G protein-coupled estrogen receptor stimulates human trophoblast cell invasion via YAP-mediated ANGPTL4 expression

Insufficient invasion of trophoblast cells into the uterine decidua is associated with preeclampsia (PE). G protein-coupled estrogen receptor (GPER) is a membrane estrogen receptor involved in non-genomic estrogen signaling. GPER is expressed in human trophoblast cells and downregulated GPER levels are noted in PE. However, to date, the role of GPER in trophoblast cells remains largely unknown. Here, we applied RNA sequencing (RNA-seq) to HTR-8/SVneo human trophoblast cells in response to G1, an agonist of GPER, and identified angiopoietin-like 4 (ANGPTL4) as a target gene of GPER. Treatment of trophoblast cells with G1 or 17β-estradiol (E2) activated Yes-associated protein (YAP), the major downstream effector of the Hippo pathway, via GPER but in a mammalian STE20-like protein kinase 1 (MST1)-independent manner. Using pharmacological inhibitors as well as loss- and gain-of-function approaches, our results revealed that YAP activation was required for GPER-stimulated ANGPTL4 expression. Transwell invasion assays demonstrated that activation of GPER-induced ANGPTL4 promoted cell invasion. In addition, the expression levels of GPER, YAP, and ANGPTL4 were downregulated in the placenta of patients with PE. Our findings reveal a mechanism by which GPER exerts its stimulatory effect on human trophoblast cell invasion by upregulating YAP-mediated ANGPTL4 expression.

Overexpression of ERAP2N in Human Trophoblast Cells Promotes Cell Death

The genes involved in implantation and placentation are tightly regulated to ensure a healthy pregnancy. The endoplasmic reticulum aminopeptidase 2 (ERAP2) gene is associated with preeclampsia (PE). Our studies have determined that an isoform of ERAP2-arginine (N), expressed in trophoblast cells (TC), significantly activates immune cells, and ERAP2N-expressing TCs are preferentially killed by both cytotoxic T lymphocytes (CTLs) and Natural Killer cells (NKCs). To understand the cause of this phenomenon, we surveyed differentially expressed genes (DEGs) between ERAP2N expressing and non-expressing TCs. Our RNAseq data revealed 581 total DEGs between the two groups. 289 genes were up-regulated, and 292 genes were down-regulated. Interestingly, most of the down-regulated genes of significance were pro-survival genes that play a crucial role in cell survival (LDHA, EGLN1, HLA-C, ITGB5, WNT7A, FN1). However, the down-regulation of these genes in ERAP2N-expressing TCs translates into a propensity for cell death. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that 64 DEGs were significantly enriched in nine pathways, including “Protein processing in endoplasmic reticulum” and “Antigen processing and presentation”, suggesting that the genes may be associated with peptide processes involved in immune recognition during the reproductive cycle.

STX2 Promotes Trophoblast Growth, Migration, and Invasion Through Activation of the PI3K-AKT Pathway in Preeclampsia

ObjectivesAbnormal trophoblast behaviors during pregnancy contribute to the development of preeclampsia (PE). Syntaxin2 (STX2) has been shown to be a crucial epithelial mediator in numerous diseases. However, the functions of STX2 and the mechanisms underlying its role in PE remain largely unknown. The aim of this study was to explore the role of STX2 on trophoblast biology and unravel the molecular mechanisms that contribute to the development and progression of PE.Materials and MethodsWe first compared the expression of STX2 in placental tissues from women with PE and women with normal pregnancies. Then, we investigated the role of STX2 on trophoblast proliferation, migration and invasion in HTR-8/SVneo and primary human trophoblast cells by loss or gain of function experiments. In addition, co-immunoprecipitation, pulldown and immunofluorescence assays were performed to investigate the co-localization of STX2 with other proteins, and to help clarify the mechanisms underlying STX2-mediated functions on trophoblasts.ResultsWe demonstrated that STX2 expression was downregulated in placental tissues of women with PE compared with those from normal pregnancies. Loss and gain of function experiments further confirmed a role for STX2 in cell proliferation, migration and invasion in trophoblasts. By co-immunoprecipitation, pulldown and immunofluorescence co-localization assays, we revealed that STX2 selectively interacted with p85, a subunit of PI3K, and directly recruited p85 to the cytomembrane, thereby activating the AKT signaling pathway. We further demonstrated that the AKT activation was abolished by the use of a PI3K inhibitor (LY294002), which negatively affected STX2-mediated functions on trophoblasts.ConclusionAll together, our findings point to a crucial role for STX2 in PE progression. Our new insights also suggest that STX2 may be a potential diagnostic tool and a novel therapeutic target for treating PE.