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Human trophoblast stem cells (hTSCs) and related trophoblast organoids are state-of-the-art culture systems that facilitate the study of trophoblast development and human placentation. Using single-cell transcriptomics, we evaluate how organoids derived from freshly isolated first-trimester trophoblasts or from established hTSC cell lines reproduce developmental cell trajectories and transcriptional regulatory processes defined in vivo. Although organoids from primary trophoblasts and hTSCs overall model trophoblast differentiation with accuracy, specific features related to trophoblast composition, trophoblast differentiation, and transcriptional drivers of trophoblast development show levels of misalignment. This is best illustrated by the identification of an expanded progenitor state in stem cell-derived organoids that is nearly absent in vivo and transcriptionally shares both villous cytotrophoblast and extravillous trophoblast characