There are three isoforms of the RAR receptor designated RARα, RARβ and RARγ, encoded by
three separate genes located on chromosomes 17p21.1, 3p24 and 12q17, respectively (Brand
et al, 1988; Mattei et al, 1988). Like RARs, three isoforms of RXRs (RXRα, RXRβ and RXRγ)
have been identified and each is encoded by separate a gene located on chromosomes 9q34.3,
27
6q21.3 and 1q22, respectively (Hoopes et al, 1992; Mangelsdorf et al, 1990; Yu et al, 1991).
Regardless of the type of retinoid receptor, all have two distinct functional domains: a DNA
binding domain and a ligand binding domain (Mangelsdorf, 1994; Reichel and Jacob, 1993).
Retinoid receptors form heterodimers or homodimers and bind to cis-acting DNA
elements in the genome. The retinoic acid receptor consensus sequence (RARE) is a direct
repeat (DR) of the half-site AGTTCA separated by 2 or 5 nucleotides (DR-2, DR-5) and is
recognized by RAR-RXR heterodimers. For RXR homodimers, the retinoid X receptor
consensus sequence (RXRE) is a DR-1 of AGGTCA (Mangelsdorf, et al., 1995). RARs form
heterodimers with RXR isoforms while RXRs are promiscuous forming heterodimers with other
nuclear receptor family members including vitamin D receptor (VDR), peroxisome proliferator
activated receptor (PPAR), and thyroid receptor (TR) in addition to forming homodimers with
itself (Mangelsdorf, et al., 1995). Currently, experimental evidence supports the view that, in the
absence of a retinoid ligand, dimerized receptors repress gene transcription by interacting with
co-repressor molecules. Upon ligand binding, this repression is released and gene transcription
occurs (Minucci and Pelicci, 1999). Retinoid responsive genes with no obvious RARE or RXRE
elements in their promoters, usually require de novo protein synthesis to induce their expression
and, as such, show slower kinetics of expression. These genes are referred to as ‘delayed
retinoid response genes’ (Arany et al, 2002; Chen et al, 2001).
The retinoid receptors have different affinities for retinoid ligands. For the purpose of this
thesis, I will focus on all-trans retinoic acid (ATRA), 9-cis retinoic acid (9-cis RA) and 13-cis
retinoic acid (13-cis RA). The RARs recognize both ATRA and 9-cis RA with similar affinity (Kd ~
1nM). The RXRs exclusively bind to 9-cis RA with affinities ranging from 14-18 nM depending
on the receptor isoform (Allenby et al, 1993). 9-cis RA has a higher affinity for RAR than RXR
receptors. Competitive binding assays with radio-labeled 9-cis RA and 50 µM 13-cis RA indicate
that 13-cis RA does not bind RXRs; however, some experiments show that 13-cis RA can bind
to RARs, specifically RARγ, with low affinity (Allenby, et al., 1993; Idres et al, 2002). In addition
to the RARs and RXRs that have defined ligands and interactions, there are also RAR-related
orphan receptors (RORa,b,c, etc.) that, as of yet, have no identified biological ligands.
The vast majority of tissues express at least one RAR and RXR receptor. Expression of
the multiple RAR and RXR isotypes varies by tissue and stage of development suggesting that
each receptor performs some unique functions; although, some functional redundancy has also
been identified (for Review see (Chambon, 1994; Chiba et al, 1997; Krezel et al, 1998). Skin
expresses all 6 isoforms of the RAR and RXR receptors, with RARγ and RXRα the predominant
28
isoforms (Boehm et al, 2004; Chakravarti et al, 2006; Elder et al, 1992; Roos, et al., 1998).
Within the sebaceous gland, expression of RARβ and RXRα has been detected (Boehm, et al.,
2004; Downie, et al., 2004).
.
1.4.1.3 Retinoid binding proteins and retinoid metabolizing enzymes
In addition to binding to retinoid receptors, the functions of retinoic acids are also
controlled by retinoid binding proteins (RBP). Numerous retinoid binding proteins have been
identified and are classified as extracellular binding proteins (lipocalins) or intracellular, cytosolic
binding proteins. Each RBP demonstrates ligand-binding specificity (Table 3). RBPs are
involved in retinoid absorption, transport within the blood and regulating levels of “free” retinoids
(Napoli, 1996). Cellular Retinoic Acid Binding Protein I and II (CRABP I, II) have been identified
in human skin and bind to both 9-cis RA and ATRA but neither binds to 13-cis RA (Napoli, 1996;
Roos, et al., 1998; Rosdahl et al, 1997).
29
Table 3: Retinoid binding proteins with ligand identification. Table taken from Napoli, JL.
Biochemical pathways of retinoid transport, metabolism, and signal transduction. (1996) Clinical
Immunology and Immunopathology. 80(3):S52-62
Class/Protein
MW
(kDa)
Primary ligands
Loci
Prospective function
Extracellular lipid-binding proteins (lipocalins)
RBP
21
Retinol
Serum
Retinol transporter
β-lactoglobulin
18.3
Retinol?
Milk
Retinol transporter?
E-RABP
18.5
RA = 9cRA
Epididymis
RA/9cRA transporter
Intracellular lipid-binding proteins
holo: substrate for LRAT and
RoDH
apo: stimulates REH; inhibits
LRAT
CRBP
14.6
Retinol » retinal
Many (e.g., liver,
kidney, testis)
CRBP(II)
14.6
Retinol = retinal
Intestine
holo: substrates for LRAT and
retinal reductase
CRABP
15
RA » 9cRA >
13cRA » 9,13cRA
Many (e.g.,
testis, lung,
kidney)
holo: substrate for RA
metabolism; sequesters RA
and possibly RA metabolites
CRABP(II)
15.7
Same as for CRABP but with
different affinities for RAs?
CRALBP
33
IRBP
145
RA » 9cRA > 9cRA
Adult Skin,
» 9,13cRA
Embryo
Others
RPE (retinal
11-cis-retinal, 11pigment
cis-retinol
epithelium)
Retinol, many
others
Protects retinoids from
isomerization
Lipid transporter
Retinaldehyde dehydrogenases (RALDH) and cytochrome p450 (CYP)-dependent 4hydroylases also play a role in regulating the activity of retinoids within cells. Retinaldehyde
dehydrogenase converts retinaldehyde to the active retinoic acid form. Within the central
nervous system RALDH2 expression is critical to generating the gradient of retinoic acid
expression required for hindbrain patterning (Glover et al, 2006). To date, characterization of
RALDH isoforms in human skin has not been done. CYP-dependent 4-hydroxylases convert
retinoic acid forms to their 4-oxo-retinoic acid and 4-hydroxy-retinoic forms, which are more
likely to be secreted from cells, shutting down the retinoid activity (Ramp et al, 1994; Williams
and Napoli, 1985). However, it is known that these 4-oxo-RA products are themselves, capable
of gene activation in human skin (Baron et al, 2005).
30
1.4.1.4 Retinoid Function
Retinoids have been called the master regulators of differentiation and play critical roles
in development (McCaffery and Drager, 2000). Retinoic acid is essential for both embryonic
and adult growth. Retinoids control patterning of the central nervous system influencing
development of the hindbrain and spinal cord; the development of the cardiovascular system;
development of the kidney, eye, ear; and the olfactory pathway, among others (Figure 6)
(Glover, et al., 2006; Hyatt and Dowling, 1997; LaMantia et al, 2000; Maden, 2006; Mendelsohn
et al, 1999; Mollard et al, 2000; Romand et al, 2006; Vermot et al, 2003). In short, retinoids
control the processes of proliferation, differentiation and apoptosis throughout an organism’s
life. Several studies indicate that the effects of retinoids on cell proliferation, differentiation and
apoptosis are retinoid-specific and cell-type specific.
Figure 6: Functions involving retinoids Figure taken from Napoli, JL. Biochemical pathways
of retinoid transport, metabolism, and signal transduction. (1996) Clinical Immunology and
Immunopathology. 80(3):S52-62
1.4.1.4.1 Proliferation
Progression through the cell cycle, from the Gap1 (or growth phase 1) to mitosis (M), is
tightly regulated by the levels and activity of specialized groups of proteins known as cyclins,
31
cyclin-dependent kinases (CDK) and cyclin-dependent kinase inhibitors (CDKI) (Figure 7)
(Golias et al, 2004).
Retinoids influence each one of these groups of proteins. For example, retinoic acid
induces growth arrest in myeloid cell lines by up-regulation of p21/CIP1 and p27/KIP1 (CDKIs);
down-regulation of cyclin E and cyclin D1/D3, cyclin A and cyclin B; decreased CDK activity;
and de-phosphorylation of pRb (Dimberg and Oberg, 2003). In EBV-immortalized B
lymphocytes, ATRA-, 9-cis RA- and 13-cis RA- triggered growth arrest is associated with
multiple changes in G1 regulatory proteins including decreased activity of CDK2, CDK4 and
CDK6; decreased levels of cyclin D3 and cyclin A; as well as increased expression of p27/KIP1
(Zancai et al, 1998).
Figure 7: Simplified view of cell cycle control with focus on G1 and S phase of the cell
cycle. Diagram from http://www.scielo.br/img/fbpe/rimtsp/v44n1/a07fig03.gif
Within the sebaceous gland, 13-cis RA has been shown to decrease proliferation of
sebocytes as evidenced by decreased 3H-thymidine labeling after 12-weeks of treatment when
compared to baseline (Landthaler, et al., 1980). No studies to date have examined the nature
of this decrease in proliferation.
32
1.4.1.4.2 Differentiation
In the simplest definition, differentiation is a series of biochemical and structural changes
by which cells become specialized in form and function. The most studied example of retinoids
controlling differentiation is the patterning and formation of the hindbrain during embryonic
development (for Review, (Glover, et al., 2006)). In this case, the timing of and the gradient
expression of retinoic acid determines the antero-posterior as well as the dorso-ventral axes of
the hindbrain (Avantaggiato et al, 1996; Glover, et al., 2006). Excess retinoic acid in the
anterior region results in a “posteriorizing” of the hindbrain whereas decreased retinoic acid in
the posterior region results in an “anteriorizing” of the hindbrain (Glover, et al., 2006; Thompson
et al, 1969), which upsets the normal developmental patterns leading to deleterious defects. In
addition, the gradient of retinoic acid expression influences the expression of the Hox genes,
which are critical in rhombomere segment determination (Simeone et al, 1990; Wilkinson et al,
1989; Wood et al, 1994). Furthermore, the pattern formation of the hindbrain is specific to the
type of retinoid (i.e.: retinoic acid (ATRA)). Exogenous administration of 9-cis RA in zebrafish
has a more pronounced effect than ATRA in “posteriorizing” the hindbrain (Zhang et al, 1996).
This central nervous system example illustrates the fact that the amount of retinoic acid and the
specific isoform of retinoic acid present can have profound effects on differentiation.
Retinoids also influence differentiation in skin. The earliest studies noted that deficiency
of vitamin A in the diet of laboratory animals led to changes in the normal epithelium, with
specific loss of the mucous secretory epithelium, while replenishing the vitamin A restored the
normal phenotype (Wolbach and Howe, 1925). Retinoids inhibit the differentiation of
keratinocytes as evidenced by decreased keratin 1, keratin 10, transglutaminase, loricrin, and
filaggrin expression (Fisher and Voorhees, 1996). De Luca et al. have studied the effects of
retinoids in mouse endocervical epithelia undergoing squamous metaplasia as a result of
retinoid deficiency. Their studies show that vitamin A deficiency causes a simple-columnar
epithelium to gradually become squamous metaplasia and that vitamin A concentration is a
factor in maintaining a simple or more stratified epithelial morphology (De Luca et al, 1995). In
addition to retinoid effects on epithelium, it can also influence the dermis. For example, it
modulates the expression of the genes for hyaluronate and collagen, two major constituents of
the dermis. It increases their expression, synthesis and concentration in the skin, helping to
reduce wrinkle formation (Sorg et al, 2005). Within the sebaceous gland, retinoids inhibit
33
differentiation as determined by decreases in sebum secretion with 13-cis RA treatment
(Strauss, et al., 1980).
1.4.1.4.3 Apoptosis
The definition of apoptosis is based on morphological characteristics. Apoptosis is a
highly-regulated, well-orchestrated series of events culminating in nuclear condensation, DNA
fragmentation, membrane-blebbing, cell shrinkage, and eventually phagocytosis of the dying
cell (Wyllie et al, 1980). Two pathways leading to apoptosis have been well characterized:
death receptor (extrinsic) and mitochondrial (intrinsic) apoptotic pathways. These two pathways
converge with activation of caspase 3 and, in some cells, by activation of the protein Bid, a Bcl-2
family member.
Death receptors on the cell’s surface detect extracellular stimuli and upon binding of
their respective ligands, rapidly activate an intracellular caspase signaling cascade that results
in apoptosis. The ligands for death receptors include tumor necrosis factor related apoptosisinducing ligand (TRAIL) and Fas ligand (FasL, CD95L). All death receptors including Fas
(CD95) and TRAIL-R1/R2 have an intracellular region termed the “death domain” (DD). It is this
specific 80 amino acid sequence that allows the transmission of the apoptotic signal. DDs in the
receptor recruit intracellular adaptor molecules (also containing DDs) that have “death effector
domains” (DEDs). DEDs recruit and activate the “initiator” caspases 8 and 10 by cleavage.
Initiator caspases proceed to activate “effector” caspases 3, 6, and 7, which by cleavage of their
specific substrates (ie: PARP, α-fodrin) result in apoptosis of the cell. For example, TRAILmediated apoptosis is initiated by binding of TRAIL to a cell surface receptor, TRAILR1 (DR4) or
TRAILR2 (DR5), which then recruits caspase 8 via the adaptor molecules, TNF-R1 associated
death domain protein (TRADD) and Fas-associated death domain (FADD). Activated caspase 8
directly activates caspase 3, caspase 6, or caspase 7 or activates the intrinsic apoptosis
pathway via cleavage and activation of Bid (Figure 8) (Slee et al, 1999; Smith et al, 2003; Wehrli
et al, 2000).
The mitochondrial apoptotic pathway is activated by intracellular damage sensed by the
mitochondria itself resulting in permeability of the outer mitochondrial membrane and release of
cytochrome c. When cytochrome c is released into the cytoplasm, it binds to apoptotic protease
activating factor 1 (APAF-1) and through a conformational change, caspase 9 is recruited to this
34
complex and is activated. Caspase 9 (initiator caspase) is able to cleave caspase 3 causing
further activation of the signaling cascade. The Bcl-2 (B cell lymphoma) family of proteins is
intricately involved in intrinsic apoptosis by controlling the mitochondria permeability transition.
This family contains both pro-apoptotic and pro-survival proteins. The delicate balance between
these two opposing forces is important to whether or not a cell undergoes apoptosis (Figure 8)
(Green and Kroemer, 2005; Lucken-Ardjomande and Martinou, 2005).
Figure 8: Diagram of extrinsic and intrinsic apoptosis pathways. Type 1: extrinsic (death
receptor) pathway. Type II: intrinsic (mitochondrial) pathway. Figure modified from I. Petak, J.A.
Houghton. Pathology Oncology Research, Vol 7(2), 95-106, 2001.
It is well established that retinoids induce apoptosis in numerous cell types, both normal
cells and tumor cell lines. For example, 13-cis RA reduces the survival and genesis of murine
hippocampal neurons in vivo (Crandall et al, 2004; Sakai et al, 2004). ATRA has been shown to
35
induce apoptosis in primary and metastatic melanoma cells as well as inducing growth arrest
followed by apoptosis in orbital fibroblasts isolated from Graves’ disease patients (Pasquali et
al, 2003; Zhang and Rosdahl, 2004). In leukemia cells, 9-cis RA inhibited cell growth and
induced apoptosis to a greater extent than 13-cis RA or ATRA; however, in adult T cell leukemia
cells, all three retinoids were equally effective (Fujimura et al, 2003; Koistinen et al, 2002).
These studies are only a few of many in the literature that demonstrate that the actions of
retinoids are unique and specific to the model used.
Natural and synthetic retinoids induce apoptosis by activation of the extrinsic or intrinsic
pathways or simultaneous activation of both pathways in a retinoid- and cell-type specific
manner. Examples of retinoids and their mechanisms of apoptosis induction are listed in
Table 4. The cellular targets of retinoids leading to activation of the cell death pathways are just
beginning to be revealed.
Table 4: Retinoid induced apoptotic mechanisms
Retinoid
Receptor
Agonist
Mechanism
ATRA
RAR
↓ Bcl-2, Bax, survivin, mitochondrial
membrane potential
(Fujimura, et al., 2003)
(Pratt et al, 2006)
9-cis RA
RAR, RXR
↓ Bcl-2; Nur77/RXR nucleo-mitochondiral
translocation
(Fujimura, et al., 2003)
(Lee et al, 2005)
Bexarotene
RXR
↑ TRAIL, transglutaminase
Fenretinide
non-classical
↑reactive oxygen species, ceramide induction
(Boehm et al,
1995),(Altucci et al,
2004)
(Wu et al, 2001)
AGN193198
non-classical
↑ caspase activity
(Keedwell et al, 2004)
CD 437
non-classical
MX3550-1
non-classical
13-cis RA
?
↑caspase; Nur77/RXR nucleo-mitochondiral
translocation
↑ caspase activity ↓ mitochondrial membrane
potential
↓ Bcl-xL, Bcl-2, mitochondrial membrane
potential; ↑ Bax, cleaved caspase 8
(Zhang, 2007)
(Chun et al, 2005)
(Fujimura, et al., 2003)
(Arce et al, 2005;
Rigobello et al, 1999;
Tosi et al, 1999)
36
1.5 Retinoids in dermatology
The use of retinoids in dermatology dates back to 1925 when abnormal keratinization
was noticed in vitamin A-deficient animals by Wolbach and Howe (Wolbach and Howe, 1925).
Because of their lipophilic properties as well as their abilities to affect proliferation and
differentiation, retinoids have been prescribed for numerous skin conditions including ichthyosis,
psoriasis, age spots, some skin cancers and acne.
1.5.1 Significance of research project
Isotretinoin, a known teratogen, is the second drug in the United States (after
thalidomide) to have its use, as of March 1, 2006, become restricted within an FDA-mandated
registry system, iPLEDGE, involving patients, physicians, pharmacies and wholesalers. With
these new restrictions and the drug’s potent teratogenicity, it is extremely important to develop
additional treatments for acne. Progress in this area has been hampered by a lack of
understanding of the mechanism of action of 13-cis RA in the sebaceous gland. Elucidating the
cellular processes, and possible cellular pathways, that are affected by 13-cis RA in sebocytes
is a step toward understanding the overall molecular mechanism of action of this drug, which
may lead to the identification of alternative strategies for the treatment of acne.
Chapter 2
13-cis Retinoic Acid Induces Apoptosis and Cell Cycle Arrest in Human SEB-1 Sebocytes
AM Nelson, KL Gilliland, Z Cong, DM Thiboutot.
Journal of Investigative Dermatology (2006) 126: 2178-2189
2.1 Chapter Abstract
Isotretinoin (13-cis Retinoic Acid) is the most potent inhibitor of sebum production, a key
component in the pathophysiology of acne, yet its mechanism of action remains largely
unknown. The effects of 13-cis retinoic acid, 9-cis retinoic acid, and all-trans retinoic acid on cell
proliferation, apoptosis, and cell cycle proteins were examined in SEB-1 sebocytes and
keratinocytes. 13-cis retinoic acid causes significant dose-dependent and time-dependent
decreases in viable SEB-1 sebocytes. A portion of this decrease can be attributed to cell cycle
arrest as evidenced by decreased DNA synthesis, increased p21 protein expression, and
decreased cyclin D1. Although not previously demonstrated in sebocytes, we report that 13-cis
RA induces apoptosis in SEB-1 sebocytes as shown by increased Annexin V- FITC staining,
increased TUNEL staining, and increased cleaved caspase 3 protein. Furthermore, the ability
of 13-cis retinoic acid to induce apoptosis cannot be recapitulated by 9-cis retinoic acid or alltrans retinoic acid, and it is not inhibited by the presence of a retinoid acid receptor (RAR) panantagonist AGN 193109. Taken together these data indicate that 13-cis RA causes cell cycle
arrest and induces apoptosis in SEB-1 sebocytes by a retinoid acid receptor (RAR) independent
mechanism, which contributes to its sebosuppressive effect and the resolution of acne.
38
2.2 Introduction
Isotretinoin (13-cis Retinoic Acid, (13-cis RA)) is the most potent inhibitor of sebum
production, a key component in the pathophysiology of acne. It is the only retinoid that
dramatically reduces the size and secretion of sebaceous glands (Goldstein et al, 1982;
Landthaler, et al., 1980; Strauss, et al., 1980). Despite the fact that isotretinoin is extremely
effective against acne, surprisingly little is known regarding its molecular mechanism of action;
although, advances are being made in this area. This unique retinoid has been shown to
competitively inhibit the 3α-hydroxysteroid activity of retinol dehydrogenase leading to
decreased androgen synthesis in vitro as well as inhibit the migration of polymorphonuclear
leukocytes into the skin supporting its role in the reduction of inflammation that is associated
with acne (Karlsson, et al., 2003; Wozel, et al., 1991).
Numerous studies indicate that 13-cis RA and other retinoids affect cell cycle
progression, differentiation, apoptosis and cell survival in a variety of cell types including human
breast cancers, oral squamous cell carcinomas, lymphocytes and murine neurons (Cariati et al,
2000; Crandall, et al., 2004; Giannini et al, 1997; Pomponi et al, 1996; Sakai, et al., 2004; Toma
et al, 1997). Like previous studies in other cell types, 13-cis RA has been shown to decrease
sebocyte proliferation and inhibit sebocyte differentiation as indicated in histology specimens,
primary sebocytes and SZ95 immortalized human sebocytes (Doran et al, 1980; Jones, et al.,
1980; Landthaler, et al., 1980; Ridden et al, 1990; Strauss, et al., 1980; Zouboulis, et al., 1999;
Zouboulis, et al., 1991; Zouboulis, et al., 1991). Although increased levels of caspase 3 were
noted in SZ95 sebocytes 24 hours following treatment with 13-cis RA and inhibition of cell
growth was evident at 7 days, other markers failed to indicate that SZ95 sebocytes were
undergoing apoptosis (Wrobel et al, 2003; Zouboulis et al, 1993). We hypothesized that 13-cis
RA reduces sebocyte counts by cell cycle arrest and/or apoptosis and that these effects might
not be apparent within a 24-hour treatment period.
In this chapter, we report that after 48 and 72 hours of treatment with 13-cis RA, but not
9-cis retinoic acid (9-cis RA) and all-trans retinoic acid (ATRA), inhibits growth and induces
apoptosis in immortalized human SEB-1 sebocytes but not in HaCaT keratinocytes or normal
human epidermal keratinocytes (NHEK). Furthermore, the retinoid acid receptor (RAR) pan
antagonist, AGN 193109, does not block the apoptosis induced by 13-cis RA suggesting an
RAR-independent apoptotic mechanism. We hypothesize that the ability of 13-cis RA to induce
39
cell cycle arrest and apoptosis in sebocytes contributes to the overall effect on suppression of
sebum production and improvement in acne.
2.3 Results
2.3.1 13-cis RA exhibits a more rapid onset of growth inhibition of SEB-1 sebocytes
compared to 9-cis RA and ATRA.
There is a significant dose-dependent decrease in cell count after 48 and 72 hours of
treatment with 13-cis RA. At 48 hours, 13-cis RA concentrations of 0.1, 0.5, and 1 µM
decreased cell count by 19, 22, 30%, respectively, when compared to vehicle (p < 0.05). After
72 hours, cell numbers were decreased by 19, 43, and 39% with 13-cis RA concentrations of
0.1 µM (p < 0.01), 0.5 µM (p < 0.0001), and 1 µM (p < 0.05), respectively (Figure 9a). No
significant differences in cell number were noted at 24 hours of treatment.
The effects of 9-cis RA and ATRA on SEB-1 sebocytes were noted beginning at 72
hours. Decreases of 39 and 43% were noted with 9-cis RA (0.5 and 1 µM, respectively) (p <
0.05). ATRA treatment (0.1 and 1 µM) significantly decreased cell number by 14 and 37%,
respectively (p < 0.05) (Figure 9b,c). Overall, each of these three retinoids decreased SEB-1
sebocyte cell numbers at 72 hours, albeit to varying degrees, but effects were noted beginning
at 48 hours with 13-cis RA.
40
Figure 9: 13-cis RA, 9-cis RA and ATRA differentially inhibit SEB-1 sebocyte
proliferation. (a-c) Time-dependent inhibition of SEB-1 sebocyte proliferation with
individual retinoid compounds. SEB-1 cells were cultured in the presence of ethanol
vehicle alone (0.01% or less; control), 0.1 µM, 0.5 µM or 1 µM concentrations of 13-cis
RA, 9-cis RA, ATRA for 24, 48 or 72 hours. Attached cells were collected, stained with
trypan blue, and counted manually by hemacytometer. Data represent mean ± SEM, n =
12. Statistical analysis was performed by ANOVA Two-Factor with Replication. * p <
0.05, **p < 0.01, *** p < 0.0001.
41
2.3.2 13-cis RA significantly inhibits DNA synthesis in SEB-1 sebocytes.
13-cis RA (0.1, 0.5, and 1 µM) significantly decreased thymidine incorporation by
approximately 3-fold at 72 hours (p < 0.01). No significant changes were noted at 24 or 48
hours. A 1.85-fold increase in 3H thymidine incorporation was noted when cells were treated
with 1 µM 9-cis RA for 48 hours. ATRA concentrations of 0.5 and 1 µM decreased thymidine
incorporation by approximately 1.8-fold at 24 and 72 hours, respectively (Figure 10a-c).
42
Figure 10: 13-cis RA inhibits DNA synthesis to a greater extent than 9-cis RA or
ATRA. (a-c) SEB-1 sebocytes were treated with ethanol vehicle (0.01% or less; control) or
0.1, 0.5, 1 µM concentrations of 13-cis RA, 9-cis RA or ATRA for 24, 48 or 72 hours. 1µCi
3
H thymidine was added to each sample 8 hours prior to collection. Cells were washed and
collected for liquid scintillation counting. Data represent mean ± SEM, n ≥12. Statistical
analysis was performed by ANOVA Two-Factor with Replication. *p < 0.005 and **p <
0.01.
43
2.3.3 13-cis RA, but not 9-cis RA or ATRA, increases p21 levels in SEB-1 sebocytes.
To further test the hypothesis that 13-cis RA changes cell cycle progression, expression
of p21, a cell cycle inhibitor, was examined by western blot. p21 is a general cyclin dependent
kinase inhibitor that blocks progression through the G1/S phase of the cell cycle. 13-cis RA
significantly increased p21 protein expression after 48 and 72 hours (Figure 11a). Specifically,
p21 levels increased on average 2.64-fold and 3.13-fold when cells were treated with 0.1 and 1
µM 13-cis RA, respectively, for 48 hours ( p = 0.008 and 0.05). After 72 hours of treatment, all
concentrations tested increased p21 protein expression. Increases in p21 of 1.47-, 2.27-, and
3.01-fold were noted with 0.1, 1, and 10 µM concentrations of 13-cis RA, respectively. No
significant differences in p21 expression were noted at 24 hours (data not shown). When SEB-1
sebocytes were treated with 9-cis RA or ATRA in concentrations of 0.1, 0.5 and 1 µM, no
significant increases in p21 protein were noted at 48 or 72 hours (Figure 11b,c).
44
Figure 11: 13-cis RA increases p21 and decreases cyclin D1 proteins. (a) SEB-1 cells
were treated with 0.1 µM, 1µM, 10 µM 13-cis RA or vehicle. (b-c) Parallel experiments
were performed with 0.1 µM 0.5 µM, or 1 µM concentrations of 9-cis RA and ATRA. Blots
were incubated with primary antibodies to p21 and β-actin for loading control normalization
and analyzed by densitometry. (d) SEB-1 cells were treated with 0.1 µM, 1µM, 10 µM 13cis RA or vehicle and blots were incubated with primary antibodies to cyclin D1 and βactin. Magic Mark XP (MM) indicates band size. Blots are representative of a minimum of
three western blots. Graphs represent normalized values relative to vehicle (control)
expression of a minimum of three independent western blots. Mean ± SEM. * p < 0.05 ** p
= 0.01
45
2.3.4 13-cis RA, but not 9-cis RA or ATRA, decreases cyclin D1 protein in SEB-1
sebocytes.
To further explore the possibility that 13-cis RA induces G1 arrest in SEB-1 sebocytes,
cyclin D1 protein was examined by western blot. Cyclin D family members are expressed and
function in controlling the progression from G1 to S phase in the cell cycle (Baldin et al, 1993).
Overexpression of cyclin D1 shortens the duration of the G1 phase and is rate limiting in phase
progression (Quelle et al, 1993). Therefore, cyclin D1 is a likely candidate to confirm the actions
of 13-cis RA in inhibiting cell cycle progression by influencing the G1 to S phase transition.
In SEB-1 sebocytes, 13-cis RA in concentrations of 0.1, 1 and 10 µM significantly
decreased cyclin D1 protein at 72 hours. No significant effects of 13-cis RA were noted at 24 or
48 hours (24-hour data not shown). 9-cis RA or ATRA concentrations of 0.1, 0.5 and 1 µM did
not reduce cyclin D1 protein levels at 72 hours (data not shown) (Figure 11d).
2.3.5 13-cis RA induces apoptosis in SEB-1 sebocytes but not in HaCaT keratinocytes or
NHEK.
To determine if the effect of 13-cis RA on apoptosis is cell-type specific, time course
experiments were conducted in SEB-1 sebocytes, HaCaT keratinocytes and normal human
epidermal keratinocytes (NHEK). In SEB-1 sebocytes, no significant differences in apoptosis
were noted in cells treated with 13-cis RA for 2, 4, 6 or 24 hours. A marginal, yet significant,
increase in the percentage of cells in early apoptosis was noted in SEB-1 cells treated with 0.1
µM 13-cis RA: 2.03% to 2.49% at 48 hours and 2.19% to 2.84% at 72 hours (p < 0.01 at both
time points). Significant increases in the percentage of cells in late apoptosis were noted at 48
and 72 hours with increasing concentrations of 13-cis RA (Figure 12a, late apoptosis shown).
46
Figure 12: 13-cis RA induces late apoptosis in SEB-1 sebocytes but not in HaCaT
keratinocytes or NHEK. (a) SEB-1 cells were treated with vehicle (negative control), 13cis RA (0.1 µM or 1 µM), or staurosporine (S) (positive control) for indicated times. (b)
HaCaT cells were treated with vehicle, 13-cis RA (0.1 µM or 1 µM), or staurosporine (S) for
the indicated times. (c) NHEK cells were treated with vehicle, 13-cis RA (0.1 µM or 1 µM),
or staurosporine (S) for indicated times. In all experiments, cells were prepared according
to manufacturer’s protocol for Annexin V-FITC / PI staining. (BD ApoAlert, BD Biosciences)
Data was analyzed with Cell Quest Software and represent mean ± SEM, n ≥ 12.
Statistical analysis was performed with ANOVA Two Factor with Replication. *p<0.01,
**p<0.00001.
47
Specifically, 0.1 µM 13-cis RA increased the percentage of late apoptosis: 4.06% to
5.22% at 48 hours and 5.31% to 8.11% at 72 hours. 13-cis RA at 1 µM concentration caused
increases from 3.64% to 5.08% and from 7.57% to 12.18% at 48 and 72 hours, respectively
(Figure 12a). Nanomolar concentrations of 13-cis RA did not induce apoptosis at any of the
time points examined (data not shown).
In HaCaT keratinocytes, no significant differences in the percentage of cells in early or
late stage apoptosis or necrosis were noted in cells treated with 0.1 µM 13-cis RA at all time
points examined. 13-cis RA (1 µM) significantly increased the percentage of cell in early and
late stage apoptosis at 24 and 48 hours. Yet, these increases were very minor, with the total
percentage of cells in apoptosis with 13-cis RA treatment being less than 2% of the cells
(Figure 12b). In experiments with NHEK, no significant differences were noted in cells treated
with 13-cis RA, with the exception of an increase from 5.25% to 6.2% in late stage apoptosis at
2 hours with 1 µM 13-cis RA (Figure 12c).
Apoptosis was significantly induced by staurosporine in SEB-1 sebocytes, HaCaT
keratinocytes and NHEK showing that all three cell types are capable of undergoing apoptosis.
No significant differences were noted between standard culture medium and ethanol vehicle
controls in any cell type at any time point during these studies indicating that the concentrations
of ethanol used in these experiments did not induce apoptosis.
2.3.6 13-cis RA specifically increases levels of cleaved caspase 3 in SEB-1 sebocytes.
SEB-1 sebocytes were treated with 13-cis RA and four independent western blots were
run to detect cleaved caspase 3. No cleaved caspase 3 was noted at 24 hours in negative
control lanes or in cells treated with 13-cis RA. 13-cis RA significantly increased cleaved
caspase 3 levels at 48 and 72 hours in SEB-1 sebocytes (Figure 13a). Specifically, 0.1 µM 13cis RA and 1 µM 13-cis RA increased expression of cleaved caspase 3 an average of 3.58-fold
and 3.33-fold (p < 0.01), respectively, at 48 hours. Small fold increases were noted at 72 hours
that were not statistically significant. Although the magnitude of the increase in cleaved caspase
3 was greatest with 10 µM 13-cis RA at 48 hours, these results were not statistically significant.
This is, most likely, due to the variability induced by the limited survival of the cells at this higher
concentration.
48
Figure 13: 13-cis RA induces cleaved caspase 3 expression in SEB-1 sebocytes. (a)
SEB-1 sebocytes were treated with vehicle, 0.1 µM, 1 µM, or 10 µM 13-cis RA. (b-c)
Parallel experiments were performed with 0.1 µM, 0.5 µM, or 1 µM concentrations of 9-cis
RA or ATRA. Blots were incubated with primary antibodies to cleaved caspase 3 (1:1000)
and actin (1:1000) for loading control normalization and analyzed by densitometry. p17 and
p19 are cleaved caspase 3 active fragments. Blots are representative of a minimum of 4
independent experiments. Graph represents normalized values relative to vehicle (control)
expression for 4 independent western blots. Data represent mean ± SEM * p < 0.01.
To determine if the induction of apoptosis is specific to 13-cis RA, SEB-1 sebocytes
were also treated with 0.1, 0.5 and 1 µM concentrations of 9-cis RA and ATRA. Again, no
cleaved caspase 3 was detected at 24 hours post treatment in negative controls or with any
49
concentration of either retinoid. Furthermore, and unlike the case with 13-cis RA, no significant
increases in cleaved caspase 3 were noted with either 9-cis RA or ATRA at 48 and 72 hours
(Figure 13b,c).
For additional confirmation that the apoptotic effect of 13-cis RA is specific to sebocytes,
western blots for cleaved caspase 3 were performed on NHEK. No cleaved caspase 3 could be
detected at any time point examined with NHEK cells treated with 13-cis RA. However, cleaved
caspase 3 was detected with 1 µM staurosporine treatment further confirming that these cells
are capable of undergoing apoptosis (Figure 14).
Figure 14: Cleaved caspase 3 is not detected in NHEK treated with 13-cis RA. NHEK were
treated with vehicle, 0.1 µM, 0.5 µM, or 1 µM concentrations of 13-cis RA or 1 µM staurosporine
(S; positive control). Blots were incubated with primary antibodies to cleaved caspase 3 (1:1000)
and β-actin (1:1000) for loading control normalization and analyzed by densitometry.
Representative blot is shown.
2.3.7 13-cis RA, but not 9-cis RA or ATRA, increases TUNEL staining in SEB-1 sebocytes.
To further test the hypothesis that 13-cis RA induces apoptosis in SEB-1 sebocytes and
to confirm the results from the annexin V-FITC FACS experiments, we examined the effects of
13-cis RA on SEB-1 sebocytes by TUNEL assay. 13-cis RA (0.1 and 1 µM) increased the
percentage of TUNEL-positive cells by 3.5- and 5.67-fold, respectively (p ≤ 0.01) at 48 hours,
while each concentration increased the percentage of TUNEL-positive cells by approximately
13-fold at 72 hours (p ≤ 0.01) (Figure 15a,b). No differences were noted at 24 hours (data not
shown). To compare the actions of 13-cis RA to its isomerization products, SEB-1 sebocytes
were also treated with the same concentrations of 9-cis RA and ATRA and no significant
increases in TUNEL-stained cells were noted at any time point examined (Figure 15a,b). Both
9-cis RA and ATRA had 1-3% TUNEL positive cells at all time points. Fenretinide is a synthetic
retinoid known to induce apoptosis by ceramide or reactive oxygen species generation.
50
Fenretinide treatment in SEB-1 sebocytes significantly increased the percentage of TUNELpositive cells in a dose-dependent manner at 48 and 72 hours (ranging from 15% to 85%
positive cells) (Figure 15a,b). No significant increase in TUNEL staining was noted with retinoid
X receptor (RXR) pan agonist, CD 3254, at 48 hours. However, 50 nM CD 3254 significantly
increased TUNEL-positive cells from 3% to 48% at 72 hours (p < 0.01) (data not shown).
51
Figure 15: The increase in TUNEL staining with 13-cis RA is not inhibited in the
presence of RAR pan antagonist AGN 193109. (a) Representative images of control, 0.1
µM, 1 µM, and 10 µM 13-cis RA, 9-cis RA, ATRA, and fenretinide treatments at 72 hours. (48
hour data not shown) (b) Quantification of the percentage of TUNEL positive stained cells per
treatment at 48 and 72 hours. (9-cis RA not shown) Data represent mean + SEM, n = 6-12.
Statistical analyses were performed with ANOVA Two Factor with Replication. * p < 0.01 **
p < 0.001 (c) Representative images of negative control, 1 µM 13-cis RA, AGN 193109, and
13-cis RA combined with 10 µM AGN 193109 at 72 hours. (48 hour data not shown) (d)
Quantification of the percentage of TUNEL positive cells at 72 hours. Data represent mean +
SEM, n = 12. Statistical analyses were performed with ANOVA Two Factor with Replication.
* p < 0.05 when compared to control; + not statistically different. (e) QPCR verification of
RAR antagonist AGN 193109 activity in SEB-1 sebocytes. Bars represent the efficiency
corrected normalized average fold change of TIG1 under the experimental conditions as
determined by REST-XL software. n = 4.
52
2.3.8 Apoptosis induction by 13-cis RA in SEB-1 sebocytes is not blocked by RAR
antagonist AGN 193109
To determine if the effects of 13-cis RA on apoptosis are mediated by retinoic acid
receptors (RARs), SEB-1 sebocytes were treated with 1 µM 13-cis RA in the presence of 10 µM
AGN 193109, an RAR pan antagonist, and the TUNEL assay was performed. 13-cis RA alone
significantly increased the percentage of TUNEL positive cells by approximately 5-fold at 48 and
72 hours. (p < 0.05). These increases were not inhibited in the presence of AGN 193109 at 48
and 72 hours (Figure 15d,e). To verify the activity of AGN 193109 within our cells at the time
points examined in the TUNEL assay, we performed quantitative PCR for RAR responsive
gene, tazarotene-induced gene 1 (TIG1). RAR activation induces the expression of TIG1
(Nagpal et al, 1996). In the presence of 1 µM 13-cis RA alone, TIG1 expression was
approximately 13- and 17-fold higher than controls at 48 and 72 hours, respectively. With the
addition of AGN 193109, TIG1 gene expression dramatically decreases at 48 and 72 hours and
is lower than vehicle treated controls (Figure 15e).
2.3.9 13-cis RA is isomerized to ATRA over time in SEB-1 sebocytes.
To study the kinetics of 13-cis RA uptake in SEB-1 sebocytes and its possible
isomerization to ATRA or 9-cis RA, SEB-1 sebocytes were treated with 13-cis RA and subjected
to HPLC analysis. 13-cis RA remains relatively stable in standard culture medium for
approximately 24 hours (Figure 16a). The concentration of 13-cis RA in standard culture
medium alone is similar to the concentration in medium removed from SEB-1 sebocytecontaining plates (Figure 16a,b). The concentration within SEB-1 sebocytes increases to a
maximum of 350 ng/mL at 12 hours, at which point the concentration declines for the duration of
the experiment (Figure 16c). The concentration of ATRA in the medium alone and from plates
containing SEB-1 sebocytes was much lower than 13-cis RA concentrations at the
corresponding time points. The concentration of ATRA within SEB-1 sebocytes begins to rise at
12 hours and continues through the remaining time periods. 9-cis RA concentrations are
53
minimal at best, both in medium alone and in medium from SEB-1 containing plates during the
time course. Within SEB-1 sebocytes, 9-cis RA concentrations range from 1.4 ng/mL at 0 hour
to a maximum or 12ng/mL at 72 hours; these concentrations are magnitudes lower than either
13-cis RA or ATRA at the same time periods.
54
Figure 16: 13-cis RA is isomerized to ATRA within SEB-1 sebocytes. HPLC analysis of
(a) SEB-1 medium alone, (b) medium removed from SEB-1 sebocyte-containing plates and
(c) SEB-1 sebocytes after 5 µM 13-cis RA treatment for the indicated times. Points are the
average of duplicate samples.
55
2.4 Discussion
Determining the actions of isotretinoin on the sebaceous gland is essential in advancing
our understanding of the molecular mechanism of action of this drug and in our search for safer
therapeutic alternatives. Several studies indicate that the effects of retinoids on cell proliferation,
cell cycle, and apoptosis are retinoid or cell-type specific. For example, growth inhibition with
13-cis RA has been reported in human breast cancer cell lines, primary glioblastoma cells,
Epstein-Barr Virus-immortalized B lymphocytes, and oral squamous cell carcinoma cell lines
(Bouterfa et al, 2000; Giannini, et al., 1997; Pomponi, et al., 1996; Toma, et al., 1997). In some
cases the effects noted with 13-cis RA or 9-cis RA were not duplicated by ATRA (Bouterfa, et
al., 2000). Most studies in other cell types suggest that retinoids cause a block in the G1/S
phase of the cell cycle, triggering decreased S phase and an increased percentage of cells in
the G0/G1 phase (Crandall, et al., 2004; Giannini, et al., 1997; Toma, et al., 1997). It is also
well established that retinoids induce apoptosis in numerous cell types, both normal cells and
tumor cell lines, although not previously demonstrated in sebocytes. For example, in doses
comparable to those given for the treatment of acne in humans, 13-cis RA reduces the survival
and genesis of murine hippocampal neurons in vivo (Crandall, et al., 2004; Sakai, et al., 2004).
ATRA has been shown to induce apoptosis in primary and metastatic melanoma cells (Zhang
and Rosdahl, 2004) as well as inducing growth arrest followed by apoptosis in orbital fibroblasts
isolated from Graves’ disease patients (Pasquali, et al., 2003). In OCI/AML-2 retinoid-sensitive
cell line subclones, derived from leukemia cells, 9-cis RA inhibited cell growth and induced
apoptosis to a greater extent than 13-cis RA or ATRA (Koistinen, et al., 2002). These studies
demonstrate that the actions of retinoids are unique and specific to the model used.
The exact mechanism of action of 13-cis RA in the treatment of acne remains largely
unknown. 13-cis RA has little to no ability to bind to cellular retinol-binding proteins or the RA
nuclear receptors (RARs and RXRs) (Allenby, et al., 1993; Fogh K. et al, 1993; Levin et al,
1992). It has been suggested that 13-cis RA may, in fact, act as a pro-drug that is isomerized
intracellularly to ATRA, which can bind to and activate RAR, leading to the overall inhibition of
sebocyte proliferation (Tsukada et al, 2000). Our studies confirm that 13-cis RA is primarily
isomerized to ATRA in SEB-1 sebocytes beginning at 24 hours. It is well established, however,
that 13-cis RA is superior to either 9-cis RA or ATRA for sebosuppression (Geiger et al, 1996;
Hommel et al, 1996; Ott et al, 1996). Alternatively, 13-cis RA may act in a receptor independent
manner by influencing cellular signaling pathways through direct protein interactions as
56
demonstrated with other retinoids or by enzyme inhibition (Hoyos et al, 2000; Imam et al, 2001;
Karlsson, et al., 2003; Zorn and Sauro, 1995).
Previous studies have examined the actions of 13-cis RA, 9-cis RA and ATRA on
cultured human sebocytes, SZ95 SV40-immortalized sebocytes, and rat preputial cells
(Tsukada, et al., 2000; Wrobel et al, 2003; Zouboulis, et al., 1991; Zouboulis, et al., 1993). 13cis RA at concentrations greater than 10-7 µM and ATRA (10-6 to 10-5 M) significantly decreased
human sebocyte proliferation after 7 and 14 days (Zouboulis, et al., 1991; Zouboulis, et al.,
1993). Studies of immortalized human sebocytes SZ95, showed that 13-cis RA, 9-cis RA and
ATRA at concentrations of 10-7 M, all significantly reduced proliferation by approximately 50%
after 9 days of treatment (Tsukada, et al., 2000). In primary rat preputial cells, ATRA and other
RAR-selective agonists significantly decreased cell numbers after 9 days (Kim et al, 2000).
Processes such as cell cycle arrest or apoptosis may explain the histological data in
human skin biopsies that demonstrate a drastic decrease in size, shape, and lipid content of the
sebaceous glands after 16 weeks of isotretinoin treatment (Goldstein, et al., 1982). Since
proliferation studies in SZ95 sebocytes suggested that the effects of 13-cis RA and other
retinoids may be noted after 7-9 days of treatment, we designed experiments to examine the
early effects of 13-cis RA, 9-cis RA and ATRA on proliferation, cell cycle progression and
apoptosis focusing on 24, 48 or 72 hours of treatment. Our proliferation studies show that 13-cis
RA causes a dose-dependent decrease in cell count after 48 and 72 hours whereas 9-cis RA
and ATRA show significant decreases beginning at 72 hours. We would expect that if our
experiments were extended, the magnitude of this decrease would be greater as previously
reported in SZ95 sebocytes after 9 days (Tsukada, et al., 2000). Overall, 13-cis RA at the
concentrations tested in our studies act sooner in inhibiting proliferation than either 9-cis RA or
ATRA. These data are supported by studies demonstrating an approximate 3-fold decrease in
3H-thymidine incorporation in SEB-1 sebocytes that were treated with 13-cis RA for 72 hours.
This decrease is nearly 2-fold greater than the decreases produced by 9-cis RA or ATRA. This
experiment suggests that 13-cis RA is more potent at growth inhibition than either 9-cis RA or
ATRA in SEB-1 sebocytes.
Further supporting the hypothesis that 13-cis RA causes a block in the G1/S phase of
the cell cycle as demonstrated in other cell types, we show that 13-cis RA increases p21 protein
and decreases cyclin D1 protein expression at 48 and 72 hours. Cyclin D1 protein expression
decreases by approximately 50% by 72 hours which coincides with our 3H-thymidine studies
where 13-cis RA had the most striking effect at 72 hours. Furthermore, cyclin D1 protein was
not decreased with 9-cis RA or ATRA at 72 hours, which is also consistent with our 3H-
57
thymidine incorporation studies. No significant increases in p21 protein were noted with 9-cis
RA or ATRA; although increasing trends were noted. Taken together these experiments show
that in SEB-1 sebocytes, 13-cis RA is much more effective than 9-cis RA or ATRA in both
decreasing the proportion of cells synthesizing DNA and inducing a G1/S phase block by
increasing p21 and decreasing cyclin D1 protein expression.
Studies in SZ95 sebocytes did not demonstrate apoptosis in cells treated for up to 24
hours with 13-cis RA (10-8 to 10-5 M) and assayed by DNA fragmentation and lactate
dehydrogenase release (Wrobel, et al., 2003). At 24 hours, no changes in apoptosis were
noted when SZ95 sebocytes were treated with 10-7 M 13-cis RA as assessed annexin V
staining, cell death assays or FACS analysis and reverse transcription (RT)-PCR for the
apoptotic proteins, Bcl-2 and Bax. Interestingly, in SZ95 sebocytes, 13-cis RA increased the
levels of caspase 3 as detected by FACS analysis at 24 hours. Accordingly, in our studies of
SEB-1 sebocytes, no increase in apoptosis was noted 24 hours after 13-cis RA as assayed by
annexin V-FITC FACS. However, increases in early and late stage apoptosis were noted at 48
and 72 hours with concentrations of 13-cis RA similar to those used in SZ95 sebocytes,
although the magnitude of the percentage of cells is small compared to the positive control,
staurosporine. In contrast, the magnitude of the changes in apoptosis induced by 13-cis RA was
much greater in the TUNEL assay. By extending our treatment times beyond 24 hours, we were
able to detect the induction of apoptosis by 13-cis RA, which was verified by increased
expression of cleaved caspase 3. Furthermore, the increase in apoptosis was limited to 13-cis
RA as no significant increases in apoptosis were noted when SEB-1 sebocytes were treated
with 9-cis RA or ATRA.
