Dataset Information

Roles of Estrogen Receptor-? and the Coactivator MED1 During Human Endometrial Decidualization.

ABSTRACT: The steroid hormones 17?-estradiol and progesterone are critical regulators of endometrial stromal cell differentiation, known as decidualization, which is a prerequisite for successful establishment of pregnancy. The present study using primary human endometrial stromal cells (HESCs) addressed the role of estrogen receptor-? (ESR1) in decidualization. Knockdown of ESR1 transcripts by RNA interference led to a marked reduction in decidualization of HESCs. Gene expression profiling at an early stage of decidualization indicated that ESR1 negatively regulates several cell cycle regulatory factors, thereby suppressing the proliferation of HESCs as these cells enter the differentiation program. ESR1 also controls the expression of WNT4, FOXO1, and progesterone receptor (PGR), well-known mediators of decidualization. Whereas ESR1 knockdown strongly inhibited the expression of FOXO1 and WNT4 transcripts within 24 hours of the initiation of decidualization, PGR expression remained unaffected at this early time point. Our study also revealed a major role of cAMP signaling in influencing the function of ESR1 during decidualization. Using a proteomic approach, we discovered that the cAMP-dependent protein kinase A (PKA) phosphorylates Mediator 1 (MED1), a subunit of the mediator coactivator complex, during HESC differentiation. Using immunoprecipitation, we demonstrated that PKA-phosphorylated MED1 interacts with ESR1. The PKA-dependent phosphorylation of MED1 was also correlated with its enhanced recruitment to estrogen-responsive elements in the WNT4 gene. Knockdown of MED1 transcripts impaired the expression of ESR1-induced WNT4 and FOXO1 transcripts and blocked decidualization. Based on these findings, we conclude that modulation of ESR1-MED1 interactions by cAMP signaling plays a critical role in human decidualization.

SUBMITTER: Kaya Okur HS

PROVIDER: S-EPMC4771698 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Roles of Estrogen Receptor-α and the Coactivator MED1 During Human Endometrial Decidualization.

Kaya Okur Hatice S HS Das Amrita A Taylor Robert N RN Bagchi Indrani C IC Bagchi Milan K MK

Molecular endocrinology (Baltimore, Md.) 20160205 3

The steroid hormones 17β-estradiol and progesterone are critical regulators of endometrial stromal cell differentiation, known as decidualization, which is a prerequisite for successful establishment of pregnancy. The present study using primary human endometrial stromal cells (HESCs) addressed the role of estrogen receptor-α (ESR1) in decidualization. Knockdown of ESR1 transcripts by RNA interference led to a marked reduction in decidualization of HESCs. Gene expression profiling at an early st ...[more]

PMID: 26849466

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Project description:Steroid receptor coactivator-3 (SRC-3; also known as NCOA3 or AIB1) is a member of the multifunctional p160/SRC family of coactivators, which also includes SRC-1 and SRC-2. Clinical and cell-based studies as well as investigations on mice have demonstrated pivotal roles for each SRC in numerous physiological and pathophysiological contexts, underscoring their functional pleiotropy. We previously demonstrated the critical involvement of SRC-2 in murine embryo implantation as well as in human endometrial stromal cell (HESC) decidualization, a cellular transformation process required for trophoblast invasion and ultimately placentation. We show here that, like SRC-2, SRC-3 is expressed in the epithelial and stromal cellular compartments of the human endometrium during the proliferative and secretory phase of the menstrual cycle as well as in cultured HESCs. We also found that SRC-3 depletion in cultured HESCs results in a significant attenuation in the induction of a wide-range of established biomarkers of decidualization, despite exposure of these cells to a deciduogenic stimulus and normal progesterone receptor expression. These molecular findings are supported at the cellular level by the inability of HESCs to morphologically transform from a stromal fibroblastoid cell to an epithelioid decidual cell when endogenous SRC-3 levels are markedly reduced. To identify genes, signaling pathways and networks that are controlled by SRC-3 and potentially important for hormone-dependent decidualization, we performed RNA-sequencing on HESCs in which SRC-3 levels were significantly reduced at the time of administering the deciduogenic stimulus. Comparing HESC controls with HESCs deficient in SRC-3, gene enrichment analysis of the differentially expressed gene set revealed an overrepresentation of genes involved in chromatin remodeling, cell proliferation/motility, and programmed cell death. These predictive bioanalytic results were confirmed by the demonstration that SRC-3 is required for the expansion, migratory and invasive activities of the HESC population, cellular properties that are required in vivo in the formation or functioning of the decidua. Collectively, our results support SRC-3 as an important coregulator in HESC decidualization. Since perturbation of normal homeostatic levels of SRC-3 is linked with common gynecological disorders diagnosed in reproductive age women, this endometrial coregulator-along with its new molecular targets described here-may open novel clinical avenues in the diagnosis and/or treatment of a non-receptive endometrium, particularly in patients presenting non-aneuploid early pregnancy loss.

