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Cutting Edge: Progesterone Directly
Upregulates Vitamin D Receptor Gene
Expression for Efficient Regulation of T Cells
by Calcitriol
Shankar Thangamani, Myughoo Kim, Youngmin Son,
Xinxin Huang, Heejoo Kim, Jee H. Lee, Jungyoon Cho,
Benjamin Ulrich, Hal E. Broxmeyer and Chang H. Kim
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Copyright © 2015 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2015; 194:883-886; Prepublished online 29
December 2014;
doi: 10.4049/jimmunol.1401923
http://www.jimmunol.org/content/194/3/883
The
Cutting Edge
Journal of
Immunology
Cutting Edge: Progesterone Directly Upregulates Vitamin
D Receptor Gene Expression for Efficient Regulation of
T Cells by Calcitriol
Shankar Thangamani,*,1 Myughoo Kim,*,1 Youngmin Son,* Xinxin Huang,†
Heejoo Kim,* Jee H. Lee,* Jungyoon Cho,* Benjamin Ulrich,*
Hal E. Broxmeyer,† and Chang H. Kim *,†,‡,x
T
he biologically active form of vitamin D (vit.D; 1,25dihydroxyvitamin D3, also called calcitriol) is a nuclear hormone ligand mediating its effects mainly
through the vit.D receptor (VDR)–retinoid X receptor nuclear hormone receptor system (1). VDR is widely expressed
in the body. Within the immune system, T cells express VDR
and are an important target of calcitriol for immune regulation (2). Calcitriol induces regulatory T cells (Tregs) but
suppresses the generation of effector T cells (3–8). Vit.D
metabolites inhibit the maturation of dendritic cells and make
them tolerogenic cells (9, 10). Progesterone (P4) is another
nuclear hormone critical for preparation and maintenance of
pregnancy (11). Whereas vit.D is obtained through dietary
*Laboratory of Immunology and Hematopoiesis, Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907;
†
Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202; ‡Weldon School of Biomedical Engineering, Purdue
University, West Lafayette, IN 47907; and xPurdue Center for Cancer Research, Purdue
University, West Lafayette, IN 47907
1
S.T. and M.K. are cofirst authors.
Received for publication August 4, 2014. Accepted for publication December 1, 2014.
This work was supported in part by National Institutes of Health Grants R01AI074745,
R01DK076616, 1R01AI080769, and 1S10RR028293 and by a grant from the National
Multiple Sclerosis Society (to C.H.K.).
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401923
absorption and UV-induced synthesis in the skin and sequentially activated in the liver and kidneys to become calcitriol, P4 is produced at high levels directly from ovaries and
placenta and at low levels in the adrenal gland and the testes
(12). In a manner similar to vit.D, P4 increases Tregs but
suppresses inflammatory effector T cells (13).
In this study, we report that P4 induces VDR expression in
CD4+ Th cells, an effect mediated by P4-induced binding of
P4 receptor (PR) to canonical PR-binding elements (PREs) in
the human VDR (hVDR) gene. VDR induction by P4 allows
T cells to be more efficiently regulated by calcitriol for enhanced promotion of Tregs but suppression of potentially
inflammatory effector T cells.
Materials and Methods
Cell isolation and animal study
Cord blood (CB) CD4 + CD25 2 and adult peripheral blood
CD4+CD252CD45RO2CD692 naive CD4+ T cells were isolated as described before (14). Total spleen CD4+ T cells were isolated from pregnant
mice (15–18 d postcoitus), and naive CD4+ T cells were isolated from the
spleen of nonpregnant mice as described before (15). Female C57BL/6 mice
were injected s.c. with medroxyprogesterone (2 mg; Depo-Provera from
Pfizer) and sacrificed 4 d later. All human subject and animal studies were
approved by Institutional Review Committees at Purdue University.