The effects of 13-cis RA on apoptosis and growth inhibition may or may not be mediated
by retinoid receptors. It is possible that the effects of 13-cis RA on apoptosis and growth
inhibition may be mediated by other isomerization products such as 4-oxo-isotretinoin or 4hydroxy-isotretinoin (Orfanos and Zouboulis, 1998). The 4-oxo metabolites of retinoids have
been shown to be functionally active in human keratinocytes and fibroblasts by their ability to
induce changes in gene expression (Baron, et al., 2005). Our data show that RAR panantagonist AGN 193109 sufficiently blocks RAR activation in the presence of 13-cis RA as
measured by a significant decrease in TIG1 gene expression, yet does not block apoptosis
induced by 13-cis RA in SEB-1 sebocytes, thus supporting the hypothesis that apoptosis
induction via 13-cis RA is independent of RAR activation. Alternatively, apoptosis maybe
58
mediated through RXR nuclear receptor activation (Zhao et al, 2004). Using RXR pan-agonist,
CD 3254, at a concentration of 50 µM, a significant increase in the percentage of TUNELpositive SEB-1 was noted at 72 hours. Although our HPLC data indicate very low levels of 9-cis
RA (a maximum of 12 ng/mL at 72 hours), RXR activation by 9-cis RA is possible (Allenby, et
al., 1993) or 13-cis RA may be metabolized to another as yet unidentified metabolite that is
capable of RXR activation.
Alternatively, 13-cis RA may have effects that are independent of retinoid receptors.
Interestingly, we showed that fenretinide; a synthetic retinoid known to induce apoptosis by
primarily, RAR- and RXR-independent means is able to induce significant apoptosis in our SEB1 sebocytes. In fact, the degree of apoptosis induced by fenretinide at 48 hours is very similar to
that observed with 13-cis RA treatment at 72 hours. Fenretinide induces apoptosis by elevating
reactive oxygen species, and increases in activation of ceramide and caspases (Wu, et al.,
2001). In addition, a retinoid-related molecule, AGN 193198 induces apoptosis without
activation of the classical retinoid receptors (Balasubramanian et al, 2005; Keedwell, et al.,
2004). It is possible that 13-cis RA acts similarly to fenretinide or AGN 193198 via receptorindependent mechanisms; although additional experiments are required to test this hypothesis.
Since the actions of retinoids differ in various cell types and the effects of 13-cis RA are
most profound on sebaceous glands in vivo, it is likely that the induction of apoptosis and cell
cycle arrest is specific to sebocytes. 13-cis RA failed to induce apoptosis in HaCaT
keratinocytes or NHEK. It is possible that with higher concentrations of 13-cis RA or longer
treatment times that apoptosis may be induced in keratinocytes. Although there is no evidence
in the literature of 13-cis RA-induced apoptosis in keratinocytes, ATRA and tazarotene (RAR β/γ
selective agonist), have been shown to induce apoptosis in HaCaT keratinocytes (Louafi et al,
2003; Papoutsaki et al, 2004). Taken together, these experiments support the hypothesis that
13-cis RA specifically induces apoptosis in SEB-1 sebocytes and not in keratinocytes.
In conclusion, our data indicate that 13-cis RA inhibits growth and induces apoptosis in
SEB-1 sebocytes and not keratinocytes at concentrations that are therapeutically achievable in
human plasma (Adamson, 1994; Almond-Roesler et al, 1998; Rollman and Vahlquist, 1986).
Previous studies in human sebocytes and immortalized sebocytes have also documented
growth inhibition with 13-cis RA, however, we have extended these studies to show that this
growth inhibition is most likely due to influencing the G1/S phase of the cell cycle as evidenced
by decreased DNA synthesis, increased p21 protein and decreased cyclin D1 protein. In
addition, we report for the first time, that 13-cis RA also induces apoptosis in SEB-1 sebaceous
59
cells. The ability to induce apoptosis is specific to sebocytes, not keratinocytes, and is distinct
from the effects observed with 9-cis RA or ATRA that may account, in part, for the superior
efficacy of 13-cis RA in reducing sebum production. Furthermore, the induction of apoptosis by
13-cis RA does not appear to involve RAR nuclear receptors. Elucidating the cellular processes
that are affected by 13-cis RA in sebocytes is a step toward understanding the overall molecular
mechanism of action of this drug, which may lead to the identification of alternative strategies for
the treatment of acne.
2.5 Materials and Methods
2.5.1 Cell Culture
The SEB-1 human sebocyte cell line was generated by transfection of secondary
sebocytes with SV40 Large T antigen as previously described (Thiboutot, et al., 2003). SEB-1
sebocytes were cultured and maintained in standard culture medium containing: 5.5mM low
glucose Dulbecco’s Modified Eagle Medium (DMEM) 3:1 Ham’s F12, 2.5% fetal bovine serum
(FBS) , hydrocortisone (0.4µg/mL), adenine (1 X 10-8 M), insulin (10ng/mL), epidermal growth
factor (3 ng/mL), cholera toxin (1.2 X 10-10 M) and antibiotics.
HaCaT keratinocytes were cultured and maintained in 5.5 mM low glucose DMEM, 5%
FBS and antibiotics. HaCaT keratinocytes served as a control cell line in annexin V-FITC FACS
apoptosis assays. Normal human epidermal keratinocytes (NHEK)-neonatal, pooled (NHEKneo, Clonetics Keratinocyte System, Cambrex Bioscience, Walkersville, MD) were cultured in
keratinocyte growth medium-2 (KGM-2) (Cambrex Bioscience, Walkersville, MD). NHEK
keratinocytes served as control cells in annexin V-FITC FACS apoptosis assays and western
blots for cleaved caspase 3.
60
2.5.2 Effects of retinoids on SEB-1 proliferation
Retinoid compounds were purchased through SIGMA (St. Louis, MO): 13-cis RA (R
3255), 9-cis RA (R 4653) and ATRA (R 2625). Stock solutions of retinoids were handled under
dimmed yellow light, dissolved in 100% ethanol to a concentration of 10 mM and stored under
N2 gas at -20ºC until use. The RAR pan-antagonist AGN 193198 was obtained from Allergan
(gift, Dr Rosh Chandraratna), dissolved in DMSO at a concentration of 10 mM and stored at 70ºC until use. Treatments were made from retinoid stock solutions diluted to the appropriate
concentration in standard culture mediums under dimmed yellow light. Staurosporine (S 5921,
SIGMA, St Louis, MO) was solubilized in 100% ethanol at a concentration of 10 mM, stored at 20ºC and diluted to desired final concentration in appropriate cell culture medium for a positive
control in apoptosis assays.
SEB-1 sebocytes (passage 20-23) were seeded at 4 X 104 cells per 35-mm plate and
grown until approximately 40% confluent. Plates were treated with 0.1, 0.5 or 1 µM
concentrations of 13-cis RA, 9-cis RA, ATRA or ethanol vehicle (0.01% or less; control) in
triplicate for 24, 48 and 72 hours. Cells were detached using trypsin (0.05%), collected, and
diluted in standard cell culture medium for manual cell counts using a hemacytometer. Cell
viability was assessed using Trypan Blue dye exclusion. Each proliferation assay was
performed three independent times. Analysis of variance (ANOVA) Two Factor with Replication
was used for analysis. Results were considered significant if p < 0.05.
2.5.3 3H thymidine incorporation assay
SEB-1 sebocytes (passages 21-26) were seeded at 2.5 X 104 cells per well in 12-well
plates and grown until 30-40% confluent. Wells were rinsed with phosphate-buffered saline
(PBS) prior to the addition of 0.1, 0.5 or 1 µM concentrations of 13-cis RA, 9-cis RA, ATRA or
ethanol vehicle (0.01% or less) alone in triplicate wells in standard culture medium. 3H thymidine
(1µCi/well) was added a minimum of 8 hours prior to the end of the treatment period. At the end
of the treatment period, medium was removed and cells were rinsed twice with PBS, detached
using trypsin (0.05%) and collected for liquid scintillation counting. Each assay was performed a
61
minimum of three independent times. Statistical significance was determined with ANOVA Two
Factor with Replication. Results were considered significant if p < 0.05
2.5.4 Western blot analysis for p21, cyclin D1 and cleaved caspase 3
To confirm the results from cell proliferation and apoptosis assays, protein levels of p21,
cyclin D1 and cleaved caspase 3 were examined using western blot analysis in our various cell
lines. p21, a cyclin dependent kinase inhibitor, blocks progression through the G1/S phase of
the cell cycle. Cyclin D1 is specifically required for progression into S phase. Caspase 3, the key
executioner caspase, is synthesized in the cell as a pro-caspase, which then becomes cleaved
and activated when cells undergo apoptosis. Primary antibodies for p21 Waf/Cip1 (DCS60),
cyclin D1 (DCS6), cleaved caspase 3 (Asp175) and β-actin as well as secondary anti-rabbit IgG
horseradish peroxidase antibody were purchased from Cell Signaling Technology (Beverly,
MA). Actin primary antibody and anti-mouse horseradish peroxide-linked secondary antibody
were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
SEB-1 sebocytes (passage 20-26) were grown in 100-mm plates in standard culture
medium until 50-75% confluent. Plates were rinsed with PBS and treated with 13-cis RA (0.1, 1
and 10 µM); 9-cis RA (0.1, 0.5 and 1 µM); ATRA (0.1, 0.5 and 1 µM); ethanol vehicle (0.01% or
less) as a negative control; or 1 µM staurosporine as a positive control. Cells were treated for
24, 48 or 72 hours. NHEK cells (passage 3) were grown in 100-mm plates in standard culture
medium until approximately 50-75% confluent. Plates were rinsed with PBS and treated with 13cis RA (0.1, 0.5, and 1 µM); ethanol vehicle (0.01% or less); or 1 µM staurosporine for 2, 4, 6,
18, 24, 48 or 72 hours.
Total cell protein lysates from adherent and floating cells of SEB-1 sebocytes and NHEK
were collected, flash frozen in liquid nitrogen and stored at -80ºC until needed. Protein
concentration of each sample was determined by the BCA Protein Assay (Pierce, Rockford, IL).
Equal amounts of protein were run on NuPage 10% or 4-12% Bis-Tris Gels with MES Running
Buffer (Invitrogen Life Technologies, Carlsbad, CA). Gels were transferred to polyvinylidene
difluoride membrane, blocked for 1 hour at room temperature in 5% non-fat dry milk and
incubated with 1:1,000 dilution of cleaved caspase 3 antibody (Asp175) rabbit monoclonal
antibody, 1:1,000 dilution of cyclin D1 mouse monoclonal antibody or 1:8,000-15,000 dilution of
62
p21 mouse monoclonal antibody. Secondary anti-rabbit and –mouse horseradish peroxidase
linked antibodies were used to detect primary antibodies. SuperSignal West Pico
Chemiluminescent Substrate (Pierce, Rockford, IL) was used for protein detection. Blots were
stripped with Restore Western Blot Stripping Buffer (Piece, Rockford, IL) and reprobed with βactin or actin for a loading control. Films of blots were analyzed and quantified by densitometry
with QuantityOne Software (Bio-Rad, Hercules, CA) after background subtraction. Western blots
were repeated a minimum of three independent times. Data was analyzed with Student’s t-test
and results were considered significant if p < 0.05.
2.5.5 Annexin V-FITC/Propidium Iodide FACS Apoptosis Assay
To determine if 13-cis RA induces apoptosis in SEB-1 sebocytes and the time course of
this effect, the annexin V-FITC FACS assay was chosen (Martin et al, 1995). Apoptosis assays
were performed in SEB-1 sebocytes, HaCaT keratinocytes and NHEK that were treated with 13cis RA. SEB-1 sebocytes (passages 22-26) and HaCaT keratinocytes (passages 23-29) were
seeded at 8 X 104 cells per 35-mm plate in their standard culture mediums and allowed to grow
for 3 days, feeding once prior to treatment. Treatments consisted of standard culture medium or
ethanol vehicle (0.01% or less) as negative controls; 1 µM staurosporine as a positive control;
and 13-cis RA at final concentrations of 0.1 or 1 µM in SEB-1 sebocytes and HaCaT
keratinocytes for the initial studies. For follow-up studies examining a possible 13-cis RA dose
response, SEB-1 sebocytes were subjected to 0.1, 1, 10 nM 0.1, 1 and 10 µM as well as
previously mentioned controls. All samples were run in triplicate and treatments were carried out
for 2, 4, 6, 24, 48 and 72 hours. In parallel experiments, NHEK (passage 3) were grown in
KGM-2 growth medium until 70% confluent. Treatments consisted of 0.1, 0.5, and 1 µM 13-cis
RA; ethanol vehicle (0.01% or less) and 1 µM staurosporine. Samples were run in triplicate and
assayed at 2, 4, 6, 18, 24, 48, and 72 hours. Each sample was prepared according to BD
ApoAlert Annexin V Protocol (Cat no. K2025-1, BD Biosciences, Clontech, Palo Alto, CA). Ten
thousand events (cells) were collected per sample using flow cytometry and debris was
excluded by scatter gating. Single annexin V-FITC and propidium iodide-stained control
samples determined quadrants for data analysis. Data analysis was by Cell Quest software
(Becton Dickinson, Canada) and percentage of cells in early apoptosis, late apoptosis, necrosis,
63
and viable (unaffected) quadrants were calculated and compared by ANOVA Two Factor with
Replication. Assay was performed three independent times. Results were considered significant
if p < 0.05.
2.5.6 TdT-Mediated dUTP Nick End Labeling (TUNEL) Staining
SEB-1 sebocytes (passages 22-28) cultured in 12-well plates in standard medium until
approximately 30-40% confluent. Wells were rinsed with PBS and were treated in triplicate with
ethanol vehicle (0.01% or less) control, 13-cis RA, 9-cis RA or ATRA each in concentrations of
0.1, 1 or 10 µM. Retinoids were diluted in standard culture medium and treatments were carried
out for 24, 48 and 72 hours. In parallel experiments, SEB-1 sebocytes (passages 22-24) were
cultured as above and treated in triplicate with ethanol vehicle (0.01% or less), DMSO vehicle
(0.01% or less), both vehicles together, 1 µM 13-cis RA alone, 10 µM AGN 193109 alone or 1
µM 13-cis RA + 10 µM AGN 193109 combination. Additional experiments were performed with
fenretinide, a synthetic retinoid known to induce apoptosis via an RAR independent mechanism.
(Wu, et al., 2001) Fenretinide (4-hydroxyphenyl-retinamide) was handled under dimmed yellow
light and dissolved in 100% ethanol to create a 10 mM stock solution that was stored at -20ºC
(H 7779, SIGMA, St Louis, MO). SEB-1 sebocytes were treated in triplicate with 0.1, 1, 10 µM
concentrations. Furthermore, experiments were performed with RXR pan-agonist CD 3254
(Galderma R&D, Sophia Antipolis, France) CD 3254 was handled under normal light conditions
and dissolved in DMSO to create a 10 mM stock solution that was stored at -20ºC until use.
SEB-1 sebocytes were treated in triplicate with 1 and 50 nM concentrations.
All compounds were diluted in standard culture mediums and applied for 48 and 72
hours. Each well was considered one sample. Samples were prepared by manufacturer’s
instructions for In Situ Cell Death Detection Assay (Roche Applied Science, Indianapolis, IN).
Additional assay controls included negative controls of labeling solution only and DNase Itreated wells as positive wells. Results were quantified by counting positive stained cells in 3
representative fields per well for each of the treatments carried out in triplicate. Each assay was
performed three independent times; fenretinide and CD 3254 experiments were repeated twice.
64
Data analysis was performed using ANOVA Two Factor with Replication and considered
significant if p < 0.05.
2.5.7 Quantitative Polymerase Chain Reaction (QPCR)
To verify RAR antagonist activity in TUNEL experiments, quantitative PCR was used to
document down-regulation of the RAR target gene, tazarotene induced gene 1 (TIG1, retinoic
acid receptor responder 1). SEB-1 sebocytes were handled, maintained and treated with 13-cis
RA and RAR pan-antagonist AGN 193109 under conditions that were identical to those used in
the TUNEL assays. Total RNA was isolated and QPCR performed as previously described
(Trivedi et al, 2006). Primer-probe sets for TATA-binding protein (TBP; reference gene) and
retinoic acid responder 1 (TIG1) were purchased from Applied Biosystems (Foster City, CA).
Controls included “no template” and “no amplification” samples. The Relative Expression
Software Tool (REST-XL) was used for data analysis.
2.5.8 HPLC
13-cis RA is reported to isomerize to ATRA in other cell types including SZ95
immortalized sebocytes (Tsukada, et al., 2000). To eliminate the possibility of an alternative
pattern of isomerization and to study the kinetics of 13-cis RA uptake into SEB-1 sebocytes, we
utilized liquid-liquid extraction, reverse phase HPLC with UV detection. SEB-1 sebocytes
(passage 22) were grown to 80% confluence in 100-mm tissue culture plates. For “medium
only” controls, SEB-1 medium alone was placed in empty 100-mm plates. 5 µM 13-cis RA was
applied to SEB-1 sebocytes and “medium only” control plates in duplicate for 0, 2, 4, 6, 12, 18,
24, 48 and 72 hours. Experimental samples included medium collected from “medium only”
control plates, medium from SEB-1 sebocyte plates and SEB-1 sebocyte cell pellet. Sample
preparation was by liquid-liquid extraction with ethyl acetate. Ethyl acetate was evaporated and
the residue was re-dissolved in a mixture of acetonitrile and purified water (80/20, vol/vol) before
injection. Internal standard (acitretin), 13-cis RA, 9-cis RA and ATRA standards as well as
quality control solutions were made and analyzed to generate the calibration curve. Samples
65
were injected into Agilent 1100 Series HPLC System (Agilent Technologies, Palo Alto, CA)
using Nucleosil® 100-5 C18 (250 X 4mm2) HPLC columns (Macherey-Nagel Inc., Düren,
Germany). Samples were eluted in a gradient solution composed of purified water and
acetonitrile containing 0.2% acetic acid. Retinoid compounds were detected by UV detection at
350 nm.
We thank Drs. Johannes Voegel and Jean-Claude Caron of Galderma R&D for provision
of compounds and HPLC analysis and Dr. Rosh Chandraratna of Allergan Inc. for provision of
compounds. We also thank Nate Sheaffer of the Cell Science/Flow Cytometry Core Facility of
the Section of Research Resources, Penn State College of Medicine, for excellent technical
assistance with all FACS experiments and Anne Stanley of the Molecular Core Facility for
assistance with densitometry analysis. Finally we thank Chelsea Billingsley for providing
technical assistance.
This work is supported by NIH NIAMS R01 AR047820 to D.M.T. and the Jake Gittlen Cancer
Research Foundation at the Pennsylvania State University College of Medicine.
Chapter 3
Array profiling of skin from patients on isotretinoin provides insights into potential
mediators of its apoptotic effect on sebaceous glands.
3.1 Chapter Abstract
Although 13-cis retinoic acid (isotretinoin) is the most potent agent in the treatment of
acne, its mechanism of action is still unknown. This is the first study to examine the effects of
oral 13-cis retinoic acid on gene expression in the skin of acne patients. Gene expression
analysis was performed on seven patients before and at one-week of treatment and also eight
patients before and after 8-weeks of treatment as well as in our cell culture models: SEB-1
sebocytes and HaCaT keratinocytes for insights into cell specific effects of 13-cis RA.
Histological estimation of sebaceous gland size was done and mRNA was isolated for gene
array expression analysis using the Affymetrix system. Significant gene changes at 8-weeks
are consistent with the known decreases in sebaceous gland lipid production induced by 13-cis
RA. Significantly changed genes at one-week isotretinoin therapy can provide insight into the
initial effects induced by this drug and these genes can be broadly categorized as tumor
suppressors, protein processors or genes involved in transfer or binding of ions, amino acids,
lipids or retinoids, including lipocalin 2. Lipocalins are small molecular weight proteins that
regulate processes such as immune response, retinol transport, and prostaglandin synthesis.
The lipocalin 2 gene product, neutrophil gelatinase associated lipocalin (NGAL), is known to
function in innate immunity and can induce apoptosis. Immunohistochemistry on patient skin
biopsies revealed increased NGAL after isotretinoin treatment with localization to the basal layer
of the sebaceous gland and upper sebaceous duct. Our previous studies indicated that 13-cis
RA can induce apoptosis in sebocytes. Purified NGAL protein induced significant apoptosis at
48 hours post-treatment in SEB-1 sebocytes, lending support to NGAL mediating the apoptosis
actions of 13-cis RA. Together, these data provide rationale for further study of candidate
genes, including lipocalin 2, that mediate retinoid response in the skin.
67
3.2 Introduction
13-cis retinoic acid (isotretinoin, 13-cis RA) is the most effective drug for the treatment of
acne. It is a known teratogen whose use in the United States, as of March 1, 2006, is restricted
within a registry system. The mechanism by which 13-cis RA reduces the size and secretion of
sebaceous glands, normalizes follicular keratinization and improves acne is largely unknown. Its
profound effect and improvement in acne was an unexpected finding during clinical trials for its
use in ichthyosis (Peck, 1979). The lack of an animal model for acne has also deterred
advances in the understanding of its mechanism of action. Because there are no safe
alternatives to 13-cis RA that demonstrate comparable efficacy, insights into its mechanism of
action are essential for alternative drug discovery.
Transcriptional profiling represents a new powerful tool to examine changes in gene
expression. When combined with advances in bioinformatics, transcriptional profiling can
generate data that may target future hypothesis-driven investigation to specific genes or
pathways. The goal of the present study is to gain broad insight into the potential pathways by
which 13-cis RA exerts its clearing effect in acne.
This is the first study to examine the effects of oral 13-cis RA on gene expression in the
skin of acne patients. Array analysis was performed on skin samples which were taken from the
backs of two cohorts of acne patients at baseline and after one-week or eight-weeks of
isotretinoin therapy. The data generated distinct differences in the patterns of gene expression
depending on the duration of therapy. In particular, marked decreases in the expression of
genes involved in lipid metabolism were found at 8-weeks, which is in agreement with the
marked histological decrease in sebaceous gland size. After one-week of treatment, a
completely different profile emerged with significant changes in genes involved in differentiation,
tumor suppression, serine proteases, serine protease inhibitors and solute transfer including
lipocalin 2. In addition, gene array analysis was performed on our SEB-1 sebocytes and HaCaT
keratinocytes, providing clues to possible cell-specific mechanisms of 13-cis RA.
We explored in more detail the actions of lipocalin 2 in human skin and in our cell culture
models, SEB-1 sebocytes and HaCaT and NHEK keratinocytes. Lipocalins are small molecular
weight proteins that regulate processes such as immune response, retinol transport, and
prostaglandin synthesis. The lipocalin 2 gene product, neutrophil gelatinase associated lipocalin
(NGAL), is known to function in innate immunity and can induce apoptosis (Devireddy et al,
2005; Flo et al, 2004). Induction of lipocalin 2/NGAL may be one mechanism through which 13-
68
cis RA-induced apoptosis occurs in sebaceous glands. These data provide important clues to
the effects of 13-cis RA that could advance our understanding of retinoid action not only in acne,
but in other retinoid-responsive conditions such as psoriasis, leukemia and other cancers.
3.3 Results
3.3.1 Patient selection and procedures
A total of 15 patients that were prescribed isotretinoin by their dermatologist for their
severe acne were enrolled in the study after signing informed consent forms. Early studies
indicated that 13-cis RA drastically decreased sebaceous gland size after 16 weeks of treatment
(Goldstein, et al., 1982); therefore, we chose an 8-week time point to examine the change in
skin histology and gene expression in patients receiving the treatment for their severe acne.
Eight patients had 5-mm punch biopsies of skin from their upper backs at baseline and at
approximately 8 weeks into therapy. Sample analysis indicated a marked decrease in
sebaceous gland size and decreased expression of numerous genes involved in lipid
metabolism that are characteristic of sebaceous glands. In an effort to detect earlier gene
changes, a second cohort of 7 patients was recruited to have biopsies preformed at baseline
and one-week into treatment. Information regarding patient age, sex, the time of biopsy, and the
dose of isotretinoin at the time of biopsy is presented in Table 5. The second biopsies from
patients in Group 1 were performed during a regularly scheduled dermatology visit
approximately 8 weeks into therapy. Due to scheduling issues, there was variation in the length
of time at which the second biopsy was performed (9.12 ± 1.1 weeks). The doses of 13-cis RA
depicted in Table 5 were those that the patients were receiving at the time of their second
biopsy.
69
Table 5: Isotretinoin patient demographics
Group 1: 8 week study
Group 2: 1 week study
Sex
Dose
mg/kg/d
Biopsy
(weeks)
22
F
1
8
17
M
1
Subject #
Age
1
2
Subject #
Age
Sex
Dose
mg/kg/d
Biopsy
(days)
8
9
15
M
0.5
7
3
32
F
1
10
10
17
M
0.5
7
4
15
M
1
10
11
17
M
0.5
7
5
18
M
0.5
9
12
21
F
0.67
7
6
24
F
0.67
11
13
17
M
0.67
7
7
14
M
0.5
8
14
20
F
0.67
7
8
15
M
1
9
15
23
M
0.5
7
Mean ± SD
19.6 ± 6
0.83 ± 0.23
9.12 ± 1.1
Mean ± SD
18.5 ± 2.8
0.57 ± 0.09
7±0
3.3.2 Histology reveals statistically significant decrease in sebaceous gland size after 8
weeks of treatment.
Hematoxylin and eosin staining was performed on sections of the baseline and
treatment biopsies from both groups of patients (Figure 17a). In both cohorts, at baseline,
sebaceous glands from the back were large and multi-lobulated. In Group 1 patients (8-week)
treatment biopsies, sebaceous glands were markedly reduced in size by approximately 8.5-fold
from baseline (Figure 17c). Glands lost their multi-lobular structure and sparse sebocytes were
noted only in close proximity to hair follicles. This architecture and location closely mirrors the
sebaceous glands in murine skin. For Group 2 patients (1-week), the changes in architecture
were not as obvious, with only a decreasing trend in sebaceous gland size being noted
(Figure 17b,c).
70
Figure 17: 13-cis RA decreases sebaceous gland volume. (a) Hematoxylin and eosin
sections of back skin from patients before and after 8 weeks of treatment reveals a significant
decrease in sebaceous gland volume. (b) Variable changes in sebaceous gland size were noted
after one week of treatment compared to baseline biopsies (c) Area of sebaceous glands.
Statistical significance was determined by paired t-test. Representative images are shown at a
total magnification of 100X. Magnification bars = 250 µm.
71
3.3.3 Significant decreases in genes that regulate lipid metabolism were noted in the
gene array expression analysis of skin biopsies taken from patients at 8 weeks
into isotretinoin therapy.
In comparing the gene array data from the pre-treatment biopsies to the 8-week
treatment biopsies, 197 genes were significantly up-regulated and 587 genes were significantly
down regulated as determined by using a false discovery rate (FDR) of 0.05, that corresponds
to a 5% chance of false positive genes among those genes considered significantly changed.
Select genes that were up-regulated by approximately 2-fold or greater and genes that were
down-regulated greater than 4-fold are listed in Table 6. For a complete listing of all significantly
changed genes at 8-weeks, see A.1. Many of the down-regulated genes at 8-weeks are
involved in the metabolism of steroids, cholesterol and fatty acids, which is consistent with the
known decreases in sebaceous gland lipid production induced by 13-cis RA.
72
Table 6: Selected significantly changed genes after 8 weeks isotretinoin therapy
Fold Change
2.62
2.03
-6.31
-6.19
-6.06
-5.17
-5.1
-5.09
-4.9
-4.78
-4.71
-4.57
-4.51
-4.14
-4.02
-3.99
-3.73
-3.73
-3.68
-3.6
-3.59
-3.37
-3.28
-3.25
-3.16
-3.08
-3.03
-2.92
-2.88
-2.87
-2.84
-2.84
-2.83
-2.83
-2.8
-2.78
-2.77
-2.73
-2.7
-2.66
-2.65
-2.61
-2.56
-2.52
Gene Title*
microseminoprotein, betacollagen, type I, alpha 1
hydroxyacid oxidase 2 (long chain)
hydroxy-delta-5-steroid dehydrogenase, 3 beta
thioesterase domain containing 1
solute carrier organic anion transporter family, member
4C1
male sterility domain containing 1
phospholipase A2, group VII (PAF acetylhydrolase)
fatty acid desaturase 1
glycine dehydrogenase
Galanin
PDZ domain containing 1
fatty acid binding protein 7, brain
histone 1, H1c
fatty acid binding protein 7, brain
arachidonate 15-lipoxygenase, second type
mucin 1, transmembrane
insulin induced gene 1
3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1
Lipidosin
sterol O-acyltransferase
fatty acid desaturase 2
carnitine acetyltransferase
hypothetical protein MAC30
peroxisomal long-chain acyl-coA thioesterase
chitinase 3-like 1 (cartilage glycoprotein-39)
apolipoprotein C-I
hydroxysteroid (11-beta) dehydrogenase 1
transmembrane protease, serine 11E
peroxisomal trans-2-enoyl-CoA reductase
SA hypertension-associated homolog (rat)
homogentisate 1,2-dioxygenase (homogentisate oxidase)
dehydrogenase/reductase (SDR family) member 9
steroid-5-alpha-reductase, alpha polypeptide 1
cell death-inducing DFFA-like effector a
SEC14-like 4 (S. cerevisiae)
Malic enzyme 1, NADP(+)-dependent, cytosolic
phosphodiesterase 6A, cGMP-specific, rod, alpha
NAD(P) dependent steroid dehydrogenase-like
acetyl-Coenzyme A acetyltransferase 2
fatty acid 2-hydroxylase
farnesyl diphosphate synthase
glycerol kinase
3-hydroxy-3-methylglutaryl-Coenzyme A reductase
Symbol
MSMB
COL1A1
HAO2
HSD3B1
THEDC1
RARE
RAR
#
RAR
SLCO4C1
MLSTD1
PLA2G7
FADS1
GLDC
GAL
PDZK1
FABP7
HIST1H1C
FABP7
ALOX15B
MUC1
INSIG1
HMGCS1
BG1
SOAT1
FADS2
CRAT
MAC30
ZAP128
CHI3L1
APOC1
HSD11B1
TMPRSS11E
PECR
SAH
HGD
DHRS9
SRD5A1
CIDEA
SEC14L4
ME1
PDE6A
NSDHL
ACAT2
FA2H
FDPS
GK
HMGCR
RAR
RAR,RXR
RAR, RXR
RAR
RAR, RXR
RAR
RXR
RAR
RAR
RAR, RXR
RAR, RXR
RAR, RXR
RAR, RXR
RAR
RAR,RXR
RAR, RXR
RAR, RXR
RAR, RXR
RAR
RAR, RXR
RAR
RAR
RAR, RXR
RAR
*italics indicate genes involved in or linked to fatty acid or cholesterol metabolism
# retinoic acid or rexinoid receptor response element located within first 1000 base pairs of gene
73
3.3.4 Gene expression analysis of skin from patients treated with 13-cis RA for one-week
revealed significant increases in genes encoding calcium binding proteins,
retinoid signaling molecules, solute carriers and serine proteases.
Array data was not generated for patient 11 due to insufficient quantity of RNA. In the
array data from the remaining 6 patients, 38 genes were significantly up-regulated and 5 genes
were significantly down-regulated when compared to baseline biopsies using a false discovery
rate (FDR) of 0.05. Significantly changed genes are shown in Table 7. Many of the significantly
up-regulated genes are known to be retinoid-responsive genes including: retinoic acid
responder 1 [tazarotene induced gene 1 (TIG1)}, cellular retinol binding protein 1 and cellular
retinoic acid binding protein 2. In addition, calcium-binding proteins (i.e. S100 proteins), serine
proteases, serine protease inhibitors (serpins), lipocalin and solute carriers were significantly
affected by 13-cis RA.
74
Table 7: Significantly changed genes after 1 week isotretinoin therapy
Fold Change
Gene Title*
7.03
lipocalin 2 (oncogene 24p3)
6.2*
S100 calcium binding protein A7 (psoriasin 1)
Symbol
RARE
#
LCN2
RAR, RXR
S100A7
RAR, RXR
S100A9
RAR
4.53
S100 calcium binding protein A9 (calgranulin B)
3.78
solute carrier family 12 (K/Cl transporters)
3.32
cytochrome P450, family 2, subfamily B
CYP2B7P1
RAR
2.61
serine (or cysteine) proteinase inhibitor
SERPINA3
RAR,RXR
2.61
retinoic acid receptor responder (TIG 1)
RARRES1
2.35
transmembrane protease, serine 4
TMPRSS4
2.27
KIAA0125
KIAA0125
2.21
placenta-specific 8
PLAC8
2.08
Rhesus blood group, C glycoprotein
RHCG
RAR
2.04
pipecolic acid oxidase
PIPOX
RAR
1.99
S100 calcium binding protein P
S100P
1.96
ERO1-like (S. cerevisiae)
ERO1L
1.92
ATPase, H+/K+ transporting, nongastric, alpha
ATP12A
1.91
chemokine (C-C motif) ligand 2
CCL2
RAR, RXR
1.81
retinol binding protein 1, cellular
RBP1
RAR, RXR
1.69
solute carrier family 6 (amino acid transporter)
1.67
E74-like factor 3 (ets domain transcription factor)
1.62
stimulated by retinoic acid gene 6 homolog
1.57
SLC12A8
RAR
SLC6A14
ELF3
RAR
STRA6
RAR, RXR
SEC14-like 2 (S. cerevisiae)
SEC14L2
RAR, RAR
1.56
cellular retinoic acid binding protein 2
CRABP2
RAR, RXR
1.52
defensin, beta 1
DEFB1
1.51
calbindin 2, 29kDa (calretinin)
CALB2
RAR, RXR
S100A2
RAR
1.5
S100 calcium binding protein A2
1.49
fucosyltransferase 3
1.49
involucrin
1.49
1.48
1.43
CD24 antigen
1.41
growth differentiation factor 15
1.4
KIAA1462
FUT3
IVL
RAR
interleukin 27 receptor, alpha
IL27RA
RAR, RXR
cytoplasmic polyadenylation element BP 1
CPEB1
CD24
GDF15
KIAA1462
1.39
serine protease inhibitor, Kazal type 5
SPINK5
1.38
UDP-N-acetyl-alpha-D-galactosamine
GALNT6
1.27
microtubule associated monoxygenase
MICAL3
1.26
keratin 23 (histone deacetylase inducible)
-2.29
solute carrier family 26, member 3
SLC26A3
-2.27
phospholipase A2, group VII (PAF acetylhydrolase)
PLA2G7
-2.13
phosphodiesterase 6A, cGMP-specific, rod, alpha
PDE6A
RAR
-1.55
carboxypeptidase M
CPM
RXR
-1.5
cysteine and glycine-rich protein 2
RAR
KRT23
CSRP2
*italics indicated genes within chromosomal region 1q21: epidermal differentiation complex
# retinoic acid or rexinoid receptor response element located within first 1000 base pairs of gene
75
3.3.5 Gene expression analysis in SEB-1 sebocytes and HaCaT keratinocytes with 72
hour 13-cis RA treatment.
Gene expression array analysis was performed on patient skin biopsies which contained
mixed populations of cells. Our in vitro cell culture model, SEB-1, was used to examine
sebocyte-specific gene changes induced by 13-cis RA. Three control samples and three
samples treated with 0.1 µM 13-cis RA were analyzed. A total of 85 genes (78 different genes)
were significantly influenced by 13-cis RA: 58 up-regulated and 27 down-regulated genes.
Selected significantly changed genes are listed in Table 8. For a complete listing of all
significantly changed genes, see A.2. The tumor suppressor, TIG1 and lipocalin 2 demonstrated
the greatest changes in gene expression. In addition, there were changes in several genes
involved in apoptosis and innate immunity such as TNFα-induced protein 2, TRAIL, interferon
regulatory factor 1 (IRF1), interferon-induced proteins, NFκB, the death receptor, Fas and TIG3
(a.k.a. retinoic acid inducible gene 1 (RIG1)). TIG3 encodes an RNA helicase and represents a
key intracellular protein that like the TLR3, can recognize viral double stranded RNA (dsRNA).
(Sen and Sarkar, 2005; Yoneyama et al, 2004)
76
Table 8: Selected significantly changed gene in SEB-1 sebocytes after 13-cis RA
treatment
Fold
Change
Gene Title
12.25
retinoic acid receptor responder (tazarotene induced) 1
7.04
lipocalin 2 (oncogene 24p3)
Gene Symbol
#
RARE
RARRES1
LCN2
RAR, RXR
TNFAIP2
RAR, RXR
5.95
tumor necrosis factor, alpha-induced protein 2
5.91
hydroxyprostaglandin dehydrogenase 15-(NAD)
4.64
cytochrome P450, family 1, subfamily B, polypeptide 1
4.25
hydroxyprostaglandin dehydrogenase 15-(NAD)
4.18
tumor necrosis factor (ligand) superfamily, member 10 (TRAIL)
TNFSF10
RAR
3.70
serine (or cysteine) proteinase inhibitor, clade B (ovalbumin),
SERPINB3
RAR
3.43
insulin-like growth factor binding protein 3
IGFBP3
N/A
3.29
aldehyde dehydrogenase 1 family, member A3
ALDH1A3
3.22
retinoic acid receptor responder (tazarotene induced) 3
RARRES3
RXR
3.08
oxidised low density lipoprotein (lectin-like) receptor 1
OLR1
N/A
3.06
solute carrier family 1 (glial high affinity glutamate transporter)
3.00
growth differentiation factor 15
2.60
cyclin-dependent kinase inhibitor 1A (p21, Cip1)
HPGD
CYP1B1
RAR
HPGD
SLC1A3
GDF15
RAR, RXR
CDKN1A
RAR
N/A
2.42
interferon regulatory factor 1
IRF1
2.20
BTG family, member 2
BTG2
2.07
2',5'-oligoadenylate synthetase 1, 40/46kDa
OAS1
1.85
GATA binding protein 3
GATA3
1.79
protein kinase C, alpha
PRKCA
RAR, RXR
1.70
nuclear factor of kappa light polypeptide gene enhancer in B-cells 2
NFKB2
RAR
1.69
Fas (TNF receptor superfamily, member 6)
1.66
dual specificity phosphatase 8
1.60
glutathione peroxidase 2 (gastrointestinal)
FAS
RAR
DUSP8
RAR, RXR
GPX2
RAR
1.47
phosphoinositide-3-kinase, regulatory subunit 3 (p55, gamma)
PIK3R3
RXR
-3.12
dihydrofolate reductase
DHFR
N/A
-2.48
glutamate dehydrogenase 1
GLUD1
N/A
-2.27
ribonucleotide reductase M2 polypeptide
RRM2
RXR
-2.12
CD86 antigen (CD28 antigen ligand 2, B7-2 antigen)
CD86
RAR, RXR
-2.05
DNA replication complex GINS protein PSF1
PSF1
RXR
-1.81
S100 calcium binding protein A10
S100A10
-1.71
phospholipase A2, group IVA (cytosolic, calcium-dependent)
PLA2G4A
-1.59
BUB3 budding uninhibited by benzimidazoles 3 homolog (yeast)
BUB3
# retinoic acid or rexinoid receptor response element located within first 1000 base pairs of gene
In addition, we performed gene expression analysis on triplicate samples of HaCaT
keratinocytes treated with 0.1 µM 13-cis RA or vehicle control for 72 hours. 13-cis RA induced a
total of 54 significantly changed genes (47 different genes) using a false discovery rate (FDR) of
0.05: 53 up-regulated genes and only one down-regulated gene. Selected significantly changed
77
genes are listed in Table 9. For a complete listing of all significantly changed genes, see A.3.
Results show unique gene changes that suggest HaCaT keratinocytes have powerful
mechanisms in place to protect against retinoid induced apoptosis including TRIM31 that
encodes an E3 ubiquitin ligase, and P450RAI2 (CYP26A), a potent retinoic acid 4-hydroxylase.
Table 9: Selected significantly changed genes in HaCaT keratinocytes after 13-cis RA
treatment
Fold
Change
Gene Title
Gene Symbol
#
RARE
3.56
lipocalin 2 (oncogene 24p3)
3.22
carcinoembryonic antigen-related cell adhesion molecule 5
3.21
amiloride binding protein 1 (amine oxidase (copper-containing))
3.21
cytochrome P450 retinoid metabolizing protein
P450RAI-2
2.77
carcinoembryonic antigen-related cell adhesion molecule 6
CEACAM6
RXR
2.69
phospholipase A2, group X
PLA2G10
RAR
2.33
fibulin 1
2.28
latexin protein
LXN
2.11
kallikrein 6 (neurosin, zyme)
KLK6
2.08
3'-phosphoadenosine 5'-phosphosulfate synthase 2
1.96
plasminogen activator, tissue
1.82
2',5'-oligoadenylate synthetase 1, 40/46kDa
1.81
GATA binding protein 3
GATA3
1.79
insulin-like growth factor binding protein 3
IGFBP3
1.76
nebulette
NEBL
1.70
sarcospan (Kras oncogene-associated gene)
SSPN
1.70
G protein-coupled receptor, family C, group 5, member B
1.69
S100 calcium binding protein P
S100P
1.67
prostaglandin I2 (prostacyclin) synthase
PTGIS
1.64
midkine (neurite growth-promoting factor 2)
1.64
annexin A9
1.63
involucrin
1.62
insulin-like growth factor binding protein 6
IGFBP6
1.54
phosphatidic acid phosphatase type 2A
PPAP2A
1.48
lysophosphatidic acid phosphatase
ACP6
1.34
retinoic acid induced 3
RAI3
1.30
2'-5'-oligoadenylate synthetase-like
OASL
-2.10
Microfibril-associated glycoprotein-2
MAGP2
LCN2
CEACAM5
RAR, RXR
RXR
ABP1
FBLN1
PAPSS2
PLAT
RAR
RAR,RXR
RAR
OAS1
GPRC5B
MDK
RAR,RXR
RAR
RAR, RXR
RXR
ANXA9
IVL
RAR
RAR, RXR
RAR, RXR
#retinoic acid or rexinoid receptor response element located within first 1000 base pairs of gene
78
3.3.6 QPCR verification of select genes from array analyses.
Quantitative real-time PCR for a select group of genes (based on fold changes) was
performed on a subset of RNA samples (depending on the availability of remaining RNA from
patient samples) in order to verify the direction and magnitude of the fold change in the 8-week
and one-week array analysis. Additional QPCR experiments to verify select SEB-1 sebocytes
and HaCaT keratinocytes gene changes were also performed.
For verification of the changes noted in the one-array analysis, sufficient RNA was
available from patients 9, 10, 13, 14, and 15 from among the 6 patients in this group. The genes
verified included lipocalin 2 (LCN2), retinoic acid receptor responder 1 (RARRES1, TIG1),
S100A7, serine protease inhibitor A3 (SERPINA3) and phospholipase A2 group 7 (platelet
activating factor acetyl hydrolase) (Figure 18a). In the 8-week analysis, sufficient RNA was
available from patients 1, 2, 4, and 7. Verification of changes in the level of expression of 3βhydroxysteroid dehydrogenase (3βHSD1), HMG CoA reductase (HMGCR), phospholipase A2
group 7, insulin induced gene 1 (INSIG), carnitine acyltransferase (CRAT) and zinc finger
binding protein 145 (ZBTB16) were performed by QPCR (Figure 18b).
For the SEB-1 gene array, we verified LCN2, TIG1, insulin-like growth factor binding
protein 3 (IGFBP3), GATA transcription factors 3 and 6 (GATA3, 6), ZBTB16 and solute carrier
family member (SLC22A17) (Figure 18c). Four gene changes were verified for the HaCaT
keratinocyte samples: LCN2, SLC22A17, RARRES1, and IGFBP3 (Figure 18d).
Furthermore, QPCR analysis was performed on normal human epidermal keratinocytes
(NHEK) treated with 0.1 µM 13-cis RA for 72 hours, even though gene expression analysis was
not done. NHEK samples were used for comparison to HaCaT keratinocytes and as such,
QPCR was performed for LCN2, SLC22A17, RARRES1, and IGFBP3 (Figure 18e).
For all QPCR analyses, patient samples and cell line samples, the direction and
magnitude of the change in expression for the selected genes is similar to that observed in the
gene array analyses.
79
Figure 18: QPCR verification of gene array gene changes. (a) One-week (b) 8-week. Data
represent the mean ± SEM of the fold change in gene expression as determined by REST-XL
(QPCR) in 4-5 subjects compared to 6-8 subjects as determined by gene array. (c) SEB-1 (d)
HaCaT (e) NHEK. Data represent the mean ± SEM of the fold change in gene expression as
determined by REST-XL (QPCR) in 3 samples compared to 3 samples as determined by gene
array.
80
3.3.7 Cluster Analysis
Using the computer software dChip (Li and Hung Wong, 2001), we performed
hierarchical clustering of the entire set of genes that were significantly up- or down-regulated
from our one-week and 8-week isotretinoin patient gene arrays. A two-way cluster analysis was
done; the data was clustered by patient sample and also by genes exhibiting a similar
expression profile. We found that the biopsy samples from the pre-treatment samples clustered
into separate and distinct groups when compared with their respective after-treatment biopsies
from both the one- and 8-week groups (Figure 19, one week data shown). Clustering of the
patient samples (columns) is indicated at the top of the diagram. Genes with higher correlation
coefficients among the standardized gene expression values across samples are clustered
together by rows. Therefore, genes in the same cluster share similar expression patterns. A
comparable analysis was performed with the SEB-1 gene array data; results showed control
and 13-cis RA treated arrays clustering in separate and distinct groups (data not shown).
81
Figure 19: Hierarchical clustering diagram of one-week isotretinoin patient samples.
Hierarchical clustering was used to compute a dendrogram that assembled all genes and
samples into a single tree. Patient samples included skin biopsies taken prior to treatment and
at one-week of treatment. Normalized array data was imported into dChip software version 1.3.
The information files for the Affymetrix human genome HG-U133A 2.0 array was obtained from
www.dChip.org (8-week data not shown). Each row represents a single gene and each column
represents a patient sample. (B=baseline and A=after treatment). The color reflects the level of
expression when compared to the mean level of expression for the entire biopsy set. Red
indicates expression higher than the mean and blue indicates lower expression than the mean.
82
3.3.8 Functional categorization of significantly changed genes
Gene expression analysis revealed numerous genes significantly affected by 13-cis RA
treatment. In order to determine if 13-cis RA is preferentially influencing a particular subset of
genes, we categorized them by the “dChip” computer software. Genes included on each array
carry annotations that allow them to be grouped according to categories including “gene
ontology”, “protein domains”, ”chromosomal location”, and “pathway”. It is important to note that
annotations in each of these categories are not available for all genes on the arrays. Each
category contains predefined terms for classifying genes. After hierarchical clustering, dChip
assessed the significance of all functional categories within the cluster tree. The 42 genes
changed in the one-week analysis and the 784 genes whose expression was significantly
changed in the 8-week analysis were assessed for significant enrichment for the above listed
categories. The p-value for this functional categorization is the probability of seeing x genes with
a certain category occurring in a group of k genes at random, given n annotated genes on the
array, of which m genes carry that specific annotation. Groups of genes mapping to a particular
cluster with p < 0.001 were considered significant.
From the 42 significantly changed genes at one-week, we identified 5 gene ontology
terms, 3 protein domains and one chromosomal location that were enriched according to dChip
(Table 10). The chromosomal location of 1q21 is the site of the epidermal differentiation
complex. Genes that are located within this chromosomal region are indicated in italics in
Table 7. “Pathway” analysis failed for this data; most likely due to the small number of
significantly changed genes (only 42) and the fact that only three of these genes carried
”pathway” annotations.
Table 10: Functional categorization of significantly changed one-week genes.
Gene Ontologies
Ectoderm development
Epidermal development
Response to pest, pathogen, or parasite
Response to external biotic stimulus
Vitamin binding
Protein Domains
Lipocalin related
Calcium binding
Latexin
Chromosomal Location
1q21
p-value
0.0001
0.00006
0.0007
0.0007
0.0006
0.00038
0
0
0.00013
83
Within the 784 significantly changed genes at 8 weeks, we identified 98 gene ontology
terms (data not shown), 21 protein domains, 0 chromosomal locations and 10 pathways that
were significantly enriched (p < 0.001). Enriched protein domains are listed in Table 11;
“anaphylotoxin/fibulin”, “fibronectin, type 1” and “collagen triple helix repeat” protein domains
were significantly up-regulated in our list of genes, while all other protein domains were
significantly down-regulated.