Project description:Recent data from human and mouse studies strongly support an indispensable role for steroid receptor coactivator-2 (SRC-2)-a member of the p160/SRC family of coregulators-in progesterone-dependent endometrial stromal cell decidualization, an essential cellular transformation process that regulates invasion of the developing embryo into the maternal compartment. To identify the key progesterone-induced transcriptional changes that are dependent on SRC-2 and required for endometrial decidualization, we performed comparative genome-wide transcriptional profiling of endometrial tissue RNA from ovariectomized SRC-2(flox/flox) (SRC-2(f/f) [control]) and PR(cre/+)/SRC-2(flox/flox) (SRC-2(d/d) [SRC-2-depleted]) mice, acutely treated with vehicle or progesterone. Although data mining revealed that only a small subset of the total progesterone-dependent transcriptional changes is dependent on SRC-2 (?13%), key genes previously reported to mediate progesterone-driven endometrial stromal cell decidualization are present within this subset. Along with providing a more detailed molecular portrait of the decidual transcriptional program governed by SRC-2, the degree of functional diversity of these progesterone mediators underscores the pleiotropic regulatory role of SRC-2 in this tissue. To showcase the utility of this powerful informational resource to uncover novel signaling paradigms, we stratified the total SRC-2-dependent subset of progesterone-induced transcriptional changes in terms of novel gene expression and identified transcription factor 23 (Tcf23), a basic-helix-loop-helix transcription factor, as a new progesterone-induced target gene that requires SRC-2 for full induction. Importantly, using primary human endometrial stromal cells in culture, we demonstrate that TCF23 function is essential for progesterone-dependent decidualization, providing crucial translational support for this transcription factor as a new decidual mediator of progesterone action.

Project description:Decidualization is a morphological and biochemical transformation of endometrial stromal fibroblast into differentiated decidual cells, which is critical for embryo implantation and pregnancy establishment. The complex regulatory networks have been elucidated at both the transcriptome and the proteome levels, however very little is known about the post-transcriptional regulation of this process. miRNAs regulate multiple physiological pathways and their de-regulation is associated with human disorders including gynaecological conditions such as endometriosis and preeclampsia. In this study we profile the miRNAs expression throughout human endometrial stromal (hESCs) decidualization and analyze the requirement of the miRNA biogenesis enzyme Dicer during this process. A total of 26 miRNAs were upregulated and 17 miRNAs downregulated in decidualized hESCs compared to non-decidualized hESCs. Three miRNAs families, miR-181, miR-183 and miR-200, are down-regulated during the decidualization process. Using miRNAs target prediction algorithms we have identified the potential targets and pathways regulated by these miRNAs. The knockdown of Dicer has a minor effect on hESCs during in vitro decidualization. We have analyzed a battery of decidualization markers such as cell morphology, Prolactin, IGFBP-1, MPIF-1 and TIMP-3 secretion as well as HOXA10, COX2, SP1, C/EBPß and FOXO1 expression in decidualized hESCs with decreased Dicer function. We found decreased levels of HOXA10 and altered intracellular organization of actin filaments in Dicer knockdown decidualized hESCs compared to control. Our results provide the miRNA signature of hESC during the decidualization process in vitro. We also provide the first functional characterization of Dicer during human endometrial decidualization although surprisingly we found that Dicer plays a minor role regulating this process suggesting that alternative biogenesis miRNAs pathways must be involved in human endometrial decidualization.

Project description:Human endometrium derived mesenchymal stem cells (hEndSCs) offer a great promise for regenerative medicine and reproductive system disorders treatment methods based on cell therapy due to their broad differentiation potential and highly efficient proliferation. In our study, we investigated the characteristics of hEndSCs that were isolated from two sources: endometrium and menstrual blood, which both contain endometrial origin stem cells. Changes in gene and protein expression levels during long-term cultivation and decidualization potential were examined in endometrial stem cells (EndSCs) and menstrual blood stem cells (MenSCs). The decidualization process was induced on early and late passages of hEndSCs using dibutyryl cyclic-AMP (db-cAMP) and medroxyprogesterone acetate (MPA) agents. We demonstrated that after long-term cultivation of hEndSCs the expression of typical mesenchymal stromal cell surface markers such as CD44, CD73, CD90, CD105 and perivascular marker CD146 remains at a similar level throughout long-term cultivation. Additionally, hematopoietic and endothelial markers CD34, CD45 were also tested, they were negative in all cases. Analyzed stem cells gene markers, such as OCT4, SOX2, NANOG, KLF4, showed similar expression in all passages of hEndSCs. RT-qPCR results demonstrated that the expression of cell cycle control associated genes - CDK2, CCNA2, CCNE2, p21, p53 and Rb, among all groups was very similar. Expression of genes associated with senescence (ATM, JUND, TOP2A, MYC) was maintained at a similar level throughout passaging. In addition, Western blot analysis was used to assess changes in proteins' levels associated to epigenetics (EZH2, SUZ12, H3K27me3) and cell cycle control (cyclinE1, p53) during long-term cultivation. The levels of proteins associated with epigenetic changes were fluctuated slightly depending on the patient. Also, we demonstrated that in all induced hEndSCs the expression of decidualization markers Prolactin (PRL), IGFBP1 and WNT4 was upregulated. In conclusion, we demonstrated successful decidualization of stem cells derived from two reproductive system resources: endometrium and menstrual blood by using db-cAMP and MPA regardless of the length of the stem cell passaging. According these findings, we suppose that endometrium derived stem cells and menstrual blood derived stem cells could have a potency not only for endometrium tissue regeneration, but could also become a successful therapy for reproductive system disorders, including infertility or recurrent pregnancy loss.

Dataset Information

Roles of Estrogen Receptor-? and the Coactivator MED1 During Human Endometrial Decidualization.

Publications

Roles of Estrogen Receptor-α and the Coactivator MED1 During Human Endometrial Decidualization.

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