GATA2 and Progesterone Receptor Interaction in Endometrial Stromal Cells Undergoing Decidualization.

The transcription factor GATA2 is important for endometrial stromal cell decidualization in early pregnancy. Progesterone receptor (PGR) is also critical during decidualization but its interaction with GATA2 in regulating genes and pathways necessary for decidualization in human endometrium are unclear. RNA-sequencing (RNA-seq) was performed to compare gene expression profiles (n = 3), and chromatin immunoprecipitation followed by sequencing (ChIP-seq) using an antibody against GATA2 (n = 2) was performed to examine binding to target genes in human endometrial stromal cells undergoing in vitro decidualization (IVD including estrogen, progestin, and 3',5'-cyclic AMP analogue) or vehicle treatment. We identified 1232 differentially expressed genes (DEGs) in IVD vs vehicle. GATA2 cistrome in IVD-treated cells was enriched with motifs for GATA, ATF, and JUN, and gene ontology analysis of GATA2 cistrome revealed pathways that regulate cholesterol storage, p38 mitogen-activated protein kinase, and the c-Jun N-terminal kinase cascades. Integration of RNA-seq and ChIP-seq data revealed that the PGR motif is highly enriched at GATA2 binding regions surrounding upregulated genes in IVD-treated cells. The integration of a mined public PGR cistrome in IVD-treated human endometrial cells with our GATA2 cistrome showed that GATA2 binding was significantly enhanced at PGR-binding regions in IVD vs vehicle. Interrogating 2 separate ChIP-seq data sets together with RNA-seq revealed integration of GATA2 and PGR action to coregulate biologic processes during decidualization of human endometrial stromal cells, specifically via WNT activation and stem cell differentiation pathways. These findings reveal the key pathways that are coactivated by GATA2 and PGR that may be therapeutic targets for supporting implantation and early pregnancy.

Genome-wide estrogen receptor-α binding and action in human endometrial stromal cells.

OBJECTIVE: To investigate the gene targets of estradiol (E2)-estrogen receptor-α (ESR1) in human endometrial stromal cells. DESIGN: Basic science. SETTING: University research center. PATIENT(S): Premenopausal women with or without endometriosis. INTERVENTION(S): Primary cultures of human endometrial stromal cells from healthy endometrium, with or without small-interfering RNA (siRNA) knockdown of ESR1 expression, were treated with E2 or vehicle control. MAIN OUTCOME MEASURE(S): Genome-wide RNA expression by RNA sequencing was compared in endometrial stromal cells with or without siRNA knockdown of ESR1 in the presence or absence of E2. Genome-wide recruitment of ESR1 to chromatin was assessed by chromatin immunoprecipitation sequencing. Gene expression by real-time qualitative polymerase chain reaction of a potential E2-ESR1 target gene was determined in endometrial stromal cells and endometriotic stromal cells. RESULT(S): We identified several important pathways that are dependent on E2-ESR1 signaling in endometrial stromal cells, including progesterone signaling, cell-matrix adhesion, and cytoskeleton rearrangement, as well as paracrine signaling by members of the fibroblast growth factor family. We detected a total of 709 ESR1 target sites on chromatin. By integrating data on genome-wide transcriptomic changes and E2-ESR1 binding sites, we identified inositol polyphosphate phosphatase type II (INPP4B) as a candidate E2-mediated suppressor of proliferation in healthy endometrial cells. INPP4B was downregulated in endometriosis-derived stromal cells. CONCLUSION(S): E2-ESR1 activates genes involved in human endometrial stromal cell cycle regulation, progesterone response, and production of stromal growth factors. Understanding the direct role of estrogen on the endometrial stroma and identifying downstream targets of E2-ESR1 can inform the development of targeted therapies for endometriosis and diminished endometrial receptivity.

CATACOMB: An endogenous inducible gene that antagonizes H3K27 methylation activity of Polycomb repressive complex 2 via an H3K27M-like mechanism.

