A Role for Malignant Brain Tumor Domain-Containing Protein 1 in Human Endometrial Stromal Cell Decidualization.
ABSTRACT: Up to 30% of women experience early miscarriage due to impaired decidualization. For implantation to occur, the uterine endometrial stromal fibroblast-like cells must differentiate into decidual cells, but the genes required for decidualization have not been fully defined. Here, we show that Malignant Brain Tumor Domain-containing Protein 1 (MBTD1), a member of the polycomb group protein family, is critical for human endometrial stromal cell (HESC) decidualization. MBTD1 predominantly localized to HESCs during the secretory phase and the levels were significantly elevated during in vitro decidualization of both immortalized and primary HESCs. Importantly, siRNA-mediated MBTD1 knockdown significantly impaired in vitro decidualization of both immortalized and primary HESCs, as evidenced by reduced expression of the decidualization markers PRL and IGFBP1. Further, knockdown of MBTD1 reduced cell proliferation and resulted in G2/M cell cycle arrest in decidualizing HESCs. Although progesterone signaling is required for decidualization, MBTD1 expression was not affected by progesterone signaling; however, MBTD1 knockdown significantly reduced expression of the progesterone target genes WNT4, FOXOA1, and GREB1. Collectively, our data suggest that MBTD1 contributes to in vitro decidualization of HESCs by sustaining progesterone signaling. This work could have implications for designing diagnostic and therapeutic tools for recurrent pregnancy loss.
Project description:Is Growth Regulation by Estrogen in Breast Cancer 1 (GREB1) required for progesterone-driven endometrial stromal cell decidualization?GREB1 is a novel progesterone-responsive gene required for progesterone-driven human endometrial stromal cell (HESC) decidualization.Successful establishment of pregnancy requires HESCs to transform from fibroblastic to epithelioid cells in a process called decidualization. This process depends on the hormone progesterone, but the molecular mechanisms by which it occurs have not been determined.Primary and transformed HESCs in which GREB1 expression was knocked down were decidualized in culture for up to 6 days. Wild-type and progesterone receptor (PR) knockout mice were treated with progesterone, and their uteri were assessed for levels of GREB1 expression.Analysis of previous data included data mining of expression profile data sets and in silico transcription factor-binding analysis. Endometrial biopsies obtained from healthy women of reproductive age during the proliferative phase (Days 8-12) of their menstrual cycle were used for isolating HESCs. Experiments were carried out with early passage (no more than four passages) HESCs isolated from at least three subjects. Transcript levels of decidualization markers prolactin (PRL) and insulin-like growth factor-binding protein-1 (IGFBP-1) were detected by quantitative RT-PCR as readouts for HESC decidualization. Cells were also imaged by phase-contrast microscopy. To assess the requirement for GREB1, PR and SRC-2, cells were transfected with specifically targeted small interfering RNAs. Results are shown as mean and SE from three replicates of one representative patient-derived primary endometrial cell line. Experiments were also conducted with transformed HESCs.Progesterone treatment of mice and transformed HESCs led to an ~5-fold (5.6 ± 0.81, P < 0.05, and 5.2 ± 0.26, P < 0.01, respectively) increase in GREB1 transcript levels. This increase was significantly reduced in the uteri of PR knock-out mice (P < 0.01), in HESCs treated with the PR antagonist RU486 (P < 0.01), or in HESCs in which PR expression was knocked down (P < 0.05). When GREB1 expression was knocked down, progesterone-driven decidualization markers in both immortalized and primary HESCs was significantly reduced (P < 0.05 and P < 0.01). Finally, GREB1 knock down signficantly reduced expression of the PR target genes WNT4 and FOXOA1 (P < 0.05 and P < 0.01, respectively).This study used the Nuclear Receptor Signaling Atlas.Although in vitro cell culture studies indicate that GREB1 is required for endoemtrial decidualization, the in vivo role of GREB1 in endometrial function and dysfunction should be assessed by using knock-out mouse models.Identification and functional analysis of GREB1 as a key molecular mediator of decidualization may lead to improved diagnosis and clinical management of women with peri-implantation loss due to inadequate endometrial decidualization.This research was funded in part by: a National Institutes of Health (NIH)/ National Institute of Child Health and Human Development (NICHD) grant (R00 HD080742) and Washington University School of Medicine start-up funds to R.K., an NIH/NICHD grant (RO1 HD-07857) to B.W.O.M., and a NIH/NICHD grant (R01 HD-042311) to J.P.L. The authors declare no conflicts of interests.
