A progesterone-regulated transcription factor regulates stromal-epithelial communication in early pregnancy.

Differentiation of endometrial stromal cells into decidual cells is a prerequisite for successful embryo implantation. Our previous studies in the mouse have shown that bone morphogenetic protein 2 (BMP2), a morphogen belonging to the TGFβ superfamily, is essential for this differentiation process. BMP2 is markedly induced in human primary endometrial stromal cells (HESCs) as they undergo differentiation in response to steroid hormones and cAMP. The present study was undertaken to identify the BMP2-mediated molecular pathways in primary cultures of HESCs during decidualization. Using gene expression profiling, we identified wingless-related murine mammary tumor virus integration site 4 (WNT4) as a target of BMP2 regulation during decidualization. Attenuation of WNT4 expression in HESCs by small interfering RNA administration greatly reduced BMP2-induced stromal differentiation. Additionally, adenovirus-mediated overexpression of WNT4 in HESCs markedly advanced the differentiation program, indicating that it is a key regulator of decidualization. The stimulatory effect of WNT4 was accompanied by the accumulation of active β-catenin in the nuclei of decidualizing stromal cells, indicating the involvement of the canonical WNT signaling pathway. Functional inhibition of WNT4/β-catenin pathway by Dickkopf-1, an inhibitor of the canonical WNT signaling, or small interfering RNA-mediated silencing of β-catenin expression, greatly reduced the BMP2- and WNT4-induced decidualization. Gene expression profiling revealed that Forkhead box protein O1, a forkhead family transcription factor and previously reported regulator of HESC differentiation, is a common downstream mediator of both BMP2 and WNT4 signaling. Taken together, these studies uncovered a linear pathway involving BMP2, WNT4/β-catenin, and Forkhead box protein O1 that operates in human endometrium to critically control decidualization.

Embryo implantation is regulated by a variety of endometrial factors, including cytokines, growth factors, and transcription factors. Earlier studies identified the leukemia inhibitory factor (LIF), a cytokine produced by uterine glands, as an essential regulator of implantation. LIF, acting via its cell surface receptor, activates the signal transducer and activator of transcription 3 (STAT3) in the uterine epithelial cells. However, the precise mechanism via which activated STAT3 promotes uterine function during implantation remains unknown. To identify the molecular pathways regulated by STAT3, we created SW^d/d mice in which Stat3 gene is conditionally inactivated in uterine epithelium. The SW^d/d mice are infertile due to a lack of embryo attachment to the uterine luminal epithelium and consequent implantation failure. Gene expression profiling of uterine epithelial cells of SW^d/d mice revealed dysregulated expression of specific components of junctional complexes, including E-cadherin, α- and β-catenin, and several claudins, which critically regulate epithelial junctional integrity and embryo attachment. In addition, uteri of SW^d/d mice exhibited markedly reduced stromal proliferation and differentiation, indicating that epithelial STAT3 controls stromal function via a paracrine mechanism. The stromal defect arose from a drastic reduction in the production of several members of the epidermal growth factor family in luminal epithelium of SW^d/d uteri and the resulting lack of activation of epidermal growth factor receptor signaling and mitotic activity in the stromal cells. Collectively, our results uncovered an intricate molecular network operating downstream of STAT3 that regulates uterine epithelial junctional reorganization, and stromal proliferation, and differentiation, which are critical determinants of successful implantation.

Progesterone, acting through the progesterone receptors (PGRs), is one of the most critical regulators of endometrial differentiation, known as decidualization, which is a key step toward the establishment of pregnancy. Yet a long-standing unresolved issue in uterine biology is the precise roles played by the major PGR isoforms, PGR-A and PGR-B, during decidualization in the human. Our approach, expressing PGR-A and PGR-B individually after silencing endogenous PGRs in human endometrial stromal cells (HESCs), enabled the analysis of the roles of these isoforms separately as well as jointly. Chromatin immunoprecipitation-sequencing in combination with gene expression profiling revealed that PGR-B controls a substantially larger cistrome and transcriptome than PGR-A during HESC differentiation. Interestingly, PGR-B directly regulates the expression of PGR-A. De novo motif analysis indicated that, although the 2 isoforms bind to the same DNA sequence motif, there are both common and unique neighboring motifs where other transcription factors, such as FOSL1/2, JUN, C/EBPβ, and STAT3, bind and dictate the transcriptional activities of these isoforms. We found that PGR-A and PGR-B regulate overlapping as well as distinct sets of genes, many of which are known to be critical for decidualization and establishment of pregnancy. When PGR-A and PGR-B were coexpressed during HESC differentiation, PGR-B played a predominant role, although both isoforms influenced each other's transcriptional activity. This study revealed the gene networks that operate downstream of each PGR isoform to mediate critical functions, such as regulation of the cell cycle, angiogenesis, lysosomal activation, insulin receptor signaling, and apoptosis, during decidualization in the human.

The progesterone receptor (PR) plays a critical role during ovulation. Mice lacking the PR gene are anovulatory due to a failure in the rupture of the preovulatory follicles. The pathways that operate downstream of PR to control ovulation are poorly understood. Using gene expression profiling, we identified peroxisome proliferator-activated receptor γ (PPARγ) as a target of regulation by PR in the granulosa cells of the preovulatory follicles during the ovulatory process. To investigate the function of PPARγ during ovulation, we created a conditional knockout mouse in which this gene was deleted via Cre-Lox-mediated excision in granulosa cells. When these mutant mice were subjected to gonadotropin-induced superovulation, the preovulatory follicles failed to rupture and the number of eggs released from the mutant ovaries declined drastically. Gene expression analysis identified endothelin-2, interleukin-6, and cyclic GMP-dependent protein kinase II as novel targets of regulation by PPARγ in the ovary. Our studies also suggested that cycloxygenase 2-derived metabolites of long-chain fatty acids function as endogenous activating ligands of PPARγ in the preovulatory follicles. Collectively, these studies revealed that PPARγ is a key mediator of the biological actions of PR in the granulosa cells and activation of its downstream pathways critically controls ovulation.