J C E M
O N L I N E
B r i e f
R e p o r t — E n d o c r i n e
R e s e a r c h
20S-Hydroxyvitamin D3, Noncalcemic Product of
CYP11A1 Action on Vitamin D3, Exhibits Potent
Antifibrogenic Activity in Vivo
Andrzej Slominski, Zorica Janjetovic, Robert C. Tuckey, Minh N. Nguyen
Keka G. Bhattacharya, Jin Wang, Wei Li, Yan Jiao, Weikuan Gu, Monica Brown,
and Arnold E. Postlethwaite
Departments of Pathology (A.S., Z.J.), Medicine (A.S., K.G.B., A.E.P.), Pharmaceutical Sciences (J.W.,
W.L.), Orthopedic Surgery & BME-Campbell Clinic (Y.J., W.G.), and Pediatrics (M.B.), and Division of
Connective Tissue Diseases (A.E.P.), University of Tennessee Health Science Center and Veterans Affairs
Medical Center (A.E.P.), Memphis, Tennessee 38163; and School of Chemistry and Biochemistry (R.C.T.,
M.N.N.), University of Western Australia, Crawley, Western Australia 6009, Australia
Context: There is no effective treatment for systemic sclerosis and related fibrosing diseases. Recently the action of CYP11A1 on vitamin D3 was shown to produce biologically active 20S-hydroxyvitamin D [20(OH)D3] and 20,23(OH)2D3, 20,22(OH)2D3, and 17,20,23(OH)3D3.
Objectives: Because 20(OH)D3 is noncalcemic (nontoxic) in vivo at very high doses, we evaluated
its antifibrogenic activities both in vitro and in vivo. Because it is further metabolized by CYP11A1,
we also tested preclinical utilities of its hydroxyderivatives, especially 20,23(OH)2D3.
Design: Human dermal fibroblasts from scleroderma and normal donors were used to test the
efficiency of hydroxyvitamin D derivatives in inhibiting TGF-␤1-induced collagen and hyaluronan
synthesis and inhibiting cell proliferation. The in vivo activity of 20(OH)D3 was tested using bleomycin-induced sclerosis in C57BL/6 mice.
Results: 20(OH)D3 and 20,23(OH)2D3 inhibited TGF-␤1-induced collagen and hyaluronan synthesis similarly to 1,25(OH)2D3 in cultured human fibroblasts. Also, 20(OH)D3, 20,23(OH)2D3, and 1,25(OH)2D3
suppressed TGF-␤1-induced expression of COL1A2, COL3A1, and hyaluronan synthase-2 mRNA, indicating that they regulate these matrix components at the transcriptional level. 20(OH)D3,
20,23(OH)2D3, 20,22(OH)2D3, and 17,20,23(OH)3D3 inhibited proliferation of dermal fibroblasts with
comparable potency with 1,25(OH)2D3, with 20(OH)D2 being less active and 1␣(OH)D3 being almost
inactive. 20,23(OH)2D3 at 3 ␮g/kg had no effect on serum Ca⫹⫹ or fibroblast growth factor-23 levels and
did not cause any noticeable signs of morbidity. 20(OH)D3 markedly suppressed fibrogenesis in mice
given sc bleomycin as demonstrated by total collagen content and hematoxylin and eosin staining of
skin biopsies.
Conclusions: 20(OH)D3 is an excellent candidate for preclinical studies on scleroderma, with other
CYP11A1-derived products of its metabolism deserving further testing for antibrogenic activity.
(J Clin Endocrinol Metab 98: E298 –E303, 2013)
C
alcitriol [1,25(OH)2D3], aside from regulating calcium metabolism, shows potent immunomodulatory, antiinflammatory, and antifibrogenic properties (1–
3). Systemic sclerosis (SSc) and related fibrosing diseases
are devastating and they shorten the life span of the patient. There are no known effective Food and Drug Ad-
ISSN Print 0021-972X ISSN Online 1945-7197
Printed in U.S.A.
