Gene Expression In Trophoblast Cells Research Articles

Extracellular vesicles as mediators of stress response in embryo-maternal communication.

Introduction: The pivotal role of extracellular vesicles (EVs) in facilitating effective communication between the embryo and maternal cells during the preimplantation stage of pregnancy has been extensively explored. Nonetheless, inquiries persist regarding the alterations in EV cargo from endometrial cells under stress conditions and its potential to elicit specific stress responses in trophoblast cells. Thus, the aim of this study was to elucidate the involvement of EV miRNA miRNAs in transmitting stress signals from maternal cells to trophoblasts. Methods: The receptive endometrial epithelium analogue RL95-2 cells were subjected to stress induction with 200µM CoCl2 for 24h before EV isolation. JAr trophoblast spheroids, which serve as embryos, were subjected to treatment with stressed or unstressed EVs derived from RL95-2 cells for 24h. Transcriptomic alterations in the treated JAr spheroids as well as in the untreated group, as a negative control, were investigated by mRNA sequencing. Furthermore, the changes in EV miRNAs were assessed by sequencing EV samples. Results: A comprehensive analysis comparing the miRNA profiles between stressed and unstressed EVs revealed significant changes in 25 miRNAs. Furthermore, transcriptomic analysis of JAr spheroids treated with stressed RL95-2EVs versus unstressed EVs or the untreated group demonstrated 6 and 27 differentially expressed genes, respectively. Pathway enrichment analysis showed that stressed EVs induce alterations in gene expression in trophoblast cells, which is partially mediated by EV microRNAs. Discussion: Our results suggest that EVs can transfer stress signals from endometrial cells to the embryo. These discoveries shed new light on the mechanism underlying implantation failures under stress conditions. Unraveling the role of EVs in transmitting stress signals, can extend our knowledge to pave the way for targeted interventions to manage stress-related implantation failures.

GATA factors: Master regulators of gene expression in trophoblast progenitors

Mammalian reproduction is critically dependent on trophoblast cells, which ensure embryo implantation and placentation. Development of trophoblast cell lineages is a multi-step process and relies upon proper spatial and temporal gene expression, which is regulated by multiple transcription factors. However, most of the transcription factors that are implicated in trophoblast development regulate gene expression at a specific developmental stage or in a specific trophoblast subtype. In contrast, recent studies from our group and other laboratories indicate that conserved GATA family of transcription factors, GATA2 and GATA3, are important to regulate gene expression at multiple stages of trophoblast development. Furthermore, our conditional gene deletion studies revealed that functional redundancy of GATA2 and GATA3 ensures both self-renewal of trophoblast stem and progenitor cells and their differentiation to trophoblast cells of a matured placenta. Together these findings indicate that GATA2/GATA3 are the master orchestrators of gene expression in trophoblast cells and they fine tune gene regulatory network to establish distinct trophoblast cell types during placentation.

Nrf2 signalling and autophagy are involved in diabetes mellitus-induced defects in the development of mouse placenta.

It is widely accepted that diabetes mellitus impairs placental development, but the mechanism by which the disease operates to impair development remains controversial. In this study, we demonstrated that pregestational diabetes mellitus (PGDM)-induced defects in placental development in mice are mainly characterized by the changes of morphological structure of placenta. The alteration of differentiation-related gene expressions in trophoblast cells rather than cell proliferation/apoptosis is responsible for the phenotypes found in mouse placenta. Meanwhile, excess reactive oxygen species (ROS) production and activated nuclear factor erythroid2-related factor 2 (Nrf2) signalling were observed in the placenta of mice suffering from PGDM. Using BeWo cells, we also demonstrated that excess ROS was produced and Nrf2 signalling molecules were activated in settings characterized by a high concentration of glucose. More interestingly, differentiation-related gene expressions in trophoblast cells were altered when endogenous Nrf2 expression is manipulated by transfecting Nrf2-wt or Nrf2-shRNA. In addition, PGDM interferes with autophagy in both mouse placenta and BeWo cells, implying that autophagy is also involved, directly or indirectly, in PGDM-induced placental phenotypes. Therefore, we revealed that dysfunctional oxidative stress-activated Nrf2 signalling and autophagy are probably responsible for PGDM-induced defects in the placental development of mice. The mechanism was through the interference with differentiation-related gene expression in trophoblast cells.

