Phospholipase D1 as a key enzyme for decidualization in human endometrial stromal cells

Using primary cell cultures of human endometrial stromal cells (ES cells), we investigated the role of phospholipase D (PLD) in 8-Br-cAMP-induced decidualization, which involves morphological and biological differentiation processes. When treated with 0.5 mM 8-Br-cAMP for 12 days, ES cells were transformed into a decidualized morphology and produced significant amounts of prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1). Simultaneously, the activity and expression levels of PLD1 increased. In addition, removal of 8-Br-cAMP from decidualized ES cells restored the undifferentiated state, and this was accompanied by decreases in PLD1 promoter activity and PLD1 expression. Overexpression of dominant negative (DN)-PLD1 inhibited the morphological changes induced by 0.5 mM 8-Br-cAMP, whereas PLD1 overexpression induced morphological changes in the absence of 0.5 mM 8-Br-cAMP treatment. Moreover, knockdown of PLD1 by siRNA and blockage of PLD by treatment with 0.3% 1-butanol decreased PRL/IGFBP1 mRNA expression, whereas PLD1 overexpression increased PRL/IGFBP1 mRNA expression. Treatment of ES cells with phosphatidic acid (PA) for 3 days induced PRL mRNA expression and morphological changes, which implies that PA is an end-product of PLD activation-induced decidualization. In addition, pretreatment of ES cells with mepacrine decreased PRL/IGFBP1 expression and inhibited morphological change, whereas pretreatment with propranolol caused no changes, as compared to cAMP-treated cells, which suggests that PA induces decidualization through phospholipase A2 (PLA2G1B). Taken together, these results suggest that PLD1 regulates 8-Br-cAMP-induced decidualization through PLA2G1B, and that PLD1 upregulation is essential for the decidualization of ES cells.     

Profound architectural and functional readjustments of the secretory pathway in decidualization of endometrial stromal cells

Certain cell types must expand their exocytic pathway to guarantee efficiency and fidelity of protein secretion. A spectacular case is offered by decidualizing human endometrial stromal cells (EnSCs). In the midluteal phase of the menstrual cycle, progesterone stimulation induces proliferating EnSCs to differentiate into professional secretors releasing proteins essential for efficient blastocyst implantation. Here, we describe the architectural rearrangements of the secretory pathway of a human EnSC line (TERT-immortalized human endometrial stromal cells (T-HESC)). As in primary cells, decidualization entails proliferation arrest and the coordinated expansion of the entire secretory pathway without detectable activation of unfolded protein response (UPR) pathways. Decidualization proceeds also in the absence of ascorbic acid, an essential cofactor for collagen biogenesis, despite also the secretion of some proteins whose folding does not depend on vitamin C is impaired. However, even in these conditions, no overt UPR induction can be detected. Morphometric analyses reveal that the exocytic pathway does not increase relatively to the volume of the cell. Thus, differently from other cell types, abundant production is guaranteed by a coordinated increase of the cell size following arrest of proliferation.     

Inhibition of cAMP-mediated decidualization in human endometrial stromal cells by IL-1beta and laminin.

Both IL-1 and laminin are reported to inhibit progesterone receptor-mediated decidualization signals in endometrial stromal cells, but the effects of these two inhibitors on cAMP-mediated decidualization signals remain unknown. Furthermore, the interaction between IL-1-stimulated and laminin-stimulated signals has not been analyzed yet. In this study, interactions between these two decidualization-inhibitory signals and their effects on 8-Br-cAMP-mediated decidualization in human endometrial stromal cells were investigated. Prolactin release from decidualized stromal cells was measured by enzyme immunoassay in order to quantitate in vitro decidualization. Both IL-1beta and laminin were found to inhibit 8-Br-cAMP-induced decidualization in vitro in human endometrial stromal cells. IL-1beta dose-dependently inhibited decidualization of stromal cells cultured on both laminin-coated and collagen-coated dishes, while laminin inhibited the decidualization of stromal cells cultured both with and without IL-1beta. These results indicate that IL-1beta and laminin additively and mutually inhibit cAMP-mediated decidualization signals, and that they may inhibit these signals through different mechanisms.     