Copyright © 2013 by The Endocrine Society
doi: 10.1210/jc.2012-3074 Received August 13, 2012. Accepted November 27, 2012.
First Published Online January 7, 2013
Abbreviations: CYP11A1, Cytochrome P450scc; EMEM, Eagle’s MEM; EtOH, ethanol; HAS,
hyaluronan synthase; 20(OH)D2, 20-hydroxyvitamin D2; 1(OH)D3, 1␣-hydroxyvitamin D3;
20(OH)D3, 20S-hydroxyvitamin D3; 1,25(OH)2D3, 1␣,25-dihydroxyvitamin D3;
20,23(OH)2D3,20,23-dihydroxyvitamin D3; 17,20,23(OH)3D3, 17,20,23-trihydroxyvitamin D3;
P450scc, P450 side chain cleavage; RT, real time; SSc, systemic sclerosis; VDR, vitamin D
receptor.
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J Clin Endocrinol Metab, February 2013, 98(2):E298 –E303
J Clin Endocrinol Metab, February 2013, 98(2):E298 –E303
ministration-approved antifibrotic treatments for SSc and
related fibrotic diseases (4, 5). Although 1,25(OH)2D3 is
an excellent candidate for treatment of human autoimmune diseases, such as SSc (scleroderma), rheumatoid arthritis, multiple sclerosis, psoriasis, and systemic lupus erythematosus (1, 3–5), its application is severely limited by
its toxic (calcemic) effects (2, 3). Therefore, there is a continuing effort to develop noncalcemic vitamin D analogs
to treat autoimmune diseases (6, 7).
Recently a novel pathway of vitamin D metabolism,
mediated by the action of cytochrome P450 side chain
cleavage (P450scc, CYP11A1) was discovered (Supplemental Scheme 1, published on The Endocrine Society’s
Journals Online web site at http://jcem.endojournals.org),
which produces 20S-hydroxyvitamin D [20(OH)D3 and
20(OH)D2] as the first and main metabolites (8 –10), with
20(OH)D3 being further hydroxylated by CYP11A1
to 20,23(OH)2D3 and 17,20,23(OH)3D3 (9, 11). This
pathway can operate in vivo, with 20(OH)D3 serving as an
endogenous product (12). These hydroxyderivatives of vitamin D3 induce the differentiation program in human
keratinocytes and leukemia cells and inhibit nuclear factor-␬␤ activity (13–16), with 20(OH)D3 being noncalcemic in rats and mice at concentrations as high as 3.0 ␮g/kg
(15) and 30 ␮g/kg (17), respectively.
This lack of in vivo toxicity at very high doses (17) and
antiproliferative and potentially antiinflammatory activities (13, 14, 16), together with the enzymatic origin of
these derivatives (11) defining them as natural products
(12), prompted us to test their in vitro and in vivo antifibrogenic activities.
jcem.endojournals.org
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measured using an ELISA specific for type I collagen from Chondrex Corp (Redmond, Washington) (18).
Fibroblasts from 3 different donors were grown to confluency
in a series of 100-mm-diameter tissue culture dishes in Eagle’s
MEM (EMEM) supplemented with 9% charcoal-stripped fetal
calf serum and then cultured for 24 hours in serum-false EMEM
to synchronize the cells. Medium was then changed to EMEM
containing 1% charcoal-stripped fetal calf serum and ascorbic
acid (50 ␮g/mL). To culture dishes were added 20(OH)D3,
20,23(OH)2D3, and 1,25(OH)2D3 at a final concentration of
10⫺8 or 10⫺10 M or ethanol (EtOH) vehicle at 1:10 000 or 1:1
million dilution to control for final EtOH content of the cultures.
