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of June 14, 2017.
The IFN-γ−Dependent Suppressor of Cytokine
Signaling 1 Promoter Activity Is Positively
Regulated by IFN Regulatory Factor-1 and
Sp1 but Repressed by Growth Factor
Independence-1b and Krüppel-Like Factor-4,
and It Is Dysregulated in Psoriatic
Keratinocytes
Stefania Madonna, Claudia Scarponi, Rosanna Sestito,
Sabatino Pallotta, Andrea Cavani and Cristina Albanesi
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2010 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2010; 185:2467-2481; Prepublished online 19
July 2010;
doi: 10.4049/jimmunol.1001426
http://www.jimmunol.org/content/185/4/2467
The Journal of Immunology
The IFN-g–Dependent Suppressor of Cytokine Signaling 1
Promoter Activity Is Positively Regulated by IFN Regulatory
Factor-1 and Sp1 but Repressed by Growth Factor
Independence-1b and Krüppel-Like Factor-4, and It Is
Dysregulated in Psoriatic Keratinocytes
Stefania Madonna,* Claudia Scarponi,* Rosanna Sestito,* Sabatino Pallotta,†
Andrea Cavani,* and Cristina Albanesi*
T
cell-derived IFN-g is a key cytokine regulating the development of inflammatory and immune responses in the skin,
and keratinocytes, which constitutively bear the IFN-g
receptor complex, are a primary target of IFN-g (1, 2). In response
to this lymphokine, keratinocytes produce a plethora of immune
mediators, which induce and amplify inflammatory responses in the
skin (3–6). However, IFN-g can induce, in parallel, inhibitory molecules able to counteract its own proinflammatory effect, including the
suppressor of cytokine signaling (SOCS)1 (7, 8). SOCS1 belongs to
a family of eight intracellular proteins (cytokine-inducible Src homology 2-containing protein and SOCS1 to SOCS7), which regulate the
magnitude and duration of responses triggered by various cytokines by
inhibiting their molecular cascades in a classic negative feedback loop
(9, 10). In particular, SOCS1 potently inhibits IFN-g signaling by
*Laboratorio di Immunologia Sperimentale and †V Divisione di Dermatologia, Istituto Dermopatico dell’Immacolata, Istituto di Ricovero e Cura e Carattere Scientifico, Rome, Italy
Received for publication April 29, 2010. Accepted for publication June 3, 2010.
Address correspondence and reprint requests to Dr. Cristina Albanesi, Laboratory of
Experimental Immunology, Istituto Dermopatico dell’Immacolata, Istituto di Ricovero
e Cura e Carattere Scientifico, Via Monti di Creta, 104 00167 Rome, Italy. E-mail
address: [email protected]
Abbreviations used in this paper: ChIP, chromatin immunoprecipitation; D.U., densitometric unit; GAS, IFN-g activation site; GFI, growth factor independence; IRF,
IFN-regulatory factor; KIR, kinase inhibitory region; LS, lesional; NLS, not lesional;
Pre-LS, proximal-to-lesion; siRNA, small interfering RNA; SOCS, suppressor of
cytokine signaling; wt, wild-type.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001426
acting as a pseudosubstrate inhibitor of the Jak2 protein and, in turn,
impeding the STAT1 recruitment at the IFN-g receptor complex (11,
12). This modality of inhibition of IFN-g pathway was elucidated in
human keratinocytes stably transfected with the SOCS1 gene. As
a direct consequence of the lack of STAT1 activity, SOCS1transduced keratinocytes could not produce ICAM-1 and MHC class
II membrane molecules, as well as CCL2, CXCL9, and CXCL10
chemokines in response to IFN-g (7). The attenuation of the IFN-g
effects by SOCS1 was also found to be executed through the activation
of the RAS/ERKs 1 and 2 (ERK1/2) pathways, which drives prosurvival and anti-inflammatory programs in keratinocytes (8).
At the transcriptional level, IFN-g promotes SOCS1 expression
by inducing IFN regulatory factor (IRF)-1 transcription factor, which
has been found to bind three tandem GAAA units of socs1 promoter
in murine fibroblasts (13). Forced expression of IRF-1 in these cells
mimicked the stimulatory effect of IFN-g on socs1 gene, whereas
IRF-1 knockdown impaired SOCS1 mRNA induction by IFN-g (13).
By contrast, another study described Sp2, and not IRF-1, as the transcription factor necessary for IFN-g–mediated socs1 gene expression in transformed human HaCaT cell line (14). Although socs1
promoter contains IFN-g activation site (GAS) elements potentially
binding STAT1, this transcription factor was not directly responsible
for the IFN-g–induced SOCS1 mRNA expression in fibroblasts but,
instead for the induction of IRF-1 (15). However, GAS sequences
contained in socs1 promoter were recognized by STAT6, which
was responsible for SOCS1 mRNA expression in response to IL-4
(16, 17). Murine socs1 promoter was also found to be activated by
STAT5 in erythropoietin-treated murine fibroblasts, and negatively
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Epidermal keratinocytes can counteract the detrimental effects of IFN-g by inducing the expression of suppressor of cytokine signaling
(SOCS)1, which plays an important anti-inflammatory and self-protective role. To date, limited information exists on its expression and
regulation in human diseased keratinocytes. In this study, we compared the expression levels of SOCS1 in keratinocytes isolated from skin
affected by psoriasis with cells obtained from healthy donors, unveiling that keratinocytes are more prone than healthy cells to upregulate
SOCS1 mRNA expression in response to IFN-g. We explored the regulatory mechanisms involved in socs1 gene transcription, and found
that Sp1 and IFN regulatory factor-1 transcription factors are, respectively, responsible for the basal and IFN-g–induced activity of human
socs1 promoter. In parallel, we demonstrated that socs1 promoter is negatively regulated by two transcriptional repressors, namely,
growth factor independence-1b and Krüppel-like factor 4, which tightly control SOCS1 transcription on IFN-g stimulation.
Interestingly, although the expression of Sp1 and IFN regulatory factor-1 activators of socs1 promoter is unaltered, growth factor
independence-1b and Krüppel-like factor 4 are significantly reduced in psoriatic compared with healthy keratinocytes. This reduction
and the consequent unbalanced binding of transcriptional activators and repressors to socs1 promoter after IFN-g stimulation might be
responsible for the enhanced expression of SOCS1 in psoriatic cells. We suggest that SOCS1 exaggerated upregulation in psoriatic
keratinocytes could represent a mechanism through which these cells attempt to protect themselves from IFN-g effects. However, the
SOCS1 increased levels in psoriatic keratinocytes are not sufficient to completely inhibit the expression of proinflammatory genes. The
Journal of Immunology, 2010, 185: 2467–2481.
2468
SOCS1 TRANSCRIPTION IN HEALTHY AND PSORIATIC KERATINOCYTES
using the following oligonucleotide pairs: forward 59-ATTAGCCGGGTGTGGTGGCATGT-39 and reverse 59-ATTTGGGCTAGGGCCGGAGAAA-39, and cloned into pCRII-TOPO plasmid (Invitrogen, Life Technology, San Diego, CA). A series of deletion plasmids were generated by
digesting the 22400/+297 plasmid with specific restriction enzyme, and
cloning 21950/+31, 21620/+31, 21220/+31, 2819/+31, 2540/+31, or
275/+31 regions of human socs1 promoter into SmaI site of the pGL3
basic vector (Promega, Madison, WI). Promoter regions corresponding to
nucleotides 2248/+31, 2173/+31, 2112/+31, and –51/+31 were obtained
by PCR using the 2540/+31 plasmid as template, and cloned into pGL3
basic plasmid. PCR was conducted by using a common reverse primer
containing the HindIII site, specifically 59-ACGTAAGCTTGCATGCTCCGGGGCCAGGAG-39, and the following forward primers containing a
KpnI site: 59-CGATGGTACCCTCGCGAGGCGGGTGCTGGG-39 (for 2248/
+31), 59-CGATGGTACCACAGGGCCGAAGCGGTCCTC-39 (for 2173/+31),
59-CGATGGTACCGGGGCGGGGCCGGCAGGGGG-39 (for 2112/+31), and
59-CGATGGTACCTAAAAGACTGGCGCAGGGGC-39 (for 251/+31). Regions containing the three putative KLF binding sites were obtained by PCR
using the 2540/+31 plasmid as template. PCR was conducted by using the
reverse primer 59-ACGTAAGCTTGCATGCTCCGGGGCCAGGAG-39, and
the following forward primers containing a KpnI site: 59-TATAGGTACCGTGTGGAGACAGCTGGGG-39 (for 2540 D1), 59-TATAGGTACCGGAGGAGGGTGTGTCAGG-39 (for 2540 D2), and 59-TATAGGTACCCCCAAGAGGGCCTGGCGG-39 (for 2540 D3). All the plasmids were sequenced using
an automated dye-terminator method (ABI Prism FS, Applied Biosystems,
Branchburg, NY).
Transient transfections of keratinocytes and luciferase assay
Nine patients with severe chronic plaque psoriasis were included in this study.
Among these patients, three were selected for immunohistochemical studies and
six were selected to establish keratinocyte cultures. Patients had definite psoriasis
diagnosed according to standard criteria, and they had not received any systemic
or topical therapy for at least 1 mo before skin donation. Patients providing skin
for immunohistochemical studies or keratinocyte cultures had similar characteristics, including age of onset and disease duration and severity. Nine healthy
subjects undergoing plastic surgery were also included in this study. Of these,
three gave biopsies for immunohistochemical analysis and six for keratinocyte
cultures. Informed consent was obtained from all subjects, and the study was
approved by the Istituto Dermopatico dell’Immacolata Ethical Committee.
Cultured keratinocytes grown in 6-well plates were transiently transfected with
human socs1 promoter plasmids by using Lipofectin reagent (Invitrogen),
according to the manufacturer’s instructions. At 18 h posttransfection, the
cells were stimulated with IFN-g for 8 h, lysed in reporter lysis buffer
(Promega), and Firefly luciferase activity was measured using Dual-Glo Luciferase Assay System (Promega). To normalize the transfection efficiency,
a plasmid encoding the Renilla luciferase (pRL-null) was included in each
transfection. Luciferase activity was further normalized by total cellular protein content assayed using Bradford (Sigma-Aldrich, Milan, Italy). For doseresponse experiments with KLF3 (a generous gift of Prof. M. Crossley, Molecular and Microbial Biosciences, Faculty of Science, University of Sydney,
Sydney, Australia), KLF4 (kindly provided by Prof. W. Jang, Division of
Digestive Diseases, Emory University School of Medicine, Atlanta, GA),
or GFI-1b/Zn/GFI-1b (a generous gift of Prof. A. Iwama, Department of
Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan) expression plasmids, cells were transfected with the
specified socs1 promoter plasmids and pRL-null vector. Human keratinocytes
were also transfected with irrelevant or small interfering RNA (siRNA) specific for Sp1, IRF-1, KLF3, KLF4, KLF10, and GFI-1b (Dharmacon, Lafayette, CO), at a 50 nM final concentration, using INTERFERin reagent (Poly
Plus Transfection, New York, NY). After 36 h, siRNA-treated cells were
transfected again with socs1 promoter plasmids and treated, as previously
described. Alternatively, siRNA-transfected keratinocytes were analyzed for
the endogenous socs1 mRNA expression by real-time PCR.