In vitro differentiation of T cells and hVDR knockdown with small
interfering RNA
Human naive CD4+ T cells were activated with anti-CD3/28 beads (5 ml/
million cells: Miltenyi Biotec) and IL-2 (25 U/ml) or cultured in Th1/Th17/
Treg cytokine conditions in phenol red–free RPMI 1640 supplemented with
10% charcoal/dextran-treated FBS (HyClone) or in vit.D-free X-VIVO 15
medium (Lonza) as described before (14). CB T cells, activated for 24 h with
anti-CD3/28 beads and human IL-2, were transfected with control or hVDR
small interfering RNA (siRNA; 20 pmol/4 million cells, Santa Cruz Biotechnology) using an Amaxa Nucleofector (Lonza). Cells were cultured with
P4 (2 mg/ml) and/or calcitriol (1–100 nM) for 3 d for VDR mRNA expression or 5–6 d for the expression of Foxp3, CD25, CD38, latencyassociated peptide/TGF-b1, IL-17, and/or IFN-g (13).
Address correspondence and reprint requests to Prof. Chang H. Kim, Purdue University,
VPTH 126, 725 Harrison Street, West Lafayette, IN 47907. E-mail address:
[email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: CB, cord blood; ChIP, chromatin immunoprecipitation; hVDR, human VDR; P4, progesterone; PB, peripheral blood; PR, P4 receptor;
PRE, PR-binding element; siRNA, small interfering RNA; Treg, regulatory T cell; vit.D,
vitamin D; VDR, vitamin D receptor.
Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
The two nuclear hormone receptor ligands progesterone
and vitamin D (vit.D) play important roles in regulating
T cells. The mechanism that connects these two hormones in regulating T cells has not been established.
In this study, we report that progesterone is a novel inducer of vit.D receptor (VDR) in T cells and makes
T cells highly sensitive to calcitriol. At the molecular
level, the induction by progesterone is mediated by
two progesterone receptor-binding elements in the intron region after the first noncoding exon of the human
VDR gene. Increased expression of VDR by progesterone allows highly sensitive regulation of T cells by vit.D
even when vit.D levels are suboptimal. This novel regulatory pathway allows enhanced induction of regulatory T cells but suppression of Th1 and Th17 cells by
the two nuclear hormones. The results have significant
ramifications in effective regulation of T cells to prevent
adverse immune responses during pregnancy. The
Journal of Immunology, 2015, 194: 883–886.
884
CUTTING EDGE: PROGESTERONE INDUCES VDR IN T CELLS
In vitro T cell suppression assay
CB naive CD4+ T cells were stained with CFSE and 5 3 104 cells were added
to 96-well plates in the presence of anti-CD3/CD28–coated beads as target
cells along with in vitro–differentiated Tregs generated with IL-2 (25 U/ml)
and calcitriol (100 nM) and/or P4 (2 mg/ml). CFSE dilution was determined
by flow cytometry on day 4.
Microarray, RT-PCR, and Western blotting of VDR expression
The microarray analysis of CB naive CD4+ T cells was performed previously
(13). Quantitative RT-PCR was performed with the primers for human or
mouse VDR gene (Supplemental Table I). CB naive CD4+ T cells, activated
with anti-CD3/CD28 and IL-2 with or without P4 (2 mg/ml) for 3–5 d, were
examined for VDR protein expression with an mAb to hVDR (R&D Systems) and HRP-conjugated anti-mouse IgG (Santa Cruz Biotechnology).
Promoter analysis and chromatin immunoprecipitation assay
PREs on the hVDR gene were identified with TESS. A chromatin immunoprecipitation (ChIP) assay was performed as described before with the
primers listed in Supplemental Table I (15). CB CD4+ T cells, activated with
anti-CD3/CD28–coated beads in the presence of P4 (2 mg/ml) for 3–4 d,
were processed and immunoprecipitated using 4 mg rabbit mAb to human PR
(Abnova).
PRE no. 1 (59-AGAACT-39) and PRE no. 2 (59-GGGACA-39) in the regulatory region of VDR gene cloned in pGL3-VDR (+490/21267) (16) were
mutated to 59-AAAGGT-39 and 59-GAAGGA-39, respectively, with a sitedirected transformer mutagenesis kit (Clontech Laboratories). Mutant pGL3VDR vectors (20 mg) were electrotransfected into MCF-7 cells (310 V,
950 mF; Bio-Rad). The transfected cells were rested overnight, activated
with PMA (50 ng/ml) in the presence or absence of P4 (2 mg/ml) for 6 h, and
then assayed for luciferase activity with a Synergy HT reader (BioTek).