StarD7 Gene Expression in Trophoblast Cells: Contribution of SF-1 and Wnt-β-Catenin Signaling

Steroidogenic acute regulatory protein-related lipid transfer domain containing 7 (StarD7) is a poorly characterized member of the steroidogenic acute regulatory protein-related lipid transfer proteins, up-regulated in JEG-3 cells, involved in intracellular transport and metabolism of lipids. Previous studies dealing with the mechanisms underlying the human StarD7 gene expression led us to define the cis-acting regulatory sequences in the StarD7 promoter using as a model JEG-3 cells. These include a functional T cell-specific transcription factor 4 (TCF4) site involved in Wnt-β-catenin signaling. To understand these mechanisms in more depth, we examined the steroidogenic factor 1 (SF-1) contribution to StarD7 expression. Cotransfection experiments in JEG-3 cells point out that the StarD7 promoter is activated by SF-1, and this effect is increased by forskolin. EMSA using JEG-3 nuclear proteins demonstrated that SF-1 binds to the StarD7 promoter. Additionally, chromatin immunoprecipitation analysis indicated that SF-1 and β-catenin are bound in vivo to the StarD7 promoter. Reporter gene assays in combination with mutations in the SF-1 and TCF4 binding sites revealed that the StarD7 promoter is synergistically activated by SF-1 and β-catenin and that the TCF4 binding site (-614/-608) plays an important role in this activation. SF-1 amino acid mutations involved in the physical interaction with β-catenin abolished this activation; thus demonstrating that the contact between the two proteins is necessary for an efficient StarD7 transcriptional induction. Finally, these data suggest that β-catenin could function as a bridge between SF-1 and TCF4 forming a ternary complex, which would stimulate StarD7 expression. The SF-1 and β-catenin pathway convergence on StarD7 expression may have important implications in the phospholipid uptake and transport, contributing to the normal trophoblast development.

Histone Deacetylases Inhibit IFN-γ-Inducible Gene Expression in Mouse Trophoblast Cells

Trophoblast cells are the first cells to differentiate from the developing mammalian embryo, and they subsequently form the blastocyst-derived component of the placenta. IFN-gamma plays critical roles in activating innate and adaptive immunity, as well as apoptosis. In mice, IFN-gamma is produced in the pregnant uterus, and is essential for formation of the decidual layer of the placenta and remodeling of the uterine vasculature. Responses of mouse trophoblast cells to IFN-gamma appear to be selective, for IFN-gamma activates MHC class I expression and enhances phagocytosis, but fails to activate either MHC class II expression or apoptosis in these cells. To investigate the molecular basis for the selective IFN-gamma responsiveness of mouse trophoblast cells, IFN-gamma-inducible gene expression was examined in the trophoblast cell lines SM9 and M-11, trophoblast stem cells, and trophoblast stem cell-derived giant cells. IFN-gamma-inducible expression of multiple genes, including IFN regulatory factor-1 (IRF-1), was significantly reduced in trophoblast cells compared with fibroblast cells. Decreased IRF-1 mRNA expression in trophoblast cells was due to a reduced rate of IRF-1 transcription relative to fibroblast cells. However, no impairment of STAT-1 tyrosine phosphorylation or DNA-binding capacity was observed in IFN-gamma-treated mouse trophoblast cells. Importantly, histone deacetylase (HDAC) inhibitors significantly enhanced IFN-gamma-inducible gene expression in trophoblast cells, but not fibroblasts. Our collective studies demonstrate that IFN-gamma-inducible gene expression is repressed in mouse trophoblast cells by HDACs. We propose that HDAC-mediated inhibition of IFN-gamma-inducible gene expression in mouse trophoblast cells may contribute to successful pregnancy by preventing activation of IFN-gamma responses that might otherwise facilitate the destruction of the placenta.

Leptin Expression in Primary Trophoblast Cells in Response to Incubation with the Serum of Preeclamptic Women

Objective. We investigated whether the increase of leptin expression in preeclamptic placentas is additionally influenced by soluble maternal factors under hypoxic and nonhypoxic conditions. Methods. Term trophoblast cells were isolated and stimulated with sera from preeclamptic women under normoxic or hypoxic conditions. Levels of leptin mRNA and protein were evaluated by real-time RT-PCR or ELISA and Western blot analysis. Results. Leptin concentrations were increased in the serum of patients with preeclampsia and gestational diabetes. Hypoxia, insulin, and dexamethasone induced leptin expression in trophoblast cells. The incubation with sera from preeclamptic women led to a small, though, significant, increase of leptin gene expression. The effect of preeclamptic serum on leptin gene expression in trophoblast cells was lost under hypoxia. The serum of women with gestational diabetes did not increase leptin expression neither in normoxic nor hypoxic primary trophoblast cells. Conclusion. Our results can not exclude a soluble maternal factor in the serum of women with preeclampsia accounting for increased leptin expression in placental tissue in addition to hypoxia. However, an important biological role of this small increase in nonhypoxic conditions does not seem very likely.