Keratinocyte cultures and treatments
RNA isolation and PCR
Human keratinocytes were obtained from skin biopsies of healthy and
psoriatic donors, as previously reported (19). Briefly, cells were cultured in
the serum-free keratinocyte growth medium (Clonetics, Walkersville,
MD), for at least 3–5 d (at 60–80% confluence) before performing experiments. Stimulation with 200 U/ml human recombinant IFN-g (R&D Systems, Minneapolis, MN) was performed in keratinocyte basal medium
(Clonetics) for different time periods.
Total RNA was extracted using the TRIzol reagent (Invitrogen). mRNA was
reverse-transcribed into cDNA and analyzed by PCR or real-time PCR. The
mRNA expression of KLF members was analyzed by PCR conducted in
a Thermal Cycler (Applied Biosystems). Each reaction was carried out using
300 ng cDNA and the primer pairs’ specific for the KLF family members, as
previously described (20). The expression kinetics of SOCS1, KLF3, KLF4,
KLF10, IRF-1, Sp1, and GFI-1b were performed by real-time PCR, using
SYBR Green PCR reagents (Applied Biosystems) and the ABI PRISM SDS
7000 PCR Instrument (Applied Biosystems). The forward and reverse primers used for PCR were as follows: for SOCS1, 59-TTTTTCGCCCTTAGCGTGA-39 and 59-AGCAGCTCGAAGAGGCAGTC-39; for KLF3, 59CCCCGCAAGCATTGTTG-39 and 59-GGTCTCTTCCCAGGCTGCA-39;
for KLF4, 59- GGCACACCTGCGAACCC-39 and 59-TCCCAGTCACAGTGGTAAGGTTT-39; for KLF10, 59-TGAGCTGCAGTTGGAAGTCTGA
and 59-GGACTGTAAGGTGGAGTCAAACAA-39; for IRF-1, 59-AAGGCCAAGAGGAAGTCATGTG-39 and 59-CCATCAGAGAAGGTATCAGGGC-39; for Sp1, 59-GAGCTACAGAGGCACAAACGTACA-39 and 59ACTCAGGGCAGGCAAATTTCT-39; for GFI-1b, 59-CAGGAAGATGAACCGCTCTG-39 and 59-CAGGCACTGGTTTGGGAATAG-39; and for
b-actin, 59-CATCGAGCACGGCATCGTCA-39 and 59-TAGCACAGCCTGGATAGCAAC-39. The levels of gene expression were determined by
normalizing to b-actin mRNA expression. The values obtained from triplicate experiments were averaged, and data were presented as the mean 6 S.D.
Materials and Methods
Subjects
Immunohistochemistry
Biopsies of psoriatic skin, including lesional (LS), proximal-to-lesion (PreLS), and not lesional (NLS, 3-cm distant) zones of evolving plaques as well
as healthy skin were fixed in 10% formalin and embedded in paraffin. The
5-mm sections were dewaxed and rehydrated. After quenching endogenous
peroxidase, achieving Ag retrieval, and blocking nonspecific binding sites,
sections were incubated with mAbs against SOCS1 (MBL, Naka-ku,
Nagoya, Japan, 1:200 dilution) or CD3 (BD Pharmingen, Franklin Lakes,
NJ, 1:20 dilution). Secondary biotinylated mAbs and staining kits (Vector
Laboratories, Burlingame, CA) were used to develop immunoreactivity.
Construction of reporter plasmids
A DNA region containing the human socs1 promoter and corresponding to
nucleotides 22400 to +297 was amplified from human genomic DNA
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regulated by growth factor independence (GFI)-1b, a transcriptional repressor binding to sequences located in proximity to
GAS elements (18). Finally, the contribution of GC box(es) binding to Sp1 transcription factor to the basal activity of murine
socs1 promoter was demonstrated (13).
Although evidence on the function of SOCS1 in IFN-g–activated
healthy keratinocytes has accumulated in the past years (7, 8),
limited information exists on its expression and regulation in human diseased keratinocytes. In this study, we investigated on the
expression levels of SOCS1 in keratinocytes isolated from skin
affected by psoriasis compared with cells obtained from healthy
skin, and we found that SOCS1 mRNA levels are significantly increased in psoriatic keratinocyte strains on IFN-g stimulation. We
also explored the transcriptional regulatory mechanisms leading to
SOCS1 expression in IFN-g–activated human keratinocytes. To this
end, we generated mutant deletions of human socs1 promoter, and
tested their activity in untreated or IFN-g–treated keratinocytes.
Functional promoter analysis, together with depletions or overexpression of transcription factors putatively binding to socs1 promoter, demonstrated that the basal and the IFN-g–induced activity
of socs1 promoter depended on Sp1 and IRF-1 transcription factors,
respectively. In parallel, socs1 promoter was found to be negatively
regulated by two transcriptional repressors, namely, GFI-1b and
Krüppel-like factor (KLF)4, which inhibited socs1 promoter activity after IFN-g stimulation. Although the expression of transcriptional activators of socs1 promoter is unaltered, GFI-1b and
KLF4 are significantly reduced in psoriatic compared with healthy
cells. This reduction and the consequent unbalanced binding of
transcriptional activators and repressors to socs1 promoter after
IFN-g stimulation might be responsible for the enhanced expression of SOCS1 in psoriatic keratinocytes.
The Journal of Immunology
Immunoprecipitation and immunoblotting
(B-7 X), and Sp1 (PEP 2 X) supershifts were purchased from Santa Cruz
Biotechnology.
Chromatin immunoprecipitation analysis
Chromatin immunoprecipitation (ChIP) analysis was performed according
to the protocol from Upstate (Lake Placid, NY). Briefly, 20 3 106 keratinocytes were stimulated with medium or IFN-g. Cells were then fixed with
1% formaldehyde and washed twice with PBS. The cell pellet was lysed and
sonicated on ice to break the chromosomal DNA into fragments with an
average length of 100–500 bp. After centrifugation, 1% of the extract was
used for the total input control. The remaining extract was precleared with
a protein A-agarose. Immunoprecipitations were performed by adding normal anti–IRF-1 (C-20 X) and rabbit Ig, as control, anti–GFI-1b (B-7 X) and
mouse Ig, as control, anti-Sp1 (PEP-2 X) or anti-KLF4 (M-19 X) and goat Ig,
as control. The subsequent steps of washing, elution, and the purification of
DNA were conducted according to the manufacturer’s instructions. The purified DNA was resuspended in 20 ml Tris-EDTA buffer and analyzed by realtime PCR, as previously described. IRF-1 and Sp1 binding sites in socs1 promoter region were amplified by using the oligonucleotide pairs: 59-GAGGGTCCAGAAGAGAGGGAA-39 and 59-AGCTCCACTTTTGGTTTCTCTTTC-39. KLF sites were detected using the oligonucleotides 59-GAGAGGACAGGGCTCTGCCC-39 and 59-GTTCCACTTTCTGCCGCCAG-39.
Densitometry and statistical analysis
KLF4, GFI-1b, IRF-1, and Sp1 immunoblots of samples from healthy donors
and psoriatic patients were subjected to densitometry using an Imaging
Densitometer (model GS-670, Bio-Rad, Milan, Italy), supported by the Molecular Analyst software. The significance of differences between healthy
donors and psoriatic patients in SOCS1 mRNA expression and KLF4,
GFI-1b, IRF-1, and Sp1 protein levels was determined by Wilcoxon’s signed
rank test (SigmaStat, San Rafael, CA). This test was also used to calculate the
significance of differences in luciferase activity values obtained with different constructs in untreated and IFN-g–treated keratinocytes. Comparison
of SOCS1 mRNA expression or luciferase activity between keratinocytes
transfected with irrelevant and specific siRNA, and between cultures
transfected with GFI-1b–encoding and control plasmids was performed.
The p values #0.05 were considered significant.
Biotinylated-oligonucleotide pull-down assay
Results
The oligonucleotides containing the putative KLF binding sites were synthesized by MWG-Biotech (Ebersberg, Germany) and were modified with a
biotin group using Biotin 39End DNA Labeling Kit (Thermo Fisher Scientific,
Waltham, MA). The sequences for the oligonucleotides were as follows: 59GTCTACGGGTGGGGTCAGGC-39biotin for KLF(1) (wild-type [wt]), 59GCTGTCTCCACACCCGCCGG-39-biotin for KLF(2) (wt), 59-GGAGGAGGGTGTGTCAGGGC-39-biotin for KLF(3) (wt), and 59-GCTGTCTTTATATTTTCCGG-39-biotin for mutated KLF(2). The 50 mg nuclear extracts
prepared as previously described were precleared with streptavidin-magnetic
beads (30 ml/sample, Dynal Biotech, Invitrogen) for 1 h at 4˚C. After centrifugation, supernatants were incubated with 100 pmol biotinylated doublestrand oligonucleotides and 10 mg poly(dI/dC) (Amersham Pharmacia Biotec)
for 16 h at 4˚C. DNA bound proteins were collected with 30 ml streptavidin
magnetic beads for 2 h at 4˚C, resolved on a 10% SDS polyacrylamide gel,
and probed by immunoblotting with anti-KLF3 or anti-KLF4 Abs.
Psoriatic keratinocytes are more prone than healthy cells to
express SOCS1, which accumulates in the cytoplasm in
response to IFN-g
EMSA
Probes used in the EMSA were: 59-CCTCGCCGGACGCCACCGCGG
AAAGAGAAACCAAAAGTGGAGCTGG-39 and 59-CCTCGCCGGACG
CCACCGCGGAAAGATCGGTACAAAGTGGAGCTGG-39 for wt and mutated IRF-1 site, respectively; 59-TGGGGGCGGGGCCGGCAGGGGGCGGGGCCT-39 and 59-TAAAGGATTGGCCGGCAGAAAGCTTGGCCT-39
for wt and mutated Sp1 site, respectively; 59-CCAATCTGCAAGCCATTGCAAATCCCAGCCCCTCCCCAGCCTC-39 and 59-CCAATCTGCAAGCCAGCGTGCCGATCAGCCCCTCCCCAGCCTC-39 for wt and mutated
GFI-1b site, respectively. The 2819/2540 region (extracted from 2819/+31
plasmid) was also extracted by digestion and used in EMSA experiments. The
specific probes were labeled with g-[32P]ATP (PerkinElmer, Waltham, MA)
and purified using a QIAquick Nucleotide Removal Kit (Qiagen, Milan, Italy).