After 2 hours incubation, recombinant human TGF-␤1 (R & D
Systems) dissolved in PBS was added at a final concentration of
10 ng/mL, or PBS alone was added to appropriate cultures. After
an additional 16 hours culture, total RNAs were isolated from
fibroblast monolayers using Trizol reagent (Invitrogen, Carlsbad, California) and purified using an RNA easy cleanup kit
(QIAGEN, Valencia, California). RNAs were quantified using
NanoDrop-2000 (Thermo, Franklin, New Jersey), and the quality of RNA was checked using a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, California). Fifty nanograms of total RNA
were used for Taq Man real-time (RT)-PCR using primers for
human COL1A2, COL3A1, hyaluronan synthase (HAS)-1,
HAS2, and glyceraldehyde-3-phosphate dehydrogenase (Applied Biosystems Life Technologies, Grand Island, New York).
The Taqman RT-PCR reactions were carried out with an ABI
7900 real time PCR system [software: sequence detection system
(Applied Biosystems Inc, Foster City, California)] using Applied
Biosystems’ standard protocol for RT-PCR and PCR. Samples
were normalized to glyceraldehyde-3-phosphate dehydrogenase
mRNA expression levels. Data for hydroxyvitamin D derivative
treated fibroblast cultures are shown as ⌬⌬CT normalized to an
endogenous reference (PBS⫹EtOH) vehicle culture.
Testing of antiproliferative activity on human dermal fibroblasts is described in the Supplemental Data.
Preclinical model of scleroderma
Materials and Methods
The experiments were approved by local Institutional Animal Care
and Use Committee and institutional review board protocols committees. 20(OH)D3/D2 and 20,23(OH)2D3/17,20,23(OH)3D3
were produced enzymatically (9, 11). 1(OH)D3 and 1,25(OH)2D3
were from Sigma Chemical Co. (St Louis, Missouri).
In vitro experiments
Human dermal fibroblasts were isolated from biopsies of a
scleroderma patient and from normal donors and were grown as
described previously (18, 19). The confluent fibroblasts were
preincubated in serum-free media for 24 hours, and vitamin D
hydroxyderivatives (10⫺9 or 10⫺10 M) were added to 3 replicate
wells 2 hours prior to addition of recombinant human TGF-␤1
(R & D Systems, Minneapolis, Minnesota) to a final concentration of 5 ng/mL. To measure total collagen and hyaluronan production, after 48 hours of culture plate wells were pulsed for 24
hours with 1 ␮Ci [3H]proline to assess collagen production (20)
or 12.5 ␮Ci [3H]acetate to assess hyaluronan production (19).
To measure type I collagen protein, supernatants were collected
after 48 hours of treatment and collagen I immunoreactivity was
Female C57BL/6 mice 6 weeks old were purchased from Jackson Labs (Bar Harbor, Maine) and maintained on a regular laboratory chow diet. Groups of mice (5 each) received daily injections of either vehicle (50 ␮L sesame oil, ip, and 100 ␮L saline,
sc); bleomycin (180 ␮g per 100 ␮L bleomycin, sc, and 50 ␮L
sesame oil, ip); or bleomycin ⫹ 20(OH)D3 (180 ␮g per 100 ␮L
bleomycin, sc, and 3 ␮g/kg of 20(OH)D3 per 50 ␮L sesame oil,
ip) for 21 days. The skin was injected sc with bleomycin or vehicle
daily within the same 1.5-cm2 area. On day 22 all mice were
euthanized and skin biopsies of 1 cm circumference were taken
and divided in two. One part was formalin fixed and processed
for histopathological analysis, whereas the second part was frozen. The frozen skin samples were thawed and treated overnight
with pepsin (0.1 mg/mL) and 0.5 M acetic acid at 4°C to remove
terminal nonhelical telopeptides to release the collagen into solution. Total solubilized collagen was measured using a Sircol
collagen assay kit (Biocolor Ltd, Carrickfergus, United Kingdom). The calcemic effects of 20(OH)D3 and 20,23(OH)2D3
were measured as described previously (18). Data are presented
as means ⫾ SEM and were analyzed with a Student’s t test (for
2 groups) or ANOVA using Prism 4.00 (GraphPad, San Diego,
California).