Table 11: Protein domains enriched within genes significantly changed at 8 weeks.
Protein Domain Term
Anaphylatoxin/fibulin
Fibronectin, type I
Collagen triple helix repeat
Polyprenyl synthetase
Carbohydrate kinase
AMP-dependent synthetase and ligase
3-oxo-5-alpha-steroid 4-dehydrogenase
ERG4/ERG24 ergosterol biosynthesis protein
Cytochrome b5
Peptidase T1, 20S proteasome
Enoyl-CoA hydratase/isomerase
Thiolase
Short-chain dehydrogenase/reductase
3-beta hydroxysteroid
dehydrogenase/isomerase
H+-transporting two-sector ATPase, C subunit
CoA-binding domain
Transketolase, central region
Transketolase, C terminal
ATP-citrate lyase/succinyl-CoA ligase
Histone core
Insulin-induced
# of genes in
cluster/
total # of genes
with term on gene
array
4 out of 9
4 out of 10
14 out of 104
3 out of 5
6 out of 11
11 out of 30
4 out of 6
3 out of 4
11 out of 25
6 out of 25
5 out of 19
4 out of 9
11 out of 51
0.00021
0.00034
3.2E-05
0.000493
0.000001
0
0.000027
0.000203
0
0.000258
0.000543
0.000211
0.000002
4 out of 5
0.000009
5 out of 8
4 out of 4
5 out of 11
5 out of 8
4 out of 8
16 out of 80
3 out of 4
0.000121
0.000002
0.000028
0.000004
0.000121
0
0.000203
p-value
* italics : protein domains that were significantly up-regulated
In the analysis of pathways, genes involved in each of the 10 pathways (Table 12) were
down-regulated. Interestingly, each of these 10 down-regulated pathways is either directly
involved in or linked to fatty acid and cholesterol metabolism, further supporting 13-cis RA’s role
in sebum suppression. A subset of genes that mapped to these enriched pathways is indicated
in italics in Table 6.
84
Table 12: Down-regulated pathways enriched within genes significantly changed at 8
weeks
Pathway term
Electron transport chain
Fatty acid degradation
Mitochondrial fatty acid β-oxidation
Krebs-TCA cycle
Reductive carboxylate cycle (CO2 fixation)
Fatty acid synthesis
Pentose phosphate//GenMAPP
Cholesterol biosynthesis
Biosynthesis of steroids
Terpenoid biosynthesis
p-value
24 out of 135
12 out of 43
11 out of 31
10 out of 54
5 out of 14
8 out of 28
5 out of 13
21 out of 30
15 out of 30
6 out of 11
0
0.000003
0.000001
0.000835
0.000788
0.000121
0.000531
0
0
0.000012
% of genes
with RARE
76
87.5
75
75
75
75
75
75
70
66.7
3.3.9 Promoter analysis of genes
We analyzed the promoter regions of all significantly changed genes for one-week and
8-week patient array data as well as SEB-1 sebocyte and HaCaT keratinocyte arrays. The
significantly changed genes containing retinoic acid receptor (RAR) or rexinoid receptor (RXR)
response elements (RAREs) consensus sequences are indicated in their respective tables.
Some genes did not have promoter sequences available from the Cold Spring Harbor
Laboratory database and are indicated with N/A in each table. Of the selected 8-week changed
genes listed in Table 2, 26 (60%) contained RAR or RXR consensus sequences. Interestingly,
all genes located on chromosome 1q21 contain consensus sequences for retinoic acid
receptors. A separate promoter analysis for the 8-week data indicated that of the 117 genes
that mapped to the 10 enriched pathways, 75% of these genes contained RAR or RXR
response elements in their promoters (Table 12). In the one-week analysis 21 of 42 (50%)
significantly changed genes contained retinoid consensus sequences (Table 7). Of the 85
genes significantly changed in SEB-1 sebocytes, 39% (33) contained RAR or RXR consensus
sequences in their promoters (Table 8) compared to 54% (29) of the 54 significantly changed
genes in HaCaT keratinocytes (Table 9).
85
3.3.10 Comparison of gene changes at one-week and 8-week revealed only 3 common
genes.
From among the 42 significantly changed genes at one-week and the 784 significantly
changed genes at 8-weeks, only 3 common genes were found. These 3 genes were downregulated 2- to 5-fold and include solute carrier family member 26, member 3 (SLC26A3);
phospholipase A2, group VII, (PLA2G7); and phosphodiesterase 6A (PDE6A).
In comparing the functional classification of the significantly changed genes, no
commonalities were found between one-week and 8-week data with regard to “gene ontology”,
“protein domain”, “pathway” or “chromosomal location”. The relative proportion of genes
containing RAR or RXR consensus sequences is similar between one-week and 8-week data
sets, 49% and 57%, respectively.
3.3.11 Comparisons between one-week, SEB-1 sebocytes and HaCaT keratinocytes array
data revealed only one gene in common between all three arrays.
Pair-wise comparisons were made between significantly changed genes from the oneweek isotretinoin, SEB-1 sebocytes, and HaCaT keratinocytes gene arrays (Table 13).
Although not identical genes, family members of the S100 calcium binding proteins, and
phospholipase A2 proteins were significantly changed in all three arrays. Surprisingly, 9 genes
were found in common between SEB-1 sebocytes and HaCaT keratinocytes.
86
Table 13: Common significantly changed genes within one-week isotretinoin, SEB-1
sebocyte and HaCaT keratinocyte gene arrays.
One-week vs. SEB-1 sebocyte
Fold
change
One-week
Fold change
SEB-1
sebocytes
Gene Name
Symbol
1.41
3
Growth Differentiation Factor 15
GDF15
1.67
2.51
E74-like factor 3
ELF3
2.61
12.25
retinoic acid responder 1
RARRES1
One-week vs. HaCaT keratinocyte
Fold
change
One-week
Fold change
HaCaT
keratinocytes
1.49
1.63
involucrin
IVL
1.99
1.69
S100 calcium binding protein P
S100P
Gene Name
Symbol
SEB-1 sebocytes vs. HaCaT keratinocytes
Fold
change
SEB-1
sebocytes
1.85
Fold change
HaCaT
keratinocytes
Gene Name
Symbol
GATA3
1.81
GATA binding protein 3
2.07
1.82
2',5'-oligoadenylate synthetase 1, 40/46kDa
OAS1
2.07
1.64
annexin A9
ANXA9
2.18
3.22
carcinoembryonic antigen-related cell adhesion molecule 5
CEACAM5
2.29
1.64
fucosidase, alpha-L-1, tissue
FUCA1
2.74
3.68
tripartite motif-containing 31
TRIM31
3.43
1.79
insulin-like growth factor binding protein 3
IGFBP3
3.52
1.7
carcinoembryonic antigen-related cell adhesion molecule 1
CEACAM1
4.98
2.77
carcinoembryonic antigen-related cell adhesion molecule 6
CEACAM6
Within the 42, 85, and 54 significantly changed genes in the one-week, SEB-1 sebocyte
and HaCaT keratinocyte gene array data, respectively, we identified only one gene in common
among all three arrays: lipocalin 2. Lipocalin 2 is significantly up-regulated in all three arrays
with approximately 7-fold increases within one-week and SEB-1 sebocytes, and 3.5-fold
increase in HaCaT keratinocytes above their respective baseline or vehicle control samples.
We chose to further characterize lipocalin 2 in human skin and in our cell lines because
it was one of the most highly up-regulated genes by 13-cis RA in our entire study. Lipocalins are
small molecular weight proteins that regulate processes such as immune response, retinol
transport, and prostaglandin synthesis. The lipocalin 2 gene product, neutrophil gelatinase
87
associated lipocalin (NGAL), is known to function in innate immunity and can induce apoptosis
(Devireddy, et al., 2005; Flo, et al., 2004; Tong et al, 2003).
3.3.12 Immunohistochemistry and western analysis showed increased NGAL expression
after 13-cis RA treatment in patient skin and our cell lines, respectively.
When biopsies were taken from patients before and after isotretinoin treatment, a portion
of the biopsy was formalin-fixed and paraffin-embedded for future studies, while the remainder
was used for the aforementioned gene expression analysis. Based on availability, sections from
one-week patients 9, 10, 13 and 14 were used. Immunohistochemistry on patient skin biopsies
revealed increased NGAL after isotretinoin treatment with localization to the basal layer of the
sebaceous gland and upper sebaceous duct (Figure 20).
Figure 20: NGAL increased after one-week isotretinoin treatment. Immunohistochemistry
for NGAL on sections on back skin taken before and at one-week treatment reveals notable
increase in NGAL expression in sebaceous gland and hair follicle. Sections were incubated
overnight with a 1:50 dilution of mouse monoclonal lipocalin 2/NGAL antibody. Negative control
sections omitted primary antibody. All sections were counterstained with hematoxylin.
NC=negative control; pre=before treatment; post=after treatment; SG=sebaceous gland; and
Fol=follicle. Representative images are shown. Total magnification: 400X
88
NGAL protein expression within our cell lines was verified by western blotting. SEB-1
sebocytes, HaCaT and NHEK keratinocytes were treated with 13-cis RA for 72 hours. NGAL
protein expression increased approximately 10-fold in SEB-1 sebocytes with both
concentrations of 13-cis RA tested when compared to control (p < 0.05). Non-significant fold
increases of 1.17 and 1.40 with 13-cis RA concentrations of 0.1 µM and 1 µM, respectively,
were noted in HaCaT keratinocytes with very similar results in NHEK (1.51- and 1.77-fold
increases) (Figure 21). Of note is the higher level of NGAL expression within HaCaT and NHEK
keratinocytes control samples when compared to SEB-1 control samples.
Figure 21: 13-cis RA increases NGAL protein expression. SEB-1 sebocytes, HaCaT
keratinocytes and NHEK keratinocytes were treated with vehicle control or 13-cis RA (0.1 or 1
µM). Protein expression was verified by western blot. Blots were incubated with primary
antibody to lipocalin 2 and β-actin for loading control normalization followed by densitometry.
Graph represents normalized fold-change values relative to control expression for a minimum of
six independent blots. Mean ± SEM.
89
3.3.13 Isotretinoin increased apoptosis in one-week patient sections.
Our previous work indicated that 13-cis RA induced apoptosis within SEB-1 sebocytes;
most likely by an RAR independent mechanism (Nelson, et al., 2006). We were able to perform
in vivo studies by using the baseline and one-week isotretinoin treated patient biopsy samples.
The TUNEL-peroxidase assay was performed on sections from baseline and one-week
treatment for patients 9 through 15. Patient 15 was omitted from analysis because sebaceous
glands were not found in the sections (n = 6 pairs of samples). One week of isotretinoin
treatment significantly increased the percentage of cells with TUNEL positive staining from
13.9% before treatment to 45.9% after treatment, an approximate 4-fold increase (p = 0.0001)
(Figure 22). TUNEL staining is strongest in the basal nuclei and the early differentiated
sebocytes, in a region similar to the distribution of NGAL.
Figure 22: TUNEL staining in sebaceous glands increased in patient skin after one-week
isotretinoin treatment. Two representative “after isotretinoin” images are shown. Skin sections
were obtained from paraffin blocks of patients 9-15 and were subjected to TUNEL-peroxidase
assay, according to manufacturer’s instructions. Assay controls included DNase I treated
positive and negative controls with primary antibody omitted in negative control. At least 2
sections from every patient (before and after) were analyzed. Sections were counter-stained
with hematoxylin. Data represent mean ± SD, n=6 patients; paired t-test was used for statistical
analysis. Total magnification 400X.
90
3.3.14 Purified NGAL protein induces apoptosis in SEB-1 sebocytes but not in HaCaT or
NHEK keratinocytes.
The mouse form of NGAL, known as 24p3, has been shown to induce apoptosis in
murine pro-B lymphocytic FL5.12 cells as well as other hematopoietic cells (Devireddy, et al.,
2005). We assessed whether purified NGAL protein is capable of inducing apoptosis in our
human cell lines using the TUNEL assay. SEB-1 sebocytes, HaCaT and NHEK keratinocytes
were treated with increasing concentrations of purified recombinant human NGAL protein for 24
hours and cells were approximately 50% confluent at the time of assay. After 24 hours, NGAL
significantly increased the percentage of TUNEL positive cells in SEB-1 sebocytes to a
maximum of 35% with 1ng/mL NGAL treatment, but had no effect on HaCaT or NHEK
keratinocytes (Figure 23). The percentage of cells in apoptosis with NGAL treatment in HaCaT
keratinocytes never exceeded 1%, while approximately 5% of NHEK cells were TUNEL-positive
in all treatments.
Figure 23: NGAL increases TUNEL staining in SEB-1 sebocytes SEB-1 sebocytes, HaCaT
and NHEK keratinocytes were treated in duplicate with vehicle control, 1pg/mL, 10pg/mL,
1ng/mL and 10ng/mL purified recombinant human NGAL protein (R&D Systems) for 24 hours.
(a) Representative images of SEB-1 sebocytes are shown. Total magnification 200X.
(b) Quantification of the percentage of TUNEL positive stained cells per treatment at 24 hours.
Data represent mean + SEM, n = 4-6. Statistical analyses were performed with ANOVA Two
Factor with Replication. * p< 0.05, ** p < 0.01, *** p < 0.0001
91
3.3.15 Apoptosis induced by NGAL is mediated by specific NGAL receptor isoforms.
Recently, a cell surface receptor for 24p3/NGAL was identified in murine pro-B lymphocytic
FL5.12 cells and the presence of this receptor is believed to be responsible for cell-specific
susceptibility to apoptosis (Devireddy, et al., 2005). It is possible that NGAL functions similarly
in our human cell lines. Based on sequence homology, the human homolog of 24p3R is
predicted to be solute carrier member SLC22A17. We obtained the affinity purified antibody to
the mouse 24p3 receptor and positive and negative control lysates from Dr. Michael Green
(University of Massachusetts Medical School, Worcester, MA). SEB-1 sebocytes express the
24p3 R-Long (60kD) and the high molecular weight (HMW, 70kD) forms of the receptor. HaCaT
keratinocytes express the 24p3 R-Short form (30kD) while NHEK do not express any form of the
receptor (Figure 24). Interestingly, SEB-1, which expresses the 24p3R-L and HMW forms,
undergoes apoptosis in response to 13-cis RA and NGAL treatment whereas HaCaT and NHEK
do not. Treatment with 13-cis RA, which induces NGAL expression in all cell lines, halves the
expression of 24p3R-L in SEB-1, decreases expression of HMW form by 3.5 fold in SEB-1 and
increases 24p3R-S form in HaCaT keratinocytes. The functional significance of the various
receptor forms is currently unknown; although, susceptibility to NGAL-induced apoptosis seems
to correlate with expression of the high MW form (C. Gazin, Green lab, personal
communication).
92
Figure 24: Cell-specific expression of 24p3R/NGAL-R isoforms is influenced by 13-cis RA.
(a) Protein lysates (vehicle and 1µM 13-cis RA 48 hours) were immunoblotted with affinity
purified 24p3 receptor antibody. Positive (+) and negative (-) control protein lysates obtained
from Dr. Michael Green. Variable expression of receptor isoforms (short, long, and high
molecular weight forms) are noted across cell lines and in response to 13-cis RA. (b) Relative
quantification of isoforms. Blots were incubated with β-actin for loading control normalization
followed by densitometry. Graph represents normalized fold-change values relative to control
expression for three independent blots. Mean ± SD.
3.4 Discussion
The biological effects of 13-cis RA are complex and the pathway(s) by which sebum is
decreased and acne is improved have yet to be fully elucidated. Early studies in the 1980’s
demonstrated that 13-cis RA markedly diminishes the size and secretion of sebaceous glands
after 16 weeks of isotretinoin treatment (Goldstein, et al., 1982). Our study demonstrates that
13-cis RA markedly decreases sebaceous gland volume by 8 weeks of treatment and a trend
toward this reduction is apparent at one-week. This is consistent with the observations that
sebum secretion can be markedly reduced by 13-cis RA as early as 2 weeks (Hughes and
Cunliffe, 1994; Stewart et al, 1983). However, no study to date, has examined the global
changes in gene expression that accompany these histological changes in the skin of patients
treated with 13-cis RA.
93
Using gene expression analysis, we were able to demonstrate distinctly different
patterns of gene expression at one-week and 8-weeks of treatment in patients receiving
isotretinoin for severe acne. Hundreds of genes were significantly changed after 8-weeks of
isotretinoin therapy. The preponderance of genes that were down-regulated involved lipid and
sterol metabolism and are characteristically expressed, although not exclusively, within
differentiated sebaceous glands (Thiboutot, et al., 2003). These changes at 8 weeks are
consistent with the reduction in sebaceous gland size and volume that has been definitely
demonstrated. The majority of genes that were up-regulated at this time point encode structural
proteins of the extracellular matrix such as collagens, fibulin and fibronectin. These up-regulated
genes are consistent with the known effects of retinoids on the extracellular matrix as reported
in studies of photoaging (Weiss et al, 1988). It is clear that the down-regulation of genes
involved in cholesterol and fatty acid metabolism and reduction in gland size are the net in vivo
effects of 13-cis RA on sebaceous gland function.
Gene changes at one-week are of particular interest because they may provide clues
about the initial changes induced by this drug. Those early gene changes can be broadly
categorized as tumor suppressors, protein processors, and genes involved in transfer or binding
of ions, amino acids, lipids or retinoids. For example, tazarotene induced gene 1 (TIG1, retinoic
acid responder 1 (RARRES1)) encodes a tumor suppressor belonging to the latexin family of
proteins, whose promoter is methylated (CpG island) and therefore silenced in a variety of
cancers (Mizuiri et al, 2005; Shutoh et al, 2005; Youssef et al, 2004; Zhang et al, 2004). The
increase in expression of TIG1 induced by 13-cis RA may mediate the known effects of this
drug in chemoprevention of skin cancer in addition to the known suppressive effects on
sebocyte proliferation (Nelson, et al., 2006; Zouboulis, et al., 1991; Zouboulis, et al., 1993;
Zouboulis, et al., 1999). Furthermore, genes encoding both serine proteases and serine
protease inhibitors were up-regulated by 13-cis RA at one-week. Serine protease inhibitors
(serpins) are involved in tissue remodeling and control of inflammation (Silverman et al, 2001).
Increased expression of serpins have been reported in inflammatory processes such as
psoriasis and inflammatory acne lesions (Takeda et al, 2002; Trivedi, et al., 2006). Since 13-cis
RA is the most potent agent available to reduce severe inflammatory acne, it is possible that the
up-regulation of serpins, which in turn, scavenge pro-inflammatory proteins, mediates the antiinflammatory effect of 13-cis RA. In support of this hypothesis, our previous HPLC study
demonstrated that 13-cis RA can be isomerized to ATRA within sebocytes and it has been
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shown that SERPINA5 is capable of binding ATRA in vitro and may function in retinoid transport
(Jerabek et al, 2001; Krebs et al, 1999).
In addition, among the genes whose expression was significantly increased at oneweek, there was an increased representation of genes within chromosomal location 1q21.
CHR1q21 is the location of the epidermal differentiation complex and includes TIG1, fourteen of
the S100 proteins, involucrin and cellular retinoid binding protein 2 among others. Retinoids are
crucial to epidermal development and differentiation (Wolbach and Howe, 1925). S100 proteins
modulate cellular differentiation, energy metabolism, cytoskeletal membrane interactions, and
cell cycle progression (Eckert et al, 2004). S100 protein family members are up-regulated in our
one-week patient array as well as our SEB-1 sebocytes and HaCaT keratinocytes gene arrays.
Interestingly, the specific up-regulated S100 proteins are induced by oxidative or inflammatory
stress and S100A7 (psoriasin) may function as a chemo-attractant agent for immune cells
(Eckert, et al., 2004). One can speculate that initial up-regulation of S100 proteins by 13-cis RA
is responsible for the “acne-flare” response observed in some patients receiving oral or topical
retinoids for the treatment of their acne. Our array data confirm the increase in S100 protein
noted with in vitro analysis of NHEK keratinocytes treated with 13-cis RA, 9-cis RA, ATRA and
4-oxo-13-cis RA (Baron, et al., 2005).
The distinction between the patterns of gene expression induced by 13-cis RA at oneweek and 8 weeks is substantiated by the finding that only 3 genes were commonly downregulated at both time points. One of these genes, phosphodiesterase 6A (PDEA6) encodes the
cyclic-GMP specific PDE6A alpha subunit, which is expressed in cells of the retinal rod outer
segment. Mutations in PDE6A have been identified as one cause of autosomal recessive
retinitis pigmentosa, associated with night blindness (Wang et al, 2001). Although night
blindness is a known potential side effect of isotretinoin treatment, it has not been linked with
changes in rod cell PDE6A.
Gene expression analysis on patient samples is invaluable because it takes into account
changes occurring in all compartments of the skin; changes that may not be reflected in isolated
cell systems. To gain insight into any possible cell-type specific effects of 13-cis RA on gene
expression we performed gene expression analysis on SEB-1 sebocytes and HaCaT
keratinocytes. 13-cis RA actions are clearly specific to the cell type, as only 9 significantly
changed genes were in common to both SEB-1 sebocytes and HaCaT keratinocytes.
Within the one-week and SEB-1 sebocyte gene arrays, 13-cis RA significantly increased
expression of multiple members of the solute carrier family of proteins. Recent data have
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highlighted the importance of the solute carrier family of proteins in skin biology. For example,
SLC12A8, which is up-regulated within our one-week analysis, encodes for a
sodium/potassium/chloride transporter that has recently been identified as a candidate gene for
psoriasis susceptibility contained within the PSORS5 locus of CHR3q (Hewett et al, 2002;
Huffmeier et al, 2005). Although, no mutations in SLC12A8 have been linked to psoriasis, it is
interesting to note that it is retinoid responsive. ATP12A, another significantly up-regulated gene
at one-week, encodes sodium/potassium ATPase, an integral membrane protein involved in
solute transport responsible for the hydrolysis of ATP coupled with the exchange of hydrogen
and potassium ions across membranes. This particular protein regulates ion flux into
melanosomes and affects tyrosinase activity (Watabe et al, 2004). Interestingly, another cation
transporter (SLC24A5) has been identified as playing a prominent role in skin biology by
regulating flux across the melanosome membrane in zebrafish and this gene links to skin color
in humans (Lamason et al, 2005). No other studies to date have examined the other solute
carrier molecules and their relationship to normal human skin or skin disorders.
One gene was found in common between one-week, SEB-1 sebocyte and HaCaT
keratinocyte gene expression arrays, lipocalin 2 (LCN2). LCN2 was one of the most highly upregulated genes (approximately 7 fold in one-week and SEB-1 sebocytes and ~3.5 fold in
HaCaT keratinocytes.) Lipocalins are small molecular weight proteins that regulate immune
response, retinal transport, prostaglandin synthesis, renal tube morphogenesis, cell growth and
metabolism (Flo, et al., 2004; Hanai et al, 2005; Newcomer and Ong, 2000; Yang et al, 2002).
For these reasons, we studied LCN2 and its gene product neutrophil gelatinase associated
lipocalin (NGAL) within human skin, SEB-1 sebocytes, HaCaT and NHEK keratinocytes.
Lipocalin family members are found in all species including bacteria, plants and animals.
Family members have very low amino acid similarity, but have three structurally conserved
domains and are characterized by their ability to bind small hydrophobic, lipophilic molecules
like retinol. Furthermore, most lipocalin family members are secreted proteins that bind to
specific cell surface receptors. The prototypic lipocalin family member is retinol binding protein
(RBP) (Akerstrom et al, 2000). Lipocalin 2 (NGAL, oncogene 24p3, uterocalin, α2-microglobulin
related protein) is a 25kD secreted protein located on CHR9q34. Expression of LCN2/NGAL is
ubiquitous with its expression being detected within bone marrow, uterus, prostate, salivary
gland, stomach appendix, colon, trachea, and lung. NGAL expression has been documented
within skin by Seo et. al., who showed its expression is restricted to inner root sheath and
infundibulum of the hair follicle in normal skin (Seo et al, 2006). In line with previous studies,
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immunohistochemistry on our one-week patient skin sections revealed increased NGAL
expression within the basal layer of the sebaceous gland, upper sebaceous duct and hair follicle
after 13-cis RA treatment. NGAL protein expression was increased in SEB-1 sebocytes, HaCaT
and NHEK keratinocytes with 13-cis RA treatment. It is not altogether surprising that NGAL is
increased in response to 13-cis RA as it has been shown to be increased with retinoid treatment
in other model systems (Caramuta et al, 2006; Tong, et al., 2003).
Documented ligands of NGAL include bacterial peptides, leukotriene B4, cholesterol
oleate, retinol and retinoic acid (Akerstrom, et al., 2000; Kjeldsen et al, 2000). Recent studies
into NGAL’s function focus on its anti-bacterial actions. Lipocalin knockout mice have an
increased susceptibility to bacterial infections, but are otherwise healthy (Berger et al, 2006).
NGAL can be secreted in response to Toll receptor activation by bacteria. NGAL binds bacterial
siderophores and functions by sequestering iron from bacteria (Flo, et al., 2004). Since
activation of Toll-like receptor 2 by P. acnes has been demonstrated in acne lesions it would be
interesting to determine if NGAL released in response to 13-cis RA targets P. acnes. To this
end, we performed preliminary Gram+ staining on skin sections for P. acnes bacterium before
and after one-week isotretinoin therapy in patients 9, 10 and 13. Gram+ staining revealed P.
acnes located at the base of the sebaceous gland and adjacent hair follicles (if present in the
section) with a small amount extending into the middle of the sebaceous gland (data not
shown). NGAL and P. acnes staining appear to localize to similar regions within the sebaceous
gland and in 75% of sections with NGAL staining, P. acnes was also detected. Isotretinoin
therapy causes a significant reduction in the gram positive, anaerobic P. acnes bacteria
(including antibiotic resistant strains), with levels slowly returning to baseline after discontinuing
treatment (Coates, et al., 2005; Leyden, et al., 1986). It remains to be determined if NGAL
produced in response to 13-cis RA is capable of killing P. acnes, if P. acnes itself can stimulate
NGAL production or a combination of both processes is occurring.
Devireddy et. al. have demonstrated that secreted NGAL is capable of inducing
apoptosis in F12.5 murine lymphocytes (Devireddy, et al., 2005). TUNEL staining on sections
from the one-week patient biopsies demonstrated that 13-cis RA increased the percentage of
cells undergoing apoptosis by 4-fold. This is the first in vivo evidence of 13-cis RA’s abilities to
induce apoptosis within the human sebaceous gland. TUNEL staining is localized and strongest
in the basal nuclei and the early differentiated sebocytes along the perimeter of the sebaceous
glands. This distribution of TUNEL staining is remarkably similar to the localization of NGAL
expression. Therefore it is possible that NGAL may be responsible for mediating the apoptotic
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effect of 13-cis RA on sebaceous glands. Purified recombinant human NGAL protein induced
apoptosis (TUNEL positive staining) within SEB-1 sebocytes. However, no apoptosis was
detected within HaCaT or NHEK keratinocytes. Our in vitro cell culture models mirror the patient
skin biopsies in that apoptosis is present within the sebaceous glands (sebocytes) and absent
from the surrounding tissue or surface of the skin (keratinocytes). This data suggests it is
possible for NGAL to be mediating the apoptotic effect of 13-cis RA on sebocytes.
Lipocalins mediate their effects by binding to specific cell surface receptors. The recent
identification of the mouse 24p3 receptor (24p3R) led to the identification of the highly
conserved human homolog, SLC22A17, by GenBank searches (Devireddy, et al., 2005). The
presence of this receptor correlated with apoptosis induction by NGAL. We obtained the purified
24p3R antibody from Dr. Michael Green and examined the expression of this receptor in our cell
lines. We detected three different isoforms of this receptor. SEB-1 sebocytes express 24p3R-L
and high MW (molecular weight) forms of this receptor. HaCaT keratinocytes express the
24p3R-S form of the receptor; this form of the receptor is an alternatively spliced variant lacking
the first N-terminal 154 amino acid residues. NHEK keratinocytes do not express any form of
the receptor. SEB-1 sebocytes undergo apoptosis in response to NGAL treatment and express
the 24p3R-L and HMW forms of the receptor, while the keratinocytes which lack these forms of
the receptor do not undergo apoptosis. Our findings suggest that susceptibility to NGAL-induced
apoptosis correlates with expression of the 24p3R-L or HMW form of the receptor. Studies in Dr.
Green’s lab also suggest that the HMW form is responsible for apoptosis sensitivity (C. Gazin,
Green lab, personal communication). The identity/sequence of this high MW form remains
unknown; although, phosphorylation, ubiquitination or glycosylation modifications of the 24p3RL form are not suspected.
Most intriguing is the retinoid-responsiveness of this receptor. Within SEB-1 sebocytes,
13-cis RA decreases the expression of 24p3R-L by approximately half and the HMW form all
but disappears. To our knowledge, this is the first evidence of lipocalin cell surface receptors
being regulated by retinoids. The functional significance of this decrease is unknown. It is
exciting to speculate that the down-regulation of this receptor by 13-cis RA is a negative
regulatory feed-back loop, turning off the apoptotic signal induced by the 13-cis RA upregulation of NGAL. Future experiments are needed to test this hypothesis.
The data presented in this study suggest that 13-cis RA initiates a temporal pattern of
gene expression. Approximately half of the significantly changed genes in all gene arrays
contain consensus sequences for RAR or RXR receptors. This study does not address the
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question of whether the changes in gene expression are the direct result of 13-cis RA, one of its
metabolites, or if retinoid receptors are involved. Instead, this study focused on the function of
these significantly changed genes and how these functions may mediate the effect of 13-cis RA
on sebaceous glands.
It is clear that the down-regulation of genes involved in cholesterol and fatty acid
metabolism and the reduction in size of the gland are the net in vivo effects of 13-cis RA on
sebaceous gland function. Gene expression analysis at the one-week time-point provided
insight into the initial changes induced by 13-cis RA, including up-regulation of NGAL. We
further investigated NGAL localization and function and suggest that NGAL may, in part,
mediate the apoptotic action of 13-cis RA on sebaceous glands. Our study provides rationale for
further study of candidate genes, including lipocalin 2, that mediate retinoid response in the skin
with the goal of discovering safer alternatives to oral retinoid use in the treatment of acne.
3.5 Materials and Methods
3.5.1 Patient selection and tissue biopsies
All protocols were approved by the Institutional Review Board of the Pennsylvania State
University College of Medicine and were conducted according to the principles outlined in the
Declaration of Helsinki. All subjects signed the informed consent form. Subjects included males
and females ages 14 to 40 years who were scheduled by their dermatologist to receive
treatment with 13-cis RA (isotretinoin, brand not noted) for severe acne. All aspects of the
patients’ treatment with 13-cis RA apart from the skin biopsies were standard of care and were
not part of this research. Exclusion criteria included patients on medications that could alter
sebum excretion such as hormonal therapy (including oral contraceptives) or patients with
underlying medical conditions requiring treatment with systemic medications that might interfere
with the gene array analysis. In order to avoid multiple biopsies from each patient, two groups of
patients were enrolled. Group 1 had a 5-mm punch biopsy of skin taken from their back before
treatment and again after approximately 8 weeks of daily treatment with oral isotretinoin. Group
2 had biopsies before treatment and again at Day 7 of treatment (See Table 1 for treatment
details). Biopsies were placed on ice and immediately transferred to the laboratory where they
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were trimmed of fat and a small section of each biopsy was taken and paraffin-embedded for
histology and immunohistochemistry. The remaining portion of the biopsy was flash frozen in
liquid nitrogen and used for total RNA isolation.
3.5.2 Image analysis of sebaceous gland size
Following hematoxylin and eosin staining of sections from each of the biopsies, image
analysis of sebaceous gland size was performed. Briefly, images were captured using a Spot
digital camera (Diagnostic Instruments, Inc.) and measurements were obtained with Image Pro
Plus Imaging Software Version 3.0 after spatial calibration with a micrometer slide under 10X
magnification. All areas of sebaceous gland were circled using a free-hand measuring tool and
the total area of the sebaceous gland was calculated in each section from the biopsy before
treatment and from the biopsy during treatment for all subjects in the study. The mean area of
sebaceous glands was calculated for each of the following groups: 8 week baseline, 8 week
treatment, 1 week baseline and 1 week treatment. Paired t- tests (α =0.05) were performed to
look for significant differences before and during treatment for the 8-week group and for the 1week group.
3.5.3 Cell Culture
The SEB-1 human sebocyte cell line was generated by transfection of secondary
sebocytes with SV40 Large T antigen as previously described (Thiboutot, et al., 2003). SEB-1
sebocytes were cultured and maintained in standard culture medium containing: 5.5mM Low
Glucose DulBecco’s Modified Eagle Medium (DMEM) 3:1 Ham’s F12, 2.5% fetal bovine serum
(FBS), hydrocortisone 0.4 µg/mL, adenine 1.8 X 10-4 M, insulin 10 ng/mL, epidermal growth
factor (EGF) 3 ng/mL, cholera toxin 1.2 X 10-10 M, and antibiotics. HaCaT keratinocytes were
cultured and maintained in 5.5 mM Low Glucose DMEM, 5% FBS and antibiotics. Normal
Human Epidermal Keratinocytes-neonatal (pooled) [NHEK-neo, Clonetics Keratinocyte System,
Cambrex Bioscience, Walkersville, MD] were cultured in Keratinocyte Growth Medium-2 (KGM2) (Cambrex Bioscience, Walkersville, MD).
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13-cis RA (R 3255) was purchased through SIGMA (St. Louis, MO). Stock solutions of
13-cis RA were handled under dimmed yellow light, dissolved in 100% ethanol to a
concentration of 10 mM and stored under N2 gas at -20ºC until use. Purified recombinant human
NGAL protein (amino acids 21-198) was purchased from R&D Systems (Minneapolis, MN)
ready to use and was stored at -20ºC until needed. Stock solutions were diluted to desired
concentrations in standard sebocyte culture medium.
3.5.4 Gene expression microarray analysis
Total RNA was isolated from skin biopsies and DNase treated using the RNeasy Fibrous
Tissue Kit (Qiagen Inc., Valencia, CA). Total RNA was isolated from SEB-1 sebocytes and
HaCaT keratinocytes treated with 0.1 µM 13-cis RA or vehicle alone (0.001% ethanol) in three
independent samples for 72 hours using a RNeasy kit (Qiagen Inc., Valencia, CA).
Approximately 2µg of total RNA from each sample was used to generate double stranded cDNA
using a T7-oligo (dT) primer. Biotinylated cRNA, produced through in vitro transcription, was
fragmented and hybridized to an Affymetrix human U95Av2 microarray for SEB-1 sebocytes
and U133A 2.0 microarray for all others. The arrays were processed on a GeneChip Fluidics
Station 450 and scanned on an Affymetrix GeneChip Scanner (Santa Clara, CA). Expression
signals were normalized as previously described (Irizarry et al, 2003; Irizarry et al, 2003; Trivedi,
et al., 2006). Significant gene expression alterations were identified using Significance Analysis
of Microarrays (SAM) computer software (Tusher et al, 2001). SAM controls the false positives
resulting from multiple comparisons through controlling the false discovery rate (FDR)
(Benjamini and Yekutieli, 2005). FDR is defined as the proportion of false positive genes
among all genes that are considered significant.
3.5.5 Quantitative real-time polymerase chain reaction (QPCR)
Quantitative real-time PCR was performed to confirm the direction and magnitude of
changes in the expression of select genes from the array data using Applied Biosystems’
Assays-on-Demand Taqman Universal PCR Master Mix and primer probe sets with ABI’s
7900HT Fast Real-Time PCR System with 384-well plate block module (Applied Biosystems,
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Foster City, CA). Integrity of isolated RNA was verified by agarose gel electrophoresis. cDNA
was generated from 1 µg of total RNA, primed with oligo-dT, using the Superscript First-Strand
Synthesis System for reverse transcription-PCR (Invitrogen, Carlsbad, CA). Diluted cDNA
samples were run for the reference gene TATA binding protein (TBP) as well as 6 genes of
interest from the 8 week array analysis (3βHSD1, HMG CoA reductase 1, PLA2G7, INSIG1,
Carnitine acyl transferase, ZFP145) and 5 genes of interest from the 1 week array analysis
(lipocalin 2, [LCN2] RARRES1, S100A7, SERPIN A3, PLA2G7). For SEB-1, HaCaT and NHEK
cell lines, 5-8 independent samples treated with 0.1 µM 13-cis RA or vehicle alone for 72 hours
were analyzed by QPCR. Genes of interest included: LCN2, RARRES1, insulin-like growth
factor binding protein 3 (IGFBP-3), GATA3, GATA6, ZBTB16, and LCN2 cell surface receptor
(SLC22A17). Assay controls included samples omitting reverse transcriptase enzyme as well
as samples without cDNA.
3.5.6 Cluster Analysis
Hierarchical clustering of patient samples and of significantly changed genes was
performed using the normalized array data imported into dChip software version 1.3. The
information files for the Affymetrix human genome HG-U133A 2.0 array was obtained from
www.dChip.org. Separate cluster analyses were performed for one-week and 8-week patient
gene arrays. Each row represents a single gene and each column represents a patient sample.
(B=baseline and A=after treatment). The color reflects the level of expression when compared
to the mean level of expression for the entire biopsy set. Red indicates expression higher than
the mean and blue indicates lower expression than the mean.
3.5.7 Database promoter analysis of genes whose expression was significantly changed
by 13-cis RA.
In an effort to understand which genes might be directly regulated by 13-cis RA or its
metabolites 9-cis RA, which activates retinoic acid receptors (RARs) and rexinoid receptors
(RXRs), or ATRA, which specifically activates RARs, the first 1000 basepairs (promoter regions
included) of the top 136 genes with the greatest statistically significant fold change at 8-weeks
were examined for retinoic acid response elements (RAREs). The first 1000 basepairs of all
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genes significantly changed at one-week, within SEB-1 sebocytes and within HaCaT
keratinocytes were also examined.
The base pair sequences of each gene were obtained from the Cold Spring Harbor
Laboratories Promoter Database (ftp://cshl.edu/pub/science/mzhanglab/PromoterSet) These
sequences were scanned for RAREs using the predefined consensus sequences within the
Transfac database through the Transcription Element Search System (TESS)
3.5.8 Comparisons of gene expression arrays
The one-week and 8-week array data was compared to identify common genes whose
expression was influenced by 13-cis RA. The data from the functional categorization of
significantly changed genes was similarly compared to identify common gene ontologies,
protein domains, chromosomal locations or pathways that are affected by isotretinoin treatment.
A comparison was also made between the percentage of significantly changed genes
containing RAR or RXR consensus sequences. Pair-wise comparisons were also performed
between one-week patient, SEB-1 sebocyte and HaCaT keratinocyte gene arrays to identify any
significantly changed genes in common.
3.5.9 NGAL immunohistochemistry
Immunohistochemistry was performed on formalin-fixed paraffin-embedded human skin
sections using the avidin-biotin complex method and AEC development (ABC kit and AEC
Substrate Kit for Peroxidase, Vector Laboratories, Inc.; Burlingame, CA). Briefly, sections from
patient (patients 9, 10, 13, 14) pre-treatment and after treatment biopsies were subjected to
deparaffinization, rehydration and antigen retrieval prior to immunohistochemistry. Antigen
retrieval was preformed using TRILOGY buffer (Cell Marque, Hot Springs, AR). Sections were
incubated overnight with 1:50 dilution of mouse monoclonal Lipocalin 2 antibody (Abcam Inc,
Cambridge MA). Negative control slides omitted primary antibody. Sections were counterstained with hematoxylin using standard procedures.
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3.5.10 Western blotting
Mouse monoclonal antibody to lipocalin 2 (NGAL), used at 1:1000 dilution with overnight
incubation (4ºC), was obtained from Abcam, Inc., (Cambridge, MA). Affinity purified 24p3 R
(NGAL receptor) antibody, used at 1:2000 dilution overnight incubation (4ºC), was kindly
provided by Dr. Michael Green (Howard Hughes Medical Institute, MA). Anti-rabbit horseradish
peroxidase (HRP) linked secondary antibody was purchased from Cell Signaling Technology
(Beverly, MA). Secondary Anti-mouse HRP antibody was purchased from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA).
Protein levels of NGAL and its receptor were examined using western blotting in our
various cell lines as previously described (Nelson, et al., 2006). SEB-1 sebocytes (passages
22-26), HaCaT keratinocytes (passages 20-25) and NHEK (passage 3) were grown in 100-mm
plates in standard culture medium until 50-75% confluent. Plates were rinsed with phosphate
buffered saline and then treated with: 13-cis RA (0.1 µM, or 1 µM,) or ethanol vehicle (0.01% or
less) as a negative control. Cells were treated for 48 or 72 hours. For NGAL-R blot, positive
(Madin-Darby canine kidney; MDCK) and negative control lysates were obtained from Dr.
Michael Green (University of Massachusetts Medical School, Worcester, MA). Blots were
incubated with appropriate primary and secondary antibodies. SuperSignal West Pico
Chemiluminescent Substrate (Pierce, Rockford, IL) was used for protein detection. β-actin was
used as a loading control. Films of blots were analyzed and quantified by densitometry with
QuantityOne Software (Bio-Rad, Hercules, CA.) after background subtraction. Western blots
were repeated a minimum of 3-6 independent times. Data was analyzed using a Student’s t-test
and results were considered significant if p < 0.05.
3.5.11 TdT-mediated dUTP Nick End Labeling (TUNEL) staining.
Sections from one-week before and after treatment biopsies from Patients 9-15 were
used. Sections of skin were subjected to deparaffinization with xylenes, rehydration with graded
ethanol series and permeabilization with 0.1% Triton-X, 0.1% sodium citrate in phosphate
buffered saline according to manufacturer’s instructions. Sections were subjected to "In Situ Cell
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Death Detection, Peroxidase" followed by counter staining with hematoxylin. Assay controls
included DNase I treated positive and negative controls with primary antibody omitted in
negative control. At least 2 sections from every patient (before and after) were analyzed.
Results were analyzed and quantified by counting positive staining cells / total cells in
sebaceous glands. Data represents mean ± SD, n = 6 patients; paired t-test was used for
statistical analysis and considered significant if p < 0.05.
SEB-1 sebocytes (passage 22-25); HaCaT keratinocytes (passage 24-25) and NHEK
keratinocytes (passage 1) were cultured in 12-well plates in standard medium until
approximately 30-40% confluent. Wells were rinsed with phosphate buffered saline (PBS) and
were treated in duplicate with vehicle control, 1 pg/mL, 10 pg/mL, 1 ng/mL, or 10 ng/mL of
recombinant human NGAL for 24 hours. Each well was considered one sample. Samples were
prepared by manufacturer’s instructions for In Situ Cell Death Detection Kit, Fluorescein (Roche
Applied Science, Indianapolis, IN). Assay controls included DNase I treated positive and
negative controls with primary antibody omitted in negative control. Results were analyzed and
quantified by counting positive staining cells / total cells in 3 representative fields per well for
each of the treatments done in duplicate. Each assay was performed 2-3 independent times.
Data analysis was performed using ANOVA Two Factor with Replication and considered
significant if p < 0.05.
Chapter 4
Mechanisms involved in induction of apoptosis in SEB-1 sebocytes:
Activation of the extrinsic death receptor pathway by Tumor Necrosis Factor related
apoptosis inducing ligand (TRAIL)
4.1 Chapter Abstract
Our previous work has shown that 13-cis RA induces apoptosis within SEB-1 sebocytes.
Gene expression analysis indicates that 13-cis RA induces gene expression of mediators of
apoptosis, particularly those involved with the extrinsic pathway including Tumor Necrosis
Factor related apoptosis inducing ligand (TRAIL). Induction of TRAIL by 13-cis RA was
confirmed by QPCR and western blotting. Furthermore, we verified that purified recombinant
human TRAIL protein induces apoptosis within SEB-1 sebocytes confirming that TRAIL
receptors are present on the surface of SEB-1 cells and the TRAIL apoptosis pathway is intact
within our cell system. In addition, using RNA interference technology, we demonstrated that by
decreasing TRAIL mRNA and protein expression in the presence of 13-cis RA leads to
decreased cleaved caspase 3 protein, a marker for apoptosis induction. These data indicate
that 13-cis RA preferentially activates the extrinsic apoptosis pathway through up-regulation of
TRAIL and suggests that TRAIL plays a role in mediating apoptosis induced by 13-cis RA in
SEB-1 sebocytes. Elucidating the cellular processes and pathways that are affected by 13-cis
RA in sebocytes is a step toward understanding the overall molecular mechanism of action of
this drug, which may lead to the identification of alternative strategies for the treatment of acne.
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4.2 Introduction
The mechanism of sebosuppression by 13-cis RA is poorly understood. Although as
early as the 1980’s, histological studies showed that isotretinoin dramatically reduces the size
and secretion of the sebaceous gland in animal models. Previous work in our laboratory
suggests that 13-cis RA decreases sebum production by influencing cell cycle and apoptotic
pathways (Nelson, et al., 2006).
Apoptosis, or programmed cell death, is absolutely essential during development when
organisms need to eliminate unwanted cells in an orderly manner without invoking the
inflammatory response. Apoptosis is a highly-regulated, well-orchestrated series of events
culminating in nuclear condensation, DNA fragmentation, membrane-blebbing, cell shrinkage,
and eventually phagocytosis of the dying cell (Wyllie, et al., 1980). Many different cellular
stimuli can induce apoptosis including growth factor withdrawal, cellular stress, irreversible DNA
damage, cytokine signaling and interference with pro-survival pathways. Two pathways of
apoptosis have been well characterized: the death receptor (extrinsic) and the mitochondrial
(intrinsic) apoptosis pathways. These two pathways converge with activation of caspase 3 and
in some cells by activation of the protein Bid, a Bcl-2 family member.
In this chapter, we investigated the apoptotic pathways activated by 13-cis RA. We were
guided by gene expression analysis that indicated that 13-cis RA induces key genes involved in
apoptosis. We report that 13-cis RA treatment up-regulates expression of Tumor necrosis factor
Related Apoptosis Inducing Ligand (TRAIL) within SEB-1 sebocytes. Treatment with
recombinant human purified TRAIL protein increased cleaved caspase 3 protein indicating that
TRAIL induces apoptosis in SEB-1 sebocytes on its own. Utilizing siRNA knockdown, we have
successfully inhibited TRAIL expression induced by 13-cis RA. In addition, knockdown of TRAIL
protein decreased 13-cis RA induced-cleaved caspase 3 protein expression; thus indicating that
TRAIL plays a role in mediating apoptosis induced by 13-cis RA in SEB-1 sebocytes.
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4.3 Results
4.3.1 13-cis RA up-regulates genes involved in apoptosis in SEB-1 sebocytes.
Gene expression analysis of SEB-1 sebocytes treated with 0.1 µM 13-cis RA for 72
hours revealed significant up-regulation of genes involved in apoptosis (Table 14). Most
interestingly, expression of tumor necrosis factor related apoptosis inducing ligand (TRAIL,
TNFSF10) and TNF superfamily member 6, also known as Fas (CD95) receptor were
increased. Both TRAIL and Fas exert their apoptotic effects through the extrinsic (death
receptor) pathway of apoptosis, with Fas being a death receptor within the plasma membrane
and TRAIL being a potent ligand (soluble or membrane bound) that binds to TRAIL-R1 and
TRAIL-R2 receptors (Smith, et al., 2003). Up-regulation of both of these molecules by 13-cis
RA suggests that 13-cis RA induces apoptosis via a death receptor pathway in sebocytes. In
comparison, neither TRAIL nor Fas is significantly increased in HaCaT keratinocytes, nor do
these cells undergo apoptosis in response to 13-cis RA (For significantly changed genes within
HaCaT keratinocytes, see A.3).
Table 14: Genes involved in apoptosis whose expression is significantly changed by 13cis RA in SEB-1
Fold Change
12.25
7.04
4.18
3.43
3.22
3.00
2.98
2.60
2.42
1.7
1.70
Gene
tazarotene induced gene
lipocalin 2 (oncogene 24p3)
tumor necrosis factor apoptosis inducing ligand
insulin-like growth factor binding protein 3
tazarotene induced gene
growth differentiation factor 15
SRY (sex determining region Y)-box 4
cyclin-dependent kinase inhibitor 1A (p21, Cip1)
interferon regulatory factor
Fas (TNF receptor superfamily, member 6) (CD95)
nuclear factor of kappa light polypeptide gene enhancer 2
(p49/p100)
Symbol
TIG1
LCN2
TNFSF10
IGFBP3
TIG3
GDF15
SOX4
CDKN1A
IRF1
Fas
NFKB2
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4.3.2 13-cis RA increases cleaved caspase 8 to a greater extent than 9-cis RA or ATRA.