EMSAs were performed by incubating 100 ng labeled-probes with 10 mg
nuclear extracts. For competition or Ab supershift experiments, nuclear
extracts were incubated with an excess of cold competitors or specific Abs
(2 mg) for 30 min at room temperature in binding buffer (10 mM HEPES [pH
7.8], 50 mM KCl, 5 mM MgCl2, 1 mM EDTA, and 5% glycerol), then loaded
onto 5% native polyacrylamide gels for separation. The dried gels were exposed at –80˚C for more than 24 h. Abs used for IRF-1(C-20 X), GFI-1b
Although SOCS1 is known to be abundantly induced by IFN-g in
human healthy keratinocytes, to date limited information exist on
SOCS1 expression in diseased cells. Therefore, we sought to analyze the expression of SOCS1 in keratinocytes of patients affected
by psoriasis, and to compare its levels with those present in healthy
cells. To this end, human keratinocytes were isolated from skin biopsies obtained from healthy donors (n = 6) and psoriatic patients
(n = 6), and left untreated or stimulated with IFN-g for 3 h, a time
point where SOCS1 reaches the maximal expression in keratinocytes
(Fig. 1A). mRNA levels were, thus, analyzed by real-time PCR,
which revealed that SOCS1 expression was substantially higher in
IFN-g–activated keratinocytes obtained from psoriatic patients
compared with those isolated from healthy donors (p , 0.05) (Fig.
1B). The difference in SOCS1 expression in the two cell strains was
further increased after an additional stimulation of cultures with
IFN-g (Fig. 1C). This effect was evident at 3 h of restimulation
with IFN-g and persisted at least for 18 h after its readministration
(Fig. 1C). These data suggest that cultured psoriatic keratinocytes
activated with IFN-g are more prone than healthy cells to upregulate
SOCS1. SOCS1 expression was also investigated in vivo by immunohistochemistry in healthy skin (n = 3) and psoriatic plaques (n = 3).
Biopsies of psoriatic skin included LS, Pre-LS, and NLS (3-cm distant) zones of newly developed plaque lesions. As shown in Fig. 1D,
SOCS1 staining was intense throughout the epidermis of LS psoriasis, with a pre-eminent localization in the basal and spinosum layers
(Fig. 1Di). SOCS1 accumulation mainly resided in the cytoplasm of
the majority SOCS1+ cells, but it was also present in the nucleus of
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Total proteins were prepared by solubilizing cells in RIPA buffer (1% NP-40,
0.5% sodium dehoxycholate, and 0.1% SDS in PBS containing a mixture of
protease and phosphatase inhibitors). Alternatively, cytosolic and nuclear
extracts were prepared from cells grown in 75-mm flasks, which were lysed
with 1 ml cold Buffer A (20 mM HEPES [pH 7.9], 10 mM KCl, 1 mM
EDTA, 1 mM EGTA, 1.5 mM MgCl2, 0.2% NP-40, and 1 mM DTT plus
protease and phosphatase inhibitors). After centrifugation, the supernatants
containing cytoplasms were collected, whereas the pellets containing nuclei
were resuspended in 0.4 ml cold Buffer B (20 mM HEPES [pH 7.9], 0.35 M
NaCl, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1.5 mM MgCl2, 10%
glycerol, and 1 mM DTT plus protease and phosphatase inhibitors). After
incubation at 4˚C for 30 min, the suspensions were centrifuged at 14,000
rpm for 10 min, and the supernatants collected and diluted 5-fold in Buffer
C (20 mM HEPES [pH 7.9], 60 mM NaCl, 10 mM KCl, 1 mM EDTA, 1
mM EGTA, 1.5 mM MgCl2, 5% glycerol, 0.05% NP-40, and 1 mM DTT
plus protease and phosphatase inhibitors). The resulting samples were aliquoted and frozen at –80˚C. Total, cytosolic, or nuclear proteins were subjected to SDS-PAGE, and transferred to polyvinylidene difluoride (PVDF)
membranes (Amersham Pharmacia Biotech, Buckinghamshire, U.K.). The
latter were blocked and probed with various primary Abs diluted in PBS
containing 5% nonfat dried milk or 3% BSA. For immunoprecipitation, 500
mg protein extracts were incubated with protein A-Agarose beads (Amersham Pharmacia Biotech) and the specific Abs. Proteins eluted from the
beads were resolved on 8% SDS-PAGE, transferred to PVDF filters, and
probed with primary Abs. Western blotting and immunoprecipitation filters
were developed using the ECL-plus detection system (Amersham Pharmacia Biotech) or, otherwise, the SuperSignal West Femto kit (Pierce, Rockford, IL). The Abs used for the study were as follows: anti-KLF3 (N-20;
Santa Cruz Biotechnology, Santa Cruz, CA), anti-KLF4 (H-180; Santa Cruz
Biotechnology), anti-KLF10 (H-190; Santa Cruz Biotechnology), anti–
IRF-1 (M-20; Santa Cruz Biotechnology), anti-Sp1 (PEP-2; Santa Cruz
Biotechnology), anti–GFI-1b (B7; Santa Cruz Biotechnology), anti–bactin (C-11; Santa Cruz Biotechnology), anti–a-tubulin (B7; Santa Cruz
Biotechnology), and antilamin B (M-20; Santa Cruz Biotechnology). Anti–
phospho-Sp1 (Thr 453) and anti-SOCS1 Abs were provided by Abcam
(Cambridge, U.K.) and MBL, respectively. Anti-Flag M2 Ab was purchased
from Millipore (Billerica, MA).
2469
2470
SOCS1 TRANSCRIPTION IN HEALTHY AND PSORIATIC KERATINOCYTES
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FIGURE 1. IFN-g–induced SOCS1 is higher in psoriatic compared with healthy keratinocytes and it preferentially accumulates in the cytoplasm of IFNg–activated keratinocytes during psoriasis plaque development. A, SOCS1 mRNA levels were detected by real-time PCR analysis in cultured healthy
keratinocytes unstimulated or treated with 200 U/ml IFN-g for different time periods, and B, in a panel of healthy keratinocyte (Healthy KC, N) (n = 6) and
psoriatic keratinocyte (PS KC, O) (n = 6) strains left untreated or 3 h stimulated with IFN-g. C, SOCS1 mRNA levels were detected in healthy and psoriatic
keratinocyte cultures stimulated with IFN-g for 24 h and then restimulated (restim) or not with IFN-g for the indicated time periods. SOCS1 mRNA levels
were determined after normalization with b-actin mRNA values. Error bars in A and C are SD of mean values of three different donors. Horizontal lines in B
indicate mean values for each experimental group. Differences in SOCS1 mRNA expression between IFN-g–treated healthy and psoriatic keratinocytes in B
The Journal of Immunology
Identification of the sequences regulating the human socs1
promoter activity in human keratinocytes in basal conditions
and on IFN-g stimulation
The enhanced IFN-g–dependent SOCS1 expression observed in
psoriatic keratinocytes could be the result of an altered SOCS1
transcriptional regulation in these cells. Because of the lacking of
information on the molecular bases of SOCS1 gene expression in
primary cultures of human keratinocytes in response to IFN-g, we
performed a detailed functional analysis of socs1 promoter. To this
end, we first identified the regions crucial for the transcription of
socs1 gene in keratinocytes activated by IFN-g. The computational predictive analysis of the 1950-bp region upstream to
human socs1 gene revealed the existence of sequences putatively
able to bind: 1) GFI-1/GFI-1b transcription factors (two putative
binding sites in position 21110/21102 and 2800/2786); 2) members of KLF family (three potential KLF-binding sites, in position
2515/2509, 2433/2427, and 2405/2399); 3) IRF-1 (located between 2139 and 2125 bp); and 4) Sp1 (three potential binding sites
in positions 2111/2105, 295/289, and 270/264) (Fig. 2A). Putative sequences for STAT-like and AP4 transcription factors were
also found in the 21950 socs1 promoter region (in positions 2695/
2611 and 2416/2410, respectively), as previously reported (Fig.
2A) (17). The functional characterization of these predicted sequences was performed by transfecting primary cultures of human keratinocytes with plasmids containing 59-serial deletions of the socs1
promoter placed upstream a luciferase reporter gene. Then, the
cultures were treated or not with IFN-g for 8 h. As shown in Fig.
2B, the basal and the IFN-g–regulated activity of socs1 promoter
did not vary when deletions of the region comprised between
21950 and 2819 were performed. In contrast, the 2540 construct
(lacking of GFI-1/GFI-1b binding sequence in position 2800/
2786) exhibited a higher responsiveness to IFN-g (3-fold increase
respect to untreated cells) when compared with 2819 plasmid (2.0fold versus 3-fold responsiveness of 2819 and 2540 plasmid, respectively) (p , 0.05), suggesting a possible involvement of a transcriptional repressor in the 2819/2540 region regulating socs1
promoter activity after IFN-g stimulation. A further deletion of
the sequence comprised between 2540 and 2248 (containing the
three putative KLF binding sites) determined a significant increase
of the basal transcriptional activity of the promoter in transfected
cells (p , 0.05) (Fig. 2B), allowing to identify a region likely able
to bind constitutively a transcriptional repressor. Also, the deletion
of nucleotides from 2173 to 2112 (containing the predicted
IRF-1 binding sequence) resulted in a significant reduction of the
IFN-g–induced luciferase activity, with the 2112 and 2173 constructs showing 1.5- and 3.5-fold increase of luciferase activities,
respectively (Fig. 2B). These data indicated that the 2173/2112
region contained sequence importantly contributing to IFN-g
responsiveness of socs1 promoter. The deletion of the 2112/275
region (containing two putative Sp1 binding sites) determined a quite
total abrogation of luciferase expression (p , 0.001) (Fig. 2B),
suggesting the presence of binding sites for transcription factor(s)
fundamental for the basal socs1 promoter activity. A more detailed
analysis of 2540/2248 region performed using three deletion
constructs, termed 2540 D1 (2430 plasmid), 2540 D2 (2409
plasmid), and 2540 D3 (2386 plasmid) (Fig. 2C), revealed that
the deletion of the two nearest KLF-binding sites, designed as S1
(position 2515/2509) and S2 (position 2433/2427), increased
socs1 promoter activity. In contrast, the third KLF-binding site,
designed as S3 (position 2405/2399), and the putative AP4 binding sequence (position 2416/2410) did not influence socs1 promoter functionality. Thus, this set of experiments permitted us to
identify regions of socs1 promoter involved in the repression and
activation of socs1 gene expression in human keratinocytes.
Expression pattern of KLF, IRF-1, Sp1, and GFI-1b
transcription factors in resting and IFN-g–activated keratinocytes
The identified regions involved in the regulation of socs1 promoter
contained sequences putatively binding repressors, namely, GFI-1/
GFI-1b and KLF family proteins and the transcriptional activators
IRF-1 and Sp1, as assessed by computational analysis (Fig. 2A).
