Identification of molecular signaling pathways underlying human endometrial stromal cell differentiation

Human endometrial stromal cell differentiation;estrogen receptor alpha;progesterone receptor isoforms

Kaya, Hatice

关键词: Human endometrial stromal cell differentiation;    estrogen receptor alpha;    progesterone receptor isoforms;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/46949/Hatice_Kaya.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Decidualization, the differentiation of endometrial stromal cells, is essential for a successful pregnancy. Although the steroid hormones estrogen (E) and progesterone (P) are known to control decidualization, the precise mechanisms via which these hormones act to control this differentiation process are poorly understood. We used primary cultures of human endometrial stromal cells (HESC) to analyze the role of estrogen receptor alpha (ESR1) and progesterone receptor isoforms (PRA and PRB) in human decidualization.Previous studies established that HESC, when treated with the differentiation cocktail containing E, P, and a cyclic adenosine monophosphate (cAMP) analog, cease proliferation and undergo differentiation. In the present study, when ESR1 expression was silenced, the HESC continued to proliferate in the presence of the differentiation cocktail and their differentiation was severely inhibited. Gene expression profiling revealed that, in the absence of ESR1, the expression levels of several cell cycle regulatory factors were increased and those of specific cell cycle inhibitors were decreased. Our study also revealed that ESR1 promoted the expression of key regulators of HESC differentiation (PGR, FOXO1 and WNT4). Expression of these targets was dependent on the addition of cAMP, suggested a functional link between cAMP and ESR1 signaling. Using a proteomic approach, we identified MED1 as a target of cAMP-activated protein kinase (PKA) during HESC differentiation. The PKA-dependent phosphorylation of MED1 enhanced its ability to interact efficiently with ESR1. Furthermore, loss of MED1 expression inhibited HESC differentiation with parallel impairment in the expression of a subset of ESR1 target genes. Addition of cAMP increased recruitment of MED1 to the ESR1 binding regions of a target gene (WNT4). Collectively, these results indicated that, during decidualization, ESR1 suppresses HESC proliferation and promotes their differentiation via interactions with MED1, which is activated in response to cAMP signaling.Progesterone, acting through its receptors, is essential for the precise regulation of the endometrial processes required for a successful pregnancy, including decidualization. However, the specific roles of the progesterone receptor isoforms, PRA and PRB, during endometrial differentiation in the human have remained unknown. A major focus of my project was to shed light on the roles of the receptor isoforms by identifying their cistromes and correlated gene expression profiles during differentiation of human stromal cells. We expressed PRA and PRB individually after silencing endogenous progesterone receptors so that the roles of the isoforms could be analyzed independently as well as jointly. Identification of the cistromes of PRA and PRB using chromatin immunoprecipitation (ChIP) followed by high throughput sequencing (ChIP-seq), revealed that PRB cistrome was larger, covering that of PRA, at an early time point of in vitro differentiation of human endometrial stromal cells. Our de novo motif analysis showed that, both isoforms bind to the same DNA sequence motif, progesterone response element (PRE). In addition to the PRE, they had both common and distinct nearby motifs where other transcription factors might bind. Many molecules previously identified as progesterone target genes and known to regulate stromal differentiation were found to contain PRA and PRB binding sites, including BMP2, HAND2 and HOXA10,confirming the validity of our method.Our comprehensive gene expression analysis suggested that PRA and PRB regulated overlapping and distinct sets of genes. When PRA and PRB were expressed together, PRB was the prevalent isoform, while both isoforms influenced each other’s transcriptional activity. Our findings suggested that PRB was a more effective transcription factor than PRA in human stromal cell differentiation by the extent to which it interacted with the genomic binding sites and regulated downstream gene networks. Collectively, our first line of analysis provided an insight into the early molecular mechanisms regulated by progesterone receptor isoforms during stromal differentiation. With this study, we confirmed some of the known roles of progesterone, such as regulation of angiogenesis, proliferation and apoptosis. We could also distinguish signaling networks downstream of each isoform and identify new molecules, such as IRS2, as possible direct targets of progesterone signaling for the establishment of a successful pregnancy.