To examine the possibility of death receptor pathway activation in SEB-1 sebocytes in
response to 13-cis RA, caspase 8 protein expression was studied via western blot. Previous
studies indicated that maximum apoptosis occurs at 72 hours post treatment with 13-cis RA. If
activation of the death receptor pathway is responsible for this apoptosis, then caspase 8
cleavage and activation would likely occur prior to 72 hours. Therefore, we examined the
expression of full length (p57), intermediate (p43/41) and active (p18) fragments of caspase 8 at
24 and 48 hours following 13-cis RA treatment. No cleaved caspase 8 was detected at 24 hours
(data not shown). At 48 hours, 13-cis RA increases expression of active caspase 8 (p18) by
approximately 7-fold compared to control (Figure 25). Experiments with 13-cis RA will be
repeated at least one more time before statistical significance of fold change will be determined.
Previous studies demonstrated that 13-cis RA induces apoptosis in SEB-1 sebocytes
while 9-cis RA and ATRA do not. It is possible that 13-cis RA is more potent in activation of
apoptotic pathways. Therefore, expression of full length (p57), intermediate (p43/41) and active
(p18) fragments of caspase 8 at 48 hours following 9-cis RA or ATRA treatment was examined.
In contrast to 13-cis RA, 9-cis RA and ATRA only increased expression of p18 by ~2-fold
(Figure 25).
Immunoblotting was performed with 1 µM 13-cis RA, 1 µM 9-cis RA and 1 µM ATRA
protein lysates as these were available at the time of these experiments. Additional
immunoblots will be performed with 0.1 µM concentrations in future experiments.
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Figure 25: Increased active caspase 8 with 13-cis RA treatment. Protein lysates from SEB-1
sebocytes treated with 1 µM concentrations of 13-cis RA, 9-cis RA and ATRA or vehicle control
(0.01% ethanol) and 1 µM) for 48 hours were immunoblotted with mouse Caspase 8 antibody
(Cell Signaling Technology). A representative blot is shown. Graph represents mean ± SD foldchange values normalized to control for 2 independent blots (13-cis RA) and 4 independent
blots (9-cis RA and ATRA)
4.3.3 13-cis RA increases TRAIL expression in SEB-1 sebocytes
Gene expression array analysis indicates that TRAIL is up-regulated 4.18-fold compared
to control at 72 hours in SEB-1 sebocytes treated with 0.1 µM 13-cis RA. To confirm this finding
and determine the time-line of TRAIL expression, we performed quantitative PCR (QPCR).
Using QPCR, TRAIL mRNA is up-regulated by 3.792-, 3.259- and 1.883-fold after 24, 48 and 72
hours of treatment with 0.1 µM 13-cis RA, respectively. Fold-changes and statistical significance
were determined with Relative Expression Software Tool (REST-XL) computer software
program and as such, the algorithms used to compare data sets do not permit determination of
standard error (Figure 26a).
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TRAIL is a type II transmembrane protein expressed on the surface of cells. It also exists
in a soluble form. Both the membrane form and the soluble form have been shown to induce
apoptosis in a variety of tumor cell lines (Wiley et al, 1995). TRAIL protein expression at 72
hours was confirmed in SEB-1 by immunoblotting with available protein lysates from cells
treated with 1µM 13-cis RA. Preliminary experiments indicate that the expression of the
membrane and soluble forms of TRAIL protein is ~4.5-fold higher with 1µM 13-cis RA than
control at 72 hours (Figure 26b).
An additional experiment was performed at a later date in SEB-1 cells treated with 1 µM
9-cis RA and ATRA. TRAIL protein expression (membrane + soluble forms) is slightly increased
with both treatments (approximately 1.5-fold increase) when compared to control (Figure 26b).
Of note, the 9-cis RA and ATRA immunoblot was performed with slightly modified transfer
conditions and a different lot number of TRAIL antibody which may explain the differences in
control TRAIL protein levels between this blot and the 13-cis RA blot. Additional experiments
with all three treatments together on the same blot need to be run to validate these perceived
differences in TRAIL protein levels. Immunoblots will also be performed with 0.1 µM retinoid
concentrations in future experiments.
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Figure 26: TRAIL expression is increased by 13-cis RA treatment in SEB-1 sebocytes. (a)
QPCR was performed for TRAIL at 24, 48 and 72 hours. Bars represent mean fold change of 3
independent samples as determined by REST-XL software. (b) TRAIL protein expression at 72
hours. Preliminary western blot shown. Graph shows relative level of TRAIL protein for one
experiment to date.
4.3.4 TRAIL increases levels of cleaved caspase 3 in SEB-1 sebocytes
TRAIL-mediated apoptosis is initiated by binding of TRAIL to a cell surface receptor,
TRAIL-R1 (DR4) or TRAIL-R2 (DR5). TRAIL-R1 or TRAIL-R2 genes were not included on our
initial U95Av2 Affymetrix microarray chips. To determine if TRAIL can induce apoptosis in SEB1 sebocytes purified recombinant human TRAIL protein (R&D Systems, Minneapolis, MN) was
added to our cells for 24 and 48 hours. Purified TRAIL protein induced apoptosis as evidenced
by increased cleaved caspase 3 protein expression (fragment sizes p19 and p17) as early as 24
hours with statistically significant increases by 48 hours (Figure 27, 48 hour data shown). At 24
hours, cleaved caspase 3 protein expression was marginally increased from 1.1 to 2-fold
compared to control. By 48 hours, TRAIL concentrations of 6ng/mL and higher, significantly
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increased cleaved caspase 3 protein levels by at least 4-fold when compared to control. This
result indicates that TRAIL receptors are present on the surface of SEB-1 cells and the TRAIL
apoptosis pathway is intact within our cell system.
Figure 27: TRAIL increases expression of cleaved caspase 3 protein. SEB-1 sebocytes
were treated with increasing concentrations of purified recombinant human TRAIL (rhTRAIL)
protein for 48 hours. (a) Representative blot is shown. (b) Graph represents normalized values
relative to control expression of three independent western blots. Mean ± SD. * p < 0.05.
4.3.5 siRNA knockdown of TRAIL inhibits activation of caspase 3 by 13-cis RA.
RNA interference (RNAi) technology allows investigators to take advantage of normal
cellular processes that recognize and degrade double-stranded RNA in a potent anti-viral
response. By introducing specific small interfering RNA (siRNA) molecules, knockdown of target
gene expression can be achieved (Dillon et al, 2005). We utilized ON-TARGETplus
SMARTpool TRAIL and siCONTROL siRNA reagents (Dharmacon, Lafayette, CO) to examine
the role of TRAIL in 13-cis RA induced apoptosis in SEB-1 sebocytes. siRNA experiments were
conducted with two concentrations of siRNA, either 0.7 µg or 1.5 µg, to determine the optimal
concentration necessary for specific “knock-down”. Twenty-four hours after nucleofection with
TRAIL or siCONTROL (non-targeting) siRNA, cells were treated with 0.1 µM 13-cis RA to
induce TRAIL expression.
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Preliminary QPCR experiments with the 0.7 µg dose of siRNA to TRAIL indicated that
TRAIL mRNA expression is decreased by 2.8-fold 24 hours after treatment with 0.1µM 13-cis
RA compared to siCONTROL samples. At 48 and 72 hours of 13-cis RA treatment, TRAIL
expression is decreased by approximately ~1.4 fold. Using 1.5 µg of siRNA to TRAIL in the
same experiment where cells were treated with 0.1 µM 13-cis RA, TRAIL mRNA levels were
decreased by 1.5 fold at 24 hours and a maximum decrease of 1.8 fold was found at 72 hours of
13-cis RA treatment (Figure 28).
24 hr
0.7 ug
1.5 ug
48 hr
0.7 ug
1.5 ug
72 hour
0.7 ug
1.5 ug
Fold-change in TRAIL mRNA
0
-0.5
-1
-1.5
-2
-2.5
-3
Figure 28: QPCR shows siRNA knockdown of TRAIL mRNA siCONTROL and TRAIL siRNA
(2 concentrations) were nucleofected into SEB-1 sebocytes using Amaxa Nucleofection Kit T
and nucleofector device. Twenty-four hours later, 0.1 µM 13-cis RA was added to induce TRAIL
expression. Total RNA was isolated at 24, 48 and 72 hours of 13-cis RA treatment. Graph
represents fold-change in level of TRAIL mRNA for one sample.
Due to limited amount of protein obtained from siRNA nucleofected samples, only one
immunoblot could be run, therefore, the blot was incubated with both TRAIL and cleaved
caspase 3 antibodies simultaneously, to prevent any loss of protein that may occur with
“stripping” the blot. Both antibodies have been previously used successfully in separate
experiments and no extraneous bands were noted that would interfere with interpretations of the
results.
TRAIL protein levels (membrane and soluble) were decreased with 1.5 µg of
siCONTROL when compared to 0.7 µg siCONTROL (Figure 29, lanes 1 and 2, bands indicated
by), which indicates that 1.5 µg dose is too high, leading to non-specific knockdown effects.
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Furthermore, TRAIL protein levels with 1.5 µg siRNA to TRAIL were increased (lane 4),
suggesting that this high dose of siRNA is toxic to the cells. Combined, these results suggest
that the lower dose of siRNA is more effective for specific “knock-down” of TRAIL.
Levels of membrane and soluble TRAIL were examined by western blot in cells that
were nucleofected with two concentrations of siRNA to TRAIL and then treated for 24 and 48
hours with 0.1 µM 13-cis RA. The membrane form of TRAIL has a molecular weight of 32 kD
whereas the molecular weight of the soluble form is 21kD. At 24 hours, no difference in the
level of TRAIL protein (membrane and soluble) was noted between siCONTROL and siRNA to
TRAIL samples (data not shown). At 48 hours, however, TRAIL protein levels were decreased
by ~2-fold in cells nucleofected with 0.7 µg of siRNA to TRAIL when compared to the
corresponding siCONTROL sample (Figure 29, lanes 1 and 3 bands indicated by). These
results are from one experiment only and additional experiments to verify the decrease of TRAIL
mRNA and protein levels with the 0.7 µg siRNA concentration will be performed.
To determine if TRAIL “knockdown” affects apoptosis in SEB-1 sebocytes, total protein
lysates from siRNA samples were subjected to immunoblotting with cleaved caspase 3 antibody
which detects the cleaved/active forms (p19, p17) of caspase 3. No active caspase 3 protein
was detected with 24 hours of 13-cis RA treatment (data not shown). This result is similar to
previous studies in SEB-1 sebocytes. However, after 48 hours of 0.1 µM 13-cis RA treatment,
active caspase 3 protein was decreased by ~2-fold in 0.7 µg siRNA TRAIL samples when
compared to siCONTROL samples of the same dose (Figure 29, lanes 1 and 3, bands indicated
by ). Active caspase 3 was decreased by ~1.5 fold with siRNA TRAIL concentration of 1.5 µg
when compared with control of the same dose (Figure 29, lanes 2 and 4, bands indicated by ).
Results are from one experiment only and additional experiments will be performed.
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Relative level TRAIL protein
1.8
siCONTROL
siTRAIL
1.2
siCONTROL
TRAIL
Relative level cl. (active) caspase 3
2
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
1
0.8
0.6
0.4
0.2
0
0
0.7 ug
1.5 ug
siRNA concentration
0.7 ug
1.5 ug
siRNA concentration
Figure 29: siRNA knockdown of TRAIL inhibits active caspase 3 protein expression.
siCONTROL and TRAIL siRNA (2 concentrations) were nucleofected into SEB-1 sebocytes
using Amaxa Nucleofection Kit T and nucleofector device. Twenty-four hours later, 0.1 µM 13cis RA was added to induce TRAIL expression. Protein was isolated at 24, 48 and 72 hours of
13-cis RA treatment and subjected to immunoblotting with TRAIL (1:500) and cleaved caspase
3 (1:800) antibodies. Graphs represent normalized relative levels (compared to control) of
TRAIL or cleaved caspase 3 protein of one sample.
In summary, siRNA to TRAIL decreases both TRAIL mRNA and protein levels. When
TRAIL levels are decreased, cleaved caspase 3 protein levels are also decreased. These
results suggest that TRAIL may be an important mediator of13-cis RA-induced apoptosis in
SEB-1 sebocytes; however, as results are from one experiment only, additional experiments are
needed to confirm this finding.
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4.4 Discussion
The mechanism of 13-cis RA- induced apoptosis in SEB-1 sebocytes and human
sebaceous glands is poorly understood. Elucidating the possible cellular pathways that are
affected by 13-cis RA in sebocytes is a step toward understanding the overall molecular
mechanism of action of this drug, which may lead to the identification of alternative strategies for
the treatment of acne. To this end, studies in this chapter focused on determining which
apoptosis pathway is activated by 13-cis RA in SEB-1 sebocytes. Gene expression analysis in
SEB-1 sebocytes revealed significant up-regulation of a number of genes involved in apoptosis.
The up-regulation of TRAIL and Fas suggests that 13-cis RA may be activating the extrinsic
apoptosis pathway.
Death receptors on the cell’s surface detect extracellular stimuli and, upon binding of
their respective ligands, rapidly activate an intracellular caspase signaling cascade that results
in apoptosis. The ligands for death receptors include Tumor necrosis factor Related ApoptosisInducing Ligand (TRAIL) and Fas ligand (FasL, CD95L). All death receptors including Fas
(CD95) and TRAIL-R1/R2 have an intracellular region termed the “death domain (DD)”. It is this
specific 80 amino acid sequence that allows the transmission of the apoptotic signal. DDs in the
receptor recruit intracellular adaptor molecules (also containing DDs) that have “death effector
domains (DED)”. DEDs recruit and activate the “initiator” caspases 8 and 10 by cleavage.
Initiator caspases proceed to activate “effector” caspases 3, 6, and 7, which by cleavage of their
specific substrates (i.e. PARP, α-fodrin) result in apoptosis of the cell. For example, TRAILmediated apoptosis is initiated by binding of TRAIL to a cell surface receptor, TRAILR1 (DR4) or
TRAILR2 (DR5), which then recruits caspase 8 via the adaptor molecules, TNF-R1 associated
death domain protein (TRADD) and Fas-associated death domain (FADD). Activated caspase 8
directly activates caspase 3, caspase 6, or caspase 7 or activates the intrinsic apoptosis
pathway via cleavage and activation of Bid (Slee, et al., 1999; Smith, et al., 2003; Wehrli, et al.,
2000).
Our initial studies focused on caspase 8. Caspase 8 is the primary caspase recruited
upon Fas or TRAIL-R1/-R2 death receptor activation by their respective ligands (Muzio et al,
1996; Srinivasula et al, 1996). 13-cis RA increased active caspase 8 (p18) by approximately 7fold while a 2-fold increase was observed with 9-cis RA and ATRA treatment in SEB-1
sebocytes. Taken together, these findings suggest that the death receptor pathway is activated
by 13-cis RA. Since TRAIL expression was significantly increased by 13-cis RA as determined
117
by gene expression analysis (4.2 fold over control), it seemed that TRAIL was a logical
candidate as a mediator of activation of the extrinsic apoptotic pathway. We confirmed the
increase in TRAIL expression and protein by QPCR and western blotting.
TRAIL is a 281 amino acid, type II transmembrane protein expressed on the surface of
cells. This tumor necrosis factor superfamily member also exists in a soluble form. Both the
membrane form and soluble form have been shown to induce apoptosis in a variety of tumor
cell lines. (Wiley, et al., 1995) TRAIL expression is detected in numerous normal tissues
(spleen, prostate, ovary, lung, small intestine, colon, kidney and pancreas) including skin.
(Stander and Schwarz, 2005; Wiley, et al., 1995) In normal human skin, TRAIL is expressed
within the epidermis with stronger expression in the basal layers than superficial layers;
infundibulum and outer root sheath of hair follicles; and in vitro studies have shown expression
in keratinocytes and melanocytes (Stander and Schwarz, 2005). TRAIL-R1 and –R2 receptors
and decoy TRAIL-R3 and –R4 are also expressed in the epidermis although expression is
restricted to distinct layers: TRAIL-R1 (suprabasal layers), TRAIL-R2 (granular layer), and both
TRAIL-R3 and TRAIL-R4 (basal layer) (Bachmann et al, 2001; Stander and Schwarz, 2005).
Decoy receptors bind to TRAIL but lack or have truncated intracellular domains preventing
transmission of the apoptotic signal (Marsters et al, 1997; Sheridan et al, 1997).
TRAIL is unique in that it is supposedly non-reactive to normal human cells and
specifically exerts its killing effect on tumor cells (Degli-Esposti et al, 1997). However, recent
studies indicate that TRAIL induces apoptosis within 6-48 hours in primary human cells such as
adult oligodendrocytes, prostate epithelial cells, primary hepatocytes and CD4+ T cells at
concentrations of 10-100ng/mL (Armeanu et al, 2003; Herbeuval et al, 2005; Matysiak et al,
2002; Nesterov et al, 2002). To determine if TRAIL induces apoptosis in our SEB-1 sebocytes,
we utilized recombinant human TRAIL protein, which maintains its bioactive function for a
minimum of 24 hours in cell culture medium at 37ºC (personal communication, R&D Systems).
The doses of TRAIL used in our studies (1-20ng) and the time frame of apoptosis induction
noted in our cells (24-48 hours) are similar to other reports in the literature. The fact that TRAIL
is capable of increasing cleaved caspase 3 protein expression within SEB-1 sebocytes suggests
the presence of TRAIL-R1/-R2 receptors on the surface of SEB-1 sebocytes and that the TRAIL
apoptosis pathway is intact within our cell system.
RNA interference is a powerful tool for investigating protein function and target
validation. Our siRNA knockdown experiments suggest that TRAIL may mediate 13-cis RAinduced apoptosis. In fact, our preliminary studies show that a 50% decrease in TRAIL protein
118
expression achieved with siRNA correlates with a 50% decrease in cleaved caspase 3 protein
expression in the presence of 13-cis RA. Additional experiments are needed, however, to
determine if TRAIL is the sole mediator of 13-cis RA- induced apoptosis in SEB-1 sebocytes.
These studies do not directly address whether TRAIL induction is directly mediated by
13-cis RA or one of its isomers such as, 9-cis RA or ATRA, or a metabolite. Furthermore,
additional studies are needed to assess whether retinoid receptor activation is required for this
increase in TRAIL expression. The promoter region of the TRAIL gene does contain an RAR
consensus sequence as identified by TESS computer software program. However, promoter
mapping experiments conducted by Clarke et. al. demonstrated that there is no retinoic acid
response element within 2kb of the transcription start site of TRAIL (Clarke et al, 2004). This is
in agreement with our previous studies within SEB-1 sebocytes demonstrating that apoptosis
was not blocked by a RAR pan-antagonist, suggesting an RAR independent mechanism to
apoptosis induction. TRAIL up-regulation by 13-cis RA, therefore, most likely occurs by an
indirect mechanism. Interferon regulatory factor 1 (IRF1) was identified as a critical factor in
mediating TRAIL induction by retinoic acid in NB4 APL leukemia cells and SK-BR-3 breast
cancer cells (Clarke, et al., 2004). Interestingly, 13-cis RA significantly up-regulates IRF1 gene
expression (2.42 fold increase) in SEB-1 sebocytes (A.2). It may be possible that TRAIL upregulation and the resulting apoptosis in SEB-1 sebocytes are due to IRF1. To definitively test
this hypothesis, studies utilizing siRNA knockdown of IRF1 could be performed.
To determine if 13-cis RA induced TRAIL mediated apoptosis is a relevant mechanism
for 13-cis RA actions within the sebaceous gland, additional in vivo studies are needed. First,
gene expression analysis of patient skin samples obtained at one week of isotretinoin (13-cis
RA) therapy did not show up-regulation of TRAIL expression compared to baseline (Table 7);
however, TRAIL expression is detected (“present”) within these patient biopsy samples. TRAIL
up-regulation may be masked within the biopsies because of the extremely low levels of
sebaceous gland mRNA relative to the total amount of mRNA from the skin biopsies. To
validate TRAIL expression in vivo, immunohistochemistry for TRAIL on sections from baseline
and one-week after isotretinoin can be performed. It would also be important to determine if
TRAIL-R1 and –R2 receptors are expressed within the sebaceous gland.
Our data (2.3.5) indicate that sebocytes, but not keratinocytes, undergo apoptosis in
response to 13-cis RA. It would be interesting to determine if the cell-specific sensitivity to
TRAIL accounts for the difference in the apoptotic response of sebocytes and keratinocytes to
13-cis RA. TRAIL-R1 and -R2 receptors are present within HaCaT keratinocytes and HaCaTs
119
undergo apoptosis in response to (500ng/mL) (Leverkus et al, 2003). Expression of TRAIL is
“present” but is not significantly increased in HaCaT keratinocytes in response to 13-cis RA
treatment according to our gene expression analysis. It would be important to confirm these
findings via QPCR or western blotting. It is possible that 13-cis RA is not capable of inducing the
level of TRAIL necessary for apoptosis induction in HaCaT keratinocytes. We can hypothesize
that the cell-type specific sensitivity to TRAIL-induced apoptosis is one reason why 13-cis RA is
incapable of inducing apoptosis within keratinocytes.
Our studies are the first to demonstrate up-regulation of TRAIL by 13-cis RA. However,
other retinoid compounds increase TRAIL expression. For example, ATRA and other RARα
specific ligands up-regulate TRAIL expression, leading to apoptosis in acute promyelocytic
leukemia (APL) cells (Altucci and Gronemeyer, 2002; Jimenez-Lara et al, 2004). In addition,
retinoids including fenretinide, synthetic retinoid CD437, and ATRA, increase sensitivity to
TRAIL-induced apoptosis by up-regulation of TRAIL-R1 and –R2 receptors in colon cancer,
prostate cancer, and lung cancer cell lines (Kouhara et al, 2007; Sun et al, 2000; Sun et al,
2000). Recently, by increasing apoptosis, 13-cis RA alone or in combination with interferon, has
been shown to be beneficial in the treatment of some forms of leukemia (Handa et al, 1997;
Maeda et al, 1996); although the mechanism of apoptosis is unknown. Our data indicate that
13-cis RA up-regulates TRAIL in sebocytes and it may be possible that 13-cis RA up-regulates
TRAIL expression in these cases, but additional experiments will be necessary to test this
hypothesis.
In summary, 13-cis RA-induced apoptosis is mediated, in part, by the up-regulation of
TRAIL and activation of the extrinsic apoptosis pathway in SEB-1 sebocytes. How 13-cis RA upregulates TRAIL expression in sebocytes and whether or not this drug alters TRAIL expression
in vivo remains to be determined. Understanding the actions of 13-cis RA on a molecular level
in terms of TRAIL regulation has implications in dermatology as well as in cancer therapeutics.
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4.5 Materials and Methods
4.5.1 Reagents
Retinoid compounds were purchased through SIGMA (St. Louis, MO): 13-cis RA (R
3255), 9-cis RA (R 4653) and ATRA (R 2625). Stock solutions of retinoids were handled under
dimmed yellow light, dissolved in 100% ethanol to a concentration of 10 mM and stored under
N2 gas at -20ºC until use. Recombinant human TRAIL protein was purchased ready-to-use from
R&D Systems (Minneapolis, MN) and stored at -20ºC. Stock solutions were diluted to desired
concentrations in standard sebocyte culture medium containing: 5.5mM low glucose Dulbecco’s
Modified Eagle Medium (DMEM) 3:1 Ham’s F12, 2.5% fetal bovine serum (FBS) ,
hydrocortisone (0.4µg/mL), adenine (1 X 10-8 M), insulin (10ng/mL), epidermal growth factor (3
ng/mL), cholera toxin (1.2 X 10-10 M) and antibiotics.
TRAIL polyclonal rabbit antibody was purchased from Abcam, INC (Cambridge, UK).
Cleaved caspase 3 (Asp175) rabbit monoclonal antibody and anti-rabbit HRP linked secondary
antibody were purchased from Cell Signaling Technology (Beverly, MA).
4.5.2 Quantitative Polymerase Chain Reaction (QPCR)
Quantitative real-time PCR was performed to confirm the direction and magnitude of
changes in the expression of select genes from the array data using Applied Biosystems’
Assays-on-Demand Taqman Universal PCR Master Mix and primer probe sets with ABI’s
7900HT Fast Real-Time PCR System with 384-well plate block module (Applied Biosystems,
Foster City, CA). SEB-1 sebocytes (passage 22) were cultured in 60-mm plates until
approximately 70% confluent. Sebocytes were treated with ethanol vehicle (0.01%) or 0.1 µM
13-cis RA in triplicate for 24, 48 and 72 hours. Total RNA was isolated using the RNeasy kit
(Qiagen Inc., Valencia, CA) and its integrity was verified by agarose gel electrophoresis. cDNA
was generated from 1 µg of total RNA, primed with oligo-dT, using the Superscript First-Strand
Synthesis System for reverse transcription-PCR (Invitrogen, Carlsbad, CA). Diluted cDNA
samples were run for the reference gene TATA binding protein (TBP) as well as TNFSF10
(TRAIL). Assay controls included samples omitting reverse transcriptase enzyme as well as
samples without cDNA. Data were analyzed using the REST-XL© program with efficiency
121
corrected values and considered significant if p < 0.05.
4.5.3 siRNA knockdown of TRAIL by nucleofection
Nucleofection optimization and efficiency in SEB-1 sebocytes was determined by ‘Cell
Line Optimization Nucleofector Kit’ according to manufacturer’s instructions (Amaxa
Biosystems, INC. Gaithersburg, MD). Briefly, SEB-1 sebocytes in their logarithmic growth
phase were nucleofected with 2 µg of pgmaxGFP DNA construct with each combination of
nucleofector solution (V or L) and each nucleofector device program. (Amaxa Biosystems, INC.,
Gaithersburg, MD). The combination with the highest efficiency (GFP expression) and lowest
mortality was chosen for future experiments: Nucleofector Kit T with program T-20.
Efficiency of nucleofection was determined by GFP expression quantified with FACS.
SEB-1 sebocytes (4 X 106 cells) were nucleofected with 2 µg of pgmaxGFP plating 1 X 106 cells
per 100-mm culture plate. Control cells were “shocked” without GFP. Immediately after
nucleofection, 500 µL RPMI 1640 medium was added to the cells and the cells were plated in
normal sebocyte growth medium. Cells were trypsinized and collected 24, 48, 72 and 96 hours
post-nucleofection and resuspended in phosphate buffered saline (PBS) for analysis by FACS.
Mock-nucleofected SEB-1 sebocytes were used to determine negative, non-GFP containing cell
populations and gates set accordingly. The percentages of cells expressing very high levels of
GFP were 87%, 90%, 73%, and 57% at 24, 48, 72 and 96 hours post-nucleofection.
ON-TARGET plus Human TNFSF10 (TRAIL) and siCONTROL siRNA duplex
oligonucleotides were purchased from Dharmacon Research (Lafayette, CO). The transfection
was performed as suggested by Dharmacon and Amaxa Biosystems with slight modifications.
SEB-1 sebocytes, 2 X 106 cells per 100 µL reaction (solution + siRNA), were nucleofected with
54 pmols (0.7 µg) siCONTROL, 105 pmols (1.5 µg) siCONTROL, 54 pmols TRAIL and 105
pmols TRAIL to optimize siRNA amount needed for future experiments. 13-cis RA (0.1 µM) was
added 24 hours post-nucleofection to induce TRAIL gene expression. Expression of TRAIL and
extent of siRNA knockdown was verified by QPCR and western blotting for TRAIL at 48, 72 or
96 hours after nucleofection (24, 48 and 72 hour 13-cis RA treatment).
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4.5.4 Western blot analysis for TRAIL and cleaved caspase 3
SEB-1 sebocytes (passage 22-26) were grown in 100-mm plates in standard culture
medium until 50-75% confluent. Plates were rinsed with PBS and treated with 13-cis RA (1 µM);
9-cis RA (1 µM); ATRA (1 µM) or ethanol vehicle (0.01% or less) as a negative control. Cells
were treated for 24, 48 or 72 hours. TRAIL and siCONTROL siRNA nucleofected cells (1 X106)
were plated in 60-mm plates and treated with 0.1 µM 13-cis RA 24 hours post nucleofection.
Total cell protein lysates from adherent and floating SEB-1 sebocytes were collected,
flash frozen in liquid nitrogen and stored at -80ºC until needed. Protein concentration of each
sample was determined by BCA Protein Assay (Pierce, Rockford, IL). Equal amounts of protein
were run on NuPage 10% or 4-12% Bis-Tris Gels with MES Running Buffer (Invitrogen Life
Technologies, Carlsbad, CA). Gels were transferred to polyvinylidene difluoride membrane,
blocked for 1 hour at room temperature in 5% non-fat dry milk and incubated with 1:1,000
dilution of cleaved caspase 3 (Asp175) rabbit monoclonal antibody, or 1:500 dilution of TRAIL
polyclonal rabbit antibody. Secondary anti-rabbit horseradish peroxidase linked antibodies were
used to detect primary antibodies. SuperSignal West Pico Chemiluminescent Substrate (Pierce,
Rockford, IL) was used for protein detection. Blots were stripped with 0.2M NaOH and reprobed
with β-actin (1:4000). Films of blots were analyzed and quantified by densitometry with
QuantityOne Software (Bio-Rad, Hercules, CA) after background subtraction. Data was
analyzed with Student’s t-test and results were considered significant if p < 0.05.
Chapter 5
Development and Characterization of a Temperature Sensitive Sebocyte Cell Line (TSS-1)
5.1 Chapter Abstract
Current sebocyte model systems are able to mimic some important characteristics of
sebaceous glands including androgen and retinoid sensitivity. The use of primary sebocytes for
experiments is challenging due to their limited replication and incomplete terminal differentiation
in culture. In order to circumvent this problem, investigators have developed Simian Virus 40
(SV40) immortalized sebocyte cell lines in their laboratories and currently, SZ95 and SEB-1, are
the only available immortalized sebocyte cell lines. The major drawback to use of these
particular cell models is the SV40 Large T antigen immortalization, which is necessary to
achieve sufficient cell numbers, interferes with the normal differentiation program of the
sebocytes. We have begun to develop a temperature sensitive SV40-immortalized sebocyte cell
line in order to remove the influence of SV40 on our experiments. TSS-1 sebocytes are a
suitable model system of human sebocytes because they 1) are able to be grown and
maintained in culture, 2) express melanocortin 5 and androgen receptors (markers of
differentiation), 3) demonstrate increased lipogenesis with androgen stimulation, 4) show
inhibited lipogenesis with 13-cis RA treatment and 5) exhibit decreased cellular proliferation with
13-cis RA treatment, all characteristics of sebocytes in vivo. With additional characterization
studies, we are hopeful that TSS-1 sebocytes will be proven to initiate a differentiation program
that more closely resembles the in vivo program.
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5.2 Introduction
Sebaceous gland research is limited by available model systems. Sebaceous glands are
composed of sebocytes, which by their nature, undergo holocrine rupture upon maturing; thus,
hindering the collection of sufficient cells to conduct experiments. Under normal conditions,
sebocytes grow for a finite span of time; the life span of a sebocyte (in vivo) from cell division to
holocrine secretion is approximately 21-25 days (Plewig and Christophers, 1974; Plewig, et al.,
1971). In culture, isolated human sebaceous gland lobules give rise to primary sebocyte
colonies that are a few centimeters in diameter and contain approximately 1 X 105 to 1 X 106
cells per colony. Cultured sebocytes accumulate lipids, which is evident by increases in Oil Red
O staining and through immunohistochemical staining for sebocyte specific markers including
EMA and OM-1 (Abdel-Naser, 2004; Zouboulis, et al., 1991). Experimentation with primary
sebocytes in the laboratory, however, is not realistic due to the low numbers of attainable cells.
In order to circumvent this problem, investigators have developed Simian Virus 40 (SV40)
immortalized sebocyte cell lines in their laboratories (Thiboutot, et al., 2003; Zouboulis, et al.,
1999). These immortalized cell lines allow for large-scale experiments and reproducibility of
results. Currently, SZ95 and SEB-1 are the only available immortalized sebocyte cell lines.
We have developed and characterized a temperature sensitive sebocyte cell line (TSS1). Using a temperature sensitive SV40 large T antigen DNA construct for immortalization, we
are able to acquire the numbers of cells required for a given experiment and then “shut-down”
the activity of the large T antigen by shifting cells to a restricted temperature, thus allowing the
cells to ‘regain’ a more normal pattern of differentiation. It is our hope that this cell line will more
accurately represent the sebocyte differentiation program that is noted in vivo. In the following
chapter, we describe the development and current characterization of our newest immortalized
sebocyte cell line.
5.3 Results
5.3.1 Temperature Sensitive Sebocytes (TSS) persist in culture
Primary sebocytes were stably transfected and transformed with DNA construct
pRSV1609. The optimum temperature for this construct is 33ºC. Immunofluorescence staining
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with an antibody to SV40 large T antigen indicates its presence in our new ‘TSS’ sebocytes and
as a positive control, SEB-1 immortalized sebocytes. Large T antigen was absent from
spontaneously immortalized HaCaT keratinocytes (negative control) (Figure 30).
Figure 30: TSS sebocytes express SV40 large T antigen. TSS sebocytes (33ºC), SEB-1
sebocytes (37ºC) and HaCaT Keratinocytes (37ºC) were incubated with primary antibody to
large T antigen and detected by anti-mouse, FITC secondary antibody. Cells were analyzed by
fluorescence microscopy. Representative images are shown.
The pRSV1609 construct encodes a Rous Sarcoma Virus (RSV) promoter to drive
expression of a mutant SV40 large T antigen. While large T antigen is expressed at all
temperatures, the protein is non-functional at the restrictive temperature. An amino acid change
resulting from the mutation at base pair 1609 triggers protein instability at elevated temperatures
leading to its unfolding and eventual degradation of the large T antigen protein. With this
construct, large T antigen expression and functionality was optimal at 33ºC and hence, this was
our permissive temperature. The stability and overall level of expression of this protein declined
with increasing temperature (Figure 31). SEB-1 sebocytes, which contain wild-type large T
antigen, were used for comparison.
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Figure 31: SV40 large T antigen expression declines with increasing temperature
within TSS sebocytes. TSS sebocytes were grown at 33ºC. Sebocytes then remained at
33ºC or were shifted to 37ºC or 41ºC for 72 hours to “shut-off” large T antigen protein. As a
control, SEB-1 sebocytes were also grown under all three temperatures. Cells were
incubated with primary antibody to large T antigen and detected by anti-mouse, FITC
secondary antibody. Representative images are shown.
Expression of SV40 large T antigen triggers uncontrolled cell growth. The most basic
view of immortalization by large T antigen is that cell proliferation is induced by interference with
Rb function and cell death is prohibited by blocking p53 normal functions, although other
proteins and pathways may play some minor roles (for Review: (Ahuja et al, 2005)). As a check
of the functionality of this protein, we performed manual cell counts every 3 days for 21 days at
the permissive temperature and also at a range of elevated temperatures (37ºC, 39ºC and
41ºC) to determine which restrictive temperature was optimal for curbing cell growth but not
triggering cell death. TSS sebocytes continually grew at 33ºC. The most rapid growth occured at
37ºC; however, this growth leveled off 9 days post plating. No increase in TSS sebocyte growth
was noted under 39ºC conditions. With the 41ºC temperature, cell numbers did not increase
(Figure 32).
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33ºC
37ºC
39ºC
41ºC
700
600
Cells x 10
4
500
400
300
200
100
0
3
6
9
12
15
18
21
Days
Figure 32: TSS sebocyte growth declines with increasing temperature. TSS-1
sebocytes were plated and placed at 33ºC, 37ºC, 39ºC or 41ºC. Manual cells counts
were performed every three days. Data points represent average of three independent
samples at each time point.
After sufficient numbers of TSS sebocytes were obtained, we began generating clonal
cell lines through serial dilution plating. Four distinct cell lines were produced: TSS-1 through
TSS-4. During the process of clonal selection, some characterization studies were performed
with “pooled” TSS sebocytes and then repeated where indicated with each of the clonal cell
lines.
5.3.2 TSS sebocytes display a differentiated phenotype: Increased intracellular lipid and
expression of the androgen receptor and the melanocortin 5 receptor.
Sebocyte differentiation is marked by an accumulation of intracellular lipids. The
differentiation status of TSS sebocytes at 33ºC, 37ºC and 41ºC was monitored by Oil Red O
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staining for intracellular neutral lipids. For comparison, Oil Red O staining was also done on
SEB-1 sebocytes under the same conditions. The magnitude of difference in O Red O staining
between SEB-1 and TSS sebocytes was not great. However, as the temperature increased, a
corresponding increase in the amount of Oil Red O staining in both cell lines was noted with
more staining in TSS-1 vs. SEB-1 at 41ºC (Figure 33).
Figure 33: Oil Red O staining in TSS sebocytes increased with elevating temperatures.
TSS and SEB-1 sebocytes were cultured at 33ºC, 37ºC and 41ºC temperatures f6r 6 days
followed by O Red O staining to detect intracellular neutral lipids. Cells were counterstained with
hematoxylin. Representative images are shown. Magnification: 400X.
In addition to lipid accumulation, differentiated sebocytes express androgen receptor and
melanocortin 5 receptor (Miyake et al, 1994; Zhang et al, 2006). Both androgen receptor and
melanocortin 5 receptor were expressed in the TSS-1, TSS-2, TSS-3 and TSS-4 sebocyte cell
lines. Melanocortin 5 receptor expression was similar in all cells lines and was unchanged with
respect to temperature. The level of androgen receptor expression varied considerably amongst
the cell lines and was higher at the restrictive temperatures (Figure 34). Based on its higher
level of expression of the androgen receptor, the TSS-1 cell line was chosen for all future
studies.
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Figure 34: Androgen receptor and melanocortin 5 receptor were expressed in
differentiated TSS sebocyte cell lines. a) Total protein lysates from TSS-1, TSS-2, TSS-3
and TSS-4 cell lines cultured at 33ºC, 37ºC and 39ºC for three days were analyzed for
expression of androgen receptor (110kD) via western blotting. Representative blots are shown.
b) Relative levels of androgen receptor expression. Mean ± SE, n= 3. c) Total protein lysates
from TSS-1, TSS-2, TSS-3 and TSS-4 cell lines at cultured 33ºC, 37ºC and 39ºC were analyzed
for expression of the melanocortin 5 receptor (32kD) via western blotting. Controls included
lysates from SEB-1, rat preputial cells (Rat P.C.) and human placenta (Plac.) Magic Markers XP
(M.M.) were used as size indicators.
5.3.3 TSS sebocytes enter senescence following prolonged incubation at ‘restrictive’
temperatures.
By shifting the sebocytes to a higher temperature, the proliferative signal generated by
expression of the large T antigen was “shut-down”. At this higher temperature (39ºC), it was
also noted that the morphology of theTSS-1 sebocytes began to change. Our experiments
depend on the cells’ ability to produce lipids and remain metabolically active, even if not in a
proliferative state. To address the possibility that TSS-1 sebocytes enter a senescent state upon
130
shifting to the restrictive temperature, a standard β-galactosidase-associated senescence assay
was performed (Dimri et al, 1995). With this assay, senescence is detected by an increase in bgalactosidase activity on provided substrate Xgal, translating to increased “blue” color within the
cells.
Shifting TSS-1 sebocytes from 33ºC to 39ºC resulted in sebocytes entering a senescent
state after prolonged incubation at this higher temperature Figure 35. By 7 days, most TSS-1
sebocytes had entered senescence at 39ºC; some senescence was detected at 33ºC.
Figure 35: TSS-1 sebocytes entered senescence after prolonged incubation at 39C. TSS-1
sebocytes were cultured at 33ºC, 37ºC and 39ºC for 3, 5 or 7 days followed by β-galactosidase
assay procedures. Representative images are shown. Magnification: 400X.
This assay was very important to the design of our future studies. Results from
immunofluorescence studies and proliferation studies showed that sufficient SV40 large T
antigen “shut-down” was achieved with 72-hour incubation at 39ºC. This assay illustrated that
TSS-1 sebocytes will enter senescence after a prolonged period of time; therefore, limiting the
amount of time a treatment can be applied. Based on the results from these three experiments,
we designed our cell culture and treatment paradigm (Figure 36).
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Figure 36: TSS-1 sebocyte culture and treatment model. This diagram outlines the timing and
temperatures involved in using TSS-1 sebocytes. Experiments analyzing basal conditions are
conducted on Day 5. Depending on the treatment length, assays are conducted on Days 6-9. In
most cases, parallel plates at 33ºC and 39ºC are examined.
5.3.4 Total lipogenesis is increased at elevated temperatures in TSS sebocytes.
Sebocytes accumulate intracellular lipids upon differentiation. Some lipids produced are
sebocyte-specific including wax esters and squalene. As a quantitative measure of total neutral
lipid production, standard lipogenesis assays were performed with confluent TSS sebocytes
maintained at 33ºC, 37ºC, and 41ºC for 3 days. Extremely low levels of lipids were detected at
41ºC (data not shown) therefore, the assay was repeated with temperatures of 33ºC, 37ºC and
39ºC. Unexpectedly, total lipogenesis was highest with a 37ºC incubation temperature, with little
to no difference between 33ºC and 39ºC (Figure 37). The experiment was repeated using an
incubation time of one week rather than 3 days. Again, at 37ºC, TSS sebocytes showed the
greatest amount of lipogenesis, with little to no difference between 33ºC and 39ºC (data not
shown).
cpm
14
6
C-acetate incorporated/10 cells/hour
Mean ± SEM
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90
80
*
70
60
50
40
30
20
10
0
37ºC
33ºC
39ºC
Figure 37: TSS sebocyte lipogenesis was greatest at 37ºC incubation with little to no
difference between 33 and 39ºC temperatures. TSS sebocytes were cultured and total
lipogenesis was performed at 3 days (one-week data not shown) after temperature switch.
Mean ± SEM; n = 6 samples. Statistical significance was determined by ANOVA Two Factor
with Replication and considered significant if *p < 0.05
Incorporation of 14C-acetate into sebaceous lipids was assayed in the TSS-1 cell line
after 3 days and at one week incubations at 33ºC, 37ºC and 39ºC. As expected from the
previous studies in TSS sebocytes, most lipid classes were highest at 37ºC followed by 33ºC
and 39ºC (Figure 38). Of note, is the lack of difference between the levels of acetate
incorporation into sebaceous lipids at 33º and 39ºC at 3 days, possibly related to temperature
requirements of the lipogenic enzymes. At the one-week time point, the decrease in lipogenesis
at 39ºC is most likely due to cells entering senescence.
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Figure 38: Incorporation of 14C acetate into lipids was greatest at 37ºC in TSS-1. TSS-1
sebocytes were cultured and lipogenesis assays were performed at 3 days (a) or one week (b)
after temperature switch. Mean ± SD; n = 4 samples. C=cholesterol, FOH=fatty alcohol,
OA=oleic acid, TAG=triglycerides, WE=wax esters, CO=cholesterol oleate, and SQ=squalene.
Statistical significance was determined by paired t-test and considered significant if *p < 0.05.
›: All temperatures statistically different from each other.
Although lipogenesis was maximal at 37ºC, subsequent lipogenesis assays were
conducted at 39ºC; thus minimizing the effect of the SV40 large T antigen.
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5.3.5 Synthetic androgen R1881 increases and 13-cis RA decreases lipids in TSS-1
sebocytes
Clinical observation and experimental evidence illustrate that androgens stimulate
sebum production in vivo. To demonstrate that our cell culture model suitably mimics the in vivo
situation, we treated TSS-1 (39ºC) with synthetic androgen R1881 (methyltrienolone) for 24
hours and measured total lipogenesis. TSS-1 sebocytes were androgen responsive. Androgen
treatment increased total lipogenesis by 31% in TSS-1 sebocytes after 24 hours when
compared to control (p = 0.003) (Figure 39).
Figure 39: Synthetic androgen R1881 increased total lipogenesis in TSS-1 sebocytes.
TSS-1 sebocytes were cultured as illustrated in Figure 36 and treated with R1881 (1 X 10-8 M)
or vehicle alone (control) for 24 hours prior to lipogenesis assay. Assay was repeated three
independent times. Mean ± SEM; n = 9. Statistical significance was determined by ANOVA Two
Factor with Replication and considered significant if p < 0.05.
13-cis RA is the most potent inhibitor of sebum production in vivo. To examine whether
13-cis RA exhibits a similar effect in our cell culture model, In experiments separate from the
synthetic androgen treatment, TSS-1 (39ºC) sebocytes were treated with 0.1 µM 13-cis RA for
24 hours prior to total lipogenesis assay. 13-cis RA significantly decreased total lipogenesis by
22% as compared to vehicle control at 24 hours (p < 0.01) (Figure 40).
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Figure 40: 13-cis RA decreased total lipogenesis in TSS-1 sebocytes. TSS-1 sebocytes
were cultured as illustrated in Figure 36 and treated with 13-cis RA (0.1 µM) or vehicle alone for
24 hours prior to lipogenesis assay. Assay was repeated three independent times. Mean ±
SEM; n = 9. Statistical significance was determined by ANOVA Two Factor with Replication and
considered significant if p < 0.05.
5.3.6 13-cis RA decreases TSS-1 proliferation
Growth inhibition by retinoids, including 13-cis RA, has been reported in numerous cell
types; including the SEB-1 sebocyte model (Nelson, et al., 2006). To further validate the
temperature sensitive sebocyte cell line, TSS-1 sebocytes were treated with 13-cis RA (0.1, 1
and 10 µM) for 24, 48 or 72 hours. All concentrations of 13-cis RA significantly decreased cell
numbers at 48 hours (8-13% decrease from control) and 72 hours (7-31% decrease from
control) in TSS-1 (33ºC), which is similar to the pattern observed in SEB-1 sebocytes (2.3.1).
At 39ºC, cell numbers decreased at both the 48 and 72 hour time points with 13-cis RA
concentrations (0.1, 1 or 10 µM) with a maximum decrease of 22% with 10 µM 13-cis RA at 72
hours. In general, a trend in decreased cell numbers is evident; although, not all decreases
were significant (Figure 41).
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Figure 41: 13-cis RA causes growth inhibition in TSS-1 sebocytes. (a) TSS-1 (33ºC) (b)
TSS-1 (39ºC). Time-dependent inhibition of TSS-1 sebocyte proliferation. TSS-1 cells were
cultured in the presence of ethanol vehicle alone (0.01% or less; control), 0.1 µM, 1 µM or 10
µM concentrations of 13-cis RA 24, 48 or 72 hours. Attached cells were collected, stained with
trypan blue, and counted manually. Data represent mean ± SEM, n = 9. Statistical analysis was
performed by ANOVA Two-Factor with Replication. * p < 0.05, **p < 0.01.
5.3.7 13-cis RA induces apoptosis in TSS-1 sebocytes at the restrictive temperature.
Sebaceous glands are retinoid responsive. Histologically, sebaceous glands from
patients treated with 13-cis RA are markedly reduced in size and individual sebocytes appear
undifferentiated, lacking the characteristic cytoplasmic accumulation of sebaceous lipids
(Goldstein, et al., 1982; Landthaler, et al., 1980; Strauss, et al., 1980). Apoptosis may explain
137
the reduced size of the sebaceous gland and previous studies demonstrated that 13-cis RA
induces apoptosis in SEB-1 sebocytes. Therefore, the TUNEL assay was performed in TSS-1
sebocytes (39ºC) treated with 13-cis RA (0.1, 1 and 10 µM) for 48 and 72 hours. At 48 hours,
the percentage of TUNEL-positive cells significantly increased with 1 µM and 10 µM
concentrations in a dose dependent manner (Figure 42). All concentrations of 13-cis RA at 72
hours increased the percentage of TUNEL-positive cells although much less than 48 hours.
Most cells at the 72 hour time point were “floating” in medium and therefore clearly had
undergone apoptosis but were not able to be assayed.
Figure 42: TSS-1 sebocytes undergo apoptosis with 13-cis RA treatment: TUNEL Staining.
Representative images of control, 0.1 µM, 1µM, and 10 µM 13-cis RA treatment at 48 and 72
hours at 39ºC. (b) Quantification of the percentage of TUNEL positive stained cells per
treatment at 48 and 72 hours. Data represent mean + SEM, n = 6. Statistical analyses were
performed with ANOVA Two Factor with Replication. * p < 0.05 ** p < 0.001
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5.4 Discussion
Immortalization of primary cells by the large T antigen of DNA tumor virus, SV40,
artificially disrupts the normal cell cycle and differentiation patterns. Large T antigen affects two
major players in cell cycle control: Retinoblastoma (Rb) tumor suppressor and p53. Large T
antigen binds and inactivates Rb, which acts as a negative regulator of normal cell proliferation.