Before studying the function of these transcription factors, we
evaluated their expression pattern and kinetics in untreated and
IFN-g–activated keratinocytes. Regarding to KLF family, it comprises at least 14 members sharing structural and functional sim-
were significant (pp , 0.05). D, Immunohistochemistry for SOCS1 and CD3 (both stained in red) was performed on paraffin-embedded sections from
biopsies of psoriatic skin (n = 3) including LS, Pre-LS, and NLS (3-cm distant) zones of evolving plaques (i–iii and v–vii). Healthy skin was also analyzed
(iv and viii). Immunoreactivity was revealed by using avidin-biotin-peroxidase complexes and 3-amino-9-ethylcarbazole as substrate. Sections were
counterstained with Mayer’s H&E. Representative stainings of three psoriasis are shown. E, Immunohistochemistry and Western blotting permitted to
localize SOCS1 in the cytoplasm of epidermal keratinocytes of LS psoriatic skin (i) and of 6 h IFN-g–treated keratinocyte cultures, and in the nucleus of
epidermal keratinocytes of NLS psoriasis (ii) and untreated keratinocytes. Western blotting were performed on lysates obtained from three healthy donors
(P1, P2, and P3). Nuclear and cytosol fractionation of lysates was evaluated by probing blots with anti-lamin B and anti-tubulin Abs, respectively. LS,
lesional; NLS, not lesional; Pre-LS, proximal to lesion.
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scattered epidermal keratinocytes (Fig. 1Di, 1Ei). During the transition from LS to NLS area of the same skin biopsy, we observed the
reduction of SOCS1 immunoreactivity in the epidermal compartment as well as a different distribution of SOCS1 staining, which
was mainly nuclear and confined to the basal layer of the epidermis
(Fig. 1Dii, 1Eii). The transition of SOCS1 staining from cytoplasm to
nucleus of keratinocytes started to be evident in the Pre-LS zone of
psoriatic plaques (Fig. 1Dii). In addition, the pattern of expression of
SOCS1 observed in NLS psoriasis was identical to that observed in
healthy skin (Fig. 1Div). Interestingly, the more intense and cytoplasmic SOCS1 expression in psoriatic LS skin correlated with the presence in the upper dermis of infiltrating CD3+ T lymphocytes (Fig.
1Dv, 1Dvi), mainly represented in psoriatic lesions by IFN-g–
producing Th1 and T cytotoxic cells (5). In contrast, the nuclear
SOCS1 expression confined to basal layer keratinocytes of NLS
skin was concomitant to a reduced number of CD3+ T cells in the
dermis (Fig. 1Dvii). These data suggested a possible involvement of
IFN-g not only in the induction of SOCS1 in keratinocytes but also in
the regulation of its cellular distribution, which was nuclear or
cytoplasmic in absence or presence of T cells in the upper dermis,
respectively (Fig. 1D). And, in fact, SOCS1 immunoprecipitation
experiments performed on nuclear and cytosolic fractions of
untreated and IFN-g–treated cultured keratinocytes demonstrated
that SOCS1 protein prevalently resided in the nuclear compartment
when cells were resting but it accumulated in the cytoplasm after
IFN-g activation (Fig. 1E, lower panels). Thus, keratinocytes of
psoriatic patients can greatly express SOCS1 in response to IFN-g,
at levels higher than those exhibited by healthy cells. Because IFN-g
is also able to induce SOCS1 accumulation in cytoplasms of cultured
keratinocytes, it could be responsible for the different SOCS1
distribution observed in the epidermis during psoriatic plaque
formation.
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SOCS1 TRANSCRIPTION IN HEALTHY AND PSORIATIC KERATINOCYTES
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FIGURE 2. Functional analysis of socs1 promoter in human keratinocytes. A, Schematic representation of socs1 human promoter showing putative binding
sites for GFI-1/GFI-1b, STAT (GAS sequences), KLF, IRF-1, Sp1, and AP4 transcription factors, calculated by using the MatInspector software. B, 59-serial
deletions of human socs1 promoter were prepared as reported in Materials and Methods, and deleted promoters were transfected into primary cultures of
healthy keratinocytes. After 18 h, transfected cells were stimulated or not with IFN-g prior to assay Firefly luciferase activity on cellular extracts. In parallel,
Renilla luciferase activity was measured to normalize the transfection efficiency. C, 59-serial deletions of the 2540/2370 region were prepared as described
in Materials and Methods, and used to transfect keratinocyte cultures. Data shown in B and C were obtained from three independent experiments and are
expressed as mean 6 SD of Firefly luciferase values normalized to Renilla luciferase and micrograms of proteins. In B, pp , 0.05; ppp , 0.001. In C, pp , 0.05.
ilarities (21, 22), and so far, few data on KLF expression in human
epidermal keratinocytes are available. RT-PCR analysis showed
that KLF2 (lung KLF), KLF3 (basic KLF), KLF4, KLF5, KLF7
(ubiquitous KLF), KLF10 (TGF-b early gene 1), KLF11 (TGF-b
early gene 2), and KLF12 (AP2 repressor) are all expressed in
both resting and IFN-g–activated keratinocytes (Fig. 3A). In contrast,
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2473
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FIGURE 3. Expression profile and kinetics of KLF, IRF-1, SP1, and GFI-1b transcription factors in human keratinocytes. A, The expression pattern of
KLF family members was evaluated by RT-PCR analysis on total RNA prepared from untreated and 3 h IFN-g–treated keratinocyte cultures. B, KLF3,
KLF4, and KLF10 expression was detected in keratinocyte cultures treated or not with IFN-g for the indicated time periods at both mRNA (upper panels)
and protein levels by real-time PCR and Western blotting, respectively. C and D, IRF-1 and Sp1 expression was analyzed at both mRNA (upper panels) and
protein levels in untreated or IFN-g–stimulated keratinocyte cultures. E, (upper panel) GFI-1b, but not GFI-1 mRNA, was detected in IFN-g–treated
keratinocytes by real-time PCR. GFI-1b was revealed mainly in IFN-g–treated cells by immunoprecipitation. KLF3-4-10, IRF-1, Sp1, and GFI-1b proteins
were detected mainly in the nuclear fraction of cells. Similar results were obtained in three independent experiments.
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SOCS1 TRANSCRIPTION IN HEALTHY AND PSORIATIC KERATINOCYTES
KLF1 (erythroid KLF), KLF9 (basic transcription element basic 1),
KLF13, and KLF16 mRNA were only slightly or at all detected in
keratinocytes, even though the cells were treated with IFN-g (Fig.
3A). Within KLF family, two subgroups have been defined on the
basis of their sequence and function (22). Therefore, we studied representative members for each group, namely, KLF10 (first subgroup)
and KLF3 and KLF4 [members of second subgroup differing for
some structural features (21)]. As shown in Fig. 3B, IFN-g up-
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FIGURE 4. KLF4 and GFI-1b act as transcriptional repressors of SOCS1 expression. A, Keratinocyte cultures were transfected with siRNA specific for
KLF3, KLF4, KLF10, or irrelevant siRNA (NC), as described in Materials and Methods. After 36 h transfection, cells were transfected again with –540
plasmid, and subsequently stimulated (black bars) or not (white bars) with IFN-g for 8 h. B, Untreated (white bars), 3 h IFN-g–treated (gray bars), and 18 h
IFN-g–treated (black bars) keratinocytes transfected with KLF3, KLF4, KLF10, or control siRNA were analyzed for endogenous SOCS1 mRNA content by
real-time PCR. C, The 2540 plasmid and increasing amounts of KLF4 (left graph) or KLF3 (right graph) expression plasmids were transiently transfected in
untreated (white bars) or IFN-g–treated (black bars) keratinocyte cultures. D, Keratinocytes overexpressing KLF3 or KLF4 plasmids were analyzed for
endogenous SOCS1 mRNA expression by real-time PCR. The effect of GFI-1b overexpression on 2819 plasmid was evaluated in keratinocyte cultures
treated (black bars) or not with IFN-g (white bars). E, Transfection were also performed with the mutant Zn/GFI-1b plasmid (right graph). Knockdown of
endogenous KLF3, KLF4, and KLF10, shown in A and B, as well as the ectopic expression of KLF3, KLF4, GFI-1b, and Zn/GFI-1b, shown in C, D, and E were
evaluated by Western blotting analyses. All data were obtained from three independent experiments Data shown in A, C, and E are expressed as mean 6 SD of
Firefly luciferase values normalized to Renilla luciferase and micrograms of total proteins. SOCS1 mRNA levels shown in B and D were normalized to b-actin
mRNA. In all panels, pp , 0.001.
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2475
regulated KLF3, KLF4, and KLF10 at both mRNA and protein level.
KLF3, KLF4, and KLF10 were mainly localized in the nuclear fraction, with the exception of KLF4, which was detected also in the
cytoplasm (Fig. 3B). Other than KLF proteins, IFN-g also induced
IRF-1 mRNA and protein (Fig. 3C), and upregulated the phosphorylation in threonin 453 of Sp1 (p-Sp1) without affecting its mRNA and
total protein expression (Fig. 3D). Finally, IFN-g upregulated GFI-1b,
but not GFI-1, mRNA, and protein (Fig. 3E). IRF-1, p-Sp1, and
GFI-1b were mostly confined to the nuclear fractions.
KLF4 and GFI-1b function as repressors, whereas IRF-1 and
Sp1 as activators of SOCS1 transcription
FIGURE 5. IRF-1 and Sp1 are determinant for the IFN-g–induced and
basal SOCS1 expression, respectively. A, Keratinocyte cultures were
treated with irrelevant siRNA or with siRNA specific for IRF-1, Sp1, or
both, as described in Materials and Methods. Cells were then transiently
transfected with –540 plasmid, and stimulated or not with IFN-g for 8 h.
The effects of depletion of Sp1 and IRF-1 or both on socs1 promoter were
evaluated by assaying luciferase activities. Data are expressed as mean 6
SD of Firefly luciferase values normalized to Renilla luciferase and micrograms of total proteins. pp , 0.001; ppp , 0.05. B, Endogenous SOCS1
mRNA expression in IRF-1– and Sp1-depleted keratinocytes, and in control cells, was determined by real-time PCR. SOCS1 mRNA levels were
normalized to b-actin mRNA. pp , 0.001; ppp , 0.05. Endogenous
knockdown of IRF-1 and Sp1 was evaluated by Western blotting analysis.
down determined a significant reduction of the basal socs1 promoter
activity without affecting the fold induction of socs1 promoterdriven luciferase in IFN-g–treated over untreated samples (Fig.
5A). Simultaneous depletion of both IRF-1 and Sp1 in keratinocytes
abrogated the global socs1 promoter activity (Fig. 5A). Consistently,
IRF-1–silenced keratinocytes could not upregulate SOCS1 mRNA
expression on IFN-g stimulation (Fig. 5B), confirming the fundamental role of IRF-1 in mediating the IFN-g–induced expression of
SOCS1. The important role of Sp1 in driving the basal socs1
promoter activity was finally confirmed in Sp1-depleted keratinocytes, which showed only very low levels of SOCS1 mRNA (Fig.
5B). As a whole, these findings identify KLF4 and GFI-1b as
repressors and IRF-1 together with Sp1 as activators of SOCS1
mRNA expression.