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20(OH)D3 Inhibits Fibrogenic Activity
J Clin Endocrinol Metab, February 2013, 98(2):E298 –E303
Results and Discussion
The profibrotic activities of TGF-␤1
have been implicated in the etiology of
systemic sclerosis and related fibrosing
diseases as well as the bleomycin skin
fibrosis preclinical model of scleroderma (4, 5, 19, 21). Both 20(OH)D3
and 1,25(OH)2D3 at 10⫺10 M concentration significantly inhibited TGF-␤1induced total collagen protein and
hyaluronan production by confluent
cultures of normal human fibroblasts
with comparable efficacy (Figure 1A).
The fibroblasts remained confluent,
maintained normal morphology, and
their viability after treatments was
greater than 90% by trypan blue exclusion. A similar inhibition was seen at
10⫺9 M (not shown). This effect of
20(OH)D3 on collagen production was
further confirmed in dermal fibroblasts
grown from involved skin from a patient with diffuse cutaneous SSc using
an ELISA test, which showed that secreted type I collagen protein production was inhibited (Figure 1B). In additional experiments using another
Figure 1. Vitamin D hydroxyderivatives inhibit total collagen and hyaluronan production
normal dermal fibroblast line, we obstimulated by TGF-␤1. Scleroderma human dermal fibroblast line 08 (A and B) or normal
served inhibitory effects of 20(OH)D3,
human dermal fibroblasts line 442 (C) were exposed to 20(OH)D3, 20,23(OH)2D3, or
1,25(OH)2D3 and then treated with TGF-␤1 as described. Total collagen (A and C) and
20,23(OH)2D3, and 1,25(OH)2D3 on
hyaluronan (A) and collagen type 1 (B) were measured versus PBS ⫹ EtOH and TGF-␤1 ⫹
collagen synthesis (Figure 1C).
EtOH controls, respectively. Data are presented as mean ⫾ SEM of 3 replicates, and P values
In studies using additional normal
for statistically significant differences between control and treatments are shown above the
bars. Results were confirmed using 2 additional normal human dermal fibroblast lines (data
human fibroblasts, induced by TGFnot shown).
␤1, hydroxyderivatives of vitamin D
caused the suppression of collagen and
Testing calcemic effects
Female C57BL/6 mice, 6 weeks old, were purchased from
hyaluronan production at the transcriptional level (Figure
Jackson Labs and maintained on a regular laboratory chow diet.
2). Specifically fibroblasts exhibited up-regulation of mRMice were divided into groups of 3 to receive either sterile sesame
NAs for COL1A2 (P ⬍ .05), COL3A1 (P ⬍ .01), and
oil (50 ␮L; Sigma Chemical Co) or 3 ␮g/kg 20,23(OH)2D3 disHAS2 (P ⬍ .01) after 16 hours treatment with TGF-␤1
solved in 50 ␮L sterile sesame oil by daily ip injection for 14 days,
as described previously (17, 18). On day 15 mice were euthanized
that was significantly suppressed by either 10⫺8 or 10⫺10
and sera were taken for measurement of total Ca⫹⫹ by atomic
M of 20(OH)D3, 20,23(OH)2D3, and 1,25(OH)2D3,
absorption spectrophotometry and fibroblast growth factor-23
which served as positive control for classical active form of
by ELISA (Immuntopics, San Clemente, California), as described
vitamin D (Figure 2, A–C). The experiment was repeated
previously (18).
with similar results (not shown). Under the conditions
Docking analyses
used, HAS1 mRNA expression was low and did not
Docking analyses were performed as described in the Supchange after TGF-␤1 treatment, preventing a reliable evalplemental Data using the crystal structure of the vitamin D reuation of vitamin D analogs modulatory effects (not
ceptor (VDR).
shown).
