The androgen and progesterone receptors regulate distinct gene networks and cellular functions in decidualizing endometrium

Abstract

Progesterone is indispensable for differentiation of human endometrial stromal cells (HESCs) into decidual cells, a process that critically controls embryo implantation. We now show an important role for androgen receptor (AR) signaling in this differentiation process. Decreased posttranslational modification of the AR by small ubiquitin-like modifier (SUMO)-1 in decidualizing cells accounted for increased responsiveness to androgen. By combining small interfering RNA technology with genome-wide expression profiling, we found that AR and progesterone receptor (PR) regulate the expression of distinct decidual gene networks. Ingenuity pathway analysis implicated a preponderance of AR-induced genes in cytoskeletal organization and cell motility, whereas analysis of AR-repressed genes suggested involvement in cell cycle regulation. Functionally, AR depletion prevented differentiation-dependent stress fiber formation and promoted motility and proliferation of decidualizing cells. In comparison, PR depletion perturbed the expression of many more genes, underscoring the importance of this nuclear receptor in diverse cellular functions. However, several PR-dependent genes encode for signaling intermediates, and knockdown of PR, but not AR, compromised activation of WNT/beta-catenin, TGFbeta/SMAD, and signal transducer and activator of transcription (STAT) pathways in decidualizing cells. Thus, the nonredundant function of the AR in decidualizing HESCs, centered on cytoskeletal organization and cell cycle regulation, implies an important role for androgens in modulating fetal-maternal interactions. Moreover, we show that PR regulates HESC differentiation, at least in part, by reprogramming growth factor and cytokine signal transduction.

Fig. 1.

DHT selectively enhances decidual PRL expression in a time-dependent manner. A, Primary HESCs were cultured in the presence of 8-br-cAMP, P4, and DHT as indicated. The medium was collected and cells harvested every 48 h. The data represent the mean PRL (upper panel) and IGFBP-1 (lower panel) concentrations (±sd) in the supernatant, with normalization for RNA content at each time point, of triplicate cultures. Significant differences were found in terms of PRL secretion between cultures treated with cAMP/P4 and cAMP/P4/DHT at 4, 6, and 8 d (*, P < 0.05). B, Primary HESCs were decidualized with 8-Br-cAMP, P4, and increasing concentrations of DHT (0.001, 0.01, 0.1, and 1 μm). The medium was collected and assayed for PRL concentration. The data are expressed as percentage increase over cAMP+P4 alone (mean ±sd). C, RTQ-PCR analysis was carried out for PRL mRNA levels in cultures treated as in A. The results show mean PRL mRNA levels (±sd) normalized to L19 mRNA of three independent cultures. D, HESCs were treated as in A for 48 h followed by transfection with dPRL-3000/Luc. Subsequently, the cells were maintained in the same culture conditions for 24 h. Luciferase and galactosidase assays were performed, and the results represent the mean (±sd) of triplicate measurements of one representative experiment. E, Primary HESCs were cultured in the presence of 8-br-cAMP, P4, DHT, and bicalutamide (Casodex; CDX) as indicated and PRL secretion determined as described above. PRL secretion was significantly different between cAMP/P4/DHT- and cAMP/P4/DHT/CDX-treated cultures (*, P < 0.05). F, HESCs, decidualized with a combination of 8-Br-cAMP, P4, and DHT for 48 h, were either mock-transfected or transfected with NT or AR siRNAs. The treatments were continued for 72 h and the supernatants assayed for PRL. The data represent the relative effect of DHT (percent) on decidual PRL secretion (mean ± sd) of triplicate cultures normalized to total protein content of each well (*, P < 0.01).

Fig. 2.