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We next investigated the role of KLF3, KLF4, KLF10, GFI-1b,
IRF-1, and Sp1 in regulating socs1 gene expression in resting
and IFN-g–activated keratinocytes. To this end, we transfected
keratinocyte cultures with siRNA specific for these transcription
factors, and evaluated the effect of their depletion on the activity
of ectopic socs1 promoter (2540 plasmid, Fig. 2B) as well as on
endogenous SOCS1 mRNA expression. Concerning KLF family
members, KLF4-depleted unstimulated keratinocytes showed a significant increase of 2540 plasmid activity compared with cells
transfected with irrelevant siRNA (Fig. 4A, p , 0.001). In parallel,
inhibition of KLF4 expression did not influence socs1 promoter
responsiveness to IFN-g after 8 h stimulation. In contrast, siRNAmediated knockdown of KLF3 or KLF10 did not alter the 2540
promoter activity nor expression of endogenous SOCS1 mRNA
in both resting and IFN-g–treated keratinocytes (Fig. 4A, 4B).
Consistently with data obtained using ectopic socs1 promoter,
KLF4 knockdown determined a significant increase of endogenous SOCS1 mRNA in untreated cultures (p , 0.001 versus
keratinocytes transfected with irrelevant siRNA). Of note, KLF4
silencing did not influence SOCS1 expression at 3 h of IFN-g
stimulation but it determined an increment (4-fold induction compared with nanocurcumin (NC)-treated cells) of SOCS1 mRNA at
18 h, time point at which SOCS1 mRNA expression substantially
decrease in NC-treated cells (Fig. 4B). These data suggest that
KLF4, but not of KLF3 and KLF10, represses not only the basal
expression of SOCS1 but also its induction after 18 h of IFN-g
stimulation. The function of KLF4 as repressor of SOCS1 was
further evaluated by overexpressing KLF4 in keratinocytes
cultures and analyzing the activity of 2540 socs1 promoter
(2540 plasmid, Fig. 2B) as well as endogenous SOCS1 mRNA
levels. As shown in Fig. 4C, socs1 promoter activity was strongly
inhibited in keratinocytes transfected with KLF4 plasmid in both
untreated (p , 0.001 versus mock-transfected cells) and IFN-g–
treated (p , 0.001) cells. In contrast, the basal and IFN-g–
induced socs1 promoter activity was unaffected or only slightly
decreased by KLF3 plasmid overexpression (Fig. 4C), confirming
the specific effect of KLF4 in modulating SOCS1 transcription. Accordingly, KLF4, but not KLF3 overexpression, robustly reduced
the endogenous SOCS1 mRNA expression in both untreated and
IFN-g–treated cells (Fig. 4D). GFI-1b overexpression also resulted
in a reduction of socs1 promoter activity (2819 plasmid), but only
in keratinoytes stimulated with IFN-g (Fig. 4E). As expected,
keratinocytes transfected with the mutated form of GFI-1b plasmid
(pFLAG-Zn/GFI-1b, depleted of the SNAG repression domain)
showed unaltered 2819 socs1 promoter activity (Fig. 4E).
We next studied the function of IRF-1 and Sp1 transcription factors, whose putative binding sequences were found at 2139/2125
and 2111/264 positions of socs1 promoter, respectively. IRF-1
depletion in keratinocytes by mRNA silencing resulted in a marked
drop of socs1 promoter activity in response to IFN-g but not in
resting conditions (Fig. 5A, p , 0.001). In contrast, Sp1 knock-
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SOCS1 TRANSCRIPTION IN HEALTHY AND PSORIATIC KERATINOCYTES
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FIGURE 6. Binding of KLF4, IRF-1, Sp1, and GFI-1b binding to socs1 promoter in basal condition and on IFN-g stimulation. A, Binding of KLF3 and
KLF4 to biotin-labeled KLF(1), KLF(2), and KLF(3) oligonucleotides was analyzed in oligonucleotide pull-down assays (left panel), following a procedure
described in Materials and Methods. Oligonucleotide pull-down assay permitted us to demonstrate that KLF(2) site was specifically recognized by KLF4 in
untreated and 18 h IFN-g–treated cells (left panel). KLF4 binding to socs1 promoter was confirmed by ChIP experiments performed on untreated or IFN-g–
treated keratinocyte cultures (right panel). B, IRF-1 binding to putative IRF-1 sequence contained in socs1 promoter (2139/2125 region) was verified by
EMSA experiments (left panel), performed with nuclear extracts from keratinocytes untreated (lane 1) or treated with IFN-g for the indicated time periods
(lanes 2–6). IRF-1 complex (C1) disappeared in the presence of an anti–IRF-1 Ab (lane 9) or in the presence of a 103 or 503 molar excess of cold wt oligo
(lanes 11 and 12). IRF-1 binding to endogenous socs1 promoter was assessed by ChIP performed on untreated or IFN-g–treated keratinocyte cultures (right
The Journal of Immunology
KLF4 binding to socs1 promoter decreases after 3 h of IFN-g
stimulation but returns high at 18 h
IRF-1, Sp1, and GFI-1b binding to socs1 promoter occurs with
high-affinity on IFN-g treatment
The deletion of 2173/2112 region containing a putative IRF-1site as well as the silencing of endogenous IRF-1 strongly reduced
the inducibility of socs1 promoter by IFN-g in keratinocytes (Figs.
2B, 5A, 5B). Therefore, we next investigated on the ability of
endogenous IRF-1 to bind to socs1 promoter. To this end, EMSA
experiments were performed by incubating the IRF-1 binding sequence contained in the 2139/2125 region of socs1 promoter with
keratinocyte nuclear extracts collected at different time points after
IFN-g treatment. As shown in Fig. 6B, a gel-retarded DNA-protein
complex (C1) slightly detected in unstimulated cells, could be induced after 1 h of IFN-g stimulation (lane 2). This complex peaked
at 3–6 h of IFN-g treatment (Fig. 6C, lanes 3 and 4) and contained
IRF-1, as assessed by performing supershift assays with an anti–
IRF-1 Ab (Fig. 6C, lane 9) and competition experiments with a molar excess of wt or mutated unlabeled probes (Fig. 6C, lanes 11–14).
Consistently, IRF-1 was able to bind to endogenous socs1 promoter
after IFN-g treatment, as demonstrated by ChIP (Fig. 6B). Through
this analysis, we found that IRF-1 was not basally bound to socs1
promoter, but it was engaged after 3 h of IFN-g stimulation and
quite totally released at 18 h. (Fig. 6B). We next investigated on the
capacity of Sp1 transcription factor to bind to 2112/275 region of
socs1 promoter. EMSA experiments demonstrated that the probe
containing putative Sp1 sites was able to form two complexes
(C2 and C3) in untreated cultures (Fig. 6C, lane 1). The intensities
of these complexes increased after IFN-g treatment reaching a peak
at 6 h and decreasing thereafter (Fig. 6C, lanes 2–5). C2 and C3
complex formation was impaired in the presence of an anti-Sp1, but
not when incubated with an irrelevant Ab (Fig. 6C, lanes 7 and 9),
and it was inhibited by a 503 molar excess of unlabeled wt competitor (Fig. 6C, lane 8). The interaction kinetics of endogenous Sp1
to the 2112/275 region of socs1 promoter was also assessed by
ChIP experiments, which confirmed the upregulation of Sp1 binding to socs1 promoter at 3 h of IFN-g stimulation (Fig. 6C). Finally,
the occurrence of GFI-1b binding to the predicted sites in the 2800/
2786 region of socs1 promoter was evaluated by EMSA experiments by using the entire 2819/2540 region or an oligonucleotide corresponding to GFI-1b binding sequence. As shown in Fig. 6D,
2819/2540 region of socs1 promoter formed a complex with
nuclear extracts only after IFN-g stimulation, with a peak of interaction at 1–3 h of treatment (Fig. 6D, lanes 1–5). This binding
was specific, because an excess of wt, but not mutated competitor
inhibited the complex formation (Fig. 6D, lanes 6 and 7). Similar
results were obtained when the oligonucleotide containing GFI-1b
binding site was used as probe. In fact, the formation of GFI1b/oligo complex reached a peak at 3 h of IFN-g treatment and
decreased thereafter (Fig. 6D, lanes 8–12). The complex contained GFI-1b because its formation was impaired when an
anti–GFI-1b Ab was coincubated with nuclear extracts (Fig. 6D,
lane 14). ChIP experiments further confirmed the binding of
GFI-1b to endogenous socs1 promoter after 3 h of IFN-g stimulation. (Fig. 6D). Consistently with luciferase experiments showing that deletion of 21220/2819 region (containing another
putative GFI-1/GFI-1b) did not influence socs1 promoter activity
(Fig. 2B), no binding specific for GFI-1b was detected when this
region was used as probe in EMSA experiments (data not shown).
The enhanced IFN-g–dependent socs1 promoter activity in
psoriatic keratinocytes correlates with a reduced expression of
KLF4 and GFI-1b transcription factors
In this paper, we showed that psoriatic keratinocytes have a more
prominent ability to express SOCS1 in response to IFN-g compared
with healthy cells. This attitude is more evident when psoriatic
cells are restimulated with IFN-g (Fig. 1B, 1C). To investigate on
the mechanisms responsible for this peculiar expression of SOCS1
in psoriatic cells, we compared socs1 promoter activity as well as
expression levels of KLF4, GFI-1b, IRF-1, and Sp1 transcription
factors in a panel of healthy and psoriatic cell strains activated with
IFN-g. socs1 promoter activity was studied in parallel in healthy
and diseased keratinocytes transfected with the 2819 plasmid. Transfected cultures were activated with IFN-g, and, then, assayed for
luciferase levels. As shown in Fig. 7A, psoriatic keratinocytes
exhibited an enhanced socs1 promoter activity in response to IFN-g
compared with healthy cells (p , 0.05). This difference substantially
increased when cultures were subjected to an additional stimulation
with IFN-g (psoriatic versus healthy keratinocytes, p , 0.001). This
latter finding parallels data showed in Fig. 1C. Next, we compared the
panel). C, EMSA (left panel) also revealed the binding of Sp1 to putative Sp1 sequences contained in socs1 promoter (2111/264 region). Sp1 formed two
complexes (C2 and C3) in both basal and IFN-g–stimulated conditions (lanes 1–5), which were inhibited by an anti-Sp1 Ab (lane 7) or by a 503 molar
excess of cold wt oligo (lane 8). ChIP experiments showing Sp1 binding to endogenous socs1 promoter are shown in the right panel of C. D, EMSA (left
panel) showed that GFI-b bound to 2819/2540 region of socs1 promoter (lanes 1–5) and to putative GFI-1b sequence contained in socs1 promoter (2800/
2786 region) (lanes 8–12) only after IFN-g treatment of cultures. GFI-1b binding (C4) was inhibited by a 503 molar excess of cold wt oligo (lane 6) and
an anti–GFI-1b (lane 14). GFI-1b binding to endogenous socs1 promoter was assessed by ChIP performed on untreated or IFN-g–treated keratinocyte
cultures (right panel). Representative graphs of real-time PCR of ChIP products and relative amplicons visualized on agarose gels are shown. ChIP results
in graphs are expressed as fold enrichment relative to levels of background signal (IgG).