Regulation of Gene Expression in Mouse Trophoblast Cells by Interferon-gamma

We have previously shown that interferon-gamma (IFN-γ) activates phagocytosis and induces nitric oxide production in cultured mouse trophoblast cells. In the present study we examined the effect of this cytokine on ectoplacental cone and gene expression in trophoblast cells. Ectoplacental cones were obtained during the postimplantation period on gestational day 7.5 from CD-1 mice and exposed to 100 U/mL IFN-γ. Ectoplacental cone morphology, cell proliferation and death were also determined upon IFN-γ treatment. Complementary DNA macroarray and semiquantitative RT-PCR were used to analyze gene expression. IFN-γ treatment did not alter ectoplacental cone morphology, trophoblast cell proliferation or death. However, using gene array technology, we observed that IFN-γ affected the developing trophoblast, altering the level of mRNA expression, which resulted in upregulation of 35 genes and downregulation of seven others. The upregulation of transcription factors and immune response-associated genes suggests that IFN-γ is involved in processes beyond immunological homeostasis and plays an important role in placental development and function.

DNA Methylation-dependent Epigenetic Regulation of Dimethylarginine Dimethylaminohydrolase 2 Gene in Trophoblast Cell Lineage

Trophoblast cell lineage is established through the first cellular differentiation in mammalian embryogenesis, and its developmental potential is restricted to the extraembryonic tissues contributing solely to the placenta. Several lines of evidence suggest a relative lack of importance of DNA methylation in gene regulation in the extraembryonic tissues when compared with embryonic ones. Here we analyzed the dynamics of epigenetic status in the upstream region of mouse Ddah2 gene, which was found to be specifically repressed in a stem cell population of trophoblast cell lineage. We found a tissue-dependent differentially methylated region in the regulatory region of the Ddah2 gene. This region was hypermethylated in trophoblast stem cells and was hypomethylated in differentiated cells both in vivo and in vitro. This change was well correlated with Ddah2 expression. In addition, in vitro methylation confined to the differentially methylated region was sufficient to repress promoter activity in the reporter assay. Furthermore, a repressive pattern of histone modifications was formed around the differentially methylated region in undifferentiated trophoblast stem cells with repressed Ddah2. Our data suggest that DNA methylation-mediated chromatin remodeling is involved in the regulation of the Ddah2 gene expression and thus is important even in trophoblast cell lineage.

Regulation of major histocompatibility complex class II gene expression in trophoblast cells

Trophoblast cells are unique because they are one of the few mammalian cell types that do not express major histocompatibility complex (MHC) class II antigens, either constitutively or after exposure to IFN-γ. The absence of MHC class II antigen expression on trophoblast cells has been postulated to be one of the essential mechanisms by which the semi-allogeneic fetus evades immune rejection reactions by the maternal immune system. Consistent with this hypothesis, trophoblast cells from the placentas of women suffering from chronic inflammation of unknown etiology and spontaneous recurrent miscarriages have been reported to aberrantly express MHC class II antigens. The lack of MHC class II antigen expression on trophoblast cells is due to silencing of expression of the class II transactivator (CIITA), a transacting factor that is essential for constitutive and IFN-γ-inducible MHC class II gene transcription. Transfection of trophoblast cells with CIITA expression vectors activates both MHC class II and class Ia antigen expression, which confers on trophoblast cells both the ability to activate helper T cells, and sensitivity to lysis by cytotoxic T lymphocytes. Collectively, these studies strongly suggest that stringent silencing of CIITA (and therefore MHC class II) gene expression in trophoblast cells is critical for the prevention of immune rejection responses against the fetus by the maternal immune system. The focus of this review is to summarize studies examining the novel mechanisms by which CIITA is silenced in trophoblast cells. The elucidation of the silencing of CIITA in trophoblast cells may shed light on how the semi-allogeneic fetus evades immune rejection by the maternal immune system during pregnancy.

GCMa Regulates the Syncytin-mediated Trophoblastic Fusion

The human placental trophoblast cell can be classified as either a cytotrophoblast or a syncytiotrophoblast. Cytotrophoblasts can function as stem cells for the development of the syncytiotrophoblast layer via cell fusion. An envelope gene of the human endogenous retrovirus family W (HERV-W) called syncytin is specifically expressed in the syncytiotrophoblast layer. Syncytin is a fusogenic membrane protein; therefore, it can mediate the fusion of cytotrophoblasts into the syncytiotrophoblast layer, which is essential for pregnancy maintenance. GCMa is a placenta-specific transcription factor and is required for placental development. To study the placenta-specific fusion mediated by syncytin, we tested whether GCMa is involved in this process by regulating syncytin gene expression. In this report, we demonstrate that GCMa was able to regulate syncytin gene expression via two GCMa-binding sites upstream of the 5'-long terminal repeat of the syncytin-harboring HERV-W family member in BeWo and JEG3 cells but not in HeLa cells. Furthermore, adenovirus-directed expression of GCMa enhanced syncytin gene expression and syncytin-mediated cell fusion in BeWo and JEG3 cells but not in HeLa cells. Therefore, the integration site of the syncytin-harboring HERV-W family member in the human genome is close to the functional GCMa-binding sites by which GCMa can specifically transactivate syncytin gene expression in trophoblast cells. Our results may help to explain the mechanism underlying the cell fusion event specific for syncytiotrophoblast formation.