Laminin decreases PRL and IGFBP-1 expression during in vitro decidualization of human endometrial stromal cells

The expression of laminin, a major constituent of endometrial cell basement membranes, is increased during differentiation of human endometrial stromal cells (decidualization). To determine whether laminin plays a role in decidualization, we studied the effects of laminin substrate on the synthesis and release of prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1), two major secretory proteins of decidualized stromal cells. Endometrial stromal cells were plated on laminin as well as several other extracellular matrix (ECM) proteins (types 1 and IV collagen or fibronectin) and on plastic, and cultured in media containing medroxyprogesterone acetate (MPA) and estradiol. Cells cultured on plastic or ECM proteins displayed similar morphological changes indicative of decidualization. However, the release of PRL and IGFBP-1 from cells cultured on plastic and ECM proteins (types 1 and IV collagen and fibronection) was approximately 2.1-fold and 2.8-fold greater respectively, than from cells cultured on laminin. The decrease in PRL and IGFBP-1 expression in cells cultured on laminin was not due to differences in initial cell attachment efficiency or final DNA content. In addition, laminin had no effect on the content of laminin protein or fibronectin mRNA levels, indicating that the effects of laminin on PRL and IGFBP-1 were specific. PGE₂ stimulated the release of PRL and IGFBP-1 from cells cultured on laminin to levels comparable to those from cells cultured on plastic or other ECM proteins. This indicates that the decrease in PRL and IGFBP-1 release by laminin was not due to a generalized unresponsiveness. In contrast to the effects of laminin during decidualization, PRL expression was not altered by laminin in terminally differentiated decidual cells isolated at term. Our results support a role for laminin in selectively regulating PRL and IGFBP-1 gene expression during in vitro decidualization of human endometrial stromal cells. © 1995 Wiley-Liss, Inc.     

Leukemia inhibitory factor enhances endometrial stromal cell decidualization in humans and mice

Adequate differentiation or decidualization of endometrial stromal cells (ESC) is critical for successful pregnancy in humans and rodents. Here, we investigated the role of leukemia inhibitory factor (LIF) in human and murine decidualization. Ex vivo human (H) ESC decidualization was induced by estrogen (E, 10⁻⁸ M) plus medroxyprogesterone acetate (MPA, 10⁻⁷ M). Exogenous LIF (≥50 ng/ml) induced STAT3 phosphorylation in non-decidualized and decidualized HESC and enhanced E+MPA-induced decidualization (measured by PRL secretion, P<0.05). LIF mRNA in HESC was down-regulated by decidualization treatment (E+MPA) whereas LIF receptor (R) mRNA was up-regulated, suggesting that the decidualization stimulus ‘primed’ HESC for LIF action, but that factors not present in our in vitro model were required to induce LIF expression. Ex vivo first trimester decidual biopsies secreted >100 pg/mg G-CSF, IL6, IL8, and MCP1. Decidualized HESC secreted IL6, IL8, IL15 and MCP1. LIF (50 ng/ml) up-regulated IL6 and IL15 (P<0.05) secretion in decidualized HESC compared to 0.5 ng/ml LIF. In murine endometrium, LIF and LIFR immunolocalized to decidualized stromal cells on day 5 of gestation (day 0 = day of plug detection). Western blotting confirmed that LIF and the LIFR were up-regulated in intra-implantation sites compared to inter-implantation sites on Day 5 of gestation. To determine the role of LIF during in vivo murine decidualization, intra-peritoneal injections of a long-acting LIF antagonist (PEGLA; 900 or 1200 µg) were given just post-attachment, during the initiation of decidualization on day 4. PEGLA treatment reduced implantation site decidual area (P<0.05) and desmin staining immuno-intensity (P<0.05) compared to control on day 6 of gestation. This study demonstrated that LIF was an important regulator of decidualization in humans and mice and data provides insight into the processes underlying decidualization, which are important for understanding implantation and placentation.     