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We previously showed 2540/2430 region of socs1 promoter
(containing putative KLF binding sites) negatively regulates
SOCS1 promoter activity (Fig. 2B, 2C), and KLF4 functions as
repressor of SOCS1 expression (Fig. 4A–D). To determine
whether KLF4 is physically capable to bind the putative S1 and
S2 sites in 2540/2430 socs1 promoter region, we first performed
pull-down experiments using biotin-labeled oligonucleotides, corresponding to S1 [KLF(1)], S2 [KLF(2)], or S3 [KLF(3) located in
2405/2399 position] sites, which were incubated with nuclear
extracts of untreated keratinocytes. Western blotting analysis of
proteins pulled-down with oligonucleotides revealed that endogenous KLF4, but not KLF3, efficiently interacted with KLF(2)
sequence, whereas it exhibited a low-binding affinity toward
KLF(1) and KLF(3) probes (Fig. 6A, upper pull-down assays).
To characterize the kinetics of KLF4 binding to KLF (2), we incubated the biotin-labeled KLF(2) oligonucleotide with nuclear
lysates from keratinocytes treated with IFN-g for 3 h or 18 h.
KLF4 binding to KLF(2) sequence was constitutive in resting
keratinocytes, it decreased after 3 h of IFN-g treatment and
returned high at 18 h (Fig. 6A, lower pull-down assays). As expected, no KLF4 binding was detected when a mutant form of KLF
(2) oligonucleotide was used in the assay (Fig. 6A). KLF4 binding
to socs1 promoter was also evaluated in resting and IFN-g–treated
keratinocytes by ChIP analysis. As shown in Fig. 6A, KLF4 basally
but not after 3 h of IFN-g stimulation, bound to the promoter region
containing KLF binding sites. Similarly to what observed in pulldown experiments, KLF4 affinity for socs1 promoter returned high
at 18 h of IFN-g treatment (Fig. 6A).
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SOCS1 TRANSCRIPTION IN HEALTHY AND PSORIATIC KERATINOCYTES
protein expression of KLF-4, GFI-1b, IRF-1, and Sp1 between psoriatic (n = 6) and healthy (n = 6) keratinocytes after 3 h of IFN-g stimulation. Fig. 7B shows that the levels of KLF4 and GFI-1b transcriptional repressors in psoriatic keratinocytes are considerably lower than
those detected in healthy cells. In contrast, the amounts of both
IRF-1 and p-Sp1 activators are quite similar in psoriatic and healthy
cells (Fig. 7B). As a whole, these data demonstrate that the high SOCS1
mRNA expression observed in psoriatic keratinocytes is the result of
an enhanced socs1 promoter activity, which, in turn, could be dependent on the reduced expression of KLF4 and GFI-1b repressors observed in diseased cells in response to IFN-g.
Discussion
Psoriasis is an inflammatory skin disease pathogenetically driven
by T cells highly expressing type I proinflammatory cytokines (23,
24). Therefore, psoriatic skin lesions are characterized by a prominent presence of IFN-g that preferentially targets resident
epidermal keratinocytes. Because of their altered capacity to
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FIGURE 7. Psoriatic keratinocytes exhibit an higher socs1 promoter
activity in response to IFN-g than healthy cells and a concomitant reduced
expression of KLF4 and GFI-1b repressors. A, Healthy and psoriatic keratinocytes were transiently transfected with the 2819 construct. After
transfection, cultures treated with IFN-g for 24 h, and, then, restimulated
or not with IFN-g for 8 h. Data are expressed as mean 6 SD of Firefly
luciferase values normalized to Renilla luciferase and micrograms of total
proteins. pp , 0.001. B, IRF-1, phospo-Sp1, KLF4, and GFI-1b protein
levels were analyzed by Western blotting in IFN-g–treated keratinocyte
strains prepared from psoriatic (n = 4) (O) and healthy (n = 4) (N) skin.
Bands relative to IRF-1, phospo-Sp1, KLF4, and GFI-1b proteins on
immunoblots were analyzed by densitometry and protein expression was
reported as D.U. pp , 0.001; ppp , 0.05. D.U., densitometric unit.
respond to IFN-g, but also to other cytokines locally released
during psoriasis development, keratinocytes show abnormal hyperproliferation and differentiation, both responsible for the marked
hyperplasia of psoriatic epidermis (25). In addition, in response to
IFN-g, keratinocytes initiate a program of enhanced or de novo
expression of a plethora of inflammatory mediators involved in the
recruitment and local activation of immune cells in psoriatic skin
(26–29). Similar to other cell types, in human keratinocytes IFN-g
signaling triggers a series of molecular cascades initiated by Jak1
and Jak2 proteins, which culminates with the activation of transcription factors, mainly STAT1 and IRF-1, regulating a number of
IFN-g–dependent genes (30). However, human keratinocytes can
counteract the detrimental effects resulting from a prolonged
exposure to IFN-g by inducing the expression of SOCS1 and
SOCS3, which function as endogenous anti-inflammatory and
self-protective molecules (7, 8, 31). In fact, SOCS1 strongly inhibits IFN-g signaling by impeding STAT1 activation and, in turn, the
expression of STAT1-dependent inflammatory genes (7). It is
known SOCS1 expression is strongly induced at transcriptional
level by IFN-g in several cell types, including human keratinocytes,
but to date limited information exist on SOCS1 expression in the
epidermis of diseased skin. In this study, we compared SOCS1
expression in keratinocytes of patients affected by psoriasis with
SOCS1 present in healthy cells, unveiling that IFN-g–activated
psoriatic keratinocytes are more prone than healthy cells to
upregulate SOCS1 mRNA. Indeed, due to their altered genetic
background, psoriatic keratinocytes respond peculiarly and
excessively to IFN-g by expressing aberrant levels of certain
proinflammatory genes, including CCL2, CXCL10 chemokines,
and ICAM-1 adhesion molecule (32). Therefore, it is not surprising
that psoriatic keratinocytes attempt to inhibit IFN-g effect by
potentiating SOCS1 expression. Interestingly, other than SOCS1,
psoriatic keratinocytes showed an enhanced expression of SOCS3,
another member of SOCS family able to attenuate IFN-g signaling
but at lower extent than SOCS1 [(9), data not shown]. The attitude of
psoriatic keratinocytes to upregulate SOCS1 became more evident
after their restimulation with IFN-g, a condition mimicking the
in vivo exposure of the epidermis to cyclic waves of IFN-g, and
emphasizing the high susceptibility of psoriatic cells to this
cytokine. Interestingly, IFN-g not only regulated SOCS1 mRNA
expression but also SOCS1 cellular distribution, which was
nuclear or cytoplasmic in absence or presence of T cell-derived
IFN-g, as revealed in the epidermis of developing psoriatic
plaques as well as in IFN-g–activated keratinocyte cultures. Data
on SOCS1 nuclear localization in resting keratinocytes of
asymptomatic psoriatic skin fit with recent findings demonstrating
that SOCS1 can accumulate into the nucleus of various cell types
due to the presence of a nuclear localization aminoacid sequence
located between its SOCS box and Src homology 2 domain (33, 34).
Nuclear SOCS1 in keratinocytes could have functions totally
different from cytoplasmic SOCS1, including a role in the turnover and/or protein accumulation of some transcription factors
(i.e., STAT1 and Sp1), as demonstrated for other cell types (35).
Aimed at explaining the cause of enhanced SOCS1 induction in
psoriatic keratinocytes, we started to explore the transcriptional
regulatory mechanisms responsible for SOCS1 expression in
healthy IFN-g–activated keratinocytes. The functional analysis of
human socs1 promoter and manipulation of transcription factors
putatively binding to specific sequences of this regulatory region
revealed that Sp1 and IRF-1 were, respectively, responsible for the
basal and IFN-g–induced promoter activity, whereas GFI-1b and
KLF4 acted as repressors. In particular, we demonstrated that deletion of 2112/275 region of socs1 promoter (containing two Sp1
binding sites) as well as depletion of endogenous Sp1 determined
The Journal of Immunology
a quite total abrogation of basal socs1 promoter activity and SOCS1
mRNA expression. In parallel, deletion of IRF-1 binding sequence
in 2173/2112 region or silencing of endogenous IRF-1 mRNA
totally inhibited the IFN-g–dependent activity of socs1 promoter
or SOCS1 mRNA expression, respectively. These data are consistent with findings obtained with mouse socs1 promoter showing the
essential contribution of three GC boxes (located in the 2105/+18
region of mouse socs1 gene) and of the so-called VIRE (variant
IFN-g responsive element, located in the 288/260 region) sequence to promoter activity in resting and IFN-g–treated 3T3
murine fibroblasts, respectively. Data showing that Sp1 was
phosphorylated after IFN-g and that it efficiently bound to human
socs1 promoter after IFN-g treatment, let us to hypothesize
a possible Sp1 involvement in socs1 promoter regulation not only
in basal conditions but also during IFN-g stimulation. It is likely
phosphorylated Sp1 could function as minor coactivactor or assist
IRF-1 recruitment and/or binding to socs1 promoter in response to
IFN-g. Indeed, previous studies showed that IRF-1–mediated
transcriptional activation of target promoters requires the cooperation of adjacent Sp1 elements (36) or the direct or indirect interaction with Sp1, as demonstrated for cycline-dependent kinase 2 and
IL-6 gene promoters (37, 38). Recently, it has been shown an essential role for the transcription factor Sp2 in the regulation of socs1
promoter in IFN-g–treated HaCaT keratinocyte line (14). Although
depletion of endogenous Sp2 greatly reduced SOCS1 induction in
response to IFN-g, no direct interaction of Sp2 with its putative
binding sequence(s) in human socs1 promoter (indeed found in the
first exon of socs1 gene) was demonstrated. To this matter, it is
plausible that Sp2 could have an indirect role in SOCS1 transcriptional regulation in IFN-g–treated HaCaT cells, presumably
regulating expression and/or function of other transcription factors
binding socs1 promoter as well as influencing stability of SOCS1
mRNA.
In this study, we also demonstrated that a member of KLF family,
namely, KLF4, acts as repressor of SOCS1 expression in resting
keratinocytes. Interestingly, on IFN-g treatment, KLF4 binding to
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FIGURE 8. Schematic model of SOCS1
transcriptional regulation in healthy and psoriatic
keratinocytes. A and B, In resting conditions,
thus in absence of IFN-g signaling activation,
healthy (A, scheme I) and psoriatic (B, scheme
I) keratinocytes express only low levels of
SOCS1 mRNA due to a dominant repression of
KLF4 over Sp1 activation of socs1 promoter.