PIAS1 attenuates AR sumoylation and regulates endogenous androgen responses in differentiating HESCs. A, DHT antagonizes AR down-regulation in decidualizing HESCs. Primary cultures were treated with a combination of 8-Br-cAMP, P4, DHT, and MPA, as indicated. Whole-cell lysates, extracted every 48 h for 8 d, were immunoprobed for AR expression. B, Primary cultures transfected with AR and EGFP-SUMO-1 were treated with vehicle or decidualized with 8-Br-cAMP with or without P4 for 48 h and then pulsed with DHT, as indicated. Total protein lysates were probed for AR expression. Single and double EGFP-SUMO-1-modified AR species are indicated. β-Actin served as a loading control. C, HESCs were transfected with AR, EGFP-Sumo-1, and either NT siRNA or siRNA targeting PIAS1. Cells were left untreated for 2 d and then treated with DHT as indicated. D, Parallel cultures were treated with 8-Br-cAMP and DHT for 48 h and then subsequently transfected with pSG5 or pSG5-PIAS1 (wt or mt) and were analyzed for PRL mRNA expression by RTQ-PCR (lower panel) and immunoprobed for AR and PIAS1 (upper panel), as indicated. E, Undifferentiated HESCs were transfected with NT or PIAS1 siRNAs and treated 2 d later with vehicle or DHT for 48 h. The results show mean (±sd) PRL transcript levels normalized to L19 mRNA of three independent cultures.

Fig. 3.

AR and PR knockdown perturbs the expression of distinct gene sets in decidualizing HESCs. A, Validation of AR and PR silencing. The upper panel shows RTQ-PCR analysis of AR and PR transcript levels in cells transfected with NT, AR, or PR siRNAs before treatment with 8-Br-cAMP and MPA for 72 h. AR and PR mRNA levels were normalized to that of L19 mRNA, and the results are the mean (±sem) of four separate cultures measured in triplicate. The results are fold change relative to transcript levels in undifferentiated cells transfected with NT siRNA (dotted line). The lower panel shows Western blot analysis of AR and PR expression in protein lysates from parallel cultures. β-Actin served as a loading control. B, RTQ-PCR analysis of PRL transcript levels, normalized to L19 mRNA, in cells first transfected with NT, AR, or PR siRNA followed by differentiation with 8-Br-cAMP and MPA for 72 h. The results are fold induction of PRL mRNA expression relative to the levels in undifferentiated cells transfected with NT siRNA. *, P < 0.01. C, Venn diagram showing the number of differentially expressed genes in decidualizing cells upon AR or PR knockdown.

Fig. 4.

Validation of putative AR- and PR-dependent genes. For microarray validation, three separate cultures were first transfected with NT, AR, or PR siRNA and then treated with 8-Br-cAMP and MPA for 72 h, and mRNA levels of the indicated putative target genes were measured in triplicate for each sample by RTQ-PCR. The data normalized to L19 are expressed as fold change (±sem) relative to expression levels in undifferentiated HESCs transfected with NT siRNA (dotted lines). *, P < 0.05; **, P < 0.001.

Fig. 5.

PR regulates STAT, TGFβ/SMAD, and WNT/β-catenin signaling in decidualizing cells. Whole-cell lysates or nuclear protein fractions from HESCs, transfected first with NT, AR, or PR siRNA and then treated with 8-Br-cAMP and MPA for 72 h, were immunoprobed for various signal intermediates, as indicated. β-Actin served as a loading control.

Fig. 6.

AR controls cytoskeletal organization, cell motility, and proliferation in differentiating HESCs. A, Phalloidin staining of F-actin in undifferentiated HESCs transfected with NT siRNA (a) and cells decidualized with cAMP and MPA for 72 h after transfection with NT siRNA (b), AR siRNA (c), or PR siRNA (d). B, Motility of HESCs transfected and treated as in A was analyzed by time-lapse microscopy, quantified, and expressed in arbitrary units. The results are the mean (±sd) of triplicate analyses. Different letters above the error bars indicate that those groups are significantly different from each other at P < 0.01. C, Protein lysates obtained from parallel cultures were immunoprobed for phosphorylated MLC2. β-Actin served as a loading control. D, Primary cultures were first transfected in six-well plates with NT, AR, or PR siRNAs, replated in 96-well plates, and treated with 8-Br-cAMP and MPA, and cell viability was measured at the indicated time points. The results show the relative fold change in cell number, and the data are the mean (±sd) of triplicate measurements. E, Protein lysates from HESCs transfected with NT, AR, or PR siRNA, then treated with 8-Br-cAMP and MPA for 72 h, were subjected to Western blot analysis for total and phosphorylated RB (p-RB) expression.