Progesterone directly up-regulates vitamin D receptor gene expression for efficient regulation of T cells by calcitriol

Abstract

The two nuclear hormone receptor ligands progesterone and vitamin D (vit.D) each play important roles in regulating T cells. The mechanism that connects these two hormones in regulating T cells has not been established. We report here 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 non-coding 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 Tregs 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.

Introduction

The biologically active form of vitamin D (vit.D) (1,25(OH)₂-D₃, also called calcitriol) is a nuclear hormone ligand mediating its effects mainly through the vit.D receptor (VDR)-RXR 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). While vit.D is obtained through dietary 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).

Here, we report that P4 induces VDR expression in CD4⁺ T helper cells, an effect mediated by P4-induced binding of progesterone receptor (PR) to canonical progesterone receptor binding elements (PRE) in the human VDR 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⁻ and adult peripheral blood (PB) CD4⁺CD25⁻CD45RO⁻CD69⁻ naïve CD4⁺ T cells were isolated as described before (14). Total spleen CD4⁺ T cells were isolated from pregnant mice (15–18 dpc), and naïve CD4⁺ T cells were isolated from the spleen of unpregnant mice as described before (15). Female C57BL/6 mice were injected s.c. with medroxyprogesterone (2 mg, Depo-Provera, Pfizer) and sacrificed 4 days later. All human subject and animal studies were approved by institutional review committees at Purdue University.

In vitro differentiation of T cells and hVDR knock-down with siRNA

Human naïve CD4⁺ T cells were activated with anti-CD3/28 beads (5 μl/million cells: Miltenyi) 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 hIL-2, were transfected with control or hVDR siRNA (20 pmol/4 million cells, Santa Cruz Biotech) using an Amaxa nucleofector (Lonza). Cells were cultured with P4 (2 μg/ml) and/or calcitriol (1–100 nM) for 3 days for VDR mRNA expression or 5–6 days for the expression of FoxP3, CD25, CD38, LAP-TGF-β1, IL-17, and/or IFN-γ (13).

In vitro T cell suppression assay

CB naïve CD4⁺ T cells were stained with carboxyfluorescein succinimidyl ester (CFSE) and 5×10⁴ 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 μg/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). qRT-PCR was performed with the primers for human or mouse VDR gene (Supp. Table 1). CB naïve CD4⁺ T cells, activated with anti-CD3/CD28 and IL-2 with or without P4 (2 μg/ml) for 5 days, were examined for VDR protein expression with a monoclonal antibody to human VDR (R&D Systems) and horseradish peroxide-conjugated anti-mouse IgG (Santa Cruz Biotechnology).

Promoter analysis and chromatin immunoprecipitation assay (ChIP)

PREs on the human VDR gene were identified with TESS. A ChIP assay was performed as described before with the primers listed in Supp. Table 1 (15). CB CD4⁺ T cells, activated with anti-CD3/CD28-coated beads in the presence of P4 (2 μg/ml) for 3–4 days, were processed and immunoprecipitated using 4 μg of rabbit monoclonal antibody to human PR (Abnova).

Mutagenesis and reporter assay

PRE#1 (AGAACT) and PRE#2 (GGGACA) in the regulatory region of VDR gene cloned in pGL3-VDR (+490/−1267) (16) were mutated to AAAGGT and GAAGGA respectively with the Site-Directed Transformer mutagenesis kit (Clontech Laboratories). Mutant pGL3-VDR vectors (20 μg) were electro-transfected into MCF-7 cells (310 V, 950 μF, Bio-Rad). The transfected cells were rested overnight, activated with PMA (50 ng/ml) in the presence or absence of P4 (2 μg/ml) for 6 h, and then assayed for luciferase activity with a Synergy HT reader (Bio-Tek).

Statistical analysis

Significant differences between indicated groups were determined by Student’s paired two-tailed t test.

Results and Discussion

P4 induces VDR expression in T cells

Analysis of microarray data on human CB naïve CD4⁺ T cells activated with P4 (13) revealed that one of the major genes that are induced by P4 is the VDR gene (Fig. 1A). To verify this finding, CB naïve 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 around 2 μ/ml of 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 PB 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.

Fig. 1.