Statistical analysis
Significant differences between indicated groups were determined by a Student
paired two-tailed t test.
Results and Discussion
P4 induces VDR expression in T cells
Analysis of microarray data on human CB naive CD4+ T cells
activated with P4 (13) revealed that one of the major genes
induced by P4 is the VDR gene (Fig. 1A). To verify this
finding, CB naive CD4+ T cells were activated in the presence
of P4, and VDR expression at mRNA and protein levels was
examined. P4 substantially increased the expression of VDR
mRNA in activated T cells in a dose-dependent manner
(Fig. 1B). Optimal induction occurred at ∼2 mg/ml P4,
concentrations that were detected in placental tissues during
pregnancy (17). Thus, this concentration range is physiologically relevant. The induction of VDR gene expression was
suppressed by RU486, a PR antagonist (Fig. 1C). A similar
induction was observed in adult peripheral blood CD4+
T cells. P4 also induced the expression of VDR protein in CB
T cells (Fig. 1D). The VDR induction by P4 occurred in
Treg-, Th1-, and Th17-polarizing cytokine conditions
(Fig. 1E). The VDR induction by P4 was observed in mouse
T cells as well (Fig. 1F). It was also increased in spleen CD4+
T cells and uterus in pregnant or progestin-injected mice
(Fig. 1F). These results indicate that P4 induces VDR gene
expression in T cells in heterogeneous conditions or species.
P4 responsive elements on the hVDR gene mediate P4-induced VDR
expression
To gain insights into the molecular mechanism of the VDR
induction by P4, we examined the DNA sequence of the VDR
gene for the presence of the canonical PREs. We found five
PREs in the 59 regulatory region of the VDR gene spanning
FIGURE 1. P4 induces VDR expression in human CD4+ T cells. (A)
Multiplot microarray data showing P4-regulated genes in CB T cells. (B and
C) Expression of hVDR mRNA was determined by quantitative RT-PCR in
cultured human naive CD4+ T cells. CB T cells were used unless indicated
otherwise. (D) Expression of VDR protein in cultured CB CD4+ T cells was
determined by Western blotting. (E) P4 induces hVDR mRNA expression in
various cytokine conditions. (F) VDR expression in mouse T cells and uteri.
Naive CD4+ T cells were activated with anti-CD3/28 and IL-2 in charcoaltreated serum-containing (A and C–E) or vit.D-free medium (B and F) in the
presence or absence of P4 (2 mg/ml unless indicated otherwise) or RU486
(100 mg/ml). Combined or representative data from three to five separate
experiments are shown in (B)–(F). *p , 0.05 between indicated groups or
between controls.
into the first exon and intron regions (Fig. 2A). A luciferase
reporter containing this regulatory region was highly responsive to P4 (Fig. 2B), suggesting that this region contains
functional PREs. A ChIP assay revealed that only one site
(site D) between exon 1A and 1B had a clear PR binding
activity (Fig. 2C). When the two PREs in site D were mutated, the VDR gene promoter activity was largely abolished
(Fig. 2D).
P4-induced VDR potentiates the effect of calcitriol on suppression of
Th1 and Th17 cells
Vit.D metabolites suppress the generation of Th1 and Th17
cells (5, 8, 18). VDR directly binds the Ifng gene promoter to
suppress Th1 cells (19) and induce C/EBP protein expression
to suppress Th17 cells (8). We found that P4 decreases the
effective concentration of calcitriol in suppressing the induction of Th1 and Th17 cells (Fig. 3A). Interestingly, the
suppression by calcitriol in the presence of P4 was largely
abolished when the VDR gene was knocked down with siRNA
(Fig. 3B, 3C). Thus, P4-induced VDR functions to increase
the sensitivity of T cells to calcitriol.