Project description:Nucleotide-binding oligomerization domain (NACHT), leucine rich repeat (LRR) and pyrin domain (PYD) 7 containing protein, NLRP7, is a member of the NLR family which serves as innate immune sensors. Mutations and genetic variants of NLRP7 have been found in women with infertility associated conditions, such as recurrent hydatidiform mole, recurrent miscarriage, and preeclampsia. Decidualization of endometrial stromal cells is a hallmark of tissue remodeling to support embryo implantation and proper placental development. Given defective decidualization has been implicated in miscarriage as well as preeclampsia, we aimed to explore the link between the NLRP7 gene and decidualization.Endometrial samples obtained from pregnant women in the first trimester and non-pregnant women were used to study NLRP7 expression pattern. The human telomerase reverse transcriptase (hTERT)-immortalized human endometrial stromal cells (T-HESCs) were used to study the effect of NLRP7 on decidualization. Decidualization of T-HESCs was induced with 1 μM medroxyprogesterone acetate (MPA) and 0.5 mM 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP). siRNA was used to knock down NLRP7 while lentiviral vectors were used to overexpress NLRP7 in cells. NLRP7 expression was detected by immunofluorescence, qRT-PCR, and Western blotting. Decidualization markers, Insulin-like growth factor-binding protein 1 (IGFBP-1) and prolactin (PRL), were detected by qRT-PCR and ELISA. Nuclear translocation of NLRP7 was detected by the subcellular fractionation and confocal microscopy. The effect of NLRP7 on progesterone receptor (PR) activity was evaluated by a reporter system.NLRP7 was up-regulated in the decidual stromal cells of human first-trimester endometrium. After in vitro decidualization, T-HESCs presented with the swollen phenotype and increased expressions of IGFBP-1 and PRL. Knockdown or over-expression of NLRP7 reduced or enhanced the decidualization, respectively, according to the expression level of IGFBP-1. NLRP7 was found to translocate in the nucleus of decidualized T-HESCs and able to promote PR activity.NLRP7 was upregulated and translocated to the nucleus of the endometrial stromal cells in an in vitro decidualization model. Overexpressed NLRP7 promoted the IGFBP-1 expression and PR reporter activation. IGFBP-1 expression decreased with the knockdown of NLRP7. Therefore, we suggest that NLRP7 contributes to in vitro decidualization of endometrial stromal cells.
Project description:The actions of glucocorticoids at the feto-maternal interface are not well understood. Here, we show that decidualization of human endometrial stromal cells (HESCs) in response to progesterone and cAMP signaling is associated with a strong induction of 11?-hydroxysteroid dehydrogenase type 1 (11?HSD1) expression and enzyme activity. Decidualization also triggered a gradual decrease in glucocorticoid receptor (GR) expression and reciprocal increase in mineralocorticoid receptor (MR) levels. Gene expression profiling of differentiating HESCs after small interfering RNA (siRNA)-mediated knockdown of either GR or MR identified 239 and 167 significantly regulated genes, respectively. Interestingly, GR-repressed genes were enriched for Krüppel-associated box domain containing zinc-finger proteins, transcriptional repressors involved in heterochromatin formation. In agreement, GR knockdown was sufficient to enhance trimethylated H3K9 levels in decidualizing cells. Conversely, we identified several MR-dependent genes implicated in lipid droplet biogenesis and retinoid metabolism. For example, the induction in differentiating HESCs of DHRS3, encoding a highly conserved enzyme that catalyzes the oxidation/reduction of retinoids and steroids, was enhanced by aldosterone, attenuated in response to MR knockdown, and abolished upon treatment with the MR antagonist RU26752. Furthermore, we demonstrate that decidualization is associated with dynamic changes in the abundance and distribution of cytoplasmic lipid droplets, the formation of which was blocked by RU26752. In summary, progesterone drives local cortisol biosynthesis by decidual cells through induction of 11?-hydroxysteroid dehydrogenase type 1 (11?HSD1), leading to transcriptional regulation of distinct GR and MR gene networks involved in epigenetic programming and lipid and retinoid metabolism, respectively.