Statistical analyses
Although in this report, we have not explored mechaDifferences in values obtained with the vitamin D hydroxynisms
by which hydroxyvitamin D derivatives suppress
derivatives were compared with appropriate controls using an
TGF-␤ induced up-regulation of collagen gene expression,
ANOVA, with P ⱕ .05 considered statistically different.
J Clin Endocrinol Metab, February 2013, 98(2):E298 –E303
Figure 2. Vitamin D hydroxyderivatives down-regulate mRNAs of
types I and III collagens and hyaluronan synthase 2 in TGF␤1stimulated fibroblasts. Expression of COL1A2 mRNA in line Fib1
treated with 10⫺10 M vitamin D hydroxy-derivatives (A); COL3A1
mRNA in line Fib1 treated with 10⫺8M vitamin D hydroxy-derivatives
(B); and HAS2 in line Z4 treated with 10⫺8 M vitamin D
hydroxyderivatives (C) are shown. Results are expressed as mean ⫾ SD,
with P values of EtOH ⫹ TGF-␤1 vs different treatments as indicated
above connecting lines.
studies reported by others show that another hydroxyvitamin D derivative, 22-oxacalcitrol down-regulates levels
of phosphorylated mothers against decapentaplegic homolog 2/3 (Smad2/3) and inhibits binding of phosphoSmad 3 to the proximal promoter region of the TGF␤-1
gene, thus attenuating TGF-␤1 autoinduction (22, 23).
This could reduce TGF-␤1 influence on SP1 and AP-1
binding sequences regulating pro␣ 1 (I) collagen promoter
and pro␣2 (I) collagen gene expression (24).
jcem.endojournals.org
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Figure 3. 20(OH)D3 attenuates the bleomycin-induced fibrosis in
C57BL/6 mice. A, Hematoxylin and eosin-stained section (⫻80
magnification) show significant attenuation of bleomycin-induced
fibrosis by 3 ␮g/kg of 20(OH)D3. SP, panniculus carnosus. B, 20(OH)D3
inhibits bleomycin-induced production of total solubilized collagen
from skin fragments. Total collagen production is presented as
mean ⫾ SEM per treatment in micrograms per milligram of wet tissue,
with indicated P values vs control. Scale bar, 75 ␮m.
It must be noted that 20(OH)D3 is noncalcemic in rats
(15) and mice (17) at very high doses of at least 3 ␮g and
30 ␮/kg, respectively. Since 20,23(OH)2D3 is a first and a
major product of subsequent metabolism of 20(OH)D3
(Supplemental Scheme 1), we tested its calcemic activity in
C57BL/6 mice. 20,23(OH)2D3 at 3 ␮g/kg had no effect on
serum Ca⫹⫹ or fibroblast growth factor-23 levels (Supplemental Table 1) and did not show any noticeable signs
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20(OH)D3 Inhibits Fibrogenic Activity
of morbidity. Similarly, histological analyses of heart and
kidney showed no toxicity (not shown). In contrast,
1,25(OH)2D3 at an even lower dose of 2 ␮g/kg increased
Ca⫹⫹ up to 14.6 ⫾ 0.5 mg/dL with secondary morbidity
(17). Thus, 20(OH)D3 and 20,23(OH)2D3 are excellent
candidates for further preclinical testing in models of human fibrosing diseases.
Because 20(OH)D3 is the main product of P450sccmediated metabolism of vitamin D3 that dissociates from
the catalytic site of the enzyme and is produced endogenously by skin epidermal keratinocytes (12), we focused
our studies on testing its activity using a model of bleomycin-induced dermal sclerosis in mice. Although the
mice injected with bleomycin showed skin hardening only
on palpation, the skin of mice receiving concomitant
20(OH)D3 appeared to be normal by gross examination.