Decreased NDRG1 expression is associated with pregnancy loss in mice and attenuates the in vitro decidualization of endometrial stromal cells

Embryo implantation is an essential step for a successful pregnancy, and any defect in this process can lead to a range of pregnancy pathologies. The objective of this study was to explore the role of N‐myc downregulated gene 1 (NDRG1) in embryo implantation. It was found that uterine NDRG1 expression has a dynamic pattern during the estrous cycle in nonpregnant mice and that uterine NDRG1 expression was elevated during the implantation process in pregnant mice. The distinct accumulation of NDRG1 protein signals was observed in the primary decidual zone adjacent to the implanting embryo during early pregnancy. Furthermore, uterine NDRG1 expression could be induced by activated implantation or artificial decidualization in mice. Decreased uterine NDRG1 expression was associated with pregnancy loss in mice and was associated with recurrent miscarriages in humans. The in vitro decidualization of both mouse and human endometrial stromal cells (ESCs) was accompanied by increased NDRG1 expression and downregulated NDRG1 expression in ESCs effectively inhibited decidualization. Collectively, these data suggest that NDRG1 plays an important role in decidualization during the implantation process, and the abnormal expression of NDRG1 may be involved in pregnancy loss.     

Raf-1 levels determine the migration rate of primary endometrial stromal cells of patients with endometriosis

Endometriosis is a disease characterized by the localization of endometrial tissue outside the uterine cavity. The differences observed in migration of human endometrial stromal cells (hESC) obtained from patients with endometriosis versus healthy controls were proposed to correlate with the abnormal activation of Raf-1/ROCKII signalling pathway. To evaluate the mechanism by which Raf-1 regulates cytoskeleton reorganization and motility, we used primary eutopic (Eu-, n = 16) and ectopic (Ec-, n = 8; isolated from ovarian cysts) hESC of patients with endometriosis and endometriosis-free controls (Co-hESC, n = 14). Raf-1 siRNA knockdown in Co- and Eu-hESC resulted in contraction and decreased migration versus siRNA controls. This phenotype was reversed following the re-expression of Raf-1 in these cells. Lowest Raf-1 levels in Ec-hESC were associated with hyperactivated ROCKII and ezrin/radixin/moesin (E/R/M), impaired migration and a contracted phenotype similar to Raf-1 knockdown in Co- and Eu-hESC. We further show that the mechanism by which Raf-1 mediates migration in hESC includes direct myosin light chain phosphatase (MYPT1) phosphorylation and regulation of the levels of E/R/M, paxillin, MYPT1 and myosin light chain (MLC) phosphorylation indirectly via the hyperactivation of ROCKII kinase. Furthermore, we suggest that in contrast to Co-and Eu-hESC, where the cellular Raf-1 levels regulate the rate of migration, the low cellular Raf-1 content in Ec-hESC, might ensure their restricted migration by preserving the contracted cellular phenotype. In conclusion, our findings suggest that cellular levels of Raf-1 adjust the threshold of hESC migration in endometriosis.     

Dehydroepiandrosterone inhibits glucose flux through the pentose phosphate pathway in human and mouse endometrial stromal cells, preventing decidualization and implantation

Endometrial stromal cells (ESC) must undergo a hormone-driven differentiation to form decidual cells as a requirement of proper embryo implantation. Recent studies from our laboratory have demonstrated that decidualizing cells require glucose transporter 1 expression and an increase in glucose use to complete this step. The present study focuses on the glucose-dependent molecular and metabolic pathways, which are required by ESC for decidualization. Inhibition of glycolysis had no effect on decidualization. However, blockade of the pentose phosphate pathway (PPP) with pharmacologic inhibitors 6-aminonicotinamide or dehydroepiandrosterone (DHEA), and short hairpin RNA-mediated knockdown of glucose-6-phosphate dehydrogenase, the rate-limiting step in the PPP, both led to strong decreases in decidual marker expression in vitro and decreased decidualization in vivo. Additionally, the studies demonstrate that inhibition is due, at least in part, to ribose-5-phosphate depletion, because exogenous nucleoside administration restored decidualization in these cells. The finding that PPP inhibition prevents decidualization of ESC is novel and clinically important, because DHEA is an endogenous hormone produced by the adrenal glands and elevated in a high proportion of women who have polycystic ovary syndrome, the most common endocrinopathy in reproductive age women. Together, this data suggest a mechanistic link between increased DHEA levels, use of glucose via the PPP, and pregnancy loss.     

