Snail mediates GDF-8-stimulated human extravillous trophoblast cell invasion by upregulating MMP2 expression

Abstract

Background Extravillous trophoblast (EVT) cell invasion is a tightly regulated process that requires for a normal pregnancy. The epithelial-mesenchymal transition (EMT) has been implicated in EVT cell invasion. Growth differentiation factor-8 (GDF-8), a member of the transforming growth factor-beta (TGF-β) superfamily, is expressed in the human placenta and promotes EVT cell invasion by upregulating the expression of matrix metalloproteinase 2 (MMP2). However, the underlying molecular mechanism of GDF-8-induced MMP2 expression remains undetermined. Therefore, the present study aims to examine the role of Snail and Slug, the EMT-related transcriptional regulators, in GDF-8-stimulated MMP2 expression and cell invasion in HTR-8/SVneo human EVT cell line and primary cultures of human EVT cells. Methods HTR-8/SVneo and primary cultures of human EVT cells were used to examine the effect of GDF-8 on MMP2 expression and explore the underlying mechanism. For gene silencing and overexpression, the HTR-8/SVneo cell line was used to make the experiments more technically feasible. The cell invasiveness was measured by Matrigel-coated transwell invasion assay. Results GDF-8 stimulated MMP2 expression in both HTR-8/SVneo and primary EVT cells. The stimulatory effect of GDF-8 on MMP2 expression was blocked by the inhibitor of TGF-β type-I receptors, SB431542. Treatment with GDF-8 upregulated Snail and Slug expression in both HTR-8/SVneo and primary EVT cells. The stimulatory effects of GDF-8 on Snail and Slug expression were blocked by pretreatment of SB431542 and siRNA-mediated knockdown of SMAD4. Interestingly, using the siRNA knockdown approach, our results showed that Snail but not Slug was required for the GDF-8-induced MMP2 expression and cell invasion in HTR-8/SVneo cells. The reduction of MMP2 expression in the placentas with preeclampsia (PE) was also observed. Conclusions These findings discover the physiological function of GDF-8 in the human placenta and provide important insights into the regulation of MMP2 expression in human EVT cells.

Growth differentiation factor myostatin regulates epithelial‐mesenchymal transition genes and enhances invasion by increasing serine protease inhibitors E1 and E2 in human trophoblast cells

“…MSTN has previously been shown to induce the phosphorylation of receptor regulated SMAD2/3 in human trophoblast cells. [23][24][25][26] Moreover, we 23 and others 24 have shown that the invasive effects of MSTN on human trophoblast cells are mediated by ALK5 and not ALK4. Previous studies have characterized cell-type specific differential involvement of ALK4/5 in MSTN-dependent roles; MSTN preferentially binds to ALK4 in myoblast cells as opposed to binding ALK5 in nonmyogenic tissues.…”

“…This is likely related to technical and interpatient variability as well as the small sample size and duration of MSTN treatment (i.e., single time-point). For example, the rapid induction (3-6 h) of Snail and Slug mRNA by MSTN reported by Chen et al might explain why these EMT transcription factors did not appear as DEGs in our analysis at 24 h. 26 A major contributor to interpatient variability is the inability to predict pregnancy outcome of the first trimester samples used for trophoblast isolation, which could yield a mix of normal and diseased placentas. 27 Indeed, we did not have access to any of the associated clinical data (e.g., maternal age, parity, reason for pregnancy termination, history of placenta-mediated diseases) for the samples used in this study.…”

“…22 MSTN has been shown to increase trophoblast proliferation, 18 migration, 18 and Matrigel invasion. [23][24][25][26] However, very little is known about its mechanisms of action in trophoblast cells.…”

GDF-8 improves in vitro implantation and cryo-tolerance by stimulating the ALK5-SMAD2/3 signaling in bovine IVF embryo development

“…Additionally, it has been demonstrated that activating this signaling pathway stimulates the expression of MMP2 , thereby mediating the upregulation of invasive capabilities in human extra villous trophoblast cells ( Fang et al, 2021 ). Moreover, it facilitates trophoblast mobility, regulates invasion, and promotes the expression of FSTL3, thereby influencing invasive capabilities ( Xie et al, 2020 ; Fang et al, 2021 ; Chen et al, 2023 ). According to previous studies, GDF-8 binds to the receptor ALK5, activating the SMAD2/3 complex.…”