Measurement of total collagen in skin biopsies showed
that 20(OH)D3 inhibited normalized collagen content in
mice given sc bleomycin (Figure 3B). Hematoxylin and
eosin-stained slides of skin biopsies from mice with bleomycin-induced scleroderma also showed a marked reduction in collagen after treatment with 20(OH)D3 compared
with those treated with sesame oil (Figure 3A). The bleomycin scleroderma model is characterized by an early infiltration of monocytes and neutrophils, which is replaced
by intense fibrosis that increases from days 7–21 with minimal numbers of macrophages at day 21 (21). We were
unable to discern a difference in the small number of
mononuclear cells in skin sections of the different treatment groups. In view of the in vitro and in vivo results in
this report, we conclude that 20(OH)D3 is an excellent
candidate for further testing of its therapeutic utility in
scleroderma or other skin-fibrosing diseases.
Because 20(OH)D acts as a partial agonist on the
VDR (15, 16), we performed docking experiments using
the crystal structure of VDR, which showed that
20,23(OH)2D3 and 20(OH)D3 bind to the VDR in poses
that overlap very well with native ligand, 1,25(OH)2D3,
with relatively high docking scores (Supplemental Figure
1 and Supplemental Table 2). This supports that VDR is
the main target for 20(OH)D3 and 20,23(OH)2D3 bioregulation. However, the final proof would require future
careful testing using VDR⫺/⫺ mice.
Because 20(OH)D3 and 20,23(OH)2D3 are further hydroxylated by P450scc in vitro and ex vivo to several minor products (Supplemental Scheme 1) (11, 12), we tested
their activity in human dermal fibroblasts. Supplemental
Figure 2 shows inhibition of proliferation of neonatal
fibroblasts by 20(OH)D3, 20,23(OH)2D3, 20,22(OH)2D3,
and 17,20,23(OH)3D3 with a potency similar to
1,25(OH)2D3, with 20(OH)D2 being less active and
1␣(OH)D3 being almost inactive. This shows that subse-
J Clin Endocrinol Metab, February 2013, 98(2):E298 –E303
quent metabolites of the P450scc-mediated pathway retain
the biological activity of the parent 20(OH)D3 and that vitamin D3 hydroxyderivatives are more promising than vitamin D2 hydroxyderivatives in terms of therapeutic utility in
fibrosing diseases.
Recently we detected production of 20(OH)D3,
20,23(OH)2D3, 20,22(OH)2D3, and 17,20,23(OH)3D3 in
human epidermal keratinocytes, whether using endogenous or exogenous vitamin D3 as the substrate. The antifibrogenic activity of these novel vitamin D hydroxyderivatives documented in this paper opens up new
exciting possibilities for the keratinocyte-based pathway
as a regulator of dermal functions. In skin pathology
one can envision topical application of noncalcemic
20(OH)D3 and 20,23(OH)2D3 to treat local fibrosing diseases such as morphea (localized scleroderma), keloid, or
fibromatosis for which there is no effective treatment.
In conclusion, 20(OH)D3 is an excellent candidate for
preclinical studies on scleroderma, with other CYP11A1derived products of its metabolism deserving further testing for its ability to prevent and/or reverse fibrosis.
Acknowledgments
The excellent technical assistance of Patricia Wheller, MS, is
acknowledged.
Address all correspondence and requests for reprints to:
Arnold E. Postlethwaite, MD, Department of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee
38163. E-mail: [email protected]; or Andrzej Slominski, MD,
PhD. E-mail: [email protected].
This work was supported by National Institutes of Health/
National Institute of Arthritis and Musculoskeletal and Skin Diseases Grants R01A052190 (to A.S.) and National Institutes of
Health Grant 1R01AR056666-01A2 (to A.S.), a Merit Review
Award from the US Department of Veterans Affairs (to A.E.P.).
Additional partial support was also obtained from the Goodman
Chair of Excellence in Medicine (to A.E.P.) of the University of
Tennessee Health Science Center.
Disclosure Summary: The authors declare no conflict of
interest.
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