Under normal growth conditions, the Rb tumor suppressor and its subsequent phosphorylation,
controls the transition from the G1 phase to the S phase of the cell cycle. Hypophosphorylated
Rb binds E2F transcription factor members inhibiting the transcription of E2F regulated genes
including cyclin family members and genes involved in DNA replication and repair (Ahuja, et al.,
2005; Berthet et al, 2006; Ohtani et al, 1995). The association of Rb with E2F prevents
progression through the cell cycle (Zhang et al, 1999). Phosphorylation of Rb by cyclin
dependent kinases results in dissociation from E2F, allowing the cell to proceed into S phase
(Berthet, et al., 2006; Nevins, 2001). DNA tumor viruses, including SV40 large T antigen,
specifically recognize the hypophosphorylated forms of Rb thereby removing the ability of Rb to
regulate E2F activities and allowing uncontrolled progression into S phase (Cooper, 1995). In
addition, T antigen inhibits the function of p53 by blocking its DNA binding surface, thus
inhibiting its ability to control gene expression (Bargonetti et al, 1992; Lilyestrom et al, 2006).
The most basic view of immortalization by large T antigen is that cell proliferation is induced by
interference with Rb function and cell death is prohibited by blocking p53 normal functions,
although other proteins and pathways may play some minor roles (for Review: (Ahuja, et al.,
2005)).
Even though SV40 large T antigen immortalization of sebocytes allows for large
numbers of cells to be acquired, it hinders the native sebocyte differentiation program.
Differentiation of primary sebocytes involves slowing of cellular growth and accumulation of
lipids within the cell (Rosenfield, 1989). This can be artificially induced in cell culture models
with SV40 immortalization by allowing culture plates to reach a confluent state and the addition
of adipogenic hormones including methylisobutylxanthine, dexamethasone and insulin (MDI) to
cause accumulation of lipids. These compounds are known to trigger differentiation of 3T3-L1
pre-adipocytes into adipocytes (Student et al, 1980), a process that is analogous to the
sebocyte differentiation program. Addition of these compounds adds to the complexity of
understanding the sebocyte’s native differentiation program as the compounds themselves may
affect other aspects of cellular development.
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By using a temperature sensitive large T antigen construct, expression of the large T
antigen can be controlled, allowing the sebocytes to “regain” a differentiation program more
similar to sebocytes in vivo than other SV40 immortalized cell lines. The specific temperature
sensitive construct (pRSV1609) obtained from Dr. Judy Tevethia (Penn State University College
of Medicine, Hershey, PA) has an arginine to lysine amino acid change at position 357 and has
been renamed ts357R-K (Loeber et al, 1989). In normal African green monkey kidney fibroblast
CV-1 cells, the ts357R-K mutant was able to replicate at the permissive temperature (32ºC) and
when shifted to 39ºC, replication was significantly reduced and completely blocked at 41ºC
(Loeber, et al., 1989). These results precisely mirror the cell proliferation studies we performed
with our TSS sebocyte cell line. Furthermore, characterization of the structural regions of large
T antigen protein indicates that this amino acid substitution is in the middle of the p53 binding
domain (Loeber, et al., 1989; Schmieg and Simmons, 1988). Previous studies have
demonstrated that ts357R-K at the permissive temperature is able to bind to p53 preventing its
function; however, at higher temperatures, p53 is not bound by ts357R-K and instead is able to
regain its DNA binding abilities (Ray et al, 1996). A shift to the higher temperature, therefore,
blocks SV40 inhibition of p53 function and allows the cell to regain control of p53 regulated gene
expression. Inactivation of pRb alone is not sufficient for full transformation and cells will lose
some features associated with transformation. Studies to verify that SV40 large T antigen does
not bind p53 in our TSS-1 sebocyte cell line at 39ºC are currently in progress.
In general, sebocytes in vitro undergo an incomplete terminal differentiation when
compared to in vivo. Therefore, it is necessary to characterize the degree of differentiation
within our new cell line. In rodents, melanocortins increase sebum production. Transgenic mice
deficient in the melanocortin-5 receptor have hypoplastic sebaceous glands and reduced sebum
production (Chen, et al., 1997). Melanocortin 5 receptor is expressed at the onset of
differentiation and in lipid-containing, completely differentiated sebocytes and, as such, is a
useful marker for accessing the differentiation status of our TSS sebocytes (Zhang, et al., 2006).
Melanocortin 5 receptor is detected in each of our clonal cell lines at all temperatures, thus
indicating the cell lines are capable of differentiation. The melanocortin-5 receptor may play a
role in the modulation of sebum production. Further experimentation is required to test this
hypothesis.
Androgens regulate sebaceous gland development and differentiation in vivo. Individuals
with a genetic deficiency of androgen receptors (complete androgen insensitivity) have no
detectable sebum secretion and do not develop acne (Imperato-McGinley, et al., 1993). In
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addition, testosterone or dihydroepiandrosterone (an adrenal precursor hormone) can increase
the size and secretion of sebaceous glands (Pochi and Strauss, 1969). Immunohistochemistry
studies have demonstrated the presence of androgen receptors within sebocytes (Blauer et al,
1991; Pelletier and Ren, 2004). Furthermore, using rat preputial cells, androgen receptor
expression has been found to increase with sebocyte differentiation (Miyake, et al., 1994).
Therefore, we examined androgen receptor expression and androgen-responsiveness within
our newly developed cell line. Our studies indicate that TSS-1 sebocytes do not express
androgen receptor at 33ºC. Androgen receptor expression does, however, increase with
increasing temperature, suggesting that at 39ºC, TSS-1 sebocytes have undergone some
degree of differentiation. In contrast, SEB-1 sebocytes do not express androgen receptor.
Based on the high level of androgen receptor expression when compared to the other TSS cell
lines, the TSS-1 cell line was chosen for additional characterization studies. As an assessment
of androgen-responsiveness, we used synthetic androgen, R1881 (methyltrienolone), and
measured total lipogenesis, which is analogous to sebum production in vivo. Synthetic androgen
R1881 increased total lipogenesis within our TSS-1 sebocytes.
Retinoids also regulate sebaceous glands by inhibiting differentiation in vivo. Histology
revealed a marked decrease in the size and secretion of the sebaceous glands after 16 weeks
isotretinoin treatment (Goldstein, et al., 1982). Retinoids (13-cis RA, ATRA and 9-cis RA) have
been shown to inhibit proliferation on cultured human sebocytes, SZ95 and SEB-1 SV40immortalized sebocyte cell lines, and rat preputial cells, although the magnitude of inhibition was
retinoid- and length of treatment-dependent (Nelson, et al., 2006; Tsukada, et al., 2000; Wrobel,
et al., 2003; Zouboulis, et al., 1991; Zouboulis, et al., 1993). TSS-1 sebocytes respond similarly
to 13-cis RA treatment with the same characteristic decrease in cell proliferation that other
human sebocyte models exhibit. Results of the proliferation studies in TSS-1 (33ºC) sebocytes
parallel the results of previous studies in SEB-1 sebocytes; most likely due to the constitutive
expression of SV40 large T antigen protein in both cell lines. Interestingly, the decrease in
proliferation of the TSS-1 sebocytes at 39ºC is not as pronounced as at 33ºC. The percentage
of TSS-1 (39ºC) sebocytes undergoing 13-cis RA-induced apoptosis is higher than the
percentage seen in SEB-1 sebocytes in previous studies. 13-cis RA, the most potent inhibitor of
sebum production in vivo, significantly decreased total lipogenesis in TSS-1 sebocytes.
In summary, TSS-1 sebocytes are a suitable model system of human sebocytes
because they 1) are able to be grown and maintained in culture, 2) express melanocortin 5 and
androgen receptors (markers of differentiation), 3) demonstrate increased lipogenesis with
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androgen stimulation, 4) show inhibited lipogenesis with 13-cis RA treatment and 5) exhibit
decreased cellular proliferation with 13-cis RA treatment, all characteristics of sebocytes in vivo.
Although this cell line has numerous positive features which are listed above, there are
drawbacks to use of the model system. First, we are limited in the amount of time a treatment
can be applied to the cells. Under our experiment paradigm, cells must be at 39ºC for 72 hours
in order to “deactivate” the uncontrolled growth signal generated by functional large T antigen.
Our initial characterization studies indicate that the TSS-1 sebocytes will enter a senescent
state after a period of 7 days at the restricted temperature. Combined, this only allows for a 3-4
day treatment period. At the present time, this time period is sufficient for all our currently
planned experiments; however, future experiments will have to be designed accordingly with
this limitation in mind.
A second potential drawback to our TSS-1 model system may be the induction of the
heat shock response. By shifting TSS-1 sebocytes to 39ºC to “shut off” SV40 function, the
sustained incubation at the higher temperature may induce the heat shock response which can
interfere with the normal differentiation program. ‘Heat shock’ can rapidly induce transcription of
heat shock proteins whose primary function is to resist thermal stress. In addition, heat shock
can up-regulate signaling pathways directly involved in cell survival and cell death and,
depending on the duration and intensity of the stress, may counteract or up-regulate the
apoptotic response (Anckar and Sistonen, 2007; Nadeau and Landry, 2007; Voellmy and
Boellmann, 2007). TSS-1 sebocytes were subjected to gene expression analysis at both
temperatures (33 and 39; data not shown). Preliminary inspection of the results indicates that
incubation at 39ºC induces expression of genes involved in the heat shock response, with
approximately 10 heat shock proteins (HSP) and heat shock factor 2 binding protein (HSF2BP)
significantly increased when compared to 33ºC control gene arrays. Interestingly, one of the
most highly up-regulated genes, thioredoxin interacting protein (TXNIP, Vitamin D3 up-regulated
protein 1, (VDUP1)) (up-regulated ~10-fold), has been shown to be induced under conditions of
oxidative or heat shock stress. Overexpression of this protein triggered decreased proliferation
and induced apoptosis within mouse fibroblasts (Junn et al, 2000). This may partially explain
the observed increase in apoptosis sensitivity to 13-cis RA because up-regulation of this gene
by increased temperature may ‘prime’ apoptotic pathways.
Current sebocyte model systems are able to mimic some important characteristics of
sebaceous glands, but sebocytes generally undergo incomplete terminal differentiation in
culture. Using a temperature sensitive SV40 construct, we were able to develop the TSS-1 cell
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line that exhibits the same important characteristics of previous models but is capable of more
complete differentiation in vitro than previous models. With additional characterization studies,
we are hopeful that TSS-1 sebocytes will be proven to initiate a differentiation program that
more closely resembles the in vivo program.
5.5 Materials and Methods
5.5.1 Sebocyte Culture
Human skin was obtained from facial surgeries under a protocol approved by the
Institutional Review Board of the Pennsylvania State University College of Medicine. Human
sebaceous glands were dissected from facial skin and sebocyte cultures were established. Cells
were co-cultured with mitomycin-C-inactivated 3T3 fibroblasts in medium containing: 5.5mM
Low Glucose DulBecco’s Modified Eagle Medium (DMEM) 3:1 Ham’s F12, 2.5% fetal bovine
serum (FBS), hydrocortisone 0.4 µg/mL, adenine 1.8 X 10-4 M, insulin 10 ng/mL, epidermal
growth factor (EGF) 3 ng/mL, cholera toxin 1.2 X 10-10 M, and antibiotics. Primary sebocytes
were detected as “outgrowths” from the sebaceous glands surrounding by the clearly distinct
3T3 fibroblasts. EDTA (0.02%) removes 3T3 fibroblasts from culture dishes and sebocytes were
collected through trypsin digestion. Sebocytes were stored in freezing medium containing low
glucose DMEM, 10% FBS, and 10%DMSO in liquid nitrogen cryo-storage until needed.
5.5.2 Establishment of TSS Sebocytes and Individual Clonal Cell Lines
Secondary sebocyte cultures were established as above from sebaceous glands
dissected from the normal ear of an 80-y-old male and stored in liquid nitrogen. Primary
sebocytes (p3) were recovered and plated on four 35-mm tissue culture plates with Bajor’s
sebocyte medium containing: 10% FBS, high glucose, insulin and epidermal growth factor. Cells
were transfected with DNA construct, pRSV1609, containing the sequence encoding the
transforming SV40 large T protein with a RSV promoter (gift, Dr. Judy Tevethia) using Effectene
Transfection Reagent kit (Qiagen Sciences, Maryland) with 0.5 µg DNA per cell culture plate
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according to manufacturer instructions. The mutation at 1609 renders the protein non-functional
at non-permissive temperatures due to its “unfolding”; thus targeting it for degradation.
Successful transformation of sebocytes was confirmed in 5th passage sebocytes by localization
of SV40 large T antigen in the nucleus of transformed cells using immunohistochemistry and
continued passage in culture. We derived four cell lines (TSS-1 through TSS-4) using standard
clonal dilution techniques on passage 15 sebocytes. Permissive temperature of 33ºC and the
non-permissive temperature of 39ºC were empirically determined.
All experiments conducted with TSS sebocytes or individual clonal lines were done in
our standard sebocyte culture medium consisting of 5.5mM Low Glucose DulBecco’s Modified
Eagle Medium (DMEM) 3:1 Ham’s F12, 2.5% fetal bovine serum (FBS), hydrocortisone 0.4
µg/mL, adenine 1.8 X 10-4 M, insulin 10 ng/mL, epidermal growth factor (EGF) 3 ng/mL, cholera
toxin 1.2 X 10-10 M, and antibiotics.
5.5.3 Cell Growth and Viability
Cell growth and viability of TSS were calculated by plating equal numbers of cells per 35
mm plate followed by manual cell counts with a hemacytometer at days 3, 6, 9, 12, 15, 18 and
21. Cells were placed at 33ºC (permissive temperature) as well as 37ºC, 39ºC, and 41ºC to
determine the optimal non-permissive temperature. Viability was assessed using Trypan Blue
dye exclusion. Results are the average of two independent samples at each time point and
temperature.
To determine if 13-cis RA had any effect on TSS-1 sebocyte viability, cells were cultured
at 33ºC and shifted to 39ºC for 72 hours prior to treatment with vehicle control, 0.1 µM, 1 µM or
10 µM for 24, 48 and 72 hours in triplicate. Parallel studies were conducted on TSS-1
sebocytes maintained at 33ºC. Cells were detached using trypsin (0.05%), collected, and diluted
in standard cell culture medium for manual cell counts using a hemacytometer. Cell viability was
assessed using Trypan Blue dye exclusion. Each assay was repeated three independent times.
Statistical significance was determined by ANOVA Two Factor with Replication and considered
significant if p < 0.05.
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5.5.4 Immunohistochemistry and Oil Red O Staining
Transfection of sebocytes was confirmed by fluorescent immunohistochemistry with
SV40 antibody. Polyclonal mouse SV40 large T antigen antibody (901/902) was obtained from
Dr. Judy Tevethia. SEB-1 sebocytes were utilized as the positive control. Negative controls
included HaCaT keratinocytes and TSS incubated without primary antibody. Cells were cultured
and maintained under normal growing conditions. Antibody was used at 1:100 dilution with
overnight incubation followed by 1:500 dilution of secondary anti-mouse, fluorescein antibody.
Cells were examined under fluorescent microscopy and representative images are shown.
Sebaceous phenotype of TSS cells was verified by confirmation of lipid droplets with Oil
Red O staining. TSS sebocytes were cultured on slides placed at 33ºC. Six days later, cells
were maintained at 33ºC or shifted to 37ºC, 39ºC or 41ºC for 72 hours prior to staining. Cells
were fixed at room temperature with 10% formalin for 30 minutes followed by washings with
phosphate buffered saline (PBS). Oil Red O staining solution consisting of Oil Red Oil (Sigma,
St Louis, MO) and 99% isopropanol was added to slides for 15 minutes. Slides were rinsed with
50% isopropanol, then water and counterstained with hematoxylin. Slides were examined under
light microscopy (400X total magnification) for presence of Oil Red O staining. Representative
images were captured using a Spot digital camera (Diagnostic Instruments, Inc.).
5.5.5 Western Analysis
Total protein lysates were collected from TSS-1, TSS-2, TSS-3, TSS-4 and SEB-1
sebocytes. Each TSS-1 cell line was grown at 33ºC until approximately 70% confluent and then
maintained at 33ºC or shifted to 37ºC or 39ºC for 3 days. Total protein lysates were collected,
quantified and separated by electrophoresis as previously described in 2.5.4. Blots were
incubated with 1:1000 dilution of polyclonal rabbit androgen receptor antibody (Cell Signaling
Technology, Beverly, MA) or 1:500 dilution of polyclonal chicken melanocortin receptor 5
antibody (Cocalico Biologicals Inc, Reamstown, PA). Secondary anti-rabbit and –chicken
horseradish peroxidase linked antibodies were used to detect primary antibodies. SuperSignal
West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) was used for protein detection.
Blots were stripped with Restore Western Blot Stripping Buffer (Piece, Rockford, IL) and reprobed with β-actin (Cell Signaling Technology, Beverly, MA). Films of blots were analyzed and
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quantified by densitometry with QuantityOne Software (Bio-Rad, Hercules, CA) after
background subtraction. Androgen receptor western blots were repeated three independent
times. Data was analyzed with Student’s t-test and results were considered significant if p <
0.05.
5.5.6 β-galactosidase Senescence Assay
Individual clonal cell lines TSS-1, TSS-2, TSS-3 and TSS-4 were cultured and placed at
33ºC, 37ºC, or 39ºC for 3, 5, or 7 days. After the appropriate time period, cells were washed
with PBS and fixed with a PBS solution containing 2% formaldehyde, 0.2% glutaraldehyde.
Fixed cells were incubated overnight in a 37ºC “no-CO2” incubator with staining solution
containing: 1mg/mL X-gal in dimethylformamide, 40mM 0.2M citric acid/sodium phosphate
buffer, 5 mM ferrocyanide, 5 mM ferricyanide, 5M sodium chloride and 1M magnesium chloride.
After incubation, plates were washed twice with PBS. Plates were examined under light
microscopy for “degree of blue color” and representative images were captured at 400X total
magnification.
5.5.7 Lipogenesis Assay: 14C-actetate incorporation into neutral lipids
TSS (passages 17-20) and individual TSS cell lines, TSS-1, TSS-2, TSS-3 and TSS-4
were subjected to our standard lipogenesis assay to measure the amount of neutral lipids within
the cells at the different incubator temperatures (33ºC, 37ºC, 39ºC, 41ºC). SEB-1 sebocytes
(constitutive SV40 expression) grown under the same conditions were used for comparisons.
TSS and each TSS individual cell line (2 X 105 cells) were cultured in 35-mm plates and
incubated at 33ºC until confluent. Once confluent, some plates remained at 33ºC while other
plates were shifted to 37ºC, 39ºC or 41ºC for another 3 days prior to the assay to allow for SV40
large T antigen “shut-down”. Cells were fed every other day with standard sebocyte medium.
Cells were collected by trypsinization and manually counted. Cells were resuspended in DMEM
containing 1 µCi of 14C-acetate and incubated for 2 hours at 37ºC with shaking. Total lipids were
extracted with ethyl ether and counted by liquid scintillation counting. Manual cell counts were
performed to normalize data to cell number. Acetate incorporation into lipids was expressed as
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“cpm 14C-acetate incorporated/106 cells/hour.” Each experiment was repeated 2-3 independent
times. Additionally, TSS-1 sebocytes (passage 23) were subjected to a “neutral lipid pattern
analysis”. Assay was performed as above, except after lipid extraction, lipids were separated by
thin layer chromatography and individual lipids were counted by liquid scintillation counting
according to published methods (Smith et al, 2006).
Androgens are known to stimulate sebum production. To examine the androgen
responsiveness of TSS-1, cells were treated with synthetic androgen R1881 (methyltrienolone;
Perkin Elmer, Wellesley, MA) for 24 hours prior to total lipogenesis assay. In parallel
experiments, the effect of 0.1 µM 13-cis RA (Sigma, St Louis, MO) was examined. TSS-1 cells
were cultured in 35-mm culture plates and maintained at 33ºC until 80-90% confluent, feeding
as necessary. To allow for large T antigen shut-down, all plates were shifted to 39ºC for 72
hours. TSS-1 sebocytes were treated in triplicate with vehicle controls, 1 X 10-8 M R1881, or 0.1
µM 13-cis RA for 24 hours. Total lipogenesis was performed as above and experiment was
repeated 3 independent times; n = 9 samples. Statistical significance was determined with
ANOVA Two Factor with Replication and considered significant if p < 0.05.
5.5.8 TdT-Mediated dUTP Nick End Labeling (TUNEL) Staining
TSS-1 sebocytes were cultured in 12-well plates and shifted to 39ºC for 72 hours prior to
treatment. 13-cis RA (10 mM stock in ethanol) was diluted in standard culture medium and
applied for 48 and 72 hours. Culture plates were approximately 60% confluent at time of
treatment. Each well was considered one sample. Samples were prepared by manufacturer’s
instructions for In Situ Cell Death Detection Assay (Roche Applied Science, Indianapolis, IN)
Additional assay controls included DNase I-treated positive and negative controls, with negative
controls receiving labeling solution only. Results were quantified by counting positively stained
cells in 3 representative fields per well for each of the treatments carried out in triplicate. Each
assay was performed two independent times; n = 6 samples. Data analysis was performed
using ANOVA Two Factor with Replication and considered significant if p < 0.05.
Chapter 6
Discussion and Future Directions
6.1 Introduction
The initial observations of the effectiveness of isotretinoin in the treatment of acne came
as an unexpected side effect of its use in the treatment of ichthyosis, a hereditary disorder of
keratinization (Peck, 1979). Isotretinoin has been on the market since 1982 and even though it
has been prescribed for 25 years, extensive studies into its molecular mechanism of action in
human skin and sebaceous glands have not been done. The studies undertaken in this thesis
are the beginning to understanding the effects of 13-cis RA on the sebaceous gland and its
mechanism of action in sebum suppression.
6.2 Rationale, hypothesis, and results of this work
Isotretinoin is the most potent agent for sebosuppression, and yet, how this
sebosuppressive action occurs is unknown. It is well established that isotretinoin drastically
reduces the size and lipid secretion of sebaceous glands (Goldstein, et al., 1982). We
hypothesized that isotretinoin decreases the size of the sebaceous gland by activation of cell
cycle arrest and/or apoptosis pathways and that sebum suppression is most likely an indirect
result of the reduced size of the sebaceous gland. Our hypothesis was based on the histology of
the sebaceous gland after isotretinoin treatment.
In this thesis, we have shown that:
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1) 13-cis RA, unlike 9-cis RA or ATRA, induces cell cycle arrest and apoptosis in SEB-1
sebocytes. Its ability to induce apoptosis is not inhibited in the presence of functional
RAR pan antagonist AGN 193109, suggesting a non-RAR mediated mechanism.
2) Gene expression analysis of patient biopsies one-week into isotretinoin
therapy provided insight into the initial changes induced by this drug, including NGAL upregulation. Gene expression analysis on SEB-1 sebocytes and HaCaT keratinocytes
provided clues to possible cell specific actions of 13-cis RA.
3) NGAL localization within the sebaceous gland and its ability to induce apoptosis in
SEB-1 sebocytes suggest that NGAL may, in part, mediate the apoptotic action of
13-cis RA on sebaceous glands. We identified the possible human cell surface
receptor for NGAL and cell-specific expression of its isoforms correlates with
apoptosis sensitivity.
4) 13-cis RA, unlike 9-cis RA and ATRA, activates the extrinsic apoptosis pathway in
SEB-1 sebocytes through up-regulation of TRAIL.
The results of this body of work have allowed us to generate a model by which we believe 13cis RA is mediating apoptosis in sebocytes (Figure 43).
6.3 Explanation of model
Like other lipophilic retinoids, 13-cis RA can readily transverse the plasma membrane.
13-cis RA isomerizes to 9-cis RA and ATRA, which are known to activate their respective
retinoid receptors and affect gene expression. 13-cis RA up-regulates, although probably
indirectly through its isomers, metabolites or an as yet identified nuclear receptor, the
expression of genes involved in cell-cycle arrest and apoptosis. Up-regulation of TRAIL leads to
activation of the extrinsic apoptosis pathway and up-regulation of NGAL also leads to apoptosis,
but the pathway (extrinsic or intrinsic) is not yet known.
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Figure 43: Model of 13-cis RA induces apoptosis and cell cycle arrest in SEB-1
sebocytes and human sebaceous glands.
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6.4 Future Directions of this project
There are still many unanswered questions. In the following section, I will discuss some
of these important questions and how insights from our work can lead to advances in the field.
6.4.1 Why is 13-cis RA superior to 9-cis RA or ATRA in the treatment of acne?
13-cis RA is superior to either 9-cis RA or ATRA for sebosuppression and 13-cis RA has
little to no ability to bind to cellular retinol-binding proteins or RAR and RXR receptors which are
readily activated by both 9-cis RA and ATRA (Allenby, et al., 1993; Fogh K., et al., 1993; Geiger,
et al., 1996; Hommel, et al., 1996; Levin, et al., 1992; Ott, et al., 1996). In terms of apoptosis,
our work confirms that the actions of 13-cis RA are distinct from 9-cis RA or ATRA, with 13-cis
RA activating the extrinsic apoptosis pathway more than either 9-cis RA or ATRA. Furthermore
the apoptotic effect is not mediated by RAR activation, which suggests that 13-cis RA does not
function as a pro-drug for ATRA, but instead is capable of influencing cellular processes on its
own. Other investigators have proposed that the superior efficacy of 13-cis RA in acne
treatment may be related to its: 1) unique pharmacokinetic properties within the cell, 2)
alteration of cellular signaling pathways by covalent or non-covalent protein interactions, 3)
ability to in-directly affect enzyme activity, 4) generation of alternative metabolites (other than 9cis RA and ATRA) that have transcriptional activity or 5) binding to an as yet unidentified
receptor (Baron, et al., 2005; Holmes et al, 2003; Hoyos, et al., 2000; Imam, et al., 2001;
Karlsson, et al., 2003; Pettersson, et al., 2004; Zorn and Sauro, 1995)..
Understanding the pharmacokinetic properties of 13-cis RA within our SEB-1 sebocytes
is an important first step. In our SEB-1 sebocyte model, HPLC analysis showed that 13-cis RA
concentrations were maximal within SEB-1 sebocytes twelve hours after treatment and
concentrations of 9-cis RA and ATRA begin to increase after 24 hours. It is possible that the
reason 13-cis RA is superior to 9-cis RA or ATRA is that it is more favorably absorbed by
sebocytes or is more stable within sebocytes for a longer period of time. For example, ATRA
may be rapidly hydroxylated and inactivated by 4-hydroxylases or it may bind to PKC and
phosphorylate AKT initiating a prosurvival response as occurs in other cell systems (Bastien et
al, 2006; Marikar et al, 1998; Ochoa et al, 2003). Initial HPLC experiments examining the
metabolism of 13-cis RA, 9-cis RA and ATRA in sebocytes are necessary to differentiate
between these possibilities. In addition, treatment with 13-cis RA may be more effective as an
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acne therapy due to a combination of 13-cis RA, 9-cis RA and ATRA being present within the
sebocytes simultaneously and acting synergistically to induce apoptosis. Our HPLC studies
indicate that all three retinoids are present in measurable concentrations during the time period
of apoptosis induction. It may be possible that all three retinoids synergistically contribute to the
apoptotic effect and each may potentially work through a different apoptosis mechanism. To
begin to address this possibility, combination retinoid treatments and HPLC analysis, along with
our standard apoptosis assays would provide initial insight into this hypothesis. Finally, it may
be possible that currently unidentified metabolites or isomerization products of 13-cis RA
mediate its apoptotic actions in SEB-1 cells. Our HPLC studies did show two unknown “peaks”
that could represent metabolites of 13-cis RA; additional characterization studies are needed to
identify these unknown compounds.
Beyond pharmacokinetic studies, understanding which cellular pathways are affected by
13-cis RA is crucial to understanding its mechanism of action. Experiments indicated that 13-cis
RA induced apoptosis while ATRA or 9-cis RA did not; thus suggesting that apoptosis may be
responsible for 13-cis RA’s effectiveness in the treatment of acne. Our gene array data
indicated that 13-cis RA induced gene expression of mediators of both the intrinsic and extrinsic
pathways of apoptosis. Synthetic retinoid MX3350-1 activates both the intrinsic and extrinsic
apoptotic pathways independent of retinoid receptors (Chun, et al., 2005). It is possible that 13cis RA, like MX3350-1, activates both the intrinsic and extrinsic pathways of apoptosis in
sebocytes. In SEB-1 sebocytes, apoptosis occurs after 48 and 72 hours of 13-cis RA treatment
and gene expression analysis reveals 13-cis RA up-regulates genes involved in apoptosis
including potent apoptosis inducers TRAIL and FasL Due to the ‘delayed’ apoptotic response of
SEB-1, it is possible that new gene transcription and protein synthesis is involved in mediating
apoptosis. Apoptosis assays in the SEB-1 cell line using actinomycin D (DNA transcription
inhibitor) and cyclohexamide (de novo protein synthesis inhibitor) in the presence of 13-cis RA
could be performed to test this hypothesis.
13-cis RA- induced apoptosis may not require new gene products and instead directly
activate the classical extrinsic or intrinsic apoptosis pathways driven by the activation of the
multiple caspases within the cells (Figure 8). We have shown that 13-cis RA induced caspase 8
activation in sebocytes (most likely through TRAIL/TRAIL-R1/R2 activation) to a greater extent
than 9-cis RA or ATRA. This would suggest preferential activation of the extrinsic apoptotic
pathway by 13-cis RA; however, caspase 8 can activate Bid, leading to its truncation (tBid) and
association with the mitochondria triggering intrinsic pathway activation. To fully explore the
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possibility that 13-cis RA may activate both death receptor and mitochondrial mediated cell
death, potential future experiments may include protein lysates subjected to western analysis for
many key players involved this pathway such as Bid/tBid, caspase 9, and apoptosis activating
factor 1 (APAF1).
In order to design alternative therapies to exert potent effects on sebaceous gland
function, it is necessary to understand how 13-cis RA transcriptionally regulates a pathway or
induces a direct cellular effect that leads to apoptosis.
6.4.2 What RAR-independent events can lead to 13-cis RA-induced apoptosis?
Historically, retinoid compounds are known to exert their specific cellular effect by
binding to and activating classical retinoid receptors, resulting in changes in gene expression. It
is known that 13-cis RA is not capable of binding to these receptors (Allenby, et al., 1993).
Recently, within the cancer field, more attention has been focused on retinoid receptorindependent effects within the cell, such as modulation of signal transduction kinase cascades
including mitogen-activated protein kinase (MAPK) pathways: extracellular signal-relatedkinases (ERKs), p38MAPK, and stress activated protein kinase/c-Jun N terminal kinase
(SAPK/JNK) (Nakagawa, et al., 2003; Olson and Hallahan, 2004; Pettersson, et al., 2004).
Activation of these pathways can result in cell survival, growth inhibition or cell death depending
on the stimulus the cell receives. 13-cis RA or ATRA is able to induce apoptosis in
medulloblastoma cells by phosphorylation of p38MAPK. Apoptosis was triggered by synthetic
retinoid CD 437 in ovarian cancer cells by the same p38 phosphorylation; however, ATRA did
not trigger apoptosis, indicating, once again, that there is cell-specificity in retinoid actions
(Hallahan et al, 2003; Holmes, et al., 2003). ATRA induces sustained activation of ERK1/2
leading to apoptosis in MDA-MB-231 breast cancer cells while in SKBR-3 breast cancer cells
protein kinase Cα (PKCα)-ERK pathway is disturbed leading to growth arrest (Nakagawa, et al.,
2003; Pettersson, et al., 2004). Very few studies to date have evaluated 13-cis RA actions on
these pathways and, in particular, no study has examined these pathways in sebocytes.
Since 13-cis RA is known not to bind classical retinoid receptors, modulation of these
important signaling cascades is an intriguing possibility that could be tested in future studies.
Previous work in our laboratory has demonstrated that SEB-1 sebocytes do express p38,
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ERK1/2 (p44/42), and SAPK/JNK (p54/46) proteins (Smith, in preparation). The potential
activation of the p38/MAPK, ERK and SAPK/JNK pathways by 13-cis RA within SEB-1
sebocytes can be examined by western analysis for both total and phosphorylated forms of
p38MAPK, ERK 1/2 and JNK to determine if/when a pathway first becomes activated and if this
time course corresponds to apoptosis induction. If a particular pathway (i.e. p38/MAPK) is found
to be activated in the presence of 13-cis RA, follow-up experiments utilizing specific pathway
inhibitors SB203580, such as specific p38MAPKα/β inhibitor with no interference with ERK or
JNK activity, could be used in combination with 13-cis RA to determine if apoptosis is blocked.
Based on the scientific literature, we would anticipate that the p38/MAPK pathway or SAPK/JNK
pathway will be activated in response to 13-cis RA treatment. These pathways are activated by
inflammatory mediators, cytokines (TNF) and cellular stress; genes involved in these cellular
processes are affected by 13-cis RA treatment as indicated by our gene expression analysis.
These experiments are an initial step to clarifying receptor-independent mechanisms for the 13cis RA. If 13-cis RA is found to influence a particular signal transduction kinase pathway, new
avenues for drug development are open since many inhibitors of specific kinases are being
developed. These results would have implications in cancer biology as well as treatment of acne
since 13-cis RA is used in cancer chemoprevention and in the treatment of pediatric
neuroblastomas, colorectal cancer and some forms of leukemia (Handa, et al., 1997; Maeda, et
al., 1996; Recchia et al, 2007; Reynolds et al, 2003).
6.4.3 Lipogenesis vs. Apoptosis: Does 13-cis RA preferentially affect one of these
processes?
The sebaceous gland undergoes constant turnover and renewal. Sebocytes divide from
the basal layer, migrate into the center of the sebaceous gland and undergo terminal
differentiation followed by holocrine rupture leading to the secretion of the lipid product, sebum.
The molecular cues and pathways which control this natural process of elimination are still
unknown.
As previously mentioned, isotretinoin therapy significantly decreases sebum production
and secretion. In vivo, 13-cis RA is known to decrease sebum secretion (Strauss, et al., 1980).
In cultured human sebocytes, 13-cis RA effectively reduces the synthesis of triglycerides, wax
esters, and free fatty acids; however, squalene synthesis remained unchanged while cholesterol
synthesis was slightly increased (Zouboulis, et al., 1991). 13-cis RA (1 µM) has been shown to
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significantly decrease the production of cholesterol, fatty alcohol, cholesterol esters and
squalene in SEB-1 sebocytes after 72 hours of treatment (Trivedi, et al., 2006).
Histological data also illustrates that isotretinoin drastically decreases the size of the
sebaceous gland in vivo (Goldstein, et al., 1982). Initial studies indicated that isotretinoin
decreases sebocyte proliferation (Landthaler, et al., 1980) within the sebaceous gland. This
finding was confirmed in numerous in vitro cell culture experiments with primary human
sebocytes, rat preputial cells and immortalized sebocyte cell lines (Kim et al, 2000; Nelson, et
al., 2006; Tsukada, et al., 2000; Wrobel, et al., 2003; Zouboulis, et al., 1991; Zouboulis, et al.,
1993). In addition to effects on cell proliferation our studies are the first to show that isotretinoin
triggers significant apoptosis within sebaceous glands one-week into therapy as demonstrated
by increases in the percentages of TUNEL positive nuclei when compared to baseline biopsies.
Several lines of experimental evidence have shown that 13-cis RA affects lipogenesis as
well as cell cycle and apoptosis of the sebaceous gland. However, it is unknown which
process/pathway, lipogenesis or cell cycle arrest/apoptosis, is the primary target of 13-cis RA
within the sebaceous gland. Our gene expression analysis on before and after 8-weeks or oneweek isotretinoin treatment combined with parallel hematoxylin and eosin staining on these
biopsies may provide some insight to which process is being preferentially affected.
Initial gene expression analysis was performed before and after 8-weeks of isotretinoin
therapy: 197 genes were significantly up-regulated and 587 genes were significantly downregulated at 8-weeks when compared to baseline (A.1). Of the 197 genes that were
significantly increased, the majority of genes encode structural proteins of the extracellular
matrix such as collagens, fibulin and fibronectin. These up-regulated genes are consistent with
the known effects of retinoids on the extracellular matrix as reported in studies of photoaging
(Weiss, et al., 1988). Many of the down-regulated genes at 8-weeks are involved in the
metabolism of steroids, cholesterol and fatty acids, which is consistent with the known
decreases in sebaceous gland lipid production induced by 13-cis RA. It seems highly likely that
the down-regulation of genes involved in cholesterol and fatty acid metabolism maybe related to
the significant decrease in size of the sebaceous gland because hematoxylin and eosin staining
revealed significant decreases in gland size at the 8-week time point. It became clear, based on
these initial studies that the 8-week time point captures the overall net effect of isotretinoin
therapy and that specific effects of isotretinoin on the sebaceous gland occurs prior to this time
point.
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Gene changes at one-week are of particular interest because they may provide clues
about the initial changes induced by this drug. Those early gene changes can be broadly
categorized as tumor suppressors, protein processors, and genes involved in transfer or binding
of ions, amino acids, lipids or retinoids. Within the 42 significantly changed genes, there is
increased representation of genes that are involved in or related to processes of ectoderm and
epidermal development; pest, pathogen, parasite or external biotic response; and vitamin
binding. The one-week gene expression analysis did not indicate any significant changes in
genes involved in lipogenesis or key players in apoptosis pathways. Hematoxylin and eosin
staining did indicate a decreasing trend in sebaceous gland volume at one-week of isotretinoin
treatment.
Together, gene expression analysis and hematoxylin and eosin staining suggest that the
initial activity of isotretinoin triggers a decrease in sebaceous gland volume. As a result of the
‘shrinking’ sebaceous gland, decreases in genes involved in lipid metabolism are found at 8weeks. To more definitively test this hypothesis, future gene expression analysis as well as
hematoxylin and eosin staining could be performed before and at 4-weeks of isotretinoin
treatment. This proposed experiment would allow for a more substantial correlation in the time
frame of detectable changes in sebaceous gland volume compared to significant changes in
genes involved in lipid metabolism within the sebaceous gland.
Previous work in our laboratory demonstrated that insulin-like growth factor 1 (IFG-1)
through the activation of its respective cell-surface receptor increases lipogenesis in SEB-1
sebocytes through Sterol Response Element Binding Protein 1 (SREBP-1) transcription factor
dependent and independent pathways and has identified the phosphoinositol 3 kinase (PI3-K)
pathway as the primary signaling cascade involved in lipogenesis (Smith, et al., 2006)(Smith, in
preparation). If 13-cis RA has a direct effect on lipogenesis in SEB-1 sebocytes, it is possible
that it influences the actions of SREBP-1 or the PI3-Kinase pathway leading to the documented
decreases in lipogenesis (Trivedi, et al., 2006). The PI3-Kinase/AKT pathway is known to
mediate cell proliferation, differentiation and cell survival. To date, no study has examined the
effects of 13-cis RA or any other retinoid on these pathways in terms of lipogenesis. However, in
mouse cell systems, retinoic acid (ATRA) has been shown to both activate and inhibit PI3Kinase pathway resulting in differentiation and cell cycle arrest. (Bastien et al, 2005) Future
studies designed to examine SREBP-1 expression and activity as well as activation or inhibition
of the PI3-kinase pathway in the presence of 13-cis RA, may provide direct evidence of 13-cis
RA effects on the process of lipogenesis within sebaceous glands.
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6.4.4 Does TRAIL mediate isotretinoin-induced apoptosis within the sebaceous gland?
Our studies are the first to demonstrate up-regulation of TRAIL by 13-cis RA. We have
identified TRAIL as a possible mediator of 13-cis RA-induced apoptosis in SEB-1 sebocytes.
TRAIL expression is up-regulated in response to 13-cis RA and purified TRAIL protein induces
apoptosis in SEB-1 sebocytes which suggests the TRAIL signaling pathway is intact within our
cells.
First and foremost, it is important to validate our in vitro results within the sebaceous
gland. Immunohistochemistry using antibodies to TRAIL on sections of skin from patients that
were treated with isotretinoin for one-week would be an important piece of the puzzle to
determine if TRAIL is involved in mediating the effects of isotretinoin in vivo.
Future studies could examine the expression of TRAIL receptor isoforms in sebaceous
glands and SEB-1 sebocytes. Previous studies have examined the effects of retinoids on TRAIL
receptor expression, although no study to date has examined expression within sebaceous
glands. Retinoids including fenretinide, synthetic retinoid CD437, and ATRA, increase sensitivity
to TRAIL-induced apoptosis by up-regulation of TRAIL-R1 and –R2 receptors, the receptors
responsible for receiving and transmitting the TRAIL-mediated apoptotic signal (Kouhara, et al.,
2007; Sun, et al., 2000; Sun, et al., 2000). TRAIL-R1 and –R2 receptors and decoy TRAIL-R3
and –R4 are expressed in the epidermis (Bachmann, et al., 2001; Stander and Schwarz, 2005).
Experimental evidence suggests that, in normal and tumor cells, the sensitivity and/or
resistance to TRAIL mediated apoptosis is associated with the relative levels of ‘active’ death
receptors to decoy death receptors. (Daniels et al, 2005; Hesry Vincent, 2006; Kan Kondo,
2006; Qin et al, 2001; Sanlioglu et al, 2007). Immunohistochemical and western analysis of the
TRAIL receptor isoforms and their changes with respect to 13-cis RA treatment could be
performed in sebaceous glands and SEB-1 sebocytes, respectively, and would provide
important validation for TRAIL as a mediator of isotretinoin induced apoptosis within the
sebaceous gland.
Understanding the mechanism of TRAIL induction by 13-cis RA (i.e. direct or in-direct
up-regulation) can provide insight into 13-cis RA’s mechanism of action in acne therapy as well
as when used as cancer therapy. Recently, by increasing apoptosis, 13-cis RA alone or in
combination with interferon, has been shown to be beneficial in the treatment of some forms of
leukemia (Handa, et al., 1997; Maeda, et al., 1996) although the mechanism of apoptosis is
unknown. It is possible that TRAIL is responsible this increased apoptosis. The mechanism of
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TRAIL induction by retinoids is not defined, although advances are being made in this area.
Studies in our laboratory of the TRAIL gene promoter region indicates that it does contain an
RAR consensus sequence as identified by TESS computer software program. However,
promoter mapping experiments conducted by Clarke et. al. demonstrated that there is no
retinoic acid response element within 2Kb of the transcription start site of TRAIL (Clarke, et al.,
2004). The latter is in agreement with our previous studies within SEB-1 sebocytes
demonstrating that apoptosis was not blocked by a RAR pan-antagonist, suggesting an RAR
independent mechanism to apoptosis induction. TRAIL up-regulation by 13-cis RA, therefore,
most likely occurs by an indirect mechanism. In support of this hypothesis, Interferon regulatory
factor 1 (IRF1) was identified as a critical factor in mediating TRAIL induction by retinoic acid in
NB4 APL leukemia cells and SK-BR-3 breast cancer cells (Clarke, et al., 2004). Interestingly,
13-cis RA significantly up-regulates IRF1 gene expression (2.42 fold increase) in SEB-1
sebocytes (A.2). It may be possible that 13-cis RA induced TRAIL up-regulation in SEB-1
sebocytes is due to increases in IRF1 expression. To definitively test this hypothesis, studies
utilizing siRNA knockdown of IRF1 in the presence of 13-cis RA could be performed.
Furthermore, additional studies are needed to assess whether retinoid receptor
activation is required for this increase in TRAIL or IRF1 expression. Our studies have shown
that 13-cis RA induced apoptosis is most likely not mediated by RAR activation as studies with
RAR pan-antagonist AGN 193109 do not block apoptosis in the presence of 13-cis RA. If
TRAIL mediates 13-cis RA induced apoptosis, we would hypothesize that TRAIL expression is
not mediated by RAR activation. Studies proposed above would examine the role of IRF1 in
mediating TRAIL expression in response to 13-cis RA and it is equally important to determine if
RAR activation is required for increases in IRF-1 if IRF-1 mediates increases in TRAIL
expression. The IRF1 promoter is not available in the TESS database and no previous study
has directly accessed whether a RAR or RXR consensus sequence is located within the IRF-1
promoter; although previous studies have shown that IRF1 is induced by ATRA which would
suggest that it does contain a retinoid receptor response element within its promoter. Studies
utilizing RAR pan-antagonist AGN 193109 or siRNA to the RAR subtypes in the presence of 13cis RA and examining IRF1 and TRAIL mRNA and protein expression via quantitative
polymerase chain reaction (QPCR) and western blotting could be performed to address whether
RAR receptor activation is required for increased expression of both genes.
Understanding the mechanism by which 13-cis RA regulates TRAIL expression can lead
to advances in acne treatments as well as a greater understanding of its use as cancer therapy.
158
If future experiments definitively demonstrate that TRAIL mediates apoptosis in response to 13cis RA treatment in sebaceous glands, then drugs/compounds that increase TRAIL expression,
its active receptors’ expression or sensitivity to TRAIL mediated apoptosis may be useful as an
acne treatment. Histone deacetylase inhibitors and TRAIL receptor agonistic mono-antibodies
have been shown increase the TRAIL sensitivity of prostate cancer cells with minimal effect on
the normal prostate epithelium and are being used in conjunction with current prostate chemoand gene- therapies (Kasman et al, 2006; Shimada et al, 2007; Vanoosten et al, 2007).
Asoprisnil, a novel selective progesterone receptor modulator, up-regulates TRAIL and its active
receptors (TRAIL-R1/R2) in leiomyoma cells resulting in increased apoptosis, however, no
effect is noticed on the surrounding normal smooth muscle cells (Chen et al, 2006; Sasaki et al,
2007). Sebaceous glands are known to express the progesterone receptor (Kariya, et al., 2005)
and it is not yet known what role the progesterone receptor and its activation may play in the
development or resolution of acne. Additional research into these drugs and their effects on
sebaceous gland physiology may illuminate an alternative to isotretinoin for acne treatment.
6.4.5 Why are the apoptotic effects of 13-cis RA limited to sebocytes and do not occur
within keratinocytes?
Several studies indicate that the effects of retinoids on cell proliferation, differentiation,
and apoptosis are retinoid- or cell-type specific. Histological evidence indicates the effects of 13cis RA are most profound on sebaceous glands, with little to no alteration in the surrounding
dermis or epidermis of the skin (Landthaler, et al., 1980). Our studies clearly demonstrated that
13-cis RA triggers apoptosis within sebocytes and does not affect keratinocytes (Nelson, et al.,
2006) as assayed in both human patients and in vitro cell culture assays. To date, no study has
detected apoptosis within keratinocytes in response to 13-cis RA treatment, although other
retinoids including ATRA and tazarotene (RAR β/γ selective agonist) have been shown to
induce apoptosis in HaCaT keratinocytes (Louafi, et al., 2003; Papoutsaki, et al., 2004). The
reason for the cell-specificity of 13-cis RA-induced apoptosis is not yet known but initial studies
done within our laboratory and others may provide some clues.
Gene expression analysis revealed that 13-cis RA significantly affects different genes in
SEB-1 sebocytes than those affected in HaCaT keratinocytes, with only 9 significantly changed
genes in common. Clearly, 13-cis RA has cell-specific effects as less than 10% of changed
genes are in commonly changed between the two cell lines. Within significantly changed genes
159
from the HaCaT gene array, “cellular differentiation” and “morphogenesis” gene ontology
classifications were significantly enhanced (data not shown). Significantly up-regulated genes
that fall into these classifications include: involucrin, insulin-like growth factor binding proteins 3
and 6 (IGFBP3, 6), bone morphogenetic protein 3 (BMP3) and kallikrein 5 and 6 (KLK5, 6) and
all these genes are known to regulate cellular differentiation and proliferation in a variety of
different cell types including keratinocytes (Eckert et al, 2004; Eckert and Green, 1986;
Edmondson et al, 2005; Faucheux et al, 1997; Kishibe et al, 2007). This supports the idea that
13-cis RA does not induce an apoptosis pathway (as in sebocytes) but instead influences other
cellular pathways within keratinocytes. Although, the ontology term of ‘apoptosis’ was not
indicated within the significantly changed genes within SEB-1 sebocytes, numerous genes
involved in apoptosis were up-regulated including TRAIL, FasL, LCN2, IGFBP3, IRF1.
The question then becomes “why is 13-cis RA affecting these pathways and not
apoptosis?” A possible answer to this question is provided within the list of significantly
changed genes. Gene expression analysis revealed increased expression of TRIM31 that
encodes an E3 ubiquitin ligase, and P450RAI2 (CYP26A), a potent retinoic acid 4-hydroxylase
after 13-cis RA treatment. Up-regulation of these genes suggests that within HaCaT
keratinocytes, 13-cis RA is rapidly degraded/metabolized and implies that HaCaT keratinocytes
have powerful mechanisms in place to protect against the actions of retinoids. Törmä et. al.
demonstrated that HaCaT keratinocytes, when compared to normal epidermal keratinocytes,
have lower levels of retinoid binding proteins, increased metabolism of retinol and retinoic acid
and high levels of p450RAI; all of which suggest that HaCaT keratinocytes do not achieve or
maintain high levels of retinoids intracellularly (Torma et al, 1999) and it is possible that these
lower levels affect differentiation pathways and not activate apoptotic pathways.