Using biochemical characterization of fusion proteins associated with endometrial stromal sarcoma, we identified JAZF1 as a new subunit of the NuA4 acetyltransferase complex and CXORF67 as a subunit of the Polycomb Repressive Complex 2 (PRC2). Since CXORF67's interaction with PRC2 leads to decreased PRC2-dependent H3K27me2/3 deposition, we propose a new name for this gene: CATACOMB (catalytic antagonist of Polycomb; official gene name: EZHIP ). We map CATACOMB's inhibitory function to a short highly conserved region and identify a single methionine residue essential for diminution of H3K27me2/3 levels. Remarkably, the amino acid sequence surrounding this critical methionine resembles the oncogenic histone H3 Lys27-to-methionine (H3K27M) mutation found in high-grade pediatric gliomas. As CATACOMB expression is regulated through DNA methylation/demethylation, we propose CATACOMB as the potential interlocutor between DNA methylation and PRC2 activity. We raise the possibility that similar regulatory mechanisms could exist for other methyltransferase complexes such as Trithorax/COMPASS.

Endometriosis.

Pelvic endometriosis is a complex syndrome characterized by an estrogen-dependent chronic inflammatory process that affects primarily pelvic tissues, including the ovaries. It is caused when shed endometrial tissue travels retrograde into the lower abdominal cavity. Endometriosis is the most common cause of chronic pelvic pain in women and is associated with infertility. The underlying pathologic mechanisms in the intracavitary endometrium and extrauterine endometriotic tissue involve defectively programmed endometrial mesenchymal progenitor/stem cells. Although endometriotic stromal cells, which compose the bulk of endometriotic lesions, do not carry somatic mutations, they demonstrate specific epigenetic abnormalities that alter expression of key transcription factors. For example, GATA-binding factor-6 overexpression transforms an endometrial stromal cell to an endometriotic phenotype, and steroidogenic factor-1 overexpression causes excessive production of estrogen, which drives inflammation via pathologically high levels of estrogen receptor-ß. Progesterone receptor deficiency causes progesterone resistance. Populations of endometrial and endometriotic epithelial cells also harbor multiple cancer driver mutations, such as KRAS, which may be associated with the establishment of pelvic endometriosis or ovarian cancer. It is not known how interactions between epigenomically defective stromal cells and the mutated genes in epithelial cells contribute to the pathogenesis of endometriosis. Endometriosis-associated pelvic pain is managed by suppression of ovulatory menses and estrogen production, cyclooxygenase inhibitors, and surgical removal of pelvic lesions, and in vitro fertilization is frequently used to overcome infertility. Although novel targeted treatments are becoming available, as endometriosis pathophysiology is better understood, preventive approaches such as long-term ovulation suppression may play a critical role in the future.

Endometriosis and nuclear receptors.