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Mutagenesis and reporter assay
The Journal of Immunology
885
P4-induced VDR enhances the function of calcitriol in inducing Tregs
We next examined the effect of calcitriol on induction of Tregs
expressing Foxp3, CD38, and/or latency-associated peptide/
TGF-b1 (20, 21) in the presence or absence of P4. P4 increased T cell sensitivity to calcitriol in upregulating the Tregassociated molecules (Fig. 4A). Moreover, the Tregs generated
FIGURE 3. Impact of P4 and induced VDR on T cell response to calcitriol
in suppression of Th1 and Th17 cells. (A) CB CD4+ naive T cells were activated in Th1 (IL-2, IL-12, and anti–IL-4) or Th17 (IL-6, IL-21, IL-23, IL-1b,
TGF-b1, anti–IL-4, and anti–IFN-g) polarization conditions in a vit.D-free
medium containing P4 (2 mg/ml) and/or calcitriol for 5–6 d. (B) Expression
of hVDR mRNA after siRNA knockdown. (C) Impact of VDR knockdown
on T cell differentiation into effector T cells in response to calcitriol (1 nM)
and/or P4 (2 mg/ml). Pooled data with SEM are shown (n = 4). *p , 0.05
versus controls.
with calcitriol and P4 were more suppressive than were the
Tregs generated with P4 or calcitriol alone (Fig. 4B). The
Treg-inducing activity of calcitriol in the presence of P4 was
greatly abolished when the VDR gene was knocked down with
siRNA (Fig. 4C).
Our results revealed the presence of a novel regulatory
pathway linking P4 and vit.D in regulating T cells. The VDR
induced by P4 increases the sensitivity of T cells to calcitriol.
This VDR induction in T cells is mediated by two PRE sites
after the noncoding first exon of the VDR gene, which contains
a number of other cis-acting elements (22). It is known that
VDR expression in human T cells is upregulated upon TCR
signaling (23), and we think that P4 and calcitriol are probably involved in the induction because culture media including animal sera generally contain these two nuclear
hormones. Vit.D is important for normal function of the
reproductive system, and its deficiency is common in pregnant females and is linked to pregnancy complications such as
preeclampsia and miscarriage and to defective immune tolerance in the newborn (24–27). The VDR upregulation may
allow T cells to sense low levels of vit.D and become effectively regulated to prevent inflammation when vit.D levels
are decreased in the body. Therefore, the P4-induced VDR
in T cells would be important to prevent adverse immune
responses involved in pregnancy complications. The regulatory pathway may be active in other cell types as well because
it has been observed that VDR expression was increased in
endometrial tumor cells cultured with P4 (28) and in the
uterus in pregnant females, as determined in this study. In
summary, our findings reveal a novel role of P4 in expression
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FIGURE 2. PR binding to cis-acting elements in
the VDR gene promoter drives gene expression.
(A) Putative PREs and ChIP sites in the hVDR
gene are shown. (B) A luciferase assay was performed with a reporter vector containing the VDR
gene promoter. (C) A ChIP assay was performed to
identify PR binding sites in the 59 regulatory region of the hVDR gene. (D) A reporter assay with
null mutations in the PRE sites in the VDR gene.
A representative dataset of three to four separate
experiments is shown. *p , 0.05 between indicated groups. n.d., not detectable.
886
of the VDR gene in T cells for highly sensitive regulation of
T cell activity by vit.D metabolites.
Acknowledgments
We thank J. Fleet (Purdue University) for critical reading of this manuscript
and M. Kadakia (Wright State University) for providing pGL3-VDR.
Disclosures
The authors have no financial conflicts of interest.
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FIGURE 4. Impact of P4 and induced VDR on T cell response to calcitriol
for Treg generation. (A) Induction of T cells expressing indicated Treg Ags by
calcitriol in the presence or absence of P4 (2 mg/ml). (B) P4 and calcitriol
generate highly suppressive Tregs. The suppressive function on CFSE-labeled
fresh CB CD4+CD252 responder T cells was determined. CFSE dilution was
assessed on day 4. (C) Impact of siRNA-mediated VDR knockdown on Treg
induction by calcitriol (1 nM) and/or P4 (2 mg/ml). Vit.D-free medium was
used for (A) and (C). Pooled data with SEM (n = 3–4) are shown. *p , 0.05
versus control or the P4 + calcitriol group.
CUTTING EDGE: PROGESTERONE INDUCES VDR IN T CELLS