Project description:Herein, we investigated eMSC and eSF freshly isolated from endometrium from women with and without endometriosis and compared them to their respective short- and long-term cultures and subsequent decidualization response to progesterone. ABSTRACT Human endometrium undergoes cyclic tissue shedding with ensuing regeneration involving stem/progenitor cells, but the role of multipotent resident endometrial mesenchymal stem cells (eMSC) as progenitors of the endometrial stromal fibroblast (eSF) has not been definitively demonstrated. Progesterone (P4)-driven differentiation (decidualization) of eSF is abnormal in endometriosis, displaying P4-resistance in vivo and in vitro. This study investigated whether eMSC are precursors of eSF and, if so, whether the endometriosis P4-resistant phenotype is inherited from eMSC progenitors or acquired by eSF lineage cells within the endometrial niche. To this end we analyzed the transcriptomes of eMSC and eSF isolated by fluorescence activated cell sorting (FACS) from eutopic endometrium of women with (eMSCFACS.endo, eSFFACS.endo) and without (eMSCFACS.control, eSFFACS.control) endometriosis, and of primary cultures derived from these sorted populations. FACS-isolated eMSC and eSF displayed distinct sets of differentially expressed lineage-associated genes (LG) with >95% of the 200 most highly expressed LG conserved in endometriosis. eSFcontrol in culture maintained in vitro expression of 45% eSF LG; whereas eSFendo displayed less in vitro LG stability, expressing 33% eSF LG. Under the same culture conditions, eMSCcontrol underwent in vitro eSF lineage differentiation (eMSC→eSFcontrol), progressively losing stemness (down-regulated 81% eMSC LG) and acquiring an in vitro eSFcontrol phenotype (up-regulated 55% eSF LG) with minimal transcriptome differences vs. eSFcontrol cultures (19 genes all <2 fold change). Likewise eMSCendo cultures underwent in vitro eSF lineage differentiation (eMSC→eSFendo), down-regulating 77% eMSC LG and up-regulating 50% eSF LG, and there were 78 differentially expressed genes (-2.73 to +2.81 fold change) in eMSC→eSFendo vs. eSFendo. The in vitro disease phenotypes of eSFendo and eMSC→eSFendo shared 49 common genes differentially expressed vs. their control counterparts. However, 78 and 89 genes, respectively, were unique to eSFendo and eMSC→eSFendo, and pathway analysis revealed a proinflammatory phenotype in eSFendo but not eMSC→eSFendo cultures, suggesting divergent niche effects for in vivo vs. in vitro lineage differentiation. Cultures of eSFcontrol and eMSC→eSFcontrol responded to P4 with IGFBP1 secretion, while eSFendo and eMSC→eSFendo cultures did not, demonstrating P4-resistance inherited from eMSCendo. These findings substantiate eMSC as progenitors of eSF and reveal novel aspects of the eSF disease phenotype in endometriosis with P4-resistance inherited from the eMSC and a proinflammatory component acquired by the eSF lineage within the endometrial niche.

Project description:Eutopic endometrium in endometriosis has molecular evidence of resistance to progesterone (P4) and activation of the PKA pathway in the stromal compartment. To investigate global and temporal responses of eutopic endometrium to P4, we compared early (6-h), intermediate (48-h), and late (14-day) transcriptomes, signaling pathways, and networks of human endometrial stromal fibroblasts (hESFs) from women with endometriosis (hESFendo) to hESFs from women without endometriosis (hESFnonendo). Endometrial biopsy samples were obtained from subjects with and without mild peritoneal endometriosis (n = 4 per group), and hESFs were isolated and treated with P4 (1 μM) plus estradiol (E2) (10 nM), E2 alone (10 nM), or vehicle for up to 14 days. Total RNA was subjected to microarray analysis using a Gene 1.0 ST (Affymetrix) platform and analyzed by using bioinformatic algorithms, and data were validated by quantitative real-time PCR and ELISA. Results revealed unique kinetic expression of specific genes and unique pathways, distinct biological and molecular processes, and signaling pathways and networks during the early, intermediate, and late responses to P4 in both hESFnonendo and hESFendo, although a blunted response to P4 was observed in the latter. The normal response of hESF to P4 involves a tightly regulated kinetic cascade involving key components in the P4 receptor and MAPK signaling pathways that results in inhibition of E2-mediated proliferation and eventual differentiation to the decidual phenotype, but this was not established in the hESFendo early response to P4. The abnormal response of this cell type to P4 may contribute to compromised embryonic implantation and infertility in women with endometriosis. We compared early (6-h), intermediate (48-h), and late (14-day) in vitro whole-genome responses of hESF from women with endometriosis (hESFendo) to hESF from women without endometriosis (hESFnonendo) treated with P4 plus E2 (E2P4), E2 alone, or vehicle alone. Using this experimental paradigm, the data demonstrate unique phenotypes, gene expression processes, biochemical and signaling pathways, and networks suggestive of early, intermediate, and late responses of hESFnonendo and hESFendo to P4, giving insights into the complexity of events occurring normally in response to P4 and in the setting of endometriosis.

