Marius C. Jones

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.

Cell cycle arrest by FoxO transcription factors involves transcriptional repression of cyclin D, although the exact mechanism remains unclear. In this study, we used the BCR-ABL-expressing cell line BV173 as a model system to investigate the mechanisms whereby FoxO3a regulates cyclin D2 expression. Inhibition of BCR-ABL by STI571 results in down-regulation of cyclin D2 expression, activation of FoxO3a activity, and up-regulation of BCL6 expression. Using reporter gene assays, we demonstrate that STI571, FoxO3a, and BCL6 can repress cyclin D2 transcription through a STAT5/BCL6 site located within the cyclin D2 promoter. We propose that BCR-ABL inhibition leads to FoxO3a activation, which in turn induces the expression of BCL6, culminating in the repression of cyclin D2 transcription through this STAT5/BCL6 site. This process was verified by mobility shift and chromatin immunoprecipitation analyses. We find that conditional activation of FoxO3a leads to accumulation of BCL6 and down-regulation of cyclin D2 at protein and mRNA levels. Furthermore, silencing of FoxO3a and BCL6 in BCR-ABL-expressing cells abolishes STI571-mediated effects on cyclin D2. This report establishes the signaling events whereby BCR-ABL signals are relayed to cyclin D2 to mediate cell cycle progression and defines a potential mechanism by which FoxO proteins regulate cyclin D2 expression.

Menstruation, or cyclic shedding of nonpregnant endometrial tissue with associated bleeding, occurs only in humans and a few other species. This breakdown of the endometrium in response to falling ovarian progesterone levels is a complex process, characterized by local leukocyte infiltration, expression and activation of matrix metalloproteinases, and apoptosis. Spontaneous decidualization (differentiation) of the stromal compartment precedes the cyclic shedding of the endometrium in various menstruating species but the mechanisms that link these processes are not understood. In this study, we identified FOXO1 as a key transcription factor responsible for mediating apoptosis of decidualized human endometrial stromal cells (HESCs) in response to progesterone withdrawal. We demonstrate that medroxyprogesterone acetate (MPA, a synthetic progestin) enhances the expression of FOXO1 in differentiating HESCs while simultaneously inducing cytoplasmic retention and inactivation of FOXO1. Withdrawal of MPA from decidualized HESCs results in rapid nuclear accumulation of FOXO1, increased BIM expression, a proapoptotic FOXO1 target gene, and cell death. Conversely, silencing of FOXO1 expression completely abolishes cell death induced by MPA withdrawal. In summary, the observation that differentiating HESCs become dependent on progesterone signaling for survival through induction and reversible inactivation of FOXO1 suggests a novel mechanism that links decidualization of the endometrium to menstruation.

cAMP is required for differentiation of human endometrial stromal cells (HESCs) into decidual cells in response to progesterone, although the underlying mechanism is not well understood. We now demonstrate that cAMP signaling attenuates ligand-dependent sumoylation of the progesterone receptor (PR) in HESCs. In fact, decidualization is associated with global hyposumoylation and redistribution of small ubiquitin-like modifier (SUMO)-1 conjugates into distinct nuclear foci. This altered pattern of global sumoylation was not attributable to impaired maturation of SUMO-1 precursor or altered expression of E1 (SAE1/SEA2) or E2 (Ubc9) enzymes but coincided with profound changes in the expression of E3 ligases and SUMO-specific proteases. Down-regulation of several members of the protein inhibitors of activated STAT (PIAS) family upon decidualization pointed toward a role of these E3 ligases in PR sumoylation. We demonstrate that PIAS1 interacts with the PR and serves as its E3 SUMO ligase upon activation of the receptor. Furthermore, we show that silencing of PIAS1 not only enhances PR-dependent transcription but also induces expression of prolactin, a decidual marker gene, in progestin-treated HESCs without the need of simultaneous activation of the cAMP pathway. Our findings demonstrate how dynamic changes in the SUMO pathway mediated by cAMP signaling determine the endometrial response to progesterone.