Project description:Although GPR64 has an important role for male fertility, its physiological roles in the female reproductive system are still unknown. In the present study, immunohistochemical analysis reveals a spatiotemporal expression of GPR64 in the uterus during early pregnancy. Observation of remarkable induction of GPR64 expression in uterine decidual cells points to its potential physiological significance on decidualization. The decidualization of uterine stromal cells is a key event in implantation. Progesterone (P4) signaling is crucial for the decidualization of the endometrial stromal cells for successful pregnancy. Therefore, we examined ovarian steroid hormone regulation of GPR64 expression in the murine uterus. P4 induced GPR64 expression in the epithelial and stromal cells of the uterus in ovariectomized wild-type mice, but not in PRKO mice. ChIP analysis confirmed that PGR proteins were recruited on progesterone response element of Gpr64 gene in the uteri of wild-type mice treated with P4. Furthermore, the expression of GPR64 was increased in human endometrial stromal cells (hESCs) during in vitro decidualization. Interestingly, small interfering RNA (siRNA)-mediated knockdown of GPR64 in hESCs remarkably reduced decidualization. These results suggest that Gpr64 has a crucial role in the decidualization of endometrial stromal cells.
Project description:Decidualization denotes the transformation of endometrial stromal cells into specialized decidual cells. In pregnancy, decidual cells form a protective matrix around the implanting embryo, enabling coordinated trophoblast invasion and formation of a functional placenta. Continuous progesterone (P4) signaling renders decidual cells resistant to various environmental stressors, whereas withdrawal inevitably triggers tissue breakdown and menstruation or miscarriage. Here, we show that PLCL1, coding phospholipase C (PLC)-related catalytically inactive protein 1 (PRIP-1), is highly induced in response to P4 signaling in decidualizing human endometrial stromal cells (HESCs). Knockdown experiments in undifferentiated HESCs revealed that PRIP-1 maintains basal phosphoinositide 3-kinase/Protein kinase B activity, which in turn prevents illicit nuclear translocation of the transcription factor forkhead box protein O1 and induction of the apoptotic activator BIM. By contrast, loss of this scaffold protein did not compromise survival of decidual cells. PRIP-1 knockdown did also not interfere with the responsiveness of HESCs to deciduogenic cues, although the overall expression of differentiation markers, such as PRL, IGFBP1, and WNT4, was blunted. Finally, we show that PRIP-1 in decidual cells uncouples PLC activation from intracellular Ca(2+) release by attenuating inositol 1,4,5-trisphosphate signaling. In summary, PRIP-1 is a multifaceted P4-inducible scaffold protein that gates the activity of major signal transduction pathways in the endometrium. It prevents apoptosis of proliferating stromal cells and contributes to the relative autonomy of decidual cells by silencing PLC signaling downstream of Gq protein-coupled receptors.
Project description:Establishment of a successful pregnancy requires not only implantation of a healthy embryo into a receptive uterus but also progesterone receptor (PGR)-dependent transformation of endometrial stromal cells (ESCs) into specialized decidual cells. Decidual cells support the developing embryo and are critical for placentation. We have previously shown that a known transcriptional coregulator of the PGR, steroid receptor coactivator-2 (SRC-2), is a critical driver of endometrial decidualization in both human and mouse endometrium. However, the full spectrum of genes transcriptionally controlled by SRC-2 in decidualizing ESCs has not been identified. Therefore, using an RNA- and chromatin immunoprecipitation-sequencing approach, we have identified the transcriptome of decidualizing human ESCs (hESCs) that requires SRC-2. We revealed that the majority of hESC genes regulated by SRC-2 are associated with decidualization. Over 50% of SRC-2-regulated genes are also controlled by the PGR. While ontology analysis showed that SRC-2-dependent genes are functionally linked to signaling processes known to underpin hESC decidualization, cell membrane processes were significantly enriched in this analysis. Follow-up studies showed that retinoid signaling is dependent on SRC-2 during hESC decidualization. Specifically, SRC-2 is required for full induction of the retinol transporter, stimulated by retinoic acid 6 (STRA6), which is essential for hESC decidualization. Together our findings show that a critical subset of genes transcriptionally reprogramed by PGR during hESC decidualization requires SRC-2. Among the multiple genes, pathways and networks that are dependent on SRC-2 during hESC decidualization, first-line analysis supports a critical role for this coregulator in maintaining retinoid signaling during progesterone-driven decidualization.