P4 induces VDR expression in human CD4⁺ T cells. (A) Multi-plot microarray data showing P4-regulated genes in CB T cells. (B and C) Expression of hVDR mRNA was determined by qRT-PCR in cultured human naïve 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. Naïve CD4⁺ T cells were activated with anti-CD3/28 and IL-2 in charcoal-treated serum-containing (A, C, D, E) or vit.D-free medium (B, F) in the presence or absence of P4 (2 μg/ml unless indicated otherwise) or RU486 (100 μg/ml). Combined or representative data from 3–5 separate experiments are shown in panel B–F. *Significant differences between indicated groups (p<0.05).

Progesterone responsive elements on the human VDR 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 PR binding elements (PREs). We found five PREs in the 5′ regulatory region of the VDR gene spanning into the first exon and intron regions (Fig. 2A). A luciferase reporter containing this regulatory region was highly responsive to P4 (Fig. 2B), suggesting 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).

Fig. 2.

PR binding to cis-acting elements in the VDR gene promoter drives gene expression. (A) Putative PR binding elements (PREs) and ChIP sites in the human VDR 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 5′ regulatory region of the human VDR gene. (D) A reporter assay with null mutations in the PRE sites in the VDR gene. A representative data set of 3–4 separate experiments is shown. *Significant differences between indicated groups (p<0.05).

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 IFN-γ gene promoter to suppresses Th1 cells (19) and induces 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 VDR gene was knocked down with siRNA (Fig. 3B, C). Thus, P4-induced VDR functions to increase the sensitivity of T cells to calcitriol.

Fig. 3.

Impact of P4 and induced VDR on T cell response to calcitriol in suppression of Th1 and Th17 cells. (A) CB CD4⁺ naïve T cells were activated in Th1 (IL-2, IL-12 and anti-IL-4) or Th17 (IL-6, IL-21, IL-23, IL-1β, TGF-β1, anti-IL-4 and anti-IFNγ) polarization conditions in a Vit.D-free medium containing P4 (2 μg/ml) and/or calcitriol for 5–6 days. (B) Expression of hVDR mRNA after siRNA knock-down. (C) Impact of VDR knock-down on T cell differentiation into effector T cells in response to calcitriol (1 nM) and/or P4 (2 μg/ml). Pooled data with SEM are shown (n=4). *Significant differences from controls (p<0.05).

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 LAP-TGF-β1 (20, 21) in the presence or absence of P4. P4 increased T cell sensitivity to calcitriol in up-regulating the Treg-associated molecules (Fig. 4A). Moreover, the Tregs generated with calcitriol and P4 were more suppressive than 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 VDR gene was knocked down with siRNA (Fig. 4C).

Fig. 4.

Impact of P4 and induced VDR on T cell response to calcitriol for Treg generation. (A) Induction of T cells expressing indicated Treg antigens by calcitriol in the presence or absence of P4 (2 μg/ml). (B) P4 and calcitriol generate highly suppressive Tregs. The suppressive function on CFSE-labeled fresh CB CD4⁺CD25⁻ responder T cells was determined. CFSE dilution was assessed on day 4. (C) Impact of siRNA-mediated VDR knock-down on Treg induction by calcitriol (1 nM) and/or P4 (2 μg/ml). Vit.D-free medium was used for panel A and C. Pooled data with SEM (n=3–4) are shown. *Significant differences from control or the P4+calcitriol group.

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 non-coding 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 up-regulated upon TCR signaling (23), and we believe that P4 and calcitriol are probably involved in the induction because culture media including animal sera generally contains 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 pregnant complication such as preeclampsia and miscarriage and to defective immune tolerance in the newborn (24–27). The VDR up-regulation 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 pregnant complications. The regulatory pathway may be active in other cell types as well as 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 sum, our finding reveals a novel role of P4 in expression of the VDR gene in T cells for highly sensitive regulation of T cell activity by vit.D metabolites.

Abbreviations

1,25(OH)-D₃

1,25-dihydroxyvitamin D₃

ChIP

chromatin immunoprecipitation

CB

cord blood

PB

peripheral blood

P4

progesterone

PRE

progesterone-responsive element

Tregs

regulatory T cells

Vit.D

vitamin D

VDR

vitamin D receptor