After IFN-g binding to its receptor, SOCS1
mRNA starts to be highly upregulated and
at 3 h reaches a maximum of expression in
healthy cells due to IRF-1 and Sp1 binding as
well as KLF4 detachment from socs1 promoter
(A, scheme II). At this time point of IFN-g
stimulation the activity of socs1 promoter is
also counter-regulated by GFI-1b repressor (A,
scheme II). Psoriatic keratinocytes express higher
levels of SOCS1 mRNA than healthy cells at
3 h of IFN-g stimulation, likely as a result of
their reduced expression of GFI-1b repressor
(B, scheme II). At 18 h, IRF-1 activator, but
also GFI-1b, leave socs1 promoter and KLF4
definitely represses SOCS1 mRNA transcription, thus, contributing to the re-establishment
of SOCS1 homeostatic levels in healthy keratinocytes (A, scheme III). In contrast, at this
time point SOCS1 mRNA levels continue to
be high in psoriatic keratinocytes presumably
as consequence of their very low expression
of KLF4 repressor (B, scheme III).
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SOCS1 TRANSCRIPTION IN HEALTHY AND PSORIATIC KERATINOCYTES
and KLF4 can definitely repress SOCS1 mRNA transcription, thus,
contributing to the re-establishment of SOCS1 homeostatic levels in
healthy keratinocytes (Fig. 8). In contrast, SOCS1 mRNA levels
continue to be high in IFN-g–activated psoriatic keratinocytes
presumably as consequence of their very low expression of KLF4
repressor (Fig. 8).
It is reasonable to assume that SOCS1 upregulation in psoriatic
keratinocytes represents a mechanism through which these cells
protect themselves from an exaggerated stimulation by IFN-g.
Consistent with this hypothesis, mice lacking SOCS1 die of a complex inflammatory disease characterized by a massive T cell, macrophages, and eosinophil infiltration of visceral organs and the skin
(47). However, it cannot be excluded that SOCS1 overexpression in
psoriatic keratinocytes might have side effects in part contributing to
the development of psoriasis. In fact, the capability of SOCS1 to
activate RAS/ERK1/2 pathways as well as to upregulate CXCL8
production in keratinocytes (7, 8) could be strictly correlated with
the epidermal hyperplasia characterizing psoriasis. Nevertheless,
the antiapoptotic function of SOCS1 could further contributes to
psoriatic phenotype (48, 49). The effective role of SOCS1 upregulation in the epidermis could be established by generating transgenic
mice expressing SOCS1 in basal keratinocytes or, alternatively, by
using peptides mimicking its function. Concerning SOCS1 mimetic
molecules, it will be interesting to evaluate the effects of peptides
corresponding to different domains of SOCS1 responsible for the
anti-inflammatory (kinase inhibitory region [KIR]) (9) or prosurvival
(SOCS box) (8) effects of SOCS1. Indeed, SOCS1 peptides mimicking KIR region have been efficiently used to inhibit Jak1/STAT1
activation in IFN-g–treated macrophages and to prevent inflammatory processes in mouse models of allergic encephalomyelitis
(50, 51). Future studies using SOCS1-KIR mimetic peptides in
IFN-g–activated keratinocytes and in experimental models of
psoriasis [i.e., mice engrafted with psoriatic skin or skin organotypic cultures generated with psoriatic keratinocytes and fibroblasts,
(52, 53)] should better define the therapeutic efficacy of SOCS1.
Disclosures
The authors have no financial conflicts of interest.
References
1. Barker, J. N., M. H. Allen, and D. M. MacDonald. 1990. Alterations induced in
normal human skin by in vivo interferon-gamma. Br. J. Dermatol. 122: 451–458.
2. Albanesi, C., C. Scarponi, M. L. Giustizieri, and G. Girolomoni. 2005. Keratinocytes in inflammatory skin diseases. Curr. Drug Targets Inflamm. Allergy 4:
329–334.
3. Garioch, J. J., R. M. Mackie, I. Campbell, and A. Forsyth. 1991. Keratinocyte
expression of intercellular adhesion molecule 1 (ICAM-1) correlated with infiltration of lymphocyte function associated antigen 1 (LFA-1) positive cells in
evolving allergic contact dermatitis reactions. Histopathology 19: 351–354.
4. Wikner, N. E., J. C. Huff, D. A. Norris, S. T. Boyce, M. Cary, M. Kissinger, and
W. L. Weston. 1986. Study of HLA-DR synthesis in cultured human keratinocytes. J. Invest. Dermatol. 87: 559–564.
5. Albanesi, C., C. Scarponi, S. Sebastiani, A. Cavani, M. Federici, S. Sozzani, and
G. Girolomoni. 2001. A cytokine-to-chemokine axis between T lymphocytes and
keratinocytes can favor Th1 cell accumulation in chronic inflammatory skin
diseases. J. Leukoc. Biol. 70: 617–623.
6. Mascia, F., C. Cataisson, T. C. Lee, D. Threadgill, V. Mariani, P. Amerio,
C. Chandrasekhara, G. Souto Adeva, G. Girolomoni, S. H. Yuspa, and S. Pastore. 2010.
EGFR regulates the expression of keratinocyte-derived granulocyte/macrophage colony-stimulating factor in vitro and in vivo. J. Invest. Dermatol. 130: 682–693.
7. Federici, M., M. L. Giustizieri, C. Scarponi, G. Girolomoni, and C. Albanesi.
2002. Impaired IFN-gamma-dependent inflammatory responses in human keratinocytes overexpressing the suppressor of cytokine signaling 1. J. Immunol. 169:
434–442.
8. Madonna, S., C. Scarponi, O. De Pità, and C. Albanesi. 2008. Suppressor of
cytokine signaling 1 inhibits IFN-gamma inflammatory signaling in human keratinocytes by sustaining ERK1/2 activation. FASEB J. 22: 3287–3297.
9. Dimitriou, I. D., L. Clemenza, A. J. Scotter, G. Chen, F. M. Guerra, and
R. Rottapel. 2008. Putting out the fire: coordinated suppression of the innate and
adaptive immune systems by SOCS1 and SOCS3 proteins. Immunol. Rev. 224:
265–283.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
socs1 promoter decreased to return high at 18 h of IFN-g stimulation, when SOCS1 expression was quite totally downregulated
in keratinocytes. These data strongly suggest that KLF4 is involved not only in the repression of SOCS1 in basal conditions
but also in the re-establishment of homeostatic SOCS1 mRNA
levels on IFN-g activation. Although this is the first report on
KLF4 inhibitory function on human socs1 promoter, repression
by KLF4 of gene transcription has been extensively elucidated in
the past. KLF4, in fact, can negatively regulate either directly or
indirectly—by interacting with other transcription factors—the
expression of a number of genes (39–41). Surprisingly, we
found that the kinetics of KLF4 binding to socs1 promoter was
inversely correlated with its expression levels after IFN-g
activation, and KLF4 protein peaked at 3 h of IFN-g when its
binding to socs1 promoter was almost absent. Indeed, our preliminary experiments demonstrated that IFN-g could induce in
human keratinocytes the de novo KLF4 sumoylation (data not
shown), a posttranslational modification known to exert an
inhibitory effect on binding capability of the majority of KLF
proteins (42, 43). Together with KLF4, GFI-1b was also found
to act as repressor and, thus, to inhibit socs1 promoter activity, as
demonstrated by overexpressing ectopically GFI-1b in keratinocytes. However, GFI-1b inhibitory function was quite different
from that of KLF4 because it reduced socs1 promoter activity
only during IFN-g stimulation, as revealed by EMSA and ChIP
experiments together with functional promoter analysis with
deletion mutants. In addition, differently from what observed in
murine fibroblasts, where GFI-1b was highly expressed and acted
as constitutive repressor of socs1 promoter (18), GFI-1b was not
detectable in resting keratinocytes and was expressed only after
IFN-g activation, thus confining its function only in stimulatory
conditions. Therefore, it is likely that GFI-1b can avoid an excessive induction of SOCS1 mRNA in response to IFN-g, and,
together with KLF4, restore the basal levels of SOCS1 in
keratinocytes exposed to IFN-g.
An intriguing result of our study showed that psoriatic keratinocytes expressed only very low levels of GFI-1b and KLF4 repressors
but similar amounts of Sp1 and IRF-1 activators if compared with
healthy cells. This unbalance between Sp1/IRF1 activators and GFI1b/KLF4 repressors could be responsible for the very high socs1
promoter activity and SOCS1 mRNA expression observed in psoriatic keratinocytes undergoing to repeated IFN-g stimulations. Indeed,
KLF4 reduced expression in psoriatic keratinocytes could be a
consequence of their altered differentiation status, which characterized these cells in vivo and is responsible for the aberrant thickening of psoriatic epidermis (44). In fact, it is well-known that KLF4 is
involved in the differentiation processes of skin, and alterations of
its expression can cause skin barrier structural abnormalities and
dysfunctions, as demonstrated in mice lacking or overexpressing
KLF4 (45, 46). Although GFI-1b has been described to control cell
cycle and differentiation processes in myelomonocytic cells, its function in skin cells has never been elucidated.
In conclusion, due to a dominant repression of KLF4 over Sp1
activation of socs1 promoter both healthy and psoriatic keratinocytes express only low levels of SOCS1 mRNA in resting conditions
(Fig. 8). After IFN-g binding to its receptor, SOCS1 mRNA starts to
be upregulated, and rapidly (3 h) reaches a maximum of expression
in healthy cells due to IRF-1 and Sp1 binding as well as KLF4
detachment from socs1 promoter (Fig. 8). The IFN-g–dependent
activity of socs1 promoter is also counter-regulated by GFI-1b repressor (Fig. 8). Psoriatic keratinocytes express in response to IFN-g
higher levels of SOCS1 mRNA compared with healthy cells, likely
as a result of their reduced expression of GFI-1b. At late time points
of IFN-g stimulation (18 h), IRF-1 activator has left socs1 promoter
The Journal of Immunology
33. Koelsche, C., J. Strebovsky, A. Baetz, and A. H. Dalpke. 2009. Structural and
functional analysis of a nuclear localization signal in SOCS1. Mol. Immunol. 46:
2474–2480.
34. Baetz, A., C. Koelsche, J. Strebovsky, K. Heeg, and A. H. Dalpke. 2008.
Identification of a nuclear localization signal in suppressor of cytokine signaling
1. FASEB J. 22: 4296–4305.
35. Look, D. C., M. R. Pelletier, R. M. Tidwell, W. T. Roswit, and M. J. Holtzman.
1995. Stat1 depends on transcriptional synergy with Sp1. J. Biol. Chem. 270:
30264–30267.
36. Liu, J., S. Cao, L. M. Herman, and X. Ma. 2003. Differential regulation of interleukin (IL)-12 p35 and p40 gene expression and interferon (IFN)-gammaprimed IL-12 production by IFN regulatory factor 1. J. Exp. Med. 198: 1265–
1276.
37. Xie, R. L., S. Gupta, A. Miele, D. Shiffman, J. L. Stein, G. S. Stein, and A. J. van
Wijnen. 2003. The tumor suppressor interferon regulatory factor 1 interferes
with SP1 activation to repress the human CDK2 promoter. J. Biol. Chem. 278:
26589–26596.