BACKGROUND: Endometriosis is recognized as a steroid-dependent disorder; however, the precise roles of nuclear receptors (NRs) in steroid responsiveness and other signaling pathways are not well understood. OBJECTIVE AND RATIONALE: Over the past several years, a number of paradigm-shifting breakthroughs have occurred in the area of NRs in endometriosis. We review and clarify new information regarding the mechanisms responsible for: (i) excessive estrogen biosynthesis, (ii) estrogen-dependent inflammation, (iii) defective differentiation due to progesterone resistance and (iv) enhanced survival due to deficient retinoid production and action in endometriosis. We emphasize the roles of the relevant NRs critical for these pathological processes in endometriosis. SEARCH METHODS: We conducted a comprehensive search using PubMed for human, animal and cellular studies published until 2018 in the following areas: endometriosis; the steroid and orphan NRs, estrogen receptors alpha (ESR1) and beta (ESR2), progesterone receptor (PGR), steroidogenic factor-1 (NR5A1) and chicken ovalbumin upstream promoter-transcription factor II (NR2F2); and retinoids. OUTCOMES: Four distinct abnormalities in the intracavitary endometrium and extra-uterine endometriotic tissue underlie endometriosis progression: dysregulated differentiation of endometrial mesenchymal cells, abnormal epigenetic marks, inflammation activated by excess estrogen and the development of progesterone resistance. Endometriotic stromal cells compose the bulk of the lesions and demonstrate widespread epigenetic abnormalities. Endometriotic stromal cells also display a wide range of abnormal NR expression. The orphan NRs NR5A1 and NR2F2 compete to regulate steroid-synthesizing genes in endometriotic stromal cells; NR5A1 dominance gives rise to excessive estrogen formation. Endometriotic stromal cells show an abnormally low ESR1:ESR2 ratio due to excessive levels of ESR2, which mediates an estrogen-driven inflammatory process and prostaglandin formation. These cells are also deficient in PGR, leading to progesterone resistance and defective retinoid synthesis. The pattern of NR expression, involving low ESR1 and PGR and high ESR2, is reminiscent of uterine leiomyoma stem cells. This led us to speculate that endometriotic stromal cells may display stem cell characteristics found in other uterine tissues. The biologic consequences of these abnormalities in endometriotic tissue include intense inflammation, defective differentiation and enhanced survival. WIDER IMPLICATIONS: Steroid- and other NR-related abnormalities exert genome-wide biologic effects via interaction with defective epigenetic programming and enhance inflammation in endometriotic stromal cells. New synthetic ligands, targeting PGR, retinoic acid receptors and ESR2, may offer novel treatment options.

Generation of Progesterone-Responsive Endometrial Stromal Fibroblasts from Human Induced Pluripotent Stem Cells: Role of the WNT/CTNNB1 Pathway.

Defective endometrial stromal fibroblasts (EMSFs) contribute to uterine factor infertility, endometriosis, and endometrial cancer. Induced pluripotent stem cells (iPSCs) derived from skin or bone marrow biopsies provide a patient-specific source that can be differentiated to various cells types. Replacement of abnormal EMSFs is a potential novel therapeutic approach for endometrial disease; however, the methodology or mechanism for differentiating iPSCs to EMSFs is unknown. The uterus differentiates from the intermediate mesoderm (IM) to form coelomic epithelium (CE) followed by the Müllerian duct (MD). Here, we successfully directed the differentiation of human iPSCs (hiPSCs) through IM, CE, and MD to EMSFs under molecularly defined embryoid body culture conditions using specific hormonal treatments. Activation of CTNNB1 was essential for expression of progesterone receptor that mediated the final differentiation step of EMSFs before implantation. These hiPSC-derived tissues illustrate the potential for iPSC-based endometrial regeneration for future cell-based treatments.

Shear stress-induced improvement of red blood cell deformability.

Classically, it is known that red blood cell (RBC) deformability is determined by the geometric and material properties of these cells. Experimental evidence accumulated during the last decade has introduced the concept of active regulation of RBC deformability. This regulation is mainly related to altered associations between membrane skeletal proteins and integral proteins, with the latter serving to anchor the skeleton to the lipid matrix. It has been hypothesized that shear stress induces alterations of RBC deformability: the current study investigated the dynamics of the transient improvement in deformability induced by shear stress at physiologically-relevant levels. RBC were exposed to various levels of shear stress (SS) in a Couette type shearing system that is part of an ektacytometer, thus permitting the changes in RBC deformability during the application of SS to be monitored. Initial studies showed that there is an increase in deformability of the RBC subjected to SS in the range of 5-20 Pa, with kinetics characterized by time constants of a few seconds. Such improvement in deformability, expressed by an elongation index (EI), was faster with higher levels of SS and hence yielded shorter time constants: absolute values of EI increased by 3-8% of the starting level. Upon the removal of the shear stress, this response by RBC was reversible with a slower time course compared to the increase in EI during application of SS. Increased calcium concentration in the RBC suspending medium prevented the improvement of deformability. It is suggested that the improvement of RBC deformability by shear forces may have significant effects on blood flow dynamics, at least in tissues supplied by blood vessels with impaired vasomotor reserve, and may therefore serve as a compensating mechanism for the maintenance of adequate microcirculatory perfusion.