Differential display analysis of oxygen-mediated changes in gene expression in first trimester human trophoblast cells

Physiologic or pathologically induced periods of exposure to relatively low levels of oxygen during pregnancy affect the expression and function of certain genes in the placenta. In this study, the differential display technique was utilized to identify genes that are regulated in cultured cytotrophoblast cells by exposure to low levels of oxygen. Using this approach, four genes, which have been designated HRF-1, HRF-2, HRF-6, and HRF-8, were cloned and partially characterized. Northern blot analysis showed that clones HRF-1 and HRF-2 were downregulated in response to exposure to low levels of oxygen, whereas expression of HRF-6 and HRF-8 was increased. DNA sequencing and sequence analysis revealed that HRF-1 may represent an alternatively spliced or tissue-specific form of the Krüppel family zinc finger protein znfp104 gene. Clone HRF-2 showed a high degree of identity with exons 9, 10 and 11 of N33, a gene that is located within a homozygously deleted region of metastatic prostate cancer. Clones HRF-6 and HRF-8 did not exhibit significant sequence identity with known sequences in GenBank and may represent novel genes. None of these genes have previously been shown to be present in trophoblast cells, nor have their expressions been shown to be regulated by oxygen. This study demonstrates that the differential display technique is a novel and effective method to analyse oxygen-mediated changes in gene expression in trophoblast cells.

Hypoxia-mediated downregulation of human chorionic gonadotropin subunit mRNA expression in cytotrophoblastic JAr cells

Summary Oxygen tension plays a role in regulating placental differentiation and function. To begin to understand the mechanisms of hypoxia-mediated gene expression in trophoblast cells we investigated the regulation of hCG in cytotrophoblastic JAr cells cultured at low oxygen tension. We demonstrated that constitutive and inducible secretion of the hormone are respressed at the level of mRNA abundancy of both subunit genes, α and β. Transcripts, however, which encode the glycolytic enzyme GAPDH increase in the hypoxic environment indicating adaptation to the anaerobic conditions. Repression of hormone mRNAs may involve modulations in activating signal transduction pathways resulting in downregulation of transcriptional activity. Subtle changes in cellular cAMP are detectable, which, however, do not explain downregulation of α/β hCG mRNA expression at low oxygen supply. Furthermore, elevation of cAMP cannot overcome hypoxic suppression, suggesting that a cAMP/protein kinase-A-mediated signal transduction pathway is not involved. Although we detected IL-1- and hypoxia-dependent induction of IL-6 mRNA, IL-6 secretion is reduced in the hypoxic environment. IL-1-inducible hCG expression, therefore might be affected by changes in IL-6/IL-6 receptor-dependent tyrosine phosphorylation. Actinomycin-D mRNA stability assays revealed that α and βhCG mRNA half-lives do not decline in the hypoxic environment, suggesting that the decrease in transcriptional activities of both subunit genes could be the major cause of reduced hCG expression at low oxygen pressure.

Regulated expression of the trophoblast alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein. Differentiation and cAMP modulate protein and mRNA levels.

The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) has several ligands including activated alpha 2-macroglobulin, pregnancy zone protein, and very low density lipoproteins enriched with apolipoprotein E. The diversity of ligands suggests a role for the alpha 2MR/LRP in a variety of processes including tissue remodeling and lipoprotein metabolism. We examined alpha 2MR/LRP in placental trophoblasts, invasive cells that also function in lipid transport and cholesterol metabolism. alpha 2MR/LRP protein was localized by immunohistochemistry in the syncytiotrophoblast of term placenta. Cytotrophoblasts did not stain prominently. alpha 2MR/LRP (protein and message) in primary cultures of human trophoblast cells increased as cytotrophoblasts differentiated into syncytiotrophoblast. 8-Bromo-cAMP prevented this increase and suppressed alpha 2MR/LRP expression. The cyclic nucleotide had similar suppressive effects on alpha 2MR/LRP in BeWo choriocarcinoma cells. In contrast, low density lipoprotein receptor gene expression was increased. We conclude that: 1) there is a differentiation-dependent pattern of alpha 2MR/LRP expression in the human trophoblast; 2) cAMP negatively regulates alpha 2MR/LRP; 3) there is an inverse relationship between alpha 2MR/LRP and low density lipoprotein receptor gene expression in trophoblast cells.