38. Sancéau, J., T. Kaisho, T. Hirano, and J. Wietzerbin. 1995. Triggering of the
human interleukin-6 gene by interferon-gamma and tumor necrosis factor-alpha
in monocytic cells involves cooperation between interferon regulatory factor1, NF kappa B, and Sp1 transcription factors. J. Biol. Chem. 270: 27920–27931.
39. Zhang, G., H. Zhu, Y. Wang, S. Yang, M. Liu, W. Zhang, L. Quan, J. Bai, Z. Liu,
and N. Xu. 2009. Krüppel-like factor 4 represses transcription of the survivin
gene in esophageal cancer cell lines. Biol. Chem. 390: 463–469.
40. Rowland, B. D., R. Bernards, and D. S. Peeper. 2005. The KLF4 tumour suppressor is a transcriptional repressor of p53 that acts as a context-dependent
oncogene. Nat. Cell Biol. 7: 1074–1082.
41. Chen, Z. Y., J. L. Shie, and C. C. Tseng. 2002. Gut-enriched Kruppel-like factor
represses ornithine decarboxylase gene expression and functions as checkpoint
regulator in colonic cancer cells. J. Biol. Chem. 277: 46831–46839.
42. Wei, H., X. Wang, B. Gan, A. M. Urvalek, Z. K. Melkoumian, J. L. Guan, and
J. Zhao. 2006. Sumoylation delimits KLF8 transcriptional activity associated
with the cell cycle regulation. J. Biol. Chem. 281: 16664–16671.
43. Oishi, Y., I. Manabe, K. Tobe, M. Ohsugi, T. Kubota, K. Fujiu, K. Maemura,
N. Kubota, T. Kadowaki, and R. Nagai. 2008. SUMOylation of Krüppel-like
transcription factor 5 acts as a molecular switch in transcriptional programs of
lipid metabolism involving PPAR-delta. Nat. Med. 14: 656–666.
44. Tschachler, E. 2007. Psoriasis: the epidermal component. Clin. Dermatol. 25:
589–595.
45. Dai, X., and J. A. Segre. 2004. Transcriptional control of epidermal specification
and differentiation. Curr. Opin. Genet. Dev. 14: 485–491.
46. Jaubert, J., J. Cheng, and J. A. Segre. 2003. Ectopic expression of kruppel like
factor 4 (Klf4) accelerates formation of the epidermal permeability barrier.
Development 130: 2767–2777.
47. Metcalf, D., L. Di Rago, S. Mifsud, L. Hartley, and W. S. Alexander. 2000. The
development of fatal myocarditis and polymyositis in mice heterozygous for
IFN-g and lacking the SOCS-1 gene. Proc. Natl. Acad. Sci. USA 97: 9174–9179.
48. Morita, Y., T. Naka, Y. Kawazoe, M. Fujimoto, M. Narazaki, R. Nakagawa,
H. Fukuyama, S. Nagata, and T. Kishimoto. 2000. Signals transducers and
activators of transcription (STAT)-induced STAT inhibitor-1 (SSI1)/suppressor of cytokine signaling-1 (SOCS-1) suppresses tumor necrosis factor
alpha-induced cell death in fibroblasts. Proc. Natl. Acad. Sci. USA 97: 5405–
5410.
49. Qin, J. Z., V. Chaturvedi, M. F. Denning, P. Bacon, J. Panella, D. Choubey, and
B. J. Nickoloff. 2002. Regulation of apoptosis by p53 in UV-irradiated human
epidermis, psoriatic plaques and senescent keratinocytes. Oncogene 21: 2991–
3002.
50. Waiboci, L. W., C. M. Ahmed, M. G. Mujtaba, L. O. Flowers, J. P. Martin,
M. I. Haider, and H. M. Johnson. 2007. Both the suppressor of cytokine signaling
1 (SOCS-1) kinase inhibitory region and SOCS-1 mimetic bind to JAK2 autophosphorylation site: implications for the development of a SOCS-1 antagonist.
J. Immunol. 178: 5058–5068.
51. Mujtaba, M. G., L. O. Flowers, C. B. Patel, R. A. Patel, M. I. Haider, and
H. M. Johnson. 2005. Treatment of mice with the suppressor of cytokine
signaling-1 mimetic peptide, tyrosine kinase inhibitor peptide, prevents development of the acute form of experimental allergic encephalomyelitis and induces
stable remission in the chronic relapsing/remitting form. J. Immunol. 175: 5077–
5086.
52. Nickoloff, B. J. 2000. Characterization of lymphocyte-dependent angiogenesis
using a SCID mouse: human skin model of psoriasis. J. Investig. Dermatol.
Symp. Proc. 5: 67–73.
53. Gangatirkar, P., S. Paquet-Fifield, A. Li, R. Rossi, and P. Kaur. 2007.
Establishment of 3D organotypic cultures using human neonatal epidermal
cells. Nat. Protoc. 2: 178–186.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
10. Croker, B. A., H. Kiu, and S. E. Nicholson. 2008. SOCS regulation of the
JAK/STAT signalling pathway. Semin. Cell Dev. Biol. 19: 414–422.
11. Davey, G. M., W. R. Heath, and R. Starr. 2006. SOCS1: a potent and multifaceted regulator of cytokines and cell-mediated inflammation. Tissue Antigens
67: 1–9.
12. Ilangumaran, S., and R. Rottapel. 2003. Regulation of cytokine receptor signaling by SOCS1. Immunol. Rev. 192: 196–211.
13. Saito, H., Y. Morita, M. Fujimoto, M. Narazaki, T. Naka, and T. Kishimoto.
2000. IFN regulatory factor-1-mediated transcriptional activation of mouse
STAT-induced STAT inhibitor-1 gene promoter by IFN-g. J. Immunol. 164:
5833–5843.
14. Letourneur, M., L. Valentino, J. Travagli-Gross, J. Bertoglio, and J. Pierre. 2009.
Sp2 regulates interferon-g-mediated socs1 gene expression. Mol. Immunol. 46:
2151–2160.
15. Sims, S. H., Y. Cha, M. F. Romine, P. Q. Gao, K. Gottlieb, and A. B. Deisseroth.
1993. A novel interferon-inducible domain: structural and functional analysis of
the human interferon regulatory factor 1 gene promoter. Mol. Cell. Biol. 13: 690–
702.
16. Travagli, J., M. Letourneur, J. Bertoglio, and J. Pierre. 2004. STAT6 and
Ets-1 form a stable complex that modulates SOCS-1 expression by IL-4 in
keratinocytes. J. Biol. Chem. 279: 35182–35192.
17. Hebenstreit, D., P. Luft, A. Schmiedlechner, G. Regl, A. M. Frischauf,
F. Aberger, A. Duschl, and J. Horejs-Hoeck. 2003. IL-4 and IL-13 induce
SOCS-1 gene expression in A549 cells by three functional STAT6-binding
motifs located upstream of the transcription initiation site. J. Immunol. 171:
5901–5907.
18. Jegalian, A. G., and H. Wu. 2002. Regulation of Socs gene expression by the
proto-oncoprotein GFI-1B: two routes for STAT5 target gene induction by erythropoietin. J. Biol. Chem. 277: 2345–2352.
19. Scarponi, C., B. Nardelli, D. W. Lafleur, P. A. Moore, S. Madonna, O. De Pità,
G. Girolomoni, and C. Albanesi. 2006. Analysis of IFN-kappa expression in
pathologic skin conditions: downregulation in psoriasis and atopic dermatitis.
J. Interferon Cytokine Res. 26: 133–140.
20. Chiambaretta, F., F. De Graeve, G. Turet, G. Marceau, P. Gain, B. Dastugue,
D. Rigal, and V. Sapin. 2004. Cell and tissue specific expression of human
Krüppel-like transcription factors in human ocular surface. Mol. Vis. 10: 901–
909.
21. Pearson, R., J. Fleetwood, S. Eaton, M. Crossley, and S. Bao. 2008. Krüppel-like
transcription factors: a functional family. Int. J. Biochem. Cell Biol. 40: 1996–
2001.
22. Kaczynski, J., T. Cook, and R. Urrutia. 2003. Sp1- and Krüppel-like transcription
factors. Genome Biol. 4: 206.
23. Lew, W., A. M. Bowcock, and J. G. Krueger. 2004. Psoriasis vulgaris: cutaneous
lymphoid tissue supports T-cell activation and “Type 1” inflammatory gene
expression. Trends Immunol. 25: 295–305.
24. Nickoloff, B. J., J. Z. Qin, and F. O. Nestle. 2007. Immunopathogenesis of
psoriasis. Clin. Rev. Allergy Immunol. 33: 45–56.
25. Albanesi, C., O. De Pità, and G. Girolomoni. 2007. Resident skin cells in psoriasis: a special look at the pathogenetic functions of keratinocytes. Clin. Dermatol. 25: 581–588.
26. Albanesi, C., A. Cavani, and G. Girolomoni. 1999. IL-17 is produced by nickelspecific T lymphocytes and regulates ICAM-1 expression and chemokine production in human keratinocytes: synergistic or antagonist effects with IFNgamma and TNF-alpha. J. Immunol. 162: 494–502.
27. Albanesi, C., C. Scarponi, S. Sebastiani, A. Cavani, M. Federici, O. De Pità,
P. Puddu, and G. Girolomoni. 2000. IL-4 enhances keratinocyte expression of
CXCR3 agonistic chemokines. J. Immunol. 165: 1395–1402.
28. Zhang, X., X. Chen, H. Song, H. Z. Chen, and B. H. Rovin. 2005. Activation of
the Nrf2/antioxidant response pathway increases IL-8 expression. Eur. J.
Immunol. 35: 3258–3267.
29. Gillitzer, R., and M. Goebeler. 2001. Chemokines in cutaneous wound healing. J.
Leukoc. Biol. 69: 513–521.
30. Platanias, L. C. 2005. Mechanisms of type-I- and type-II-interferon-mediated
signalling. Nat. Rev. Immunol. 5: 375–386.
31. Yamasaki, K., Y. Hanakawa, S. Tokumaru, Y. Shirakata, K. Sayama, T. Hanada,
A. Yoshimura, and K. Hashimoto. 2003. Suppressor of cytokine signaling
1/JAB and suppressor of cytokine signaling 3/cytokine-inducible SH2 containing
protein 3 negatively regulate the signal transducers and activators of transcription signaling pathway in normal human epidermal keratinocytes. J. Invest.
Dermatol. 120: 571–580.
32. Giustizieri, M. L., F. Mascia, A. Frezzolini, O. De Pità, L. M. Chinni,
A. Giannetti, G. Girolomoni, and S. Pastore. 2001. Keratinocytes from patients
with atopic dermatitis and psoriasis show a distinct chemokine production profile
in response to T cell-derived cytokines. J. Allergy Clin. Immunol. 107: 871–877.
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