Since 1925, retinoids have been shown to influence and normalize differentiation in
keratinocytes but the exact mechanism of how this is accomplished is unknown (Wolbach and
Howe, 1925). Future investigations into these cellular pathways and its possible ‘retinoid
protection’ enzymes would be of interest to completely understand the actions of 13-cis RA on
skin and its beneficial effects on keratinizing disorders.
160
6.5 Conclusion
The studies in this thesis are the beginning to understanding the effects of 13-cis RA on
the sebaceous gland and its mechanism of action in sebum suppression. Our studies suggest
that 13-cis RA mediates its sebosuppressive effect through preferential induction of apoptosis in
sebaceous glands. Work in this area is far from complete. A fundamental understanding of 13cis RA actions in sebaceous glands is needed before meaningful investigations into safer drug
alternatives for acne treatment can be pursued.
ºoº
ºoº
ºoº
Appendix A
Supplemental gene expression array tables
A.1 All significantly changed genes after 8 weeks isotretinoin therapy
Some genes may be listed twice; indicates separate probe sets on Affymetrix gene array chips.
Fold
Change
2.54
2.03
2.00
1.98
1.85
1.77
1.73
1.71
1.68
1.67
1.67
1.66
1.64
1.63
1.63
1.62
1.61
1.61
1.60
1.60
1.60
1.59
1.59
1.57
1.57
1.56
1.56
1.56
1.56
1.55
1.55
1.55
1.54
1.54
1.53
Gene Title
microseminoprotein, betamicroseminoprotein, betakraken-like
--coagulation factor C homolog, cochlin (Limulus polyphemus)
lymphocyte cytosolic protein 2 (SH2 domain containing leukocyte
protein of 76kDa)
fibulin 1
hypothetical protein MGC27165
carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 6
KIAA0527 protein
fibulin 1
collagen, type VI, alpha 2
collagen, type V, alpha 1
SAM domain, SH3 domain and nuclear localisation signals, 1
filamin A, alpha (actin binding protein 280)
butyrophilin, subfamily 3, member A3
collagen, type V, alpha 1
protein phosphatase 1, regulatory (inhibitor) subunit 16B
tryptase beta 2
insulin-like growth factor binding protein 4
microfibrillar-associated protein 2
tryptase beta 2
tryptase beta 2
--slit homolog 3 (Drosophila)
Rho-related BTB domain containing 3
insulin-like growth factor binding protein 5
microfibrillar-associated protein 4
tryptase beta 2
collagen, type VI, alpha 2
collagen, type IV, alpha 2
fibulin 1
natural killer cell transcript 4
hematopoietic cell-specific Lyn substrate 1
tenascin N
Gene Symbol
MSMB
MSMB
dJ222E13.1
--COCH
LCP2
FBLN1
MGC27165
CHST6
KIAA0527
FBLN1
COL6A2
COL5A1
SAMSN1
FLNA
BTN3A3
COL5A1
PPP1R16B
TPSB2
IGFBP4
MFAP2
TPSB2
TPSB2
--SLIT3
RHOBTB3
IGFBP5
MFAP4
TPSB2
COL6A2
COL4A2
FBLN1
NK4
HCLS1
TNN
162
1.53
1.53
1.53
1.52
1.52
1.51
1.51
1.51
1.51
1.51
1.50
1.50
1.49
1.49
1.49
1.49
1.48
1.48
1.48
1.48
1.48
1.47
1.47
1.47
1.47
1.47
1.46
1.46
1.46
1.46
1.46
1.46
1.46
1.45
1.45
1.44
1.44
1.44
1.44
1.43
1.43
1.43
1.43
1.43
1.42
1.42
1.42
fibronectin 1 /// fibronectin 1
collagen, type VI, alpha 1
protease, serine, 11 (IGF binding)
collagen, type V, alpha 2
procollagen C-endopeptidase enhancer
protein tyrosine phosphatase, receptor type, C
integrin, beta 2 (antigen CD18 (p95), lymphocyte function-associated
antigen 1; macrophage antigen 1 (mac-1) beta subunit)
collagen, type V, alpha 1
laminin, alpha 2 (merosin, congenital muscular dystrophy)
tryptase beta 2
tryptase beta 2
matrix metalloproteinase 2 (gelatinase A, 72kDa gelatinase, 72kDa
type IV collagenase)
FLJ00133 protein
protein phosphatase 1, regulatory (inhibitor) subunit 16B
WNT inhibitory factor 1
--prostaglandin D2 synthase 21kDa (brain)
fibronectin 1
thrombospondin 4
complement component 1, s subcomponent
protein tyrosine phosphatase, receptor type, G
dynamin 1
complement component 3
serine (or cysteine) proteinase inhibitor, clade G (C1 inhibitor), member
1, (angioedema, hereditary)
DKFZP586K1520 protein
lymphocyte cytosolic protein 2 (SH2 domain containing leukocyte
protein of 76kDa)
fibronectin 1
insulin-like growth factor binding protein 5
forkhead box D1
platelet-derived growth factor receptor, beta polypeptide
DKFZP564J102 protein
KIAA1102 protein
collagen, type XIV, alpha 1 (undulin)
CDW52 antigen (CAMPATH-1 antigen)
ribonuclease T2
colony stimulating factor 1 receptor, formerly McDonough feline
sarcoma viral (v-fms) oncogene homolog
ankyrin 2, neuronal
major histocompatibility complex, class II, DP alpha 1
huntingtin interacting protein 1
collagen, type VI, alpha 3
CD3Z antigen, zeta polypeptide (TiT3 complex)
v-yes-1 Yamaguchi sarcoma viral related oncogene homolog
major histocompatibility complex, class II, DP beta 1
matrix metalloproteinase 23B
fibronectin 1
TBC1 (tre-2/USP6, BUB2, cdc16) domain family, member 1
Ras association (RalGDS/AF-6) domain family 2
FN1
COL6A1
PRSS11
COL5A2
PCOLCE
PTPRC
ITGB2
COL5A1
LAMA2
TPSB2
TPSB2
MMP2
FLJ00133
PPP1R16B
WIF1
--PTGDS
FN1
THBS4
C1S
PTPRG
DNM1
C3
SERPING1
DKFZP586K1520
LCP2
FN1
IGFBP5
FOXD1
PDGFRB
DKFZP564J102
KIAA1102
COL14A1
CDW52
RNASET2
CSF1R
ANK2
HLA-DPA1
HIP1
COL6A3
CD3Z
LYN
HLA-DPB1
MMP23B
FN1
TBC1D1
RASSF2
163
1.42
1.42
1.42
1.42
1.42
1.42
1.41
1.41
1.41
1.41
1.41
1.40
1.40
1.39
1.39
1.39
1.39
1.38
1.38
1.38
1.37
1.37
1.37
1.37
1.37
1.37
1.37
1.37
1.37
1.37
1.36
1.36
1.36
1.35
1.35
1.35
1.35
1.35
1.35
1.35
1.35
1.35
1.34
1.34
1.34
tenascin XB
lysyl oxidase-like 2
mannosidase, alpha, class 1A, member 1
insulin-like growth factor binding protein 5
LIM domain only 2 (rhombotin-like 1)
Src-like-adaptor
G protein-coupled receptor 124
tissue inhibitor of metalloproteinase 3 (Sorsby fundus dystrophy,
pseudoinflammatory)
chondroitin sulfate proteoglycan 2 (versican)
TRAF3-interacting Jun N-terminal kinase (JNK)-activating modulator
neurotrophic tyrosine kinase, receptor, type 2
phosphatidic acid phosphatase type 2B
tissue inhibitor of metalloproteinase 3 (Sorsby fundus dystrophy,
pseudoinflammatory)
chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1)
KIAA1102 protein
RNA binding protein with multiple splicing
Homo sapiens cDNA FLJ36690 fis, clone UTERU2008707, highly
similar to COMPLEMENT C1R COMPONENT PRECURSOR (EC
3.4.21.41).
CD3D antigen, delta polypeptide (TiT3 complex)
lymphocyte-specific protein tyrosine kinase
BTB (POZ) domain containing 3
fibroblast growth factor receptor 3 (achondroplasia, thanatophoric
dwarfism)
lipase, hepatic
slit homolog 3 (Drosophila)
polymerase I and transcript release factor
nuclear receptor subfamily 2, group F, member 1
Rac/Cdc42 guanine nucleotide exchange factor (GEF) 6
reticulon 1
--fucosyltransferase 8 (alpha (1,6) fucosyltransferase)
cystatin C (amyloid angiopathy and cerebral hemorrhage)
Homo sapiens T cell receptor beta chain BV20S1 BJ1-5 BC1 mRNA,
complete cds
up-regulated in liver cancer 1
protein S (alpha)
fibroblast growth factor receptor 1 (fms-related tyrosine kinase 2,
Pfeiffer syndrome)
plakophilin 1 (ectodermal dysplasia/skin fragility syndrome)
CUG triplet repeat, RNA binding protein 2
major histocompatibility complex, class I, E
--CD2 antigen (p50), sheep red blood cell receptor
up-regulated in liver cancer 1
HLA-G histocompatibility antigen, class I, G
adrenergic, alpha-2A-, receptor
Rho GDP dissociation inhibitor (GDI) beta
discs, large homolog 5 (Drosophila)
major histocompatibility complex, class I, F
TNXB
LOXL2
MAN1A1
IGFBP5
LMO2
SLA
GPR124
TIMP3
CSPG2
T3JAM
NTRK2
PPAP2B
TIMP3
CXCL12
KIAA1102
RBPMS
--CD3D
LCK
BTBD3
FGFR3
LIPC
SLIT3
PTRF
NR2F1
ARHGEF6
RTN1
--FUT8
CST3
--UPLC1
PROS1
FGFR1
PKP1
CUGBP2
HLA-E
--CD2
UPLC1
HLA-G
ADRA2A
ARHGDIB
DLG5
HLA-F
164
1.34
1.34
1.34
1.34
1.34
1.33
1.33
1.33
1.33
1.33
1.33
1.33
1.33
1.33
1.33
1.33
1.32
1.32
1.32
1.32
1.32
1.32
1.32
1.32
1.32
1.32
1.31
1.31
1.31
1.31
1.31
1.31
1.31
1.31
1.30
1.30
1.30
1.30
1.30
1.30
1.29
1.29
1.29
1.29
1.29
1.28
1.28
1.28
CD37 antigen
interferon-induced protein 35
major histocompatibility complex, class II, DQ beta 2
likely ortholog of mouse IRA1 protein
hypothetical protein FLJ10770
laminin, gamma 1 (formerly LAMB2)
cat eye syndrome chromosome region, candidate 1
collagen, type IV, alpha 2
phospholipase C-like 2
immunoglobulin superfamily containing leucine-rich repeat
KIAA1102 protein
Homo sapiens T cell receptor beta chain BV20S1 BJ1-5 BC1 mRNA,
complete cds
FK506 binding protein 10, 65 kDa
Lysosomal-associated multispanning membrane protein-5
CD97 antigen
drebrin 1
biglycan
latent transforming growth factor beta binding protein 1
sprouty-related, EVH1 domain containing 2
dihydropyrimidinase-like 2
protein tyrosine phosphatase, receptor type, M
SET and MYND domain containing 3
HLA-G histocompatibility antigen, class I, G
zinc finger protein
CD81 antigen (target of antiproliferative antibody 1)
EGF-containing fibulin-like extracellular matrix protein 2
lipoma HMGIC fusion partner
MADS box transcription enhancer factor 2, polypeptide C (myocyte
enhancer factor 2C)
F-box and leucine-rich repeat protein 7
hypothetical gene BC008967
CD34 antigen
S100 calcium binding protein A4 (calcium protein, calvasculin,
metastasin, murine placental homolog)
ras-related C3 botulinum toxin substrate 2 (rho family, small GTP
binding protein Rac2)
alpha-2-macroglobulin
major histocompatibility complex, class II, DQ beta 1
receptor tyrosine kinase-like orphan receptor 1
potassium channel tetramerisation domain containing 12
chondroitin sulfate proteoglycan 2 (versican)
interferon induced transmembrane protein 1 (9-27)
transforming growth factor, beta receptor II (70/80kDa)
adaptor-related protein complex 2, beta 1 subunit
KIAA1518 protein
RAB6 interacting protein 1
hypothetical protein FLJ21868
protein tyrosine phosphatase, receptor type, B
hypothetical protein FLJ11588
caldesmon 1
protein tyrosine phosphatase, non-receptor type substrate 1
CD37
IFI35
HLA-DQB2
IRA1
KIAA1579
LAMC1
CECR1
COL4A2
PLCL2
ISLR
KIAA1102
--FKBP10
LAPTM5
CD97
DBN1
BGN
LTBP1
SPRED2
DPYSL2
PTPRM
SMYD3
HLA-G
FLJ10697
CD81
EFEMP2
LHFP
MEF2C
FBXL7
BC008967
CD34
S100A4
RAC2
A2M
HLA-DQB1
ROR1
KCTD12
CSPG2
IFITM1
TGFBR2
AP2B1
KIAA1518
RAB6IP1
FLJ21868
PTPRB
FLJ11588
CALD1
PTPNS1
165
1.28
1.27
1.27
1.27
1.27
1.26
1.26
1.26
1.25
1.25
1.25
1.25
1.24
1.24
1.23
1.23
1.23
1.22
1.22
1.21
1.21
1.20
-7.92
-7.88
-6.91
-6.78
-6.58
-6.39
-6.25
-5.91
-5.83
-5.62
-5.52
-5.47
-4.90
-4.79
-4.79
-4.77
-4.23
-4.21
-3.99
-3.92
-3.91
-3.84
-3.80
-3.73
-3.72
selectin P ligand
chromosome 1 open reading frame 21
phosphoglycerate dehydrogenase
quaking homolog, KH domain RNA binding (mouse)
phospholipid transfer protein
intercellular adhesion molecule 2
fms-related tyrosine kinase 3 ligand
hypothetical protein DKFZp564K0822
ATPase, Ca++ transporting, plasma membrane 4
microtubule-associated protein 4
caldesmon 1
brain abundant, membrane attached signal protein 1
DIX domain containing 1
A kinase (PRKA) anchor protein 11
solute carrier family 39 (zinc transporter), member 14
CDC14 cell division cycle 14 homolog B (S. cerevisiae)
integrin, alpha V (vitronectin receptor, alpha polypeptide, antigen
CD51)
transducer of ERBB2, 2
ring finger protein 38
fasciculation and elongation protein zeta 2 (zygin II)
myocardin-related transcription factor B
glutamate receptor, ionotropic, N-methyl D-aspartate-like 1A
hydroxyacid oxidase 2 (long chain)
hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid deltaisomerase 1
hypothetical protein FLJ11106
solute carrier organic anion transporter family, member 4C1
fatty acid desaturase 1
fatty acid desaturase 1
PDZ domain containing 1
glycine dehydrogenase (decarboxylating; glycine decarboxylase,
glycine cleavage system protein P)
fatty acid binding protein 7, brain
hypothetical protein FLJ10462
phospholipase A2, group VII (platelet-activating factor acetylhydrolase,
plasma)
fatty acid desaturase 2
galanin
arachidonate 15-lipoxygenase, second type
fatty acid binding protein 7, brain
fatty acid desaturase 1
histone 1, H1c /// histone 1, H1c
mucin 1, transmembrane
3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (soluble)
insulin induced gene 1
lipidosin
sterol O-acyltransferase (acyl-Coenzyme A: cholesterol
acyltransferase) 1
lipidosin
dehydrogenase/reductase (SDR family) member 9
variable charge, Y-linked, 2
SELPLG
C1orf21
PHGDH
QKI
PLTP
ICAM2
FLT3LG
DKFZP564K0822
ATP2B4
MAP4
CALD1
BASP1
DIXDC1
AKAP11
SLC39A14
CDC14B
ITGAV
TOB2
RNF38
FEZ2
MRTF-B
GRINL1A
HAO2
HSD3B1
FLJ11106
SLCO4C1
FADS1
FADS1
PDZK1
GLDC
FABP7
FLJ10462
PLA2G7
FADS2
GAL
ALOX15B
FABP7
FADS1
HIST1H1C
MUC1
HMGCS1
INSIG1
BG1
SOAT1
BG1
DHRS9
VCY2
166
-3.71
-3.71
-3.67
-3.65
-3.59
-3.54
-3.49
-3.48
-3.48
-3.40
-3.38
-3.37
-3.34
-3.34
-3.34
-3.24
-3.24
-3.20
-3.17
-3.11
-3.11
-3.06
-3.05
-3.04
-3.02
-3.02
-2.97
-2.97
-2.95
-2.91
-2.83
-2.83
-2.78
-2.78
-2.78
-2.76
-2.75
-2.74
-2.64
-2.64
-2.63
-2.62
-2.60
-2.59
-2.54
-2.53
apolipoprotein C-I
carnitine acetyltransferase
SA hypertension-associated homolog (rat)
UDP glycosyltransferase 2 family, polypeptide A1
hypothetical protein MAC30
hyperpolarization activated cyclic nucleotide-gated potassium channel
3
phosphodiesterase 6A, cGMP-specific, rod, alpha
ureidopropionase, beta
hypothetical protein MAC30
mucin 1, transmembrane
hypothetical protein MAC30
SA hypertension-associated homolog (rat)
transitional epithelia response protein
hydroxysteroid (11-beta) dehydrogenase 1
peroxisomal long-chain acyl-coA thioesterase
Homo sapiens mRNA; cDNA DKFZp564P142 (from clone
DKFZp564P142)
insulin induced gene 1
solute carrier family 26, member 3
homogentisate 1,2-dioxygenase (homogentisate oxidase)
NAD(P) dependent steroid dehydrogenase-like
farnesyl diphosphate synthase (farnesyl pyrophosphate synthetase,
dimethylallyltranstransferase, geranyltranstransferase)
solute carrier family 27 (fatty acid transporter), member 2
peroxisomal trans 2-enoyl CoA reductase
DESC1 protein
cytochrome P450, family 4, subfamily F, polypeptide 8
cell death-inducing DFFA-like effector a
fatty acid 2-hydroxylase
chitinase 3-like 1 (cartilage glycoprotein-39)
chitinase 3-like 1 (cartilage glycoprotein-39)
steroid-5-alpha-reductase, alpha polypeptide 1 (3-oxo-5 alpha-steroid
delta 4-dehydrogenase alpha 1)
glycerol kinase
malic enzyme 1, NADP(+)-dependent, cytosolic
acetyl-Coenzyme A acetyltransferase 2 (acetoacetyl Coenzyme A
thiolase)
insulin induced gene 1
7-dehydrocholesterol reductase
--hypoxia-inducible protein 2
fructose-1,6-bisphosphatase 1
fatty acid synthase
--chromosome 6 open reading frame 105
histone 1, H2bc
3-hydroxy-3-methylglutaryl-Coenzyme A reductase
arginase, type II
abhydrolase domain containing 5
steroid-5-alpha-reductase, alpha polypeptide 1 (3-oxo-5 alpha-steroid
delta 4-dehydrogenase alpha 1)
APOC1
CRAT
SAH
UGT2A1
MAC30
HCN3
PDE6A
UPB1
MAC30
MUC1
MAC30
SAH
TERE1
HSD11B1
ZAP128
--INSIG1
SLC26A3
HGD
H105E3
FDPS
SLC27A2
PECR
DESC1
CYP4F8
CIDEA
FA2H
CHI3L1
CHI3L1
SRD5A1
GK
ME1
ACAT2
INSIG1
DHCR7
--HIG2
FBP1
FASN
--C6orf105
HIST1H2BC
HMGCR
ARG2
ABHD5
SRD5A1
167
-2.51
-2.50
-2.50
-2.50
-2.49
-2.49
-2.48
-2.48
-2.48
-2.45
-2.45
-2.44
-2.44
-2.43
-2.42
-2.42
-2.42
-2.42
-2.39
-2.38
-2.35
-2.33
-2.33
-2.32
-2.32
-2.30
-2.30
-2.29
-2.29
-2.29
-2.27
-2.27
-2.27
-2.26
-2.25
-2.25
-2.23
-2.22
-2.22
-2.21
-2.20
-2.20
-2.20
-2.20
-2.20
-2.18
-2.17
-2.16
abhydrolase domain containing 5
sterol-C4-methyl oxidase-like
acyl-Coenzyme A dehydrogenase, C-4 to C-12 straight chain
glycerol kinase
ELOVL family member 5, elongation of long chain fatty acids
(FEN1/Elo2, SUR4/Elo3-like, yeast)
B-cell receptor-associated protein 29
solute carrier family 27 (fatty acid transporter), member 2
glycerol kinase
chromosome 14 open reading frame 137
myogenic factor 3 /// myogenic factor 3
malic enzyme 1, NADP(+)-dependent, cytosolic
apolipoprotein C-I
CGI-100 protein
acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacylCoenzyme A thiolase)
glycerol kinase
deoxyribonuclease I-like 2
cut-like 2 (Drosophila)
NAD(P) dependent steroid dehydrogenase-like
nuclear receptor binding factor 1
cystathionase (cystathionine gamma-lyase)
paraoxonase 3
emopamil binding protein (sterol isomerase)
melanocortin 5 receptor
7-dehydrocholesterol reductase
mevalonate (diphospho) decarboxylase
dopa decarboxylase (aromatic L-amino acid decarboxylase)
branched chain aminotransferase 2, mitochondrial
superoxide dismutase 2, mitochondrial
fatty-acid-Coenzyme A ligase, long-chain 2
intraflagellar transport protein IFT20
carnitine acetyltransferase
retinol dehydrogenase 11 (all-trans and 9-cis)
G protein-coupled receptor 64
adipose differentiation-related protein
phosphatidylcholine transfer protein
steroid-5-alpha-reductase, alpha polypeptide 1
lysophosphatidic acid phosphatase
acyl-Coenzyme A oxidase 2, branched chain
ELOVL family member 5, elongation of long chain fatty acids
(FEN1/Elo2, SUR4/Elo3-like, yeast)
Krueppel-related zinc finger protein
abhydrolase domain containing 5
molybdenum cofactor sulfurase
dual specificity phosphatase 4
cytochrome b-5
retinol dehydrogenase 11 (all-trans and 9-cis)
potassium inwardly-rectifying channel, subfamily J, member 15
branched chain keto acid dehydrogenase E1, beta polypeptide (maple
syrup urine disease)
transmembrane 7 superfamily member 2
ABHD5
SC4MOL
ACADM
GK
ELOVL5
BCAP29
SLC27A2
GK
C14orf137
MYOD1
ME1
APOC1
CGI-100
ACAA2
GK
DNASE1L2
CUTL2
H105E3
CGI-63
CTH
PON3
EBP
MC5R
DHCR7
MVD
DDC
BCAT2
SOD2
FACL2
LOC90410
CRAT
RDH11
GPR64
ADFP
PCTP
SRD5A1
ACP6
ACOX2
ELOVL5
H-plk
ABHD5
MOCOS
DUSP4
CYB5
RDH11
KCNJ15
BCKDHB
TM7SF2
168
-2.15
-2.15
-2.15
-2.13
-2.12
-2.11
-2.11
-2.11
-2.11
-2.10
-2.09
-2.09
-2.08
-2.08
-2.06
-2.06
-2.06
-2.06
-2.05
-2.05
-2.04
-2.03
-2.02
-2.02
-2.01
-2.01
-1.99
-1.98
-1.97
-1.96
-1.95
-1.95
-1.94
-1.92
-1.92
-1.92
-1.92
-1.92
-1.92
-1.92
-1.91
-1.91
-1.91
-1.91
-1.90
-1.90
cytochrome b-5
cytochrome P450, family 4, subfamily F, polypeptide 2
acetyl-Coenzyme A acetyltransferase 2 (acetoacetyl Coenzyme A
thiolase)
emopamil binding protein (sterol isomerase)
zinc finger protein 43 (HTF6)
cytochrome b-5
propionyl Coenzyme A carboxylase, beta polypeptide
acyl-Coenzyme A dehydrogenase family, member 8
phosphomevalonate kinase
serum/glucocorticoid regulated kinase 2
N-acetylneuraminate pyruvate lyase (dihydrodipicolinate synthase)
Homo sapiens cDNA FLJ16053 moderately similar to MITOGENACTIVATED PROTEIN KINASE KINASE KINASE 5 (EC 2.7.1.-)
peroxisomal membrane protein 2, 22kDa
histone 1, H2ae
selenoprotein X, 1
histone 1, H2bg
sorbitol dehydrogenase
Homo sapiens transcribed sequence with moderate similarity to protein
ref:NP_084526.1 (M.musculus) h
chromosome 22 open reading frame 20
aldehyde dehydrogenase 3 family, member B2
1-acylglycerol-3-phosphate O-acyltransferase 3 /// 1-acylglycerol-3phosphate O-acyltransferase 3
solute carrier family 25 (mitochondrial carrier; peroxisomal membrane
protein, 34kDa), member 17
peroxisomal membrane protein 4, 24kDa
alpha-methylacyl-CoA racemase
isopentenyl-diphosphate delta isomerase
glucose-6-phosphate dehydrogenase
pyruvate kinase, liver and RBC
isopentenyl-diphosphate delta isomerase
transketolase (Wernicke-Korsakoff syndrome)
calcium binding protein P22
KIAA0626 gene product
aconitase 1, soluble
mevalonate kinase (mevalonic aciduria)
ras homolog gene family, member I
hypothetical protein MGC4172
hypothetical protein FLJ22679
acetyl-Coenzyme A acyltransferase 1 (peroxisomal 3-oxoacylCoenzyme A thiolase)
acetoacetyl-CoA synthetase
3-hydroxy-3-methylglutaryl-Coenzyme A reductase
peroxisomal biogenesis factor 11A
peroxisomal biogenesis factor 11A
carnitine palmitoyltransferase II
LAG1 longevity assurance homolog 4 (S. cerevisiae)
hypothetical protein LOC283537
glutathione peroxidase 3 (plasma)
hypothetical protein DKFZp547M236
CYB5
CYP4F2
ACAT2
EBP
ZNF43
CYB5
PCCB
ACAD8
PMVK
SGK2
NPL
--PXMP2
HIST1H2AE
SEPX1
HIST1H2BG
SORD
--C22orf20
ALDH3B2
AGPAT3
SLC25A17
PXMP4
AMACR
IDI1
G6PD
PKLR
IDI1
TKT
CHP
KIAA0626
ACO1
MVK
ARHI
MGC4172
FLJ22679
ACAA1
AACS
HMGCR
PEX11A
PEX11A
CPT2
LASS4
LOC283537
GPX3
DKFZp547M236
169
-1.90
-1.90
-1.89
-1.89
-1.89
-1.89
-1.89
-1.88
-1.87
-1.86
-1.86
-1.85
-1.85
-1.85
-1.85
-1.85
-1.85
-1.85
-1.85
-1.84
-1.84
-1.84
-1.83
-1.82
-1.82
-1.82
-1.81
-1.81
-1.81
-1.80
-1.79
-1.79
-1.79
-1.78
-1.78
-1.78
-1.77
-1.77
-1.77
-1.77
-1.77
-1.76
-1.76
-1.75
-1.75
-1.75
-1.75
-1.75
gamma-aminobutyric acid (GABA) A receptor, alpha 4
--elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3,
yeast)-like 4
pyruvate dehydrogenase (lipoamide) alpha 1
farnesyl-diphosphate farnesyltransferase 1
3-hydroxyisobutyryl-Coenzyme A hydrolase
lipin 1
peroxisomal biogenesis factor 3
solute carrier family 25 (mitochondrial carrier; peroxisomal membrane
protein, 34kDa), member 17
hypothetical protein HSPC111
glutathione peroxidase 3 (plasma)
2',5'-oligoadenylate synthetase 1, 40/46kDa
progesterone receptor membrane component 1
sterol-C5-desaturase (ERG3 delta-5-desaturase homolog, fungal)-like
--electron-transferring-flavoprotein dehydrogenase
chitinase 3-like 2
zinc finger protein 254
superoxide dismutase 2, mitochondrial
farnesyl-diphosphate farnesyltransferase 1
brain expressed, associated with Nedd4
synapsin II
zinc finger protein 145 (Kruppel-like, expressed in promyelocytic
leukemia)
zinc finger protein 91 (HPF7, HTF10)
--transketolase (Wernicke-Korsakoff syndrome)
enoyl Coenzyme A hydratase domain containing 1
phosphogluconate dehydrogenase /// phosphogluconate
dehydrogenase
ATP citrate lyase
electron-transferring-flavoprotein dehydrogenase
isocitrate dehydrogenase 1 (NADP+), soluble
peroxisome biogenesis factor 13
zinc finger protein 43 (HTF6)
peroxisomal biogenesis factor 3
aminolevulinate, delta-, synthase 1
glyceronephosphate O-acyltransferase
aldehyde dehydrogenase 3 family, member B2
sorbitol dehydrogenase
ATP citrate lyase
solute carrier family 15 (oligopeptide transporter), member 1
pleckstrin homology-like domain, family A, member 2
NAD kinase
histone 1, H2bk
FLJ23311 protein
sorting nexin 13
aminoacylase 1
TRAF6-inhibitory zinc finger protein
thiosulfate sulfurtransferase (rhodanese)
GABRA4
--ELOVL4
PDHA1
FDFT1
HIBCH
LPIN1
PEX3
SLC25A17
HSPC111
GPX3
OAS1
PGRMC1
SC5DL
--ETFDH
CHI3L2
ZNF254
SOD2
FDFT1
BEAN
SYN2
ZNF145
ZNF91
--TKT
ECHDC1
PGD
ACLY
ETFDH
IDH1
PEX13
ZNF43
PEX3
ALAS1
GNPAT
ALDH3B2
SORD
ACLY
SLC15A1
PHLDA2
FLJ13052
HIST1H2BK
FLJ23311
SNX13
ACY1
TIZ
TST
170
-1.75
-1.74
-1.74
-1.73
-1.73
-1.73
-1.72
-1.72
-1.72
-1.72
-1.71
-1.71
-1.71
-1.71
-1.71
-1.71
-1.70
-1.70
-1.69
-1.69
-1.69
-1.69
-1.68
-1.68
-1.68
-1.68
-1.67
-1.67
-1.67
-1.66
-1.66
-1.66
-1.65
-1.65
-1.64
-1.64
-1.64
-1.63
-1.63
-1.63
-1.63
-1.63
-1.62
-1.62
-1.62
-1.62
-1.62
lipin 1
fatty-acid-Coenzyme A ligase, long-chain 5
2',5'-oligoadenylate synthetase 1, 40/46kDa
cytochrome P450, family 4, subfamily F, polypeptide 3
chromosome 21 open reading frame 5
anterior gradient 2 homolog (Xenopus laevis)
fatty-acid-Coenzyme A ligase, long-chain 2
creatine kinase, mitochondrial 1 (ubiquitous)
zinc finger protein 165
YDD19 protein
--H2B histone family, member S
acetyl-Coenzyme A carboxylase alpha
alpha-methylacyl-CoA racemase
ATP citrate lyase
dicarbonyl/L-xylulose reductase
calsyntenin 3
desmocollin 2
solute carrier family 31 (copper transporters), member 1 /// solute
carrier family 31 (copper transporters), member 1
calmodulin-like 3
solute carrier family 25 (mitochondrial carrier; Graves disease
autoantigen), member 16
SAR1a gene homolog 2 (S. cerevisiae)
interleukin 1, beta
L-3-hydroxyacyl-Coenzyme A dehydrogenase, short chain
KIAA0626 gene product
peroxiredoxin 2
cytochrome P450, family 51, subfamily A, polypeptide 1
dihydrolipoamide branched chain transacylase (E2 component of
branched chain keto acid dehydrogenase complex; maple syrup urine
disease)
silver homolog (mouse)
peroxiredoxin 2 /// peroxiredoxin 2
transmembrane 4 superfamily member 6
nudix (nucleoside diphosphate linked moiety X)-type motif 4
cyclin-dependent kinase 5
hypothetical protein FLJ20574
N-terminal Asn amidase
hypothetical protein FLJ13263
transferrin receptor (p90, CD71)
peroxisomal biogenesis factor 16
low density lipoprotein receptor (familial hypercholesterolemia)
N-terminal Asn amidase
neuronal protein
DnaJ (Hsp40) homolog, subfamily C, member 3
homogentisate 1,2-dioxygenase (homogentisate oxidase)
solute carrier family 7 (cationic amino acid transporter, y+ system),
member 5
hypothetical protein FLJ22649 similar to signal peptidase SPC22/23
immediate early response 3
carcinoembryonic antigen-related cell adhesion molecule 1 (biliary
LPIN1
FACL5
OAS1
CYP4F3
C21orf5
AGR2
FACL2
CKMT1
ZNF165
YDD19
--H2BFS
ACACA
AMACR
ACLY
DCXR
CLSTN3
DSC2
SLC31A1
CALML3
SLC25A16
SARA2
IL1B
HADHSC
KIAA0626
PRDX2
CYP51A1
DBT
SILV
PRDX2
TM4SF6
NUDT4
CDK5
FLJ20574
LOC123803
FLJ13263
TFRC
PEX16
LDLR
LOC123803
NP25
DNAJC3
HGD
SLC7A5
FLJ22649
IER3
CEACAM1
171
-1.62
-1.61
-1.61
-1.60
-1.60
-1.60
-1.60
-1.60
-1.60
-1.59
-1.59
-1.58
-1.58
-1.58
-1.58
-1.58
-1.58
-1.57
-1.56
-1.56
-1.56
-1.56
-1.56
-1.55
-1.54
-1.54
-1.54
-1.54
-1.54
-1.54
-1.54
-1.53
-1.53
-1.52
-1.52
-1.52
-1.52
-1.52
-1.52
-1.52
-1.51
-1.51
-1.51
glycoprotein)
brain protein 44-like
histone 1, H2ag
Wiskott-Aldrich syndrome-like
phosphoinositide-3-kinase, class 2, gamma polypeptide
chromosome 9 open reading frame 16
RNA-binding protein
holocarboxylase synthetase (biotin-[proprionyl-Coenzyme Acarboxylase (ATP-hydrolysing)] ligase)
24-dehydrocholesterol reductase
ems1 sequence (mammary tumor and squamous cell carcinomaassociated (p80/85 src substrate)
acetyl-Coenzyme A acyltransferase 1 (peroxisomal 3-oxoacylCoenzyme A thiolase)
CGI-111 protein
Homo sapiens transcribed sequence: basic leucine-zipper protein
BZAP45;
peptidylprolyl isomerase F (cyclophilin F)
FBJ murine osteosarcoma viral oncogene homolog B
cytidine deaminase
aldo-keto reductase family 1, member A1 (aldehyde reductase)
solute carrier family 16 (monocarboxylic acid transporters), member 7
transmembrane 4 superfamily member 6
progesterone receptor membrane component 1
inositol polyphosphate-4-phosphatase, type II, 105kDa
3-hydroxyisobutyryl-Coenzyme A hydrolase
Homo sapiens RAB15, member RAS onocogene family, mRNA (cDNA
clone IMAGE:4866926), with apparent retained intron
peroxisomal biogenesis factor 16
L-3-hydroxyacyl-Coenzyme A dehydrogenase, short chain
carcinoembryonic antigen-related cell adhesion molecule 1 (biliary
glycoprotein)
AAIR8193
geranylgeranyl diphosphate synthase 1
hypothetical protein HSPC111
stromal cell protein
elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3,
yeast)-like 1
methylcrotonoyl-Coenzyme A carboxylase 1 (alpha)
DKFZP564B167 protein
multiple coagulation factor deficiency 2
transferrin receptor (p90, CD71)
etoposide induced 2.4 mRNA
chromosome 20 open reading frame 24
guanine nucleotide binding protein (G protein), gamma 4
protein phosphatase 1B (formerly 2C), magnesium-dependent, beta
isoform
Pirin
--melan-A
peptidylprolyl isomerase F (cyclophilin F)
keratin 16 (focal non-epidermolytic palmoplantar keratoderma)
BRP44L
HIST1H2AG
WASL
PIK3C2G
C9orf16
FLJ20273
HLCS
DHCR24
EMS1
ACAA1
CGI-111
--PPIF
FOSB
CDA
AKR1A1
SLC16A7
TM4SF6
PGRMC1
INPP4B
HIBCH
--PEX16
HADHSC
CEACAM1
UNQ8193
GGPS1
HSPC111
LOC55974
ELOVL1
MCCC1
DKFZP564B167
MCFD2
TFRC
EI24
C20orf24
GNG4
PPM1B
PIR
--MLANA
PPIF
KRT16
172
-1.51
-1.51
-1.51
-1.50
-1.50
-1.50
-1.50
-1.50
-1.49
-1.49
-1.49
-1.49
-1.49
-1.49
-1.48
-1.48
-1.48
-1.48
-1.48
-1.47
-1.47
-1.47
-1.47
-1.47
-1.47
-1.47
-1.47
-1.47
-1.47
-1.47
-1.47
-1.47
-1.46
-1.46
-1.46
-1.46
-1.46
-1.46
-1.46
-1.46
-1.45
-1.45
-1.45
-1.45
-1.45
-1.45
-1.45
-1.45
-1.45
G antigen 5
serine hydroxymethyltransferase 1 (soluble)
hypothetical protein FLJ20152
CGI-51 protein
chromosome 21 open reading frame 33
ribonuclease P1
progestin and adipoQ receptor family member III
translational inhibitor protein p14.5
histone 2, H2aa
hypothetical protein BC016005
putative protein similar to nessy (Drosophila)
fatty-acid-Coenzyme A ligase, long-chain 3
histone 3, H2a
RAB27A, member RAS oncogene family
solute carrier organic anion transporter family, member 4C1
solute carrier family 25 (mitochondrial carrier; oxoglutarate carrier),
member 11
biotinidase
acyl-Coenzyme A dehydrogenase, very long chain
cystatin B (stefin B)
CGI-100 protein
G protein-coupled receptor 143
DNA segment, Chr 15, Wayne State University 75, expressed
interleukin 13 receptor, alpha 1
mitochondrial carrier homolog 2 (C. elegans) /// mitochondrial carrier
homolog 2 (C. elegans)
uncharacterized hypothalamus protein HT009
c-Mpl binding protein
dihydrofolate reductase
RAR-related orphan receptor A
NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75kDa (NADHcoenzyme Q reductase)
platelet-activating factor acetylhydrolase 2, 40kDa
pyridoxal (pyridoxine, vitamin B6) kinase
ubiquitin carrier protein
hydroxysteroid (17-beta) dehydrogenase 7
v-Ki-ras2 Kirsten rat sarcoma 2 viral oncogene homolog
low molecular mass ubiquinone-binding protein (9.5kD)
pre-B-cell colony-enhancing factor
DKFZP566O084 protein
hypothetical protein FLJ10525
fumarate hydratase
ring finger protein 128
biotinidase
histone 2, H2aa
histone 1, H2bg
sushi-repeat protein
nudix (nucleoside diphosphate linked moiety X)-type motif 4
mitochondrial ribosomal protein S16
Nijmegen breakage syndrome 1 (nibrin)
pyruvate dehydrogenase complex, component X
CGI-65 protein
GAGE5
SHMT1
FLJ20152
CGI-51
C21orf33
RNASEP1
PAQR3
UK114
HIST2H2AA
LOC129642
C3F
FACL3
HIST3H2A
RAB27A
SLCO4C1
SLC25A11
BTD
ACADVL
CSTB
CGI-100
GPR143
D15Wsu75e
IL13RA1
MTCH2
HT009
LOC113251
DHFR
RORA
NDUFS1
PAFAH2
PDXK
E2-EPF
HSD17B7
KRAS2
QP-C
PBEF
DKFZp566O084
FLJ10525
FH
RNF128
BTD
HIST2H2AA
HIST1H2BG
SRPUL
NUDT4
MRPS16
NBS1
PDHX
CIA30
173
-1.44
-1.44
-1.44
-1.44
-1.44
-1.44
-1.44
-1.44
-1.44
-1.44
-1.44
-1.44
-1.44
-1.44
-1.44
-1.43
-1.43
-1.43
-1.43
-1.43
-1.43
-1.43
-1.43
-1.43
-1.43
-1.43
-1.42
-1.42
-1.42
-1.42
-1.42
-1.41
-1.41
-1.41
-1.41
-1.41
-1.41
-1.41
-1.41
-1.41
-1.40
-1.40
-1.40
-1.40
-1.40
-1.40
ethanolamine kinase
histone 1, H2bh
sialyltransferase 7D ((alpha-N-acetylneuraminyl-2,3-beta-galactosyl1,3)-N-acetyl galactosaminide alpha-2,6-sialyltransferase)
tumor protein D52-like 1
--GrpE-like 1, mitochondrial (E. coli)
RNA terminal phosphate cyclase domain 1
protease, serine, 8 (prostasin)
CGI-90 protein
3-hydroxybutyrate dehydrogenase (heart, mitochondrial) /// 3hydroxybutyrate dehydrogenase (heart, mitochondrial)
family with sequence similarity 3, member C
glycine cleavage system protein H (aminomethyl carrier)
ATP synthase, H+ transporting, mitochondrial F0 complex, subunit c
(subunit 9) isoform 3
membrane-associated nucleic acid binding protein
platelet-activating factor acetylhydrolase 2, 40kDa
hypothetical protein FLJ11011
solute carrier family 35 (UDP-galactose transporter), member A2
elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3,
yeast)-like 1
--peroxisomal biogenesis factor 7
Nit protein 2
gamma-glutamyl carboxylase
activated leukocyte cell adhesion molecule
FLJ20202 protein
NAD(P)H dehydrogenase, quinone 2
geranylgeranyl diphosphate synthase 1
chloride channel 3
lactamase, beta 2
signal-transducing adaptor protein-2
excision repair cross-complementing rodent repair deficiency,
complementation group 1 (includes overlapping antisense sequence)
Sjogren syndrome antigen A2 (60kDa, ribonucleoprotein autoantigen
SS-A/Ro)
KIAA0186 gene product
uracil-DNA glycosylase
histone 1, H2bd
cytosolic nonspecific dipeptidase (EC 3.4.13.18)
sortilin 1
MRS2-like, magnesium homeostasis factor (S. cerevisiae)
pyruvate dehydrogenase (lipoamide) beta
pre-B-cell colony-enhancing factor
v-raf murine sarcoma viral oncogene homolog B1
Nijmegen breakage syndrome 1 (nibrin)
hypothetical protein FLJ10849
methylcrotonoyl-Coenzyme A carboxylase 2 (beta)
ubiquitin specific protease 2
tumor rejection antigen (gp96) 1
RAB27A, member RAS oncogene family
EKI1
HIST1H2BH
SIAT7D
TPD52L1
--GRPEL1
RTCD1
PRSS8
CGI-90
BDH
FAM3C
GCSH
ATP5G3
MNAB
PAFAH2
FLJ11011
SLC35A2
ELOVL1
--PEX7
NIT2
GGCX
ALCAM
FLJ20202
NQO2
GGPS1
CLCN3
CGI-83
STAP2
ERCC1
SSA2
KIAA0186
UNG
HIST1H2BD
CN2
SORT1
MRS2L
PDHB
PBEF
BRAF
NBS1
FLJ10849
MCCC2
USP2
TRA1
RAB27A
174
-1.40
-1.40
-1.40
-1.40
-1.40
-1.40
-1.40
-1.40
-1.40
-1.40
-1.40
-1.39
-1.39
-1.39
-1.39
-1.39
-1.39
-1.39
-1.39
-1.39
-1.39
-1.38
-1.38
-1.38
-1.38
-1.38
-1.38
-1.38
-1.37
-1.37
-1.37
-1.37
-1.37
-1.37
-1.37
-1.37
-1.36
-1.36
-1.36
-1.36
-1.36
-1.36
-1.36
-1.36
-1.36
-1.36
-1.35
-1.35
-1.35
L-3-hydroxyacyl-Coenzyme A dehydrogenase, short chain
thioesterase superfamily member 2
chromosome 14 open reading frame 1
KIAA0626 gene product
mediator of RNA polymerase II transcription, subunit 8 homolog (yeast)
chromosome 1 open reading frame 27
DnaJ (Hsp40) homolog, subfamily D, member 1
cleavage and polyadenylation specific factor 5, 25 kDa
glycoprotein, synaptic 2
riboflavin kinase
histone 1, H2be
dual specificity phosphatase 4
stromal cell-derived factor 2-like 1
KIAA0033 protein
enoyl Coenzyme A hydratase 1, peroxisomal
programmed cell death 8 (apoptosis-inducing factor)
hypothetical protein FLJ22353
proteasome (prosome, macropain) subunit, alpha type, 5
biliverdin reductase B (flavin reductase (NADPH))
chromosome 14 open reading frame 87
phosphoglucomutase 1
secretory carrier membrane protein 1
CGI-04 protein
testis enhanced gene transcript (BAX inhibitor 1)
interferon-related developmental regulator 1
uncharacterized hematopoietic stem/progenitor cells protein MDS031
ribokinase
tumor protein D52-like 1
dystrophin related protein 2
pyruvate dehydrogenase (lipoamide) beta
hypothetical protein dJ473B4
myosin, light polypeptide 4, alkali; atrial, embryonic
dihydrolipoamide dehydrogenase (E3 component of pyruvate
dehydrogenase complex, 2-oxo-glutarate complex, branched chain
keto acid dehydrogenase complex)
solute carrier family 39 (zinc transporter), member 8
basigin (OK blood group)
interleukin 1 receptor, type II
nasopharyngeal epithelium specific protein 1
--HUS1 checkpoint homolog (S. pombe)
--biotinidase
PRKC, apoptosis, WT1, regulator
RAS protein activator like 1 (GAP1 like)
heat shock 70kDa protein 9B (mortalin-2)
ubiquinol-cytochrome c reductase core protein I
chromosome 9 open reading frame 16
hydroxysteroid (17-beta) dehydrogenase 12
solute carrier family 39 (zinc transporter), member 8
UDP glycosyltransferase 2 family, polypeptide B28 /// UDP
glycosyltransferase 2 family, polypeptide B28
HADHSC
THEM2
C14orf1
KIAA0626
MED8
C1orf27
DNAJD1
CPSF5
GPSN2
FLJ11149
HIST1H2BE
DUSP4
SDF2L1
KIAA0033
ECH1
PDCD8
FLJ22353
PSMA5
BLVRB
C14orf87
PGM1
SCAMP1
CGI-04
TEGT
IFRD1
MDS031
RBSK
TPD52L1
DRP2
PDHB
DJ473B4
MYL4
DLD
SLC39A8
BSG
IL1R2
NESG1
--HUS1
--BTD
PAWR
RASAL1
HSPA9B
UQCRC1
C9orf16
HSD17B12
SLC39A8
UGT2B28
175
-1.35
-1.35
-1.35
-1.35
-1.35
-1.35
-1.35
-1.35
-1.35
-1.35
-1.35
-1.35
-1.35
-1.35
-1.34
-1.34
-1.34
-1.34
-1.34
-1.34
-1.34
-1.34
-1.33
-1.33
-1.33
-1.33
-1.33
-1.33
-1.33
-1.33
-1.33
-1.33
-1.33
-1.33
-1.33
-1.33
-1.33
-1.33
-1.33
-1.32
-1.32
-1.32
-1.32
NADH dehydrogenase (ubiquinone) flavoprotein 2, 24kDa
ethanolamine kinase
cyclin-dependent kinase inhibitor 1A (p21, Cip1)
carboxypeptidase D
transaldolase 1
interleukin 1 receptor, type II
malonyl-CoA:acyl carrier protein transacylase (malonyltransferase)
v-maf musculoaponeurotic fibrosarcoma oncogene homolog F (avian)
cytochrome c oxidase subunit Vb
carboxypeptidase D
solute carrier family 25 (mitochondrial carrier; ornithine transporter)
member 15
putative L-type neutral amino acid transporter
ATPase, H+ transporting, lysosomal 21kDa, V0 subunit c'' /// ATPase,
H+ transporting, lysosomal 21kDa, V0 subunit c''
solute carrier family 11 (proton-coupled divalent metal ion transporters),
member 2
hypothetical protein DKFZp434G0522
CDK5 regulatory subunit associated protein 1
solute carrier family 35, member B1
peroxisomal farnesylated protein
fumarate hydratase
proteasome (prosome, macropain) subunit, alpha type, 4
NADH dehydrogenase (ubiquinone) Fe-S protein 8, 23kDa (NADHcoenzyme Q reductase)
eukaryotic translation initiation factor 2B, subunit 3 gamma, 58kDa
--proteasome (prosome, macropain) subunit, alpha type, 7
associated molecule with the SH3 domain of STAM
elongation factor, RNA polymerase II, 2
NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 3, 12kDa
transcription factor Dp-2 (E2F dimerization partner 2)
excision repair cross-complementing rodent repair deficiency,
complementation group 1 (includes overlapping antisense sequence)
Niemann-Pick disease, type C1
hypothetical protein MGC2574
proteasome (prosome, macropain) subunit, alpha type, 3
ORM1-like 2 (S. cerevisiae)
GrpE-like 1, mitochondrial (E. coli)
proteasome (prosome, macropain) subunit, beta type, 5 /// proteasome
(prosome, macropain) subunit, beta type, 5
solute carrier family 11 (proton-coupled divalent metal ion transporters),
member 2
solute carrier family 22 (organic cation transporter), member 1-like
sorbin and SH3 domain containing 1 /// sorbin and SH3 domain
containing 1
fructose-1,6-bisphosphatase 2
NADH dehydrogenase (ubiquinone) 1, subcomplex unknown, 2,
14.5kDa
translocase of inner mitochondrial membrane 13 homolog (yeast)
cytochrome c oxidase subunit VIIb
---
NDUFV2
EKI1
CDKN1A
CPD
TALDO1
IL1R2
MT
MAFF
COX5B
CPD
SLC25A15
KIAA0436
ATP6V0B
SLC11A2
DKFZp434G0522
CDK5RAP1
SLC35B1
PXF
FH
PSMA4
NDUFS8
EIF2B3
--PSMA7
AMSH
ELL2
NDUFB3
TFDP2
ERCC1
NPC1
MGC2574
PSMA3
ORMDL2
GRPEL1
PSMB5
SLC11A2
SLC22A1L
SORBS1
FBP2
NDUFC2
TIMM13
COX7B
---
176
-1.32
-1.32
-1.32
-1.32
-1.32
-1.32
-1.32
-1.32
-1.32
-1.31
-1.31
-1.31
-1.31
-1.31
-1.31
-1.31
-1.31
-1.31
-1.31
-1.31
-1.31
-1.30
-1.30
-1.30
-1.30
-1.30
-1.30
-1.30
-1.30
-1.30
-1.29
-1.29
-1.29
-1.29
-1.29
-1.29
-1.29
-1.28
-1.28
-1.28
-1.28
-1.28
-1.28
-1.28
-1.28
-1.27
likely ortholog of mouse exocyst component protein 70 kDa homolog
(S. cerevisiae) Exo70: exocyst component protein 70 kDa homolog (S.