p57与同源框基因HOXA10在子宫内膜基质细胞体外蜕膜化过程中的表达

本文旨在从mRNA和蛋白水平研究子宫内膜基质细胞(endometrial stromalcell,ESC)体外蜕膜化过程中p57和同源框基因HOXA10(homeobox A10 gene)的表达变化以及HOXA10的亚细胞定位,从而推测其在蜕膜化过程中的作用。本实验联合使用0.5mmol/L8-溴-cAMP和1×10?6mol/LMPA(medroxyprogesterone acetate)作用1、2、4d(D1、D2、D4)诱导ESC发生蜕膜化,相应时间点提取mRNA和蛋白质行半定量RT-PCR和免疫印迹,同时以2%低血清培养ESC1、4d作为对照(C1、C4)。用间接免疫荧光和基因转染的方法,观察蜕膜化过程中HOXA10的亚细胞定位。结果显示:(1)蜕膜化过程中HOXA10的表达进行性下降,D2开始与对照组(C4)比较具有显著性差异(P<0.05)。(2)相反,蜕膜化过程中p57的表达进行性上升,D2开始与对照组C4比较也有显著性差异(P<0.05)。(3)低血清培养ESC1、4d后,p57和HOXA10的表达没有显著性差异(P>0.05)。(4)蜕膜化过程中HOXA10始终定位于胞核,不发生胞浆胞核穿梭。以上观察结果表明:(1)p57的高表达是ESC脱离细胞周期走向分化的因素之一。(2)HOXA10的低表达可能是p57上调的原因之一。(3)孕激素受体(progesterone receptor,PR)途径参与了促进ESC脱离细胞周期而走向分化的过程。     

Ubiquitin is associated with the survival of ectopic stromal cells in endometriosis

Background  

Endometriosis is a condition that affects women of reproductive age, where the glandular and/or stromal tissues from the eutopic endometrium implant in ectopic locations. It is well established that the survival of ectopic implants is due to lower levels of apoptosis, but no consensus exists as to which pathway/s this is mediated by. The ubiquitin protein shares a similar sequence homology to an anti-apoptotic protein called BAG-1 and is expressed in the normal endometrium. Currently, no studies have been conducted to determine ubiquitin expression and its possible anti-apoptotic effects in endometriosis.     

Cytokine array analysis of peritoneal fluid between women with endometriosis of different stages and those without endometriosis

Cytokines are key mediators of intercellular communication and are likely to promote the development and progression of endometriosis. Previous studies provided evidence that endometriosis develops as a result of the pathogenetic factors in the peritoneal environment, especially the peritoneal fluid (PF). We determined different cytokine expression in peritoneal fluid between women with minimal/mild and moderate/severe endometriosis and those without endometriosis using the cytokine array. As a result, 78 cytokines were found to have a threefold change, including 74 increases and four decreases in endometriosis compared with the control group; 96 cytokines had a threefold change including 91 increases and five decreases in minimal and mild endometriosis compared with the control group; 83 cytokines had a threefold change including 14 increases and 69 decreases in moderate and severe endometriosis compared with minimal and mild endometriosis. The cytokine networks were produced by Pathway Studio software and revealed that most cytokines are involved in cell binding, interaction and protein synthesis and transportation regulation. Among them activin A, Smad7 and β-nerve growth factor are the most interesting as they may be involved in the pathogenesis of endometriosis. These results suggest that cytokines are very important factors in the development of endometriosis. The findings of differentially expressed cytokines improves our knowledge of the pathogenesis and development of endometriosis and these findings warrant further studies to develop potential targets for the diagnosis and treatment of endometriosis.