Project description:Progesterone (P4) is essential to prepare the endometrium for a successful pregnancy. While P4 resistance is a major cause of infertility that leads to endometrial disorders, such as endometriosis, the underlying epigenetic imbalance responsible for P4 resistance in the endometrium remains unclear. We demonstrated that CXXC finger protein 1 (CFP1), a major mediator of histone H3 lysine 4 trimethylation (H3K4m3), is an indispensable factor in the maintenance of epigenetic landscapes of P4-progesterone receptor (PGR) signaling networks in the uterus. Cfp1flox/flox;Pgr-Cre (Cfp1d/d) mice suffered from impaired P4 responses, leading to abnormal uterine cell proliferation and complete failure of embryo implantation and decidualization, albeit regular estrous cycle with normal hormone profiles. mRNA and chromatin immunoprecipitation sequencing analyses showed that CFP1 binding enriches promoter regions to regulate uterine mRNA landscapes not only in H3K4me3-dependent but also in H3K4me3-independent manners. Especially, CFP1 directly regulates subsets of important P4 response genes, including Gata2, Sox17, and Ihh, which mediate the activation of the smoothened signaling pathway in the uterus. P4 cannot interfere with E2 actions on uterine epithelial proliferation in Cfp1d/d mice, which was restored by supplementing a smoothened agonist (SAG). Furthermore, ectopic lesions of Cfp1d/d uterus in an endometriosis model showed P4 resistance, which was rescued by SAG. In human endometriosis, CFP1, GATA2, SOX17, and IHH, were significantly downregulated, and a positive correlation was found in the expression levels between CFP1 and these P4 targets regardless of PGR levels. Collectively, we suggest that CFP1 is an epigenetic modulator of P4-PGR signaling networks to promote uterine receptivity for embryo implantation and suppress endometriosis with P4 resistance. CFP1 is a key epigenetic factor that intervenes in the P4–epigenome–transcriptome networks for uterine function under physiologic and pathophysiologic conditions.

Project description:The cellular responses to the steroid hormones, estrogen (E2), and progesterone (P4) are governed by their cognate receptor’s transcriptional output. Although multiple mechanisms including the tissue-specific coactivators dictate these transcriptional signatures, the feed-forward mechanisms, that evolved to coordinate the cell-type specific transcriptional fulcrums for steroid receptors are not yet identified. In this report, we found that a common feed-forward mechanism between GREB1 and steroid receptors governs the differential effect of GREB1 on steroid hormones in a physiological or pathological context. We found that in physiological (receptive) endometrium, GREB1 is essential for progesterone but not estrogenic responses, whereas in endometriosis, an E2-dependent endometrial pathology, it pre-dominantly mediates estrogenic actions. Specifically, the deletion of GREB1 curtails endometrial receptivity and decidualization, owing to impaired P4-dependent stromal cell proliferation. In contrast, GREB1 is not required for E2-mediated epithelial proliferation of the receptive uterus. Of mechanism, in receptive endometrium, progesterone-induced GREB1 physically interacts with the progesterone receptor on chromatin and functions as a cofactor in a positive feedback mechanism to selectively regulate P4-responses. In pathological conditions (endometriosis), ablation of GREB1 led to the development of smaller endometriotic lesions in mice and reduced E2-driven proliferation of human endometriotic cells. Moreover, E2-induced GREB1 modulated the E2-dependent target gene expression in a feed-forward mechanism. Collectively, acting as a downstream effector, GREB1 governs either P4-responses or E2-responses depending on the physiological or pathological setting. Importantly, this differential action of GREB1 is exerted by a common feed-forward mechanism between GREB1 and steroid receptors, which conceptually advances our understanding of the mechanism(s) that underlie the distinct cell- and tissue-specific actions of steroid hormones.

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Steroid Hormones Regulate Genome-Wide Epigenetic Programming and Gene Transcription in Human Endometrial Cells with Marked Aberrancies in Endometriosis [methylation]

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