Project description: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.
Project description:Mouse studies support a role for endometrial early growth response 1 (EGR1) in uterine receptivity and decidualization, which are processes controlled by estrogen and progesterone. However, the importance of this transcription factor in similar cellular processes in human is unclear. Analysis of clinical samples indicate that endometrial EGR1 levels are decreased in the endometrium of women with recurrent implantation failure, suggesting that tight control of EGR1 levels are necessary for normal endometrial function. Therefore, we used siRNA-mediated knockdown of EGR1 expression in cultured human endometrial stromal cells (hESCs) to assess the functional role of EGR1 in hESC decidualization. Protein expression studies revealed that EGR1 is highly expressed in pre-decidual hESCs. However, EGR1 protein levels rapidly decrease following administration of an established deciduogenic hormone stimulus containing estradiol, medroxyprogesterone acetate, and cyclic adenosine monophosphate. Intriguingly, EGR1 knockdown in pre-decidual hESCs blocks the ability of these cells to decidualize later, indicating that EGR1 is required to transcriptionally program pre-decidual hESCs for decidualization. Support for this proposal comes from the analysis of transcriptome and cistrome datasets, which shows that EGR1 target genes are primarily involved in transcriptional regulation, cell signaling, and proliferation. Collectively, our studies provide translational support for an evolutionary conserved role for human endometrial stromal EGR1 in the early events of pregnancy establishment.
Project description:Endometrial decidualization represents an essential step for the successful implantation of the embryo; however, the molecular mechanism behind this differentiation process remains unclear. This study aimed to identify novel microRNAs (miRNAs) involved in the regulation of decidual gene expression in human endometrial stromal cells (HESCs). An in vitro analysis of primary undifferentiated and decidualizing HESCs was conducted. HESCs were isolated from hysterectomy specimens from normally cycling premenopausal women with uterine fibroids, who were not on hormonal treatment at the time of surgery. Primary HESCs were expanded in culture and decidualized with 8-bromo-cyclic adenosine monophosphate and medroxyprogesterone acetate. Microarray analysis identified six miRNAs differentially expressed in response to decidualization of HESCs. All but one miRNA were downregulated upon decidualization, including miR-542-3p. We demonstrated that miR-542-3p overexpression inhibits the induction of major decidual marker genes, including IGFBP1, WNT4 and PRL. In addition, miR-542-3p overexpression inhibited the morphological transformation of HESCs in response to deciduogenic cues. A luciferase reporter assay confirmed that the 3'-untranslated region of IGFBP1 mRNA is targeted by miR-542-3p. The results suggest that miR-542-3p plays an important role in endometrial decidualization by regulating the expression of major decidual marker genes.
Project description:Decidualization renders the endometrium transiently receptive to an implanting blastocyst although the underlying mechanisms remain incompletely understood. Here we show that human endometrial stromal cells (HESCs) rapidly release IL-33, a key regulator of innate immune responses, upon decidualization. In parallel, differentiating HESCs upregulate the IL-33 transmembrane receptor ST2L and other pro-inflammatory mediators before mounting a profound anti-inflammatory response that includes downregulation of ST2L and increased expression of the soluble decoy receptor sST2. We demonstrate that HESCs secrete factors permissive of embryo implantation in mice only during the pro-inflammatory phase of the decidual process. IL-33 knockdown in undifferentiated HESCs was sufficient to abrogate this pro-inflammatory decidual response. Further, sequential activation of the IL-33/ST2L/sST2 axis was disordered in decidualizing HESCs from women with recurrent pregnancy loss. Signals from these cultures prolonged the implantation window but also caused subsequent pregnancy failure in mice. Thus, Il-33/ST2 activation in HESCS drives an autoinflammatory response that controls the temporal expression of receptivity genes. Failure to constrain this response predisposes to miscarriage by allowing out-of-phase implantation in an unsupportive uterine environment.