cerevisiae)
dipeptidylpeptidase 3
RAB38, member RAS oncogene family
popeye domain containing 2
heat shock 70kDa protein 9B (mortalin-2)
secretory carrier membrane protein 1
glutamate-cysteine ligase, modifier subunit
erythroid associated factor
hypothetical protein FLJ10375
early growth response 3
solute carrier family 5 (sodium-dependent vitamin transporter), member
6
malignant T cell amplified sequence 1
interferon-related developmental regulator 1
H2A histone family, member X
DnaJ (Hsp40) homolog, subfamily A, member 3
recombination protein REC14
glucan (1,4-alpha-), branching enzyme 1 (glycogen branching enzyme,
Andersen disease, glycogen storage disease type IV)
CGI-51 protein
dodecenoyl-Coenzyme A delta isomerase (3,2 trans-enoyl-Coenzyme
A isomerase)
glutathione transferase zeta 1 (maleylacetoacetate isomerase)
--eukaryotic translation initiation factor 4E
growth hormone inducible transmembrane protein
NADH dehydrogenase (ubiquinone) Fe-S protein 7, 20kDa (NADHcoenzyme Q reductase)
mitochondrial ribosomal protein S33
B-cell receptor-associated protein 31
chromosome 14 open reading frame 2
HSPC171 protein
D-dopachrome tautomerase
potassium channel, subfamily K, member 1
ubiquitin-conjugating enzyme E2A (RAD6 homolog)
D-aspartate oxidase
histone 2, H2be
--solute carrier family 30 (zinc transporter), member 1
--Nijmegen breakage syndrome 1 (nibrin)
Krueppel-related zinc finger protein
ClpX caseinolytic protease X homolog (E. coli)
FLJ20288 protein
hypothetical protein MGC4276 similar to CG8198
translocase of inner mitochondrial membrane 23 homolog (yeast)
c-myc binding protein
trefoil factor 2 (spasmolytic protein 1)
translocase of outer mitochondrial membrane 70 homolog A (yeast)
cytochrome c oxidase subunit Va
EXO70
DPP3
RAB38
POPDC2
HSPA9B
SCAMP1
GCLM
ERAF
FLJ10375
EGR3
SLC5A6
MCTS1
IFRD1
H2AFX
DNAJA3
REC14
GBE1
CGI-51
DCI
GSTZ1
--EIF4E
GHITM
NDUFS7
MRPS33
BCAP31
C14orf2
HSPC171
DDT
KCNK1
UBE2A
DDO
HIST2H2BE
--SLC30A1
--NBS1
H-plk
CLPX
FLJ20288
MGC4276
TIMM23
MYCBP
TFF2
TOMM70A
COX5A
177
-1.27
-1.27
-1.27
-1.27
-1.26
-1.26
-1.26
-1.26
-1.26
-1.26
-1.26
-1.26
-1.26
-1.26
-1.26
-1.26
-1.26
-1.25
-1.25
-1.25
-1.25
-1.25
-1.25
-1.25
-1.25
-1.25
-1.25
-1.25
-1.24
-1.24
-1.24
-1.24
-1.24
-1.24
-1.23
-1.23
-1.23
-1.23
-1.22
-1.22
-1.21
-1.21
-1.21
-1.21
mitochondrial ribosomal protein S18A
polyposis locus protein 1
Homo sapiens transcribed sequence with strong similarity to protein
pir:A32800 (H.sapiens) A32800 chaperonin GroEL precursor - human
pantothenate kinase 3
--glutaredoxin 2
ATP synthase, H+ transporting, mitochondrial F0 complex, subunit c
(subunit 9) isoform 3
unc-5 homolog B (C. elegans)
mitochondrial translation optimization 1 homolog (S. cerevisiae)
ganglioside-induced differentiation-associated protein 1-like 1
huntingtin interacting protein 2
integral type I protein
AUT-like 2, cysteine endopeptidase (S. cerevisiae)
cytochrome c oxidase subunit Vb
T-cell leukemia translocation altered gene
mitochondrial ribosomal protein L33
diazepam binding inhibitor (GABA receptor modulator, acyl-Coenzyme
A binding protein)
ATPase, H+ transporting, lysosomal 16kDa, V0 subunit c
likely ortholog of rat vacuole membrane protein 1 /// likely ortholog of rat
vacuole membrane protein 1
cytochrome c oxidase subunit VIIa polypeptide 2 (liver)
metaxin 1
fatty acid binding protein 6, ileal (gastrotropin)
postsynaptic protein CRIPT
aspartylglucosaminidase
diazepam binding inhibitor (GABA receptor modulator, acyl-Coenzyme
A binding protein)
NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 3, 9kDa
succinate dehydrogenase complex, subunit C, integral membrane
protein, 15kDa
Homo sapiens transcribed sequence with weak similarity to protein
pir:PC4369 (H.sapiens) PC4369 olfactory receptor, HT2 - human
(fragment)
6-pyruvoyltetrahydropterin synthase
succinate-CoA ligase, GDP-forming, alpha subunit
adaptor-related protein complex 3, sigma 2 subunit
cytochrome c oxidase subunit Vb /// cytochrome c oxidase subunit Vb
zinc finger protein 430
chromosome 6 open reading frame 79
recombination protein REC14
ras homolog gene family, member E
F-box only protein 9
ATPase, H+ transporting, lysosomal 34kDa, V1 subunit D
peptidase (mitochondrial processing) beta
DNA segment on chromosome X (unique) 9879 expressed sequence
CGI-147 protein
malate dehydrogenase 1, NAD (soluble)
synaptogyrin 1
proteasome (prosome, macropain) subunit, alpha type, 1
MRPS18A
DP1
--PANK3
--GLRX2
ATP5G3
UNC5B
MTO1
GDAP1L1
HIP2
P24B
AUTL2
COX5B
TCTA
MRPL33
DBI
ATP6V0C
VMP1
COX7A2
MTX1
FABP6
CRIPT
AGA
DBI
NDUFA3
SDHC
--PTS
SUCLG1
AP3S2
COX5B
ZNF430
C6orf79
REC14
ARHE
FBXO9
ATP6V1D
PMPCB
DXS9879E
CGI-147
MDH1
SYNGR1
PSMA1
178
-1.21
-1.20
-1.20
-1.20
-1.20
-1.20
-1.18
chromosome 14 open reading frame 2
ATP synthase, H+ transporting, mitochondrial F0 complex, subunit f,
isoform 2
ubiquitin specific protease 3
cytochrome c oxidase subunit VIa polypeptide 1
NADH dehydrogenase (ubiquinone) Fe-S protein 3, 30kDa (NADHcoenzyme Q reductase)
ubiquinol-cytochrome c reductase core protein II
ATP synthase, H+ transporting, mitochondrial F1 complex, gamma
polypeptide 1
C14orf2
ATP5J2
USP3
COX6A1
NDUFS3
UQCRC2
ATP5C1
A.2 All significantly changed gene in SEB-1 sebocytes after 72 hours 13-cis RA treatment
Some genes may be listed twice; indicates separate probe sets on Affymetrix gene array chips.
Fold
Change
12.25
9.89
7.04
5.95
5.91
4.98
4.64
4.30
4.25
4.18
3.70
3.65
3.52
3.43
3.29
3.22
3.08
3.06
3.00
2.98
2.74
2.60
2.51
2.48
2.46
2.42
Gene Title
retinoic acid receptor responder (tazarotene induced) 1
retinoic acid receptor responder (tazarotene induced) 1
lipocalin 2 (oncogene 24p3)
tumor necrosis factor, alpha-induced protein 2
hydroxyprostaglandin dehydrogenase 15-(NAD)
carcinoembryonic antigen-related cell adhesion molecule 6 (non-specific
cross reacting antigen)
cytochrome P450, family 1, subfamily B, polypeptide 1
cytochrome P450, family 1, subfamily B, polypeptide 1
hydroxyprostaglandin dehydrogenase 15-(NAD)
tumor necrosis factor (ligand) superfamily, member 10
serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 3
homeo box A5
carcinoembryonic antigen-related cell adhesion molecule 1 (biliary
glycoprotein)
insulin-like growth factor binding protein 3
aldehyde dehydrogenase 1 family, member A3
retinoic acid receptor responder (tazarotene induced) 3
oxidised low density lipoprotein (lectin-like) receptor 1
solute carrier family 1 (glial high affinity glutamate transporter), member 3
growth differentiation factor 15
SRY (sex determining region Y)-box 4
tripartite motif-containing 31
cyclin-dependent kinase inhibitor 1A (p21, Cip1)
E74-like factor 3 (ets domain transcription factor, epithelial-specific )
breast carcinoma amplified sequence 1
tetratricopeptide repeat domain 9
interferon regulatory factor 1
Gene
Symbol
RARRES1
RARRES1
LCN2
TNFAIP2
HPGD
CEACAM6
CYP1B1
CYP1B1
HPGD
TNFSF10
SERPINB3
HOXA5
CEACAM1
IGFBP3
ALDH1A3
RARRES3
OLR1
SLC1A3
GDF15
SOX4
TRIM31
CDKN1A
ELF3
BCAS1
TTC9
IRF1
179
2.42
2.35
2.29
2.23
2.20
2.20
2.18
2.09
2.08
2.07
2.07
2.06
1.94
1.85
1.82
1.79
1.78
1.78
1.77
1.76
1.71
1.71
1.71
1.70
1.69
1.66
1.66
1.62
1.60
1.59
1.56
1.47
-4.68
-3.94
-3.78
-3.25
-3.12
-2.79
-2.72
-2.57
-2.48
-2.27
-2.25
-2.12
-2.05
interferon-induced protein with tetratricopeptide repeats 3
tripartite motif-containing 31
fucosidase, alpha-L- 1, tissue
interferon-induced protein with tetratricopeptide repeats 2
BTG family, member 2
interferon-induced protein with tetratricopeptide repeats 2
carcinoembryonic antigen-related cell adhesion molecule 5
proteasome (prosome, macropain) subunit, beta type, 10
vascular cell adhesion molecule 1
annexin A9
2',5'-oligoadenylate synthetase 1, 40/46kDa
--PTK6 protein tyrosine kinase 6
GATA binding protein 3
2',5'-oligoadenylate synthetase 1, 40/46kDa
protein kinase C, alpha
EPH receptor B2
RNA binding protein with multiple splicing
protein kinase D2
B-cell linker
major histocompatibility complex, class I, B /// major histocompatibility
complex, class I, C
glycine receptor, beta
integrin, beta 6
nuclear factor of kappa light polypeptide gene enhancer in B-cells 2
(p49/p100)
Fas (TNF receptor superfamily, member 6)
ATPase, aminophospholipid transporter (APLT), Class I, type 8A, member
1
dual specificity phosphatase 8
solute carrier organic anion transporter family, member 3A1
glutathione peroxidase 2 (gastrointestinal)
KIBRA protein
sequestosome 1
phosphoinositide-3-kinase, regulatory subunit 3 (p55, gamma)
keratin 6A /// keratin 6C
FK506 binding protein 5
keratin 6A /// keratin 6C
ELOVL family member 5, elongation of long chain fatty acids (FEN1/Elo2,
SUR4/Elo3-like, yeast)
dihydrofolate reductase
pro-melanin-concentrating hormone
zinc finger and BTB domain containing 16
pyruvate dehydrogenase kinase, isoenzyme 4
glutamate dehydrogenase 1
ribonucleotide reductase M2 polypeptide
transcription elongation factor A (SII)-like 4
CD86 antigen (CD28 antigen ligand 2, B7-2 antigen)
DNA replication complex GINS protein PSF1
IFIT3
TRIM31
FUCA1
IFIT2
BTG2
IFIT2
CEACAM5
PSMB10
VCAM1
ANXA9
OAS1
--PTK6
GATA3
OAS1
PRKCA
EPHB2
RBPMS
PRKD2
BLNK
HLA-B
GLRB
ITGB6
NFKB2
FAS
ATP8A1
DUSP8
SLCO3A1
GPX2
KIBRA
SQSTM1
PIK3R3
KRT6A
FKBP5
KRT6A
ELOVL5
DHFR
PMCH
ZBTB16
PDK4
GLUD1
RRM2
TCEAL4
CD86
PSF1
180
-2.03
-2.02
-1.96
-1.92
-1.91
-1.89
-1.81
-1.80
-1.79
-1.71
-1.71
-1.64
-1.59
-1.49
protein tyrosine phosphatase, non-receptor type 1
--coilin
collagen, type IV, alpha 6
gamma-aminobutyric acid (GABA) A receptor, alpha 2
--S100 calcium binding protein A10 (annexin II ligand, calpactin I, light
polypeptide (p11))
thrombospondin 1
ubiquinol-cytochrome c reductase hinge protein
phospholipase A2, group IVA (cytosolic, calcium-dependent)
protein tyrosine phosphatase-like (proline instead of catalytic arginine),
member b
CDC6 cell division cycle 6 homolog (S. cerevisiae)
BUB3 budding uninhibited by benzimidazoles 3 homolog (yeast)
hypothetical protein LOC130074
PTPN1
--COIL
COL4A6
GABRA2
--S100A10
THBS1
UQCRH
PLA2G4A
PTPLB
CDC6
BUB3
LOC130074
A.3 All significantly changed genes in HaCaT keratinocytes after 72 hour 13-cis RA
treatment
Some genes may be listed twice; indicates separate probe sets on Affymetrix gene array chips.
Fold
Change
3.68
3.56
3.22
3.21
3.21
2.77
2.69
2.61
2.56
2.48
2.33
2.28
2.21
2.19
2.11
2.08
1.96
1.91
1.90
Gene Title
tripartite motif-containing 31
lipocalin 2 (oncogene 24p3)
carcinoembryonic antigen-related cell adhesion molecule 5
amiloride binding protein 1 (amine oxidase (copper-containing))
cytochrome P450 retinoid metabolizing protein
carcinoembryonic antigen-related cell adhesion molecule 6
phospholipase A2, group X
tripartite motif-containing 31
tripartite motif-containing 31
carcinoembryonic antigen-related cell adhesion molecule 6
fibulin 1
latexin protein
mucin 4, tracheobronchial
chromosome 11 open reading frame 8
kallikrein 6 (neurosin, zyme)
3'-phosphoadenosine 5'-phosphosulfate synthase 2
plasminogen activator, tissue
chromosome 11 open reading frame 8
SLAM family member 7
Gene
Symbol
TRIM31
LCN2
CEACAM5
ABP1
P450RAI-2
CEACAM6
PLA2G10
TRIM31
TRIM31
CEACAM6
FBLN1
LXN
MUC4
C11orf8
KLK6
PAPSS2
PLAT
C11orf8
SLAMF7
181
1.87
1.82
1.81
1.80
1.79
1.79
1.76
1.70
1.70
1.70
1.69
1.67
1.66
1.65
1.64
1.64
1.64
1.64
1.63
1.62
1.60
1.57
1.54
1.54
1.51
1.49
1.48
1.48
1.47
1.45
1.39
1.35
1.34
1.30
-2.10
sushi-repeat protein
2',5'-oligoadenylate synthetase 1, 40/46kDa
GATA binding protein 3
neural precursor cell expressed, developmentally down-regulated 9
insulin-like growth factor binding protein 3
fibulin 1
nebulette
sarcospan (Kras oncogene-associated gene)
G protein-coupled receptor, family C, group 5, member B
carcinoembryonic antigen-related cell adhesion molecule 1 (biliary
glycoprotein)
S100 calcium binding protein P
prostaglandin I2 (prostacyclin) synthase /// prostaglandin I2 (prostacyclin)
synthase
carcinoembryonic antigen-related cell adhesion molecule 1 (biliary
glycoprotein)
Homo sapiens cDNA clone IMAGE:3865861, partial cds
SKI-like
fucosidase, alpha-L- 1, tissue
midkine (neurite growth-promoting factor 2)
annexin A9
involucrin
insulin-like growth factor binding protein 6
UDP-N-acetyl-alpha-D-galactosamine:polypeptide Nacetylgalactosaminyltransferase 12 (GalNAc-T12)
--phosphatidic acid phosphatase type 2A
basic helix-loop-helix domain containing, class B, 3
insulin-like growth factor binding protein 3
bone morphogenetic protein 3 (osteogenic)
lysophosphatidic acid phosphatase
myxovirus (influenza virus) resistance 1, interferon-inducible protein p78
(mouse)
integrin, beta-like 1 (with EGF-like repeat domains)
cathepsin H
vacuolar protein sorting 28 (yeast)
FLJ21963 protein
retinoic acid induced 3
2'-5'-oligoadenylate synthetase-like /// 2'-5'-oligoadenylate synthetase-like
Microfibril-associated glycoprotein-2
SRPUL
OAS1
GATA3
NEDD9
IGFBP3
FBLN1
NEBL
SSPN
GPRC5B
CEACAM1
S100P
PTGIS
CEACAM1
--SKIL
FUCA1
MDK
ANXA9
IVL
IGFBP6
GALNT12
--PPAP2A
BHLHB3
IGFBP3
BMP3
ACP6
MX1
ITGBL1
CTSH
VPS28
FLJ21963
RAI3
OASL
MAGP2
182
References
Abdel-Naser MB: Selective cultivation of normal human sebocytes in vitro; a simple modified
technique for a better cell yield. Exp Dermatol 13: 562-6, 2004.
Adamson PC: Pharmacokinetics of all-trans-retinoic acid: clinical implications in acute
promyelocytic leukemia. Semin Hematol 31: 14-7, 1994.
Aderem A, Ulevitch RJ: Toll-like receptors in the induction of the innate immune response.
Nature 406: 782-7, 2000.
Ahuja D, Saenz-Robles MT, Pipas JM: SV40 large T antigen targets multiple cellular pathways
to elicit cellular transformation. Oncogene 24: 7729-45, 2005.
Akerstrom B, Flower DR, Salier JP: Lipocalins: unity in diversity. Biochim Biophys Acta 1482: 18, 2000.
Allen M, Grachtchouk M, Sheng H, Grachtchouk V, Wang A, Wei L, et al: Hedgehog signaling
regulates sebaceous gland development. Am J Pathol 163: 2173-2178, 2003.
Allenby G, Bocquel MT, Saunders M, Kazmer S, Speck J, Rosenberger M, et al: Retinoic acid
receptors and retinoid X receptors: interactions with endogenous retinoic acids. Proc Natl Acad
Sci U S A 90: 30-4, 1993.
Almond-Roesler B, Blume-Peytavi U, Bisson S, Krahn M, Rohloff E, Orfanos CE: Monitoring of
isotretinoin therapy by measuring the plasma levels of isotretinoin and 4-oxo-isotretinoin. A
useful tool for management of severe acne. Dermatology 196: 176-81, 1998.
Altucci L, Gronemeyer H: Decryption of the retinoid death code in leukemia. J Clin Immunol 22:
117-23, 2002.
Altucci L, Wilhelm E, Gronemeyer H: Leukemia: beneficial actions of retinoids and rexinoids. Int
J Biochem Cell Biol 36: 178-82, 2004.
Alves AM, Thody AJ, Fisher C, Shuster S: Measurement of lipogenesis in isolated preputial
gland cells of the rat and the effect of oestrogen. Journal of Endocrinology 109: 1-7, 1986.
Anckar J, Sistonen L: Heat shock factor 1 as a coordinator of stress and developmental
pathways. Adv Exp Med Biol 594: 78-88, 2007.
Arany I, Whitehead WE, Grattendick KJ, Ember IA, Tyring SK: Suppression of Growth by Alltrans Retinoic Acid Requires Prolonged Induction of Interferon Regulatory Factor 1 in Cervical
Squamous Carcinoma (SiHa) Cells. Clin. Diagn. Lab. Immunol. 9: 1102-1106, 2002.
Arce F, Gatjens-Boniche O, Vargas E, Valverde B, Diaz C: Apoptotic events induced by
naturally occurring retinoids ATRA and 13-cis retinoic acid on human hepatoma cell lines
Hep3B and HepG2. Cancer Lett 229: 271-81, 2005.
183
Archer JS, Chang RJ: Hirsutism and acne in polycystic ovary syndrome. Best Practice &
Research Clinical Obstetrics & Gynaecology
Polycystic Ovary Syndrome: Challenges for the Clinician 18: 737-754, 2004.
Armeanu S, Lauer UM, Smirnow I, Schenk M, Weiss TS, Gregor M, et al: Adenoviral gene
transfer of tumor necrosis factor-related apoptosis-inducing ligand overcomes an impaired
response of hepatoma cells but causes severe apoptosis in primary human hepatocytes.
Cancer Res 63: 2369-72, 2003.
Avantaggiato V, Acampora D, Tuorto F, Simeone A: Retinoic acid induces stage-specific
repatterning of the rostral central nervous system. Dev Biol 175: 347-57, 1996.
Bachmann F, Buechner SA, Wernli M, Strebel S, Erb P: Ultraviolet Light Downregulates CD95
Ligand and Trail Receptor Expression Facilitating Actinic Keratosis and Squamous Cell
Carcinoma Formation. 117: 59-66, 2001.
Balasubramanian S, Chandraratna RA, Eckert RL: A novel retinoid-related molecule inhibits
pancreatic cancer cell proliferation by a retinoid receptor independent mechanism via
suppression of cell cycle regulatory protein function and induction of caspase-associated
apoptosis. Oncogene 24: 4257-70, 2005.
Baldin V, Lukas J, Marcote MJ, Pagano M, Draetta G: Cyclin D1 is a nuclear protein required
for cell cycle progression in G1. Genes Dev 7: 812-21, 1993.
Baldwin HE: The interaction between acne vulgaris and the psyche. Cutis 70: 133-9, 2002.
Bargonetti J, Reynisdottir I, Friedman PN, Prives C: Site-specific binding of wild-type p53 to
cellular DNA is inhibited by SV40 T antigen and mutant p53. Genes Dev 6: 1886-98, 1992.
Baron JM, Heise R, Blaner WS, Neis M, Joussen S, Dreuw A, et al: Retinoic Acid and its
4-Oxo Metabolites are Functionally Active in Human Skin Cells In Vitro. J Invest Dermatol 125:
143-53, 2005.
Bastien J, Plassat JL, Payrastre B, Rochette-Egly C: The phosphoinositide 3-kinase//Akt
pathway is essential for the retinoic acid-induced differentiation of F9 cells. Oncogene 25: 20402047, 2005.
Benjamini Y, Yekutieli D: Quantitative trait Loci analysis using the false discovery rate. Genetics
171: 783-90, 2005.
Berger T, Togawa A, Duncan GS, Elia AJ, You-Ten A, Wakeham A, et al: Lipocalin 2-deficient
mice exhibit increased sensitivity to Escherichia coli infection but not to ischemia-reperfusion
injury. Proc Natl Acad Sci U S A 103: 1834-9, 2006.
Berthet C, Klarmann KD, Hilton MB, Suh HC, Keller JR, Kiyokawa H, et al: Combined loss of
Cdk2 and Cdk4 results in embryonic lethality and Rb hypophosphorylation. Dev Cell 10: 563-73,
2006.
Bickers DR, Lim HW, Margolis D, Weinstock MA, Goodman C, Faulkner E, et al: The burden of
skin diseases: 2004: A joint project of the American Academy of Dermatology Association and
184
the Society for Investigative Dermatology. Journal of the American Academy of Dermatology 55:
490-500, 2006.
Blauer M, Vaalasti A, Pauli SL, Ylikomi T, Joensuu T, Tuohimaa P: Location of Androgen
Receptor in Human Skin. J Invest Dermatol 97:264-268. 97: pp.264-268, 1991.
Blumenberg M, Tomic-Canic M: Human epidermal keratinocyte: keratinization processes. Exs
78: 1-29, 1997.
Boehm MF, Zhang L, Zhi L, McClurg MR, Berger E, Wagoner M, et al: Design and synthesis of
potent retinoid X receptor selective ligands that induce apoptosis in leukemia cells. J Med Chem
38: 3146-55, 1995.
Boehm N, Samama B, Cribier B, Rochette-Egly C: Retinoic-acid receptor beta expression in
melanocytes. Eur J Dermatol 14: 19-23, 2004.
Bouterfa H, Picht T, Kess D, Herbold C, Noll E, Black PM, et al: Retinoids inhibit human glioma
cell proliferation and migration in primary cell cultures but not in established cell lines.
Neurosurgery 46: 419-30, 2000.
Brand N, Petkovich M, Krust A, Chambon P, de The H, Marchio A, et al: Identification of a
second human retinoic acid receptor. Nature 332: 850-3, 1988.
Brogden RN, Clissold SP: Flutamide. A preliminary review of its pharmacodynamic and
pharmacokinetic properties, and therapeutic efficacy in advanced prostatic cancer. Drugs 38:
185-203, 1989.
Caramuta S, De Cecco L, Reid JF, Zannini L, Gariboldi M, Kjeldsen L, et al: Regulation of
lipocalin-2 gene by the cancer chemopreventive retinoid 4-HPR. Int J Cancer 119: 1599-606,
2006.
Cariati R, Zancai P, Quaia M, Cutrona G, Giannini F, Rizzo S, et al: Retinoic acid induces
persistent, RARalpha-mediated anti-proliferative responses in Epstein-Barr virus-immortalized b
lymphoblasts carrying an activated C-MYC oncogene but not in Burkitt's lymphoma cell lines. Int
J Cancer 86: 375-84, 2000.
Chakravarti N, El-Naggar AK, Lotan R, Anderson J, Diwan AH, Saadati HG, et al: Expression of
retinoid receptors in sebaceous cell carcinoma. Journal of Cutaneous Pathology 33: 10-17,
2006.
Chambon P: The retinoid signaling pathway: molecular and genetic analyses. Semin Cell Biol 5:
115-25, 1994.
Chen J, Maltby KM, Miano JM: A Novel Retinoid-Response Gene Set in Vascular Smooth
Muscle Cells. Biochemical and Biophysical Research Communications 281: 475-482, 2001.
Chen W, Kelly M, Opitz-Araya X, Thomas R, Low M, Cone R: Exocrine gland dysfunction in
MC5-R deficient mice: evidence for coordinated regulation of exocrine gland function by
melanocortin peptides. Cell 91: 789-798, 1997.
185
Chen W, Ohara N, Wang J, Xu Q, Liu J, Morikawa A, et al: A Novel Selective Progesterone
Receptor Modulator Asoprisnil (J867) Inhibits Proliferation and Induces Apoptosis in Cultured
Human Uterine Leiomyoma Cells in the Absence of Comparable Effects on Myometrial Cells. J
Clin Endocrinol Metab 91: 1296-1304, 2006.
Chen W, Thiboutot D, Zouboulis C: Cutaneous androgen metabolism: basic research and
clinical perspectives. J Invest Dermatol 119: 992-1007, 2002.
Chen W, Yang C, Sheu H, Seltmann H, Zouboulis C: Expression of peroxisome proliferatoractivated receptor and CCAAT/enhancer binding protein transcription factors in cultured human
sebocytes. J Invest Dermatol 121: 441-447, 2003.
Chiba H, Clifford J, Metzger D, Chambon P: Specific and Redundant Functions of Retinoid X
Receptor/Retinoic Acid Receptor Heterodimers in Differentiation, Proliferation, and Apoptosis of
F9 Embryonal Carcinoma Cells. J. Cell Biol. 139: 735-747, 1997.
Chun KH, Pfahl M, Lotan R: Induction of apoptosis by the synthetic retinoid MX3350-1 through
extrinsic and intrinsic pathways in head and neck squamous carcinoma cells. Oncogene 24:
3669-77, 2005.
Chuong CM, Nickoloff BJ, Elias PM, Goldsmith LA, Macher E, Maderson PA, et al: What is the
'true' function of skin? Exp Dermatol 11: 159-87, 2002.
Clarke N, Jimenez-Lara AM, Voltz E, Gronemeyer H: Tumor suppressor IRF-1 mediates
retinoid and interferon anticancer signaling to death ligand TRAIL. Embo J 23: 3051-60, 2004.
Coates P, Vyakrnam S, Ravenscroft JC, Stables GI, Cunliffe WJ, Leyden JJ, et al: Efficacy of
oral isotretinoin in the control of skin and nasal colonization by antibiotic-resistant
propionibacteria in patients with acne. Br J Dermatol 153: 1126-36, 2005.
Cooper GM. Oncogenes. Boston: Jones and Bartlett, 1995.
Cooper MF, McGrath H, Shuster S: Sebaceous lipogenesis in human skin. Variability with age
and with severity of acne. Br J Dermatol 94: 165-72, 1976.
Cordrain L, Lindeberg S, Hurtado M, Hill K, Eaton S, Brand-Miller J: Acne vulgaris: a disease of
western civilization. Arch Dermatol 138: 1584-1590, 2002.
Cotterill J, Cunliffe W, Williamson B: Severity of acne and sebum excretion rate. British Journal
of Dermatology 85: 93-94
1971.
Crandall J, Sakai Y, Zhang J, Koul O, Mineur Y, Crusio WE, et al: 13-cis-retinoic acid
suppresses hippocampal cell division and hippocampal-dependent learning in mice. Proc Natl
Acad Sci U S A 101: 5111-6, 2004.
Cunliffe WJ: Acne and unemployment. Br J Dermatol 115: 386, 1986.
Cunliffe WJ, Shuster S: Pathogenesis of acne. Lancet 1: 685-7, 1969.
186
Dalziel K, Barton S, Marks R: The effects of isotretinoin on follicular and sebaceous gland
differentiation. Br J Dermatol 117: 317-23, 1987.
Daniels RA, Turley H, Kimberley FC, Liu XS, Mongkolsapaya J, Ch'En P, et al: Expression of
TRAIL and TRAIL receptors in normal and malignant tissues. Cell Res 15: 430-438, 2005.
Davies M, Marks R: Studies on the effect of salicylic acid on normal skin. Br J Dermatol 95: 18792, 1976.
Davies S, Dai D, Wolf DM, Leslie KK: Immunomodulatory and transcriptional effects of
progesterone through progesterone A and B receptors in Hec50co poorly differentiated
endometrial cancer cells. J Soc Gynecol Investig 11: 494-9, 2004.
Dawson MI, Hobbs PD. The Synthetic Chemistry of Retinoids. In: The Retinoids: Biology,
Chemistry and Medicine (Second ed.), edited by M. B. Sporn, A. B. Roberts and D. S.
Goodman. New York, New York: Raven Press, Ltd., 1994, p. 5-178.
De Luca LM, Darwiche N, JOnes CS, Scita G: Retinoids in differentiation and neoplasia. Scient
Am Sci Med 2: 28-37
1995.
Degli-Esposti MA, Dougall WC, Smolak PJ, Waugh JY, Smith CA, Goodwin RG: The novel
receptor TRAIL-R4 induces NF-kappaB and protects against TRAIL-mediated apoptosis, yet
retains an incomplete death domain. Immunity 7: 813-20, 1997.
Dermatology AAo. Myths about Acne and FAQs. Skin Care Physicians, 2006.
Dermatology AAo. The Social Impact of Acne. In: AcneNet American Academy of Dermatology,
2006.
Devireddy LR, Gazin C, Zhu X, Green MR: A cell-surface receptor for lipocalin 24p3 selectively
mediates apoptosis and iron uptake. Cell 123: 1293-305, 2005.
Dillon CP, Sandy P, Nencioni A, Kissler S, Rubinson DA, Van Parijs L: Rnai as an experimental
and therapeutic tool to study and regulate physiological and disease processes. Annu Rev
Physiol 67: 147-73, 2005.
Dimberg A, Oberg F: Retinoic acid-induced cell cycle arrest of human myeloid cell lines. Leuk
Lymphoma 44: 1641-50, 2003.
Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, et al: A Biomarker that Identifies
Senescent Human Cells in Culture and in Aging Skin in vivo. PNAS 92: 9363-9367, 1995.
Doran TI, Baff R, Jacobs P, Pacia E: Characterization of Human Sebaceous Cells in vitro. J
Invest Dermatol 1991 96: pp.341-348, 1991.
Doran TI, Vidrich A, Sun TT: Intrinsic and extrinsic regulation of the differentiation of skin,
corneal and esophageal epithelial cells. Cell 22: 17-25, 1980.
187
Downie MM, Sanders D, Maier L, Stock D, Kealey T: Peroxisome proliferator-activated receptor
and farnesoid X receptor ligands differentially regulate sebaceous differentiation in human
sebaeous gland organ cultures in vitro. Br J Dermatol 151: 766-775, 2004.
Downie MMT, Guy R, Kealey T: Advances in sebaceous gland research: potential new
approaches to acne management. International Journal of Comestic Science 26: 291-311,
2004.
Dreher A, Grevers G: [Fordyce spots. A little regarded finding in the area of lip pigmentation
and mouth mucosa]. Laryngorhinootologie 74: 390-2, 1995.
Ebling FJ, Ebling E, McCaffery V, Skinner J: The response of the sebaceous glands of the
hypophysectomized-castrated male rat to 5 -dihydrotestosterone, androstenedione,
dehydroepiandrosterone and androsterone. J Endocrinol 51: 181-90, 1971.
Ebling FJ, Ebling E, Skinner J: The influence of pituitary hormones on the response of the
sebaceous glands of the male rat to testosterone. J Endocrinol 45: 245-56, 1969.
Eckert RL, Broome AM, Ruse M, Robinson N, Ryan D, Lee K: S100 proteins in the epidermis. J
Invest Dermatol 123: 23-33, 2004.
Eckert RL, Crish JF, Efimova T, Dashti SR, Deucher A, Bone F, et al: Regulation of involucrin
gene expression. J Invest Dermatol 123: 13-22, 2004.
Eckert RL, Green H: Structure and evolution of the human involucrin gene. Cell 46: 583-589,
1986.
Edmondson SR, Thumiger SP, Kaur P, Loh B, Koelmeyer R, Li A, et al: Insulin-like growth
factor binding protein-3 (IGFBP-3) localizes to and modulates proliferative epidermal
keratinocytes in vivo. British Journal of Dermatology 152: 225-230, 2005.
Elder JT, Astrom A, Pettersson U, Tavakkol A, Krust A, Kastner P, et al: Retinoic acid receptors
and binding proteins in human skin. J Invest Dermatol 98: 36S-41S, 1992.
Faucheux C, Ulysse F, Bareille R, Reddi AH, Amedee J: Opposing Actions of BMP3 and
TGF[beta]1 in Human Bone Marrow Stromal Cell Growth and Differentiation. Biochemical and
Biophysical Research Communications 241: 787-793, 1997.
Ferrari RA, Chakrabarty K, Beyler AL, Wiland J: Suppression of sebaceous gland development
in laboratory animals by 17alpha-propyltestosterone. J Invest Dermatol 71: 320-3, 1978.
Fisher GJ, Voorhees JJ: Molecular mechanisms of retinoid actions in skin. Faseb J 10: 100213, 1996.
Flo TH, Smith KD, Sato S, Rodriguez DJ, Holmes MA, Strong RK, et al: Lipocalin 2 mediates an
innate immune response to bacterial infection by sequestrating iron. Nature 432: 917-21, 2004.
Flurh JW, Mao-Qiang M, Brown BE, Wertz PW, Crumrine D, Sundberg JP, et al: Glycerol
regulates stratum corneum hydration in sebaceous gland deficient (Asebia) mice. Journal of
Investigative Dermatology 120: 728-737, 2003.
188
Fogh K., Voorhees J. J., Astrom A.: Expression, Purification, and Binding Properties of Human
Cellular Retinoic Acid-Binding Protein Type I and Type II. Archives of Biochemistry and
Biophysics 300: 751-755, 1993.
Franz TJ, Lehman PA, Pochi P, Odland GF, Olerud J: The hamster flank organ model: is it
relevant to man? J Invest Dermatol 93: 475-9, 1989.
Fritsch M, Orfanos C, Zouboulis C: Sebocytes are the key regulators of androgen homeostasis
in human skin. J Invest Dermatol 116: 793-800, 2001.
Fujimura S, Suzumiya J, Yamada Y, Kuroki M, Ono J: Downregulation of Bcl-xL and activation
of caspases during retinoic acid-induced apoptosis in an adult T-cell leukemia cell line. Hematol
J 4: 328-35, 2003.
Ge L, Gordon J, Hsuan C, Stenn K, Prouty S: Identification of the delta-6 desaturase of human
sebaceous glands: expression and enzyme activity. J Invest Dermatol 120: 707-714, 2003.
Gebhart W, Metze D, Jurecka W: Identification of secretory immunoglobulin A in human sweat
and sweat glands. J Invest Dermatol 92: 648, 1989.
Geiger J-M, Hommel L, Harms M, Suarat J-H: Oral 13-cis Retinoic Acid is superior to 9-cis
retinoic acid in sebosuppression in human beings. Journal of the American Academy of
Dermatology 34: 513-515, 1996.
Geiger J: Retinoids and sebaceous gland activity. Dermatol 1995, 1995.
Giannini F, Maestro R, Vukosavljevic T, Pomponi F, Boiocchi M: All-trans, 13-cis and 9-cis
retinoic acids induce a fully reversible growth inhibition in HNSCC cell lines: implications for in
vivo retinoic acid use. Int J Cancer 70: 194-200, 1997.
Giguere V, Ong ES, Segui P, Evans RM: Identification of a receptor for the morphogen retinoic
acid. Nature 330: 624-9, 1987.
Gilaberte M, Puig L, Alomar A: Isotretinoin treatment of acne in a patient with Apert Syndrome.
Pediatric Dermatology 20: 443-446, 2003.
Glover JC, Renaud JS, Rijli FM: Retinoic acid and hindbrain patterning. J Neurobiol 66: 705-25,
2006.
Goldstein JA, Comite H, Mescon H, Pochi PE: Isotretinoin in the treatment of Acne. Archives of
Dermatology 118: 555-558, 1982.
Goldstein JA, Socha-Szott A, Thomsen RJ, Pochi PE, Shalita AR, Strauss JS: Comparative
effect of isotretinoin and etretinate on acne and sebaceous gland secretion. J Am Acad
Dermatol 6: 760-5, 1982.
Golias CH, Charalabopoulos A, Charalabopoulos K: Cell proliferation and cell cycle control: a
mini review. International Journal of Clinical Practice 58: 1134-1141, 2004.
189
Gomez EC: Differential effect of 13-cis-retinoic acid and an aromatic retinoid (Ro 10-9359) on
the sebaceous glands of the hamster flank organ. J Invest Dermatol 76: 68-9, 1981.
Gomez EC, Moskowitz RJ: Effect of 13-cis-retinoic acid on the hamster flank organ. J Invest
Dermatol 74: 392-7, 1980.
Graham GM, Farrar MD, Cruse-Sawyer JE, Holland KT, Ingham E: Proinflammatory cytokine
production by human keratinocytes stimulated with Propionibacterium acnes and P. acnes
GroEL. Br J Dermatol 150: 421-8, 2004.
Green DR, Kroemer G: Pharmacological manipulation of cell death: clinical applications in
sight? J Clin Invest 115: 2610-7, 2005.
Gribbon EM, Cunliffe WJ, Holland KT: Interaction of Propionibacterium acnes with skin lipids in
vitro. J Gen Microbiol 139: 1745-51, 1993.
Guy R, Ridden C, Kealey T: The improved organ maintenance of the human sebaceous gland:
modeling in vitro the effects of epidermal growth factor, androgens, estrogens, 13-cis retinoic
acid, and phenol red. J Invest Dermatol 106: 454-60, 1996.
Hallahan AR, Pritchard JI, Chandraratna RA, Ellenbogen RG, Geyer JR, Overland RP, et al:
BMP-2 mediates retinoid-induced apoptosis in medulloblastoma cells through a paracrine effect.
Nat Med 9: 1033-8, 2003.
Hanai J, Mammoto T, Seth P, Mori K, Karumanchi SA, Barasch J, et al: Lipocalin 2 diminishes
invasiveness and metastasis of Ras-transformed cells. J Biol Chem 280: 13641-7, 2005.
Handa H, Hegde UP, Kotelnikov VM, Mundle SD, Dong LM, Burke P, et al: The effects of 13-cis
retinoic acid and interferon-alpha in chronic myelogenous leukemia cells in vivo in patients.
Leuk Res 21: 1087-96, 1997.
Harper JC: Antiandrogen therapy for skin and hair disease. Dermatol Clin 24: 137-43, v, 2006.
Harris HH: Sustainable rates of sebum secretion in acne patients and matched normal control
subjects. J Am Acad Dermatol 8: 200, 1983.
Hay JB, Hodgins MB, Donnelly JB: Human skin androgen metabolism and preliminary evidence
for its control by two forms of 17 ß-hydroxysteroid oxidoreductase. J Endocrinology 93: 403-413,
1982.
Herbeuval JP, Hardy AW, Boasso A, Anderson SA, Dolan MJ, Dy M, et al: Regulation of TNFrelated apoptosis-inducing ligand on primary CD4+ T cells by HIV-1: role of type I IFN-producing
plasmacytoid dendritic cells. Proc Natl Acad Sci U S A 102: 13974-9, 2005.
Hesry Vincent P-: Sensitivity of prostate cells to TRAIL-induced apoptosis increases with tumor
progression: DR5 and caspase 8 are key players. The Prostate 66: 987-995, 2006.
Hewett D, Samuelsson L, Polding J, Enlund F, Smart D, Cantone K, et al: Identification of a
psoriasis susceptibility candidate gene by linkage disequilibrium mapping with a localized single
nucleotide polymorphism map. Genomics 79: 305-14, 2002.
190
Higaki S, Nakamura M, Morohashi M, Yamagishi T: Propionibacterium acnes biotypes and
susceptibility to minocycline and Keigai-rengyo-to. International Journal of Dermatology 43: 103107, 2004.
Holmes WF, Soprano DR, Soprano KJ: Early events in the induction of apoptosis in ovarian
carcinoma cells by CD437: activation of the p38 MAP kinase signal pathway. Oncogene 22:
6377-86, 2003.
Hommel L, Geiger JM, Harms M, Saurat JH: Sebum excretion rate in subjects treated with oral
all-trans-retinoic acid. Dermatology 193: 127-30, 1996.
Hoopes CW, Taketo M, Ozato K, Liu Q, Howard TA, Linney E, et al: Mapping of the mouse Rxr
loci encoding nuclear retinoid X receptors RXR alpha, RXR beta, and RXR gamma. Genomics
14: 611-7, 1992.
Hoyos B, Imam A, Chua R, Swenson C, Tong GX, Levi E, et al: The cysteine-rich regions of the
regulatory domains of Raf and protein kinase C as retinoid receptors. J Exp Med 192: 835-45,
2000.
Huffmeier U, Lascorz J, Traupe H, Bohm B, Schurmeier-Horst F, Stander M, et al: Systematic
linkage disequilibrium analysis of SLC12A8 at PSORS5 confirms a role in susceptibility to
psoriasis vulgaris. J Invest Dermatol 125: 906-12, 2005.
Hughes BR, Cunliffe WJ: A prospective study of the effect of isotretinoin on the follicular
reservoir and sustainable sebum excretion rate in patients with acne. Arch Dermatol 130: 315-8,
1994.
Hughes SE: Differential expression of the fibroblast growth factor receptor (FGFR) multigene
family in normal human adult tissues. The Journal of Histochemistry and Cytochemistry 45:
1005-1019, 1997.
Hyatt GA, Dowling JE: Retinoic acid. A key molecule for eye and photoreceptor development.
Invest Ophthalmol Vis Sci 38: 1471-5, 1997.
Idres N, Marill J, Flexor MA, Chabot GG: Activation of Retinoic Acid Receptor-dependent
Transcription by All-trans-retinoic Acid Metabolites and Isomers. J. Biol. Chem. 277: 3149131498, 2002.
Imam A, Hoyos B, Swenson C, Levi E, Chua R, Viriya E, et al: Retinoids as ligands and
coactivators of protein kinase C alpha. Faseb J 15: 28-30, 2001.
Imperato-McGinley J, Gautier T, Cai LQ, Yee B, Epstein J, Pochi P: The androgen control of
sebum production. Studies of subjects with dihydrotestosterone deficiency and complete
androgen insensitivity. J Clin Endocrinol Metabol 76: 524-528, 1993.
Irizarry RA, Gautier L, Cope LM. The Analysis of Gene Expression Data: Methods and
Software: Spriger Verlag, 2003.
191
Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, et al: Exploration,
normalization, and summaries of high density oligonucleotide array probe level data.
Biostatistics 4: 249-64, 2003.
Ito M: New findings on the proteins of sebaceous glands. J Invest Dermatol 82: 381, 1984.
Jerabek I, Zechmeister-Machhart M, Binder BR, Geiger M: Binding of retinoic acid by the
inhibitory serpin protein C inhibitor. Eur J Biochem 268: 5989-96, 2001.
Jimenez-Lara AM, Clarke N, Altucci L, Gronemeyer H: Retinoic-acid-induced apoptosis in
leukemia cells. Trends Mol Med 10: 508-15, 2004.
Jones H, Blanc D, Cunliffe WJ: 13-cis retinoic acid and acne. Lancet 2: 1048-9, 1980.
Jugeau S, Tenaud I, Knol AC, Jarrousse V, Quereux G, Khammari A, et al: Induction of toll-like
receptors by Propionibacterium acnes. British Journal of Dermatology 153: 1105-1113, 2005.
Junn E, Han SH, Im JY, Yang Y, Cho EW, Um HD, et al: Vitamin D3 Up-Regulated Protein 1
Mediates Oxidative Stress Via Suppressing the Thioredoxin Function. J Immunol 164: 62876295, 2000.
Kan Kondo SYTSNTTKMIYS: Cisplatin-dependent upregulation of death receptors 4 and 5
augments induction of apoptosis by TNF-related apoptosis-inducing ligand against esophageal
squamous cell carcinoma. International Journal of Cancer 118: 230-242, 2006.
Kariya Y, Moriya T, Suzuki T, Chiba M, Ishida K, Takeyama J, et al: Sex steroid hormone
receptors in human skin appendage and its neoplasms. Endocr J 52: 317-25, 2005.
Karlsson T, Vahlquist A, Kedishvili N, Torma H: 13-cis-retinoic acid competitively inhibits 3
alpha-hydroxysteroid oxidation by retinol dehydrogenase RoDH-4: a mechanism for its antiandrogenic effects in sebaceous glands? Biochem Biophys Res Commun 303: 273-8, 2003.
Kasman L, Lu P, Voelkel-Johnson C: The histone deacetylase inhibitors depsipeptide and MS275, enhance TRAIL gene therapy of LNCaP prostate cancer cells without adverse effects in
normal prostate epithelial cells. Cancer Gene Ther 14: 327-334, 2006.
Keedwell RG, Zhao Y, Hammond LA, Qin S, Tsang KY, Reitmair A, et al: A retinoid-related
molecule that does not bind to classical retinoid receptors potently induces apoptosis in human
prostate cancer cells through rapid caspase activation. Cancer Res 64: 3302-12, 2004.
Kerr JB. Atlas of Functional Histology. Bacelona, Spain: Mosby International Limited, 1999.
Kim M, Ciletti N, Michel S, Reichert U, Rosenfield R: The role of specific retinoid receptors in
sebocyte growth and differentiation in culture. J Invest Dermatol 114: 349-353, 2000.
Kim MJ, Ciletti N, Michel S, Reichert U, Rosenfield RL: The role of specific retinoid receptors in
sebocyte growth and differentiation in culture. Journal of Investigative Dermatology 114: 349-53,
2000.
192
Kim MJ, Deplewski D, Ciletti N, Michel S, Reichert U, Rosenfield RL: Limited cooperation
between peroxisome proliferator-activated receptors and retinoid X receptor agonists in
sebocyte growth and development. Mol Genet Metab 74: 362-9, 2001.
Kishibe M, Bando Y, Terayama R, Namikawa K, Takahashi H, Hashimoto Y, et al: Kallikrein 8 Is
Involved in Skin Desquamation in Cooperation with Other Kallikreins. J. Biol. Chem. 282: 58345841, 2007.
Kjeldsen L, Cowland JB, Borregaard N: Human neutrophil gelatinase-associated lipocalin and
homologous proteins in rat and mouse. Biochim Biophys Acta 1482: 272-83, 2000.
Kligman AM, Fulton JE, Jr., Plewig G: Topical vitamin A acid in acne vulgaris. Arch Dermatol 99:
469-76, 1969.
Kligman LH, Kligman AM: The effect on rhino mouse skin of agents which influence
keratinization and exfoliation. J Invest Dermatol 73: 354-8, 1979.
Knaggs H, Holland K, Morris C, Wood E, Cunliffe W: Quantification of cellular proliferation in
acne using the monoclonal antibody Ki-67. J Invest Dermatol 102: 89-92., 1994.
Knaggs H, Hughes B, Morris C, Wood E, Holland D, Cunliffe w: Immunohistochemical study of
desmosomes in acne vulgaris. Br J Dermatol 130: 731-737., 1994.
Koistinen P, Zheng A, Saily M, Siitonen T, Mantymaa P, Savolainen ER: Superior effect of 9-cis
retinoic acid (RA) compared with all-trans RA and 13-cis RA on the inhibition of clonogenic cell
growth and the induction of apoptosis in OCI/AML-2 subclones: is the p53 pathway involved? Br
J Haematol 118: 401-10, 2002.
Kouhara J, Yoshida T, Nakata S, Horinaka M, Wakada M, Ueda Y, et al: Fenretinide upregulates DR5/TRAIL-R2 expression via the induction of the transcription factor CHOP and
combined treatment with fenretinide and TRAIL induces synergistic apoptosis in colon cancer
cell lines. Int J Oncol 30: 679-87, 2007.
Krebs M, Uhrin P, Vales A, Prendes-Garcia MJ, Wojta J, Geiger M, et al: Protein C inhibitor is
expressed in keratinocytes of human skin. J Invest Dermatol 113: 32-7, 1999.
Krezel W, Ghyselinck N, Samad TA, Dup, eacute, Val, et al: Impaired Locomotion and
Dopamine Signaling in Retinoid Receptor Mutant Mice. Science 279: 863-867, 1998.
LaMantia AS, Bhasin N, Rhodes K, Heemskerk J: Mesenchymal/epithelial induction mediates
olfactory pathway formation. Neuron 28: 411-25, 2000.
Lamason RL, Mohideen MA, Mest JR, Wong AC, Norton HL, Aros MC, et al: SLC24A5, a
putative cation exchanger, affects pigmentation in zebrafish and humans. Science 310: 1782-6,
2005.
Lammer EJ, Chen DT, Hoar RM, Agnish ND, Benke PJ, Braun JT, et al: Retinoic acid
embryopathy. N Engl J Med 313: 837-41, 1985.
193
Lammer EJ, Flannery DB, Barr M: Does isotretinoin cause limb reduction defects? Lancet 2:
328, 1985.
Landthaler M, Kummermehr J, Wagner A, Plewig G: Inhibitory Effects of 13 cis-Reinoic Acid on
Human Sebaceous Glands. Archives of Dermatological Research 269: 297-3069, 1980.
Laurent SJ, Mednieks MI, Rosenfield RL: Growth of sebaceous cells in monolayer culture. In
vitro Cell Dev Biol 28A: 83-89, 1992.
Layton AM, Thiboutot DM, Bettoli V. Fast Facts-Acne. Oxford, UK: Health Press Limited, 2004
Lee K-W, Ma L, Yan X, Liu B, Zhang X-k, Cohen P: Rapid Apoptosis Induction by IGFBP-3
Involves an Insulin-like Growth Factor-independent Nucleomitochondrial Translocation of
RXR{alpha}/Nur77. J. Biol. Chem. 280: 16942-16948, 2005.
Levell MJ: Acne is not associated with abnormal plasma androgens. Br J Dermatol 120: 649,
1989.
Leverkus M, Sprick MR, Wachter T, Denk A, Brocker E-B, Walczak H, et al: TRAIL-Induced
Apoptosis and Gene Induction in HaCaT Keratinocytes: Differential Contribution of TRAIL
Receptors 1 and 2. 121: 149-155, 2003.
Levin A, Bosakowski T, Kazmer S, Grippo JF: 13-cis Retinoic Acid does not bind to retinoic
acid receptors alpha, beta and gamma. Toxicologist 12: 181, 1992.
Leyden JJ: Current issues in antimicrobial therapy for the treatment of acne. J Eur Acad
Dermatol Venereol 15 Suppl 3: 51-5, 2001.
Leyden JJ, McGinley KJ, Foglia AN: Qualitative and quantitative changes in cutaneous bacteria
associated with systemic isotretinoin therapy for acne conglobata. J Invest Dermatol 86: 390-3,
1986.
Li C, Guo H, Xu X, Weinberg W, Deng C-x: Fibroblast growth factor receptor 2 (Fgfr2) plays an
important role in eyelid and skin formation and patterning. Developmental Dynamics 222: 471483, 2001.
Li C, Hung Wong W: Model-based analysis of oligonucleotide arrays: model validation, design
issues and standard error application. Genome Biol 2: RESEARCH0032, 2001.
Li R, Luo X, Pan Q, Zineh I, Archer DF, Williams RS, et al: Doxycycline alters the expression of
inflammatory and immune-related cytokines and chemokines in human endometrial cells:
implication in irregular uterine bleeding. Hum. Reprod. 21: 2555-2563, 2006.
Lilyestrom W, Klein MG, Zhang R, Joachimiak A, Chen XS: Crystal structure of SV40 large Tantigen bound to p53: interplay between a viral oncoprotein and a cellular tumor suppressor.
Genes Dev 20: 2373-82, 2006.
Loeber G, Tevethia MJ, Schwedes JF, Tegtmeyer P: Temperature-sensitive mutants identify
crucial structural regions of simian virus 40 large T antigen. J Virol 63: 4426-30, 1989.
194
Lookingbill DP, Horton R, Demers LM, Egan N, Marks JG, Jr., Santen RJ: Tissue production of
androgens in women with acne. J Am Acad Dermatol 12: 481-7, 1985.
Louafi F, Stewart CE, Perks CM, Thomas MG, Holly JM: Role of the IGF-II receptor in mediating
acute, non-genomic effects of retinoids and IGF-II on keratinocyte cell death. Exp Dermatol 12:
426-34, 2003.
Lucken-Ardjomande S, Martinou JC: Regulation of Bcl-2 proteins and of the permeability of the
outer mitochondrial membrane. C R Biol 328: 616-31, 2005.
Maden M: Retinoids and spinal cord development. J Neurobiol 66: 726-38, 2006.
Maeda Y, Miyatake J, Sono H, Matsuda M, Tatsumi Y, Horiuchi F, et al: 13-cis retinoic acid
inhibits growth of adult T cell leukemia cells and causes apoptosis; possible new indication for
retinoid therapy. Intern Med 35: 180-4, 1996.
Mangelsdorf DJ: Vitamin A receptors. Nutr Rev 52: S32-44, 1994.
Mangelsdorf DJ, Ong ES, Dyck JA, Evans RM: Nuclear receptor that identifies a novel retinoic
acid response pathway. Nature 345: 224-9, 1990.
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, et al: The nuclear
receptor superfamily: the second decade. Cell 83: 835-9, 1995.
Marikar Y, Wang Z, Duell EA, Petkovich M, Voorhees JJ, Fisher GJ: Retinoic acid receptors
regulate expression of retinoic acid 4-hydroxylase that specifically inactivates all-trans retinoic
acid in human keratinocyte HaCaT cells. J Invest Dermatol 111: 434-9, 1998.
Marples RR, Leyden JJ, Stewart RN, Mills OH, Jr., Kligman AM: The skin microflora in acne
vulgaris. J Invest Dermatol 62: 37-41, 1974.
Marsters SA, Sheridan JP, Pitti RM, Huang A, Skubatch M, Baldwin D, et al: A novel receptor
for Apo2L/TRAIL contains a truncated death domain. Curr Biol 7: 1003-6, 1997.
Martin SJ, Reutelingsperger CP, McGahon AJ, Rader JA, van Schie RC, LaFace DM, et al:
Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis
regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med
182: 1545-56, 1995.
Matias JR, Orentreich N: The hamster ear sebaceous glands. I. Examination of the regional
variation by stripped skin planimetry. J Invest Dermatol 81: 43-6, 1983.
Mattei MG, de The H, Mattei JF, Marchio A, Tiollais P, Dejean A: Assignment of the human hap
retinoic acid receptor RAR beta gene to the p24 band of chromosome 3. Hum Genet 80: 18990, 1988.
Matysiak M, Jurewicz A, Jaskolski D, Selmaj K: TRAIL induces death of human
oligodendrocytes isolated from adult brain. Brain 125: 2469-80, 2002.
195
McCaffery P, Drager UC: Regulation of retinoic acid signaling in the embryonic nervous
system: a master differentiation factor. Cytokine Growth Factor Rev 11: 233-49, 2000.
Mendelsohn C, Batourina E, Fung S, Gilbert T, Dodd J: Stromal cells mediate retinoiddependent functions essential for renal development. Development 126: 1139-48, 1999.
Merrill B, Gat U, DasGupta R, Fuchs E: Tcf3 and Lef1 regulate lineage differentiation of
multipotent stem cells in skin. Genes & Development 15: 1688-1705, 2001.
Minucci S, Pelicci PG: Retinoid receptors in health and disease: co-regulators and the
chromatin connection. Semin Cell Dev Biol 10: 215-25, 1999.
Miyake K, Ciletti N, Liao S, Rosenfield RL: Androgen receptor expression in the preputial gland
and its sebocytes. Journal of Investigative Dermatology 103: 721-5, 1994.
Mizuiri H, Yoshida K, Toge T, Oue N, Aung PP, Noguchi T, et al: DNA methylation of genes
linked to retinoid signaling in squamous cell carcinoma of the esophagus: DNA methylation of
CRBP1 and TIG1 is associated with tumor stage. Cancer Science 96: 571-577, 2005.
Mollard R, Ghyselinck NB, Wendling O, Chambon P, Mark M: Stage-dependent responses of
the developing lung to retinoic acid signaling. Int J Dev Biol 44: 457-62, 2000.
Montagna W, Parakkal PF. The Structure and Function of Skin. New York, NY, U.S.A:
Academic Press, 1974.
Mourelatos K, Eady EA, Cunliffe WJ, Clark SM, Cove JH: Temporal changes in sebum
excretion and propionibacterial colonization in preadolescent children with and without acne. Br
J Dermatol 156: 22-31, 2007.
Muller M, Jasmin JR, Monteil RA, Loubiere R: Embryology of the hair follicle. Early Hum Dev
26: 159-66, 1991.
Munro CS, Wilkie AOM: Epidermal mosaicism producing localized acne: somatic mutation in
FGFR2. The Lancet 352: 704-705, 1998.
Muzio M, Chinnaiyan AM, Kischkel FC, O'Rourke K, Shevchenko A, Ni J, et al: FLICE, a novel
FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell 85: 817-27, 1996.
Nadeau SI, Landry J: Mechanisms of activation and regulation of the heat shock-sensitive
signaling pathways. Adv Exp Med Biol 594: 100-13, 2007.
Nagpal S, Patel S, Asano A, Johnson A, Duvic M, Chandraratna R: TIG1 and TGI2 (tazaroteneinduced genes 1 and 2) are novel retinoic acid receptor responsive genes in skin. J Invest
Dermatol 106: 818, 1996.
Nagy I, Pivarcsi A, Kis K, Koreck A, Bodai L, McDowell A, et al: Propionibacterium acnes and
lipopolysaccharide induce the expression of antimicrobial peptides and proinflammatory
cytokines/chemokines in human sebocytes. Microbes Infect 8: 2195-205, 2006.
196
Nakagawa S, Fujii T, Yokoyama G, Kazanietz MG, Yamana H, Shirouzu K: Cell growth
inhibition by all-trans retinoic acid in SKBR-3 breast cancer cells: involvement of protein kinase
Calpha and extracellular signal-regulated kinase mitogen-activated protein kinase. Mol Carcinog
38: 106-16, 2003.
Napoli JL: Biochemical pathways of retinoid transport, metabolism, and signal transduction. Clin
Immunol Immunopathol 80: S52-62, 1996.
Nelson AM, Gilliland KL, Cong Z, Thiboutot DM: 13-cis Retinoic acid induces apoptosis and cell
cycle arrest in human SEB-1 sebocytes. J Invest Dermatol 126: 2178-89, 2006.
Nesterov A, Ivashchenko Y, Kraft AS: Tumor necrosis factor-related apoptosis-inducing ligand
(TRAIL) triggers apoptosis in normal prostate epithelial cells. Oncogene 21: 1135-40, 2002.
Nevins JR: The Rb/E2F pathway and cancer. Hum Mol Genet 10: 699-703, 2001.
Newcomer ME, Ong DE: Plasma retinol binding protein: structure and function of the prototypic
lipocalin. Biochim Biophys Acta 1482: 57-64, 2000.
Nguyen H, Rendl M, Fuchs E: Tcf3 Governs Stem Cell Features and Represses Cell Fate
Determination in Skin. Cell 127: 171-183, 2006.
Nicolaides N: Skin lipids: their biochemical uniqueness. Science 186: 19-26, 1974.
Nicolaides N, Ansari MN, Fu HC, Lindsay DG: Lipid compsition on comedones compared with
that of human skin surface in acne patients. J Invest Dermatol 54: 487-95, 1970.
Norris DA, Osborn R, Robinson W, Tonnesen MG: Isotretinoin produces significant inhibition of
monocyte and neutrophil chemotaxis in vivo in patients with cystic acne. J Invest Dermatol 89:
38-43, 1987.
Ochoa WF, Torrecillas A, Fita I, Verdaguer N, Corbalan-Garcia S, Gomez-Fernandez JC:
Retinoic acid binds to the C2-domain of protein kinase C(alpha). Biochemistry 42: 8774-9, 2003.
Ohtani K, DeGregori J, Nevins JR: Regulation of the cyclin E gene by transcription factor E2F1.
Proc Natl Acad Sci U S A 92: 12146-50, 1995.
Olson JM, Hallahan AR: p38 MAP kinase: a convergence point in cancer therapy. Trends Mol
Med 10: 125-9, 2004.
Orfanos CE, Zouboulis CC: Oral retinoids in the treatment of seborrhoea and acne.
Dermatology 196: 140-7, 1998.
Ott F, Bollag W, Geiger JM: Oral 9-cis-retinoic acid versus 13-cis-retinoic acid in acne therapy.
Dermatology 193: 124-6, 1996.
Papoutsaki M, Lanza M, Marinari B, Nistico S, Moretti F, Levrero M, et al: The p73 gene is an
anti-tumoral target of the RARbeta/gamma-selective retinoid tazarotene. J Invest Dermatol 123:
1162-8, 2004.
197
Pasquali D, Bellastella A, Colantuoni V, Vassallo P, Bonavolonta G, Rossi V, et al: All-trans
retinoic acid- and N-(4-hydroxyphenil)-retinamide-induced growth arrest and apoptosis in orbital
fibroblasts in Graves' disease. Metabolism 52: 1387-92, 2003.
Peck GL: Prolonged remissions of cystic acne with 13-cis-retinoic acid. N Engl J Med 300: 329,
1979.
Peck GL, Yoder FW: Treatment of lamellar ichthyosis and other keratinising dermatoses with an
oral synthetic retinoid. Lancet 2: 1172-4, 1976.
Pelletier G, Ren L: Localization of sex steroid receptors in human skin. Histol Histopathol 19:
629-36, 2004.
Perisho K, Wertz PW, Madison KC, Stewart ME, Downing DT: Fatty acids of acylceramides
from comedones and from the skin surface of acne patients and control subjects. Journal of
Investigative Dermatology 90: 350-3, 1988.
Pettersson F, Couture M-C, Hanna N, Miller Jr. W: Enhanced retinoid-induced apoptosis of
MDA-MB-231 breast cancer cells by PKC inhibitors involved activation of ERK. Oncogene 23:
7053-7066, 2004.
Pietras RJ: Sex pheromone production by preputial gland: the regulatory role of estrogen.
Chem. Senses 6: 391-408, 1981.
Plewig G, Christophers E: Renewal rate of human sebaceous glands. Acta Derm Venereol 54:
177-82, 1974.
Plewig G, Christophers E, Braun-Falco O: Cell transition in human sebaceous glands. Acta
Derm Venereol 51: 423-8, 1971.
Plewig G, Dressel H, Pfleger M, Michelsen S, Kligman AM: Low dose isotretinoin combined
with tretinoin is effective to correct abnormalities of acne. J Dtsch Dermatol Ges 2: 31-45, 2004.
Plewig G, Luderschmidt C: Hamster ear model for sebaceous glands. J Invest Dermatol 68:
171-6, 1977.
Pochi PE, Strauss JS: Sebaceous gland response in man to the administration of testosterone,
∆4- androstenedione, and dehydroisoandrosterone. J Invest Dermatol 52: 32-36, 1969.
Pochi PE, Strauss JS: Sebaceous gland response in man to the administration of testosterone,
delta-4-androstenedione, and dehydroisoandrosterone. J Invest Dermatol 52: 32-6, 1969.
Pochi PE, Strauss JS, Downing DT: Skin surface lipid composition, acne, pubertal development,
and urinary excretion of testosterone and 17-ketosteroids in children. J Invest Dermatol 69: 4859, 1977.
Pomponi F, Cariati R, Zancai P, De Paoli P, Rizzo S, Tedeschi RM, et al: Retinoids irreversibly
inhibit in vitro growth of Epstein-Barr virus-immortalized B lymphocytes. Blood 88: 3147-59,
1996.
198
Potter JE, Prutkin L, Wheatley VR: Sebaceous gland differentiation. I. Separation, morphology
and lipogenesis of isolated cells from the mouse preputial gland tumor. J Invest Dermatol 72:
120-7, 1979.
Pratt MA, Niu MY, Renart LI: Regulation of survivin by retinoic acid and its role in paclitaxelmediated cytotoxicity in MCF-7 breast cancer cells. Apoptosis 11: 589-605, 2006.
Puhvel SM, Reisner RM, Amirian DA: Quantification of bacteria in isolated pilosebaceous
follicles in normal skin. J Invest Dermatol 65: 525-31, 1975.
Qin JZ, Bacon P, Chaturvedi V, Nickoloff BJ: Role of NF-kappaB activity in apoptotic response
of keratinocytes mediated by interferon-gamma, tumor necrosis factor-alpha, and tumornecrosis-factor-related apoptosis-inducing ligand. J Invest Dermatol 117: 898-907, 2001.
Quelle DE, Ashmun RA, Shurtleff SA, Kato JY, Bar-Sagi D, Roussel MF, et al: Overexpression
of mouse D-type cyclins accelerates G1 phase in rodent fibroblasts. Genes Dev 7: 1559-71,
1993.
Ramp U, Gerharz CD, Eifler E, Biesalski HK, Gabbert HE: Effects of retinoic acid metabolites
on proliferation and differentiation of the clonal rhabdomyosarcoma cell line BA-HAN-1C.
Biology of the Cell 81: 31-37, 1994.
Ray S, Anderson ME, Tegtmeyer P: Differential interaction of temperature-sensitive simian virus
40 T antigens with tumor suppressors pRb and p53. J Virol 70: 7224-7, 1996.
Recchia F, Saggio G, Cesta A, Candeloro G, Di Blasio A, Amiconi G, et al: Phase II study of
interleukin-2 and 13-cis-retinoic acid as maintenance therapy in metastatic colorectal cancer.
Cancer Immunol Immunother 56: 699-708, 2007.
Reichel RR, Jacob ST: Control of gene expression by lipophilic hormones. Faseb J 7: 427-36,
1993.
Reynolds CP, Matthay KK, Villablanca JG, Maurer BJ: Retinoid therapy of high-risk
neuroblastoma. Cancer Lett 197: 185-92, 2003.
Ridden J, Ferguson D, Kealey T: Organ Maintenance of Human Sebaceous Glands: In Vitro
Effects of 13-cis Retinoic Acid and Testosterone. J Cell Sci 95: pp.125-136., 1990.
Rigobello MP, Scutari G, Friso A, Barzon E, Artusi S, Bindoli A: Mitochondrial permeability
transition and release of cytochrome c induced by retinoic acids. Biochem Pharmacol 58: 66570, 1999.
Ro BI, Dawson TL: The role of sebaceous gland activity and scalp microfloral metabolism in the
etiology of seborrheic dermatitis and dandruff. J Investig Dermatol Symp Proc 10: 194-7, 2005.
Roche-Laboratories. System to Manage Accutane Related Teratogenicity -S.M.A.R.T Guide to
Best Practices. 2001.
Rollman O, Vahlquist A: Oral isotretinoin (13-cis-retinoic acid) therapy in severe acne: drug and
vitamin A concentrations in serum and skin. J Invest Dermatol 86: 384-9, 1986.
199
Romand R, Dolle P, Hashino E: Retinoid signaling in inner ear development. J Neurobiol 66:
687-704, 2006.
Roos TC, Jugert FK, Merk HF, Bickers DR: Retinoid metabolism in the skin. Pharmacol Rev 50:
315-33, 1998.
Rosdahl I, Andersson E, Kagedal B, Torma H: Vitamin A metabolism and mRNA expression of
retinoid-binding protein and receptor genes in human epidermal melanocytes and melanoma
cells. Melanoma Res 7: 267-74, 1997.
Rosen ED, Sarraf P, Troy AE, Bradwin G, Moore K, Milstone DS, et al: PPAR gamma is
required for the differentiation of adipose tissue in vivo and in vitro. Molecular Cell 4: 611-7,
1999.
Rosenfield RL: Relationship of sebaceous cell stage to growth in culture. J Invest Dermatol 92:
751-4, 1989.
Rosenfield RL, Kentsis A, Deplewski D, Ciletti N: Rat preputial sebocyte differentiation involves
peroxisome proliferator-activated receptors. Journal of Investigative Dermatology 112: 226-32,
1999.
Saint-Leger D, Cohen E: Practical study of qualitative and quantitative sebum excretion on the
human forehead. Br J Dermatol 113: 551-7, 1985.
Sakai Y, Crandall JE, Brodsky J, McCaffery P: 13-cis Retinoic acid (accutane) suppresses
hippocampal cell survival in mice. Ann N Y Acad Sci 1021: 436-40, 2004.
Sanlioglu, Koksal, Ciftcioglu, Baykara, Luleci, Sanlioglu: Differential Expression of TRAIL and
its Receptors in Benign and Malignant Prostate Tissues. The Journal of Urology 177: 359-364,
2007.
Sasaki H, Ohara N, Xu Q, Wang J, DeManno DA, Chwalisz K, et al: A novel selective
progesterone receptor modulator asoprisnil activates tumor necrosis factor-related apoptosisinducing ligand (TRAIL)-mediated signaling pathway in cultured human uterine leiomyoma cells
in the absence of comparable effects on myometrial cells. J Clin Endocrinol Metab 92: 616-23,
2007.
Sawaya ME, Price VH: Different levels of 5α-reductase type I and II, aromatase, and androgen
receptor in hair follicles of women and men with androgenetic alopecia. J invest Dermatol 109:
296-300, 1997.
Schmieg FI, Simmons DT: Characterization of the in vitro interaction between SV40 T antigen
and p53: mapping the p53 binding site. Virology 164: 132-40, 1988.
Schoonjans K, Staels B, Auwerx J: The peroxisome proliferator activated receptors (PPARs)
and their effects on lipid metabolism and adipocyte differentiation. Biochim Biophys Acta 1302:
93-109, 1996.
200
Seguin-Devaux C, Hanriot D, Dailloux M, Latger-Cannard V, Zannad F, Mertes PM, et al:
Retinoic acid amplifies the host immune response to LPS through increased T lymphocytes
number and LPS binding protein expression. Mol Cell Endocrinol 245: 67-76, 2005.
Seiberg M, Siock P, Wisniewski S, Cauwenbergh G, Shapiro SS: The effects of trypsin on
apoptosis, utriculi size, and skin elasticity in the Rhino mouse. J Invest Dermatol 109: 370-6,
1997.
Sen GC, Sarkar SN: Hitching RIG to action. Nat Immunol 6: 1074-6, 2005.
Seo SJ, Ahn JY, Hong CK, Seo EY, Kye KC, Lee WH, et al: Expression of neutrophil
gelatinase-associated lipocalin in skin epidermis. J Invest Dermatol 126: 510-2, 2006.
Shaw JC, White LE: Long-term safety of spironolactone in acne: results of an 8-year followup
study. J Cutan Med Surg 6: 541-5, 2002.
Sheridan JP, Marsters SA, Pitti RM, Gurney A, Skubatch M, Baldwin D, et al: Control of TRAILinduced apoptosis by a family of signaling and decoy receptors. Science 277: 818-21, 1997.
Shimada O, Wu X, Jin X, Nouh MA, Fiscella M, Albert V, et al: Human agonistic antibody to
tumor necrosis factor-related apoptosis-inducing ligand receptor 2 induces cytotoxicity and
apoptosis in prostate cancer and bladder cancer cells. Urology 69: 395-401, 2007.
Shutoh M, Oue N, Aung PP, Noguchi T, Kuraoka K, Nakayama H, et al: DNA methylation of
genes linked with retinoid signaling in gastric carcinoma: expression of the retinoid acid receptor
beta, cellular retinol-binding protein 1, and tazarotene-induced gene 1 genes is associated with
DNA methylation. Cancer 104: 1609-19, 2005.
Silverman GA, Bird PI, Carrell RW, Church FC, Coughlin PB, Gettins PG, et al: The serpins are
an expanding superfamily of structurally similar but functionally diverse proteins. Evolution,
mechanism of inhibition, novel functions, and a revised nomenclature. J Biol Chem 276: 332936, 2001.
Simeone A, Acampora D, Arcioni L, Andrews PW, Boncinelli E, Mavilio F: Sequential activation
of HOX2 homeobox genes by retinoic acid in human embryonal carcinoma cells. Nature 346:
763-6, 1990.
Slee EA, Adrain C, Martin SJ: Serial killers: ordering caspase activation events in apoptosis.
Cell Death Differ 6: 1067-74, 1999.
Smith KJ, Diwan H, and Skelton H: Death receptors and their role in dermatology, with particular
focus on tumor necrosis factor-related apoptosis-inducing ligand receptors. Int J Dermatol 42: 317, 2003.
Smith TM, Cong Z, Gilliland KL, Clawson GA, Thiboutot DM: Insulin-like growth factor-1 induces
lipid production in human SEB-1 sebocytes via sterol response element-binding protein-1. J
Invest Dermatol 126: 1226-32, 2006.
Smith TM, Gilliland KL, Clawson GA, Thiboutot, DM. IGF-1 induces SREBP-1 expression and
lipogenesis in SEB-1 sebocytes via PI3Kinase/AKT pathway activation. in preparation.
201
Sorg O, Kuenzli S, Kaya G, Saurat JH: Proposed mechanisms of action for retinoid derivatives
in the treatment of skin aging. J Cosmet Dermatol 4: 237-44, 2005.
Spahn CM, Prescott CD: Throwing a spanner in the works: antibiotics and the translation
apparatus. J Mol Med 74: 423-39, 1996.
Spiegelman BM, Hu E, Kim JB, Brun R: PPAR gamma and the control of adipogenesis.
Biochimie 79: 111-2, 1997.
Srinivasula SM, Ahmad M, Fernandes-Alnemri T, Litwack G, Alnemri ES: Molecular ordering of
the Fas-apoptotic pathway: the Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that
activates multiple Ced-3/ICE-like cysteine proteases. Proc Natl Acad Sci U S A 93: 14486-91,
1996.
Stander S, Schwarz T: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is
expressed in normal skin and cutaneous inflammatory diseases, but not in chronically UVexposed skin and non-melanoma skin cancer. Am J Dermatopathol 27: 116-21, 2005.
Stewart ME, Benoit AM, Stranieri AM, Rapini RP, Strauss JS, Downing DT: Effect of oral 13-cis
Retinoic Acid at three dose levels on sustainable rates of sebum secretion and on acne. Journal
of the American Academy of Dermatology 8: 532-538, 1983.
Stewart ME, Downing DT. Chemistry and function of mammalian sebaceous lipids. In:
Advances in Lipid Research, edited by P. M. Elias. San Diego: Academic Press, 1991, p. 263.
Stewart ME, Greenwood R, Cunliffe WJ, Strauss JS, Downing DT: Effect of cyproterone
acetate-ethinyl estradiol treatment on the proportions of linoleic and sebaleic acids in various
skin surface lipid classes. Archives of Dermatological Research 278: 481-5, 1986.
Strauss JS, Pochi PE. The hormonal control of human sebaceous gland. In: Advances in the
Biology of Skin: The sebaceous glands, edited by W. Montagna, R. Ellis and A. Silver. Oxford:
Pergamon Press, 1963, p. 220-254.
Strauss JS, Stranieri AM, Farrell LN, Downing DT: The effect of marked inhibition of sebum
production with 13 cis-retinoic acid on skin surface lipid composition. J Invest Dermatol 74: 66-7,
1980.
Student AK, Hsu RY, Lane MD: Induction of fatty acid synthetase synthesis in differentiating
3T3-L1 preadipocytes. J Biol Chem 255: 4745-50, 1980.
Sun SY, Yue P, Hong WK, Lotan R: Augmentation of tumor necrosis factor-related apoptosisinducing ligand (TRAIL)-induced apoptosis by the synthetic retinoid 6-[3-(1-adamantyl)-4hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) through up-regulation of TRAIL
receptors in human lung cancer cells. Cancer Res 60: 7149-55, 2000.
Sun SY, Yue P, Lotan R: Implication of multiple mechanisms in apoptosis induced by the
synthetic retinoid CD437 in human prostate carcinoma cells. Oncogene 19: 4513-22, 2000.
Takeda A, Higuchi D, Takahashi T, Ogo M, Baciu P, Goetinck PF, et al: Overexpression of
serpin squamous cell carcinoma antigens in psoriatic skin. J Invest Dermatol 118: 147-54, 2002.
202
Thiboutot D: Acne and Rosacea: New and Emerging Therapies. Dermatol Clinics 18: 63-71,
2000.
Thiboutot D: Regulation of human sebaceous glands. J Invest Dermatol 123: 1-12, 2004.
Thiboutot D, Chen W: Update and future of hormonal therapy in acne. Dermatology 206: 57-67,
2003.
Thiboutot D, Gilliland K, Light J, Lookingbill D: Androgen metabolism in sebaceous glands from
subjects with and without acne [see comments]. Archives of Dermatology 135: 1041-5, 1999.
Thiboutot D, Harris G, Iles V, Cimis G, Gilliland K, Hagari S: Characterization of the 5areductase enzyme in isolated sebaceous glands and whole skin obtained from face, scalp and
nonacne-prone regions. J Invest Dermatol 104: 607, 1995.
Thiboutot D, Jabara S, McAllister J, Sivarajah A, Gilliland K, Cong Z, et al: Human skin is a
steroidogenic tissue: Steroidogenic enzymes and cofactors are expressed in epidermis, normal
sebocytes, and an immortalized sebocyte cell line (SEB-1). J Invest Dermatol 120: 905-914,
2003.
Thiboutot D, Martin P, Volikos L, Gilliland K: Oxidative activity of the type 2 isozyme of 17βhydroxysteroid dehydrogenase (17β-HSD) predominates in human sebaceous glands. J Invest
Dermatol 111: 390-395, 1998.
Thiboutot D, Sivarajah A, Gilliland K, Cong Z, Clawson G: The melanocortin 5 receptor is
expressed in human sebaceous glands and rat preputial cells. J Invest Dermatol 115: 614-619,
2000.
Thiele JJ, Weber SU, Packer L: Sebaceous gland secretion is a major physiologic route of
vitamin E delivery to skin. J Invest Dermatol 113: 1006-10, 1999.
Thody AJ, Dijkstra H: Effect of ovarian steroids and alpha-melanocyte-stimulating hormone on
preputial gland sex attractants in the female rat [proceedings]. J Endocrinol 77: 48P-49P, 1978.
Thompson JN, Howell JM, Pitt GA, McLaughlin CI: The biological activity of retinoic acid in the
domestic fowl and the effects of vitamin A deficiency on the chick embryo. Br J Nutr 23: 471-90,
1969.
Thornton MJ, Taylor AH, Mulligan K, Al-Azzawi F, Lyon CC, O'Driscoll J, et al: The distribution
of estrogen receptor beta is distinct to that of estrogen receptor alpha and the androgen
receptor in human skin and the pilosebaceous unit. J Investig Dermatol Symp Proc 8: 100-3,
2003.
Toma S, Isnardi L, Raffo P, Dastoli G, De Francisci E, Riccardi L, et al: Effects of all-transretinoic acid and 13-cis-retinoic acid on breast-cancer cell lines: growth inhibition and apoptosis
induction. Int J Cancer 70: 619-27, 1997.
Tong Z, Wu X, Kehrer JP: Increased expression of the lipocalin 24p3 as an apoptotic
mechanism for MK886. Biochem J 372: 203-10, 2003.
203
Torma H, Rollman O, Vahlquist A: The vitamin A metabolism and expression of retinoid-binding
proteins differ in HaCaT cells and normal human keratinocytes. Arch Dermatol Res 291: 339-45,
1999.
Tosi P, Pellacani A, Visani G, Ottaviani E, Ronconi S, Zamagni E, et al: In vitro treatment with
retinoids decreases bcl-2 protein expression and enhances dexamethasone-induced cytotoxicity
and apoptosis in multiple myeloma cells. Eur J Haematol 62: 143-8, 1999.
Toyoda M, Nakamura M, Morohashi M: Neuropeptides and sebaceous glands. Eur J Dermatol
12: 422-7, 2002.
Tremblay MR, Luu-The V, Leblanc G, Noel P, Breton E, Labrie F, et al: Spironolactone-related
inhibitors of type II 17beta-hydroxysteroid dehydrogenase: chemical synthesis, receptor binding
affinities, and proliferative/antiproliferative activities. Bioorg Med Chem 7: 1013-23, 1999.
Trivedi NR, Cong Z, Nelson AM, Albert AJ, Rosamilia LL, Sivarajah S, et al: Peroxisome
proliferator-activated receptors increase human sebum production. J Invest Dermatol 126:
2002-9, 2006.
Trivedi NR, Gilliland KL, Zhao W, Liu W, Thiboutot DM: Gene array expression profiling in acne
lesions reveals marked upregulation of genes involved in inflammation and matrix remodeling. J
Invest Dermatol 126: 1071-9, 2006.
Tsukada M, Schroder M, Roos TC, Chandraratna RA, Reichert U, Merk HF, et al: 13-cis retinoic
acid exerts its specific activity on human sebocytes through selective intracellular isomerization
to all-trans retinoic acid and binding to retinoid acid receptors. J Invest Dermatol 115: 321-7,
2000.
Tusher VG, Tibshirani R, Chu G: Significance analysis of microarrays applied to the ionizing
radiation response. Proc Natl Acad Sci U S A 98: 5116-21, 2001.
Van Scott E, Yu R: Hyperkeratinization, corneocyte cohesion, and alpha hydroxy acids. J Am
Acad Dermatol 11: 867-879. 1984.
Vanoosten RL, Earel JK, Jr., Griffith TS: Histone deacetylase inhibitors enhance Ad5-TRAIL
killing of TRAIL-resistant prostate tumor cells through increased caspase-2 activity. Apoptosis
12: 561-71, 2007.
Vermot J, Niederreither K, Garnier JM, Chambon P, Dolle P: Decreased embryonic retinoic
acid synthesis results in a DiGeorge syndrome phenotype in newborn mice. Proc Natl Acad Sci
U S A 100: 1763-8, 2003.
Voellmy R, Boellmann F: Chaperone regulation of the heat shock protein response. Adv Exp
Med Biol 594: 89-99, 2007.
Wang Q, Chen Q, Zhao K, Wang L, Wang L, Traboulsi EI: Update on the molecular genetics of
retinitis pigmentosa. Ophthalmic Genet 22: 133-54, 2001.
Watabe H, Valencia JC, Yasumoto K, Kushimoto T, Ando H, Muller J, et al: Regulation of
tyrosinase processing and trafficking by organellar pH and by proteasome activity. J Biol Chem
279: 7971-81, 2004.
204
Webster G: Mechanisms of Propionibacterium acnes-mediated inflammation in acne vulgaris.
Semin Dermatol 1: 299, 1982.
Webster GF: Complement activation in acne vulgaris. Consumption of complement by
comedones. Infect Immuno 26: 183, 1979.
Webster GF: Neutrophil lysosomal release in response to Propionibacterium acnes. J Invest
Dermatol 72: 209, 1979.
Wehrli P, Viard I, Bullani R, Tschopp J, French LE: Death receptors in cutaneous biology and
disease. J Invest Dermatol 115: 141-8, 2000.
Weiss JS, Ellis CN, Headington JT, Tincoff T, Hamilton TA, Voorhees JJ: Topical tretinoin
improves photoaged skin. A double-blind vehicle-controlled study. Jama 259: 527-32, 1988.
White GM: Recent findings in the epidemiologic evidence, classification, and subtypes of acne
vulgaris. J Am Acad Dermatol 39: S34-7, 1998.
Wiley SR, Schooley K, Smolak PJ, Din WS, Huang CP, Nicholl JK, et al: Identification and
characterization of a new member of the TNF family that induces apoptosis. Immunity 3: 67382, 1995.
Wilkinson DG, Bhatt S, Cook M, Boncinelli E, Krumlauf R: Segmental expression of Hox-2
homoeobox-containing genes in the developing mouse hindbrain. Nature 341: 405-9, 1989.
Williams JB, Napoli JL: Metabolism of retinoic acid and retinol during differentiation of F9
embryonal carcinoma cells. Proc Natl Acad Sci U S A 82: 4658-62, 1985.
Williams ML, Hincenbergs M, Holbrook KA: Skin lipid content during early fetal development. J
Invest Dermatol 91: 263-8, 1988.
Wirmer J, Westhof E: Molecular contacts between antibiotics and the 30S ribosomal particle.
Methods Enzymol 415: 180-202, 2006.
Wolbach SB, Howe PR: Tissue changes following deprivation of fat soluble vitamin A. J Exp
Med 43: 753, 1925.
Wood H, Pall G, Morriss-Kay G: Exposure to retinoic acid before or after the onset of
somitogenesis reveals separate effects on rhombomeric segmentation and 3' HoxB gene
expression domains. Development 120: 2279-85, 1994.
Wozel G, Chang A, Zultak M, Czarnetzki BM, Happle R, Barth J, et al: The effect of topical
retinoids on the leukotriene-B4-induced migration of polymorphonuclear leukocytes into human
skin. Arch Dermatol Res 283: 158-61, 1991.
Wrobel A, Seltmann H, Fimmel S, Muller-Decker K, Tsukada M, Bogdanoff B, et al:
Differentiation and apoptosis in human immortalized sebocytes. J Invest Dermatol 120: 175181, 2003.
205
Wu JM, DiPietrantonio AM, Hsieh TC: Mechanism of fenretinide (4-HPR)-induced cell death.
Apoptosis 6: 377-88, 2001.
Wyllie AH, Kerr JF, Currie AR: Cell death: the significance of apoptosis. Int Rev Cytol 68: 251306, 1980.
Xia LQ, Zouboulis C, Detmar M, Mayer-da-Silva A, Stadler R, Orfanos CE: Isolation of human
sebaceous glands and cultivation of sebaceous gland-derived cells as an in vitro model. Journal
of Investigative Dermatology 93: 315-21, 1989.
Yang J, Goetz D, Li JY, Wang W, Mori K, Setlik D, et al: An iron delivery pathway mediated by a
lipocalin. Mol Cell 10: 1045-56, 2002.
Yemisci A, Gorgulu A, Piskin S: Effects and side-effects of spironolactone therapy in women
with acne. J Eur Acad Dermatol Venereol 19: 163-6, 2005.
Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, Miyagishi M, et al: The RNA
helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral
responses. Nat Immunol 5: 730-7, 2004.
Youssef EM, Chen XQ, Higuchi E, Kondo Y, Garcia-Manero G, Lotan R, et al: Hypermethylation
and silencing of the putative tumor suppressor Tazarotene-induced gene 1 in human cancers.
Cancer Res 64: 2411-7, 2004.
Yu VC, Delsert C, Andersen B, Holloway JM, Devary OV, Naar AM, et al: RXR beta: a
coregulator that enhances binding of retinoic acid, thyroid hormone, and vitamin D receptors to
their cognate response elements. Cell 67: 1251-66, 1991.
Zancai P, Cariati R, Rizzo S, Boiocchi M, Dolcetti R: Retinoic acid-mediated growth arrest of
EBV-immortalized B lymphocytes is associated with multiple changes in G1 regulatory proteins:
p27Kip1 up-regulation is a relevant early event. Oncogene 17: 1827-36, 1998.
Zhang H, Rosdahl I: Expression of p27 and MAPK proteins involved in all-trans retinoic acidinduced apoptosis and cell cycle arrest in matched primary and metastatic melanoma cells. Int J
Oncol 25: 1241-8, 2004.
Zhang HS, Postigo AA, Dean DC: Active transcriptional repression by the Rb-E2F complex
mediates G1 arrest triggered by p16INK4a, TGFbeta, and contact inhibition. Cell 97: 53-61,
1999.
Zhang J, Liu L, Pfeifer GP: Methylation of the retinoid response gene TIG1 in prostate cancer
correlates with methylation of the retinoic acid receptor beta gene. Oncogene 23: 2241-9, 2004.
Zhang L, Li WH, Anthonavage M, Eisinger M: Melanocortin-5 receptor: a marker of human
sebocyte differentiation. Peptides 27: 413-20, 2006.
Zhang LI, Anthonavage M, Huang Q, Li W-H, Eisinger M: Proopiomelanocortin Peptides and
Sebogenesis. Ann NY Acad Sci 994: 154-161, 2003.
Zhang X-k: Targeting Nur77 translocation. Expert Opinion on Therapeutic Targets 11: 69-79,
2007.
206
Zhang Z, Balmer JE, Lovlie A, Fromm SH, Blomhoff R: Specific teratogenic effects of different
retinoic acid isomers and analogs in the developing anterior central nervous system of
zebrafish. Dev Dyn 206: 73-86, 1996.
Zhao Y, Qin S, Atangan LI, Molina Y, Okawa Y, Arpawong HT, et al: Casein Kinase 1α Interacts
with Retinoid X Receptor and Interferes with Agonist-induced Apoptosis. Journal of Biological
Chemistry 279: 30844-30849, 2004.
Zorn NE, Sauro MD: Retinoic acid induces translocation of protein kinase C (PKC) and
activation of nuclear PKC (nPKC) in rat splenocytes. Int J Immunopharmacol 17: 303-11, 1995.
Zouboulis CC, Akamatsu H, Stephanek K, Orfanos CE: Androgens affect the activity of human
sebocytes in culture in a manner dependent on the localization of the sebaceous glands and
their effect is antagonized by spironolactone. Skin Pharmacology 7: 33-40, 1994.
Zouboulis CC, Korge B, Akamatsu H, Xia LQ, Schiller S, Gollnick H, et al: Effects of 13-cisretinoic acid, all-trans-retinoic acid, and acitretin on the proliferation, lipid synthesis and keratin
expression of cultured human sebocytes in vitro. J Invest Dermatol 96: 792-7, 1991.
Zouboulis CC, Korge BP, Mischke D, Orfanos CE: Altered Proliferation, Synthetic Activity, and
Differentiation of Cultured Human Sebocytes in the Absence of Vitamin A and Their Modulation
by Synthetic Retinoids. J Invest Dermatol 101: pp.628-633., 1993.
Zouboulis CC, Krieter A, Gollnick H, Mischke D, Orfanos CE: Progressive differentiation of
human sebocytes in vitro is characterized by increasing cell size and altering antigen expression
and is regulated by culture duration and retinoids. Experimental Dermatology 3: 151-60, 1994.
Zouboulis CC, Seltmann H, Neitzel H, Orfanos CE: Establishment and characterization of an
immortalized human sebaceous gland cell line (SZ95). Journal of Investigative Dermatology
113: 1011-20, 1999.
Zouboulis CC, Xia L, Akamatsu H, Seltmann H, Fritsch M, Hornemann S, et al: The human
sebocyte culture model provides new insights into development and management of seborrhoea
and acne. Dermatology 196: 21-31, 1998.
Zouboulis CC, Xia LQ, Detmar M, Bogdanoff B, Giannakopoulos G, Gollnick H, et al: Culture of
human sebocytes and markers of sebocytic differentiation in vitro. Skin Pharmacology 4: 74-83,
1991.
Zouboulis CC, Zia L, Korge B, Gollnick H, Orfanos CE. Cultivation of Human Sebocytes in vitro:
Cell Characterization and Influence of Synthetic Retinoids. In: Retinoids: 10 Years On, edited
by J.-H. Saurat Basel, Karger, 1991, p. pp.254-273.
VITA
Amanda Marie Nelson
Education
Ph.D – Molecular Medicine
Pennsylvania State University College of Medicine, Hershey PA
Bachelor of Science
Purdue University, West Lafayette, IN
2001-2007
1996-2000
Teaching Experience
Pennsylvania State University Graduate School Teaching Certificate, TWT Certificate
2007
Human Anatomy and Physiology Laboratory Teaching Assistant
Messiah College, Pennsylvania
2006
Molecular Genetics and Cell Biology Tutor
Pennsylvania State University College of Medicine, Hershey PA
2004-2005
Honors/Awards
La Roche-Posay Laboratoire Pharmaceutique
The North American Foundation Research Award
2007-2008
Induction into Phi Beta Kappa
1999
Invited Lectures
Advances in Acne Research Symposium
Society of Investigative Dermatology and Galderma R&D, Los Angeles, CA.
2007
“Insights into Retinoid Actions in Sebaceous Glands.” Galderma R&D, France.
2006
Publications
AM Nelson, KL Gilliland, W Zhao, A Zaenglein, W Lui, DM Thiboutot. Lipocalin 2/NGAL up-regulation by
13-cis retinoic acid induces apoptosis in human skin and SEB-1 sebocytes. In preparation.
SB Clarke, AM Nelson, RE George and DM Thiboutot. Pharmacologic Modulation of Sebaceous Gland
Activity: Mechanisms and Clinical Applications. Dermatologic Clinics. April 2007 Vol 25, Issue 2
AM Nelson and DM Thiboutot. Biology of Sebaceous Glands. Fitzpatrick’s Dermatology in General
Medicine: Seventh Edition. In press. (October 2007).
AM Nelson, KL Gilliland, Z cong, and DM Thiboutot. 13-cis retinoic acid induces apoptosis and cell cycle
arrest in human SEB-1 sebocytes. J Invest Dermatol 126(10):2178-89, 2006
NR Trivedi, Z Cong, AM Nelson, AJ Albert, LL Rosamilia, S Savarajah, KL Gilliland, W Liu, DT Mauger,
RA Gabbay, and DM Thiboutot. Peroxisome Proliferator-Activated Receptors Increase Human Sebum
Production. J Invest Dermatol 126(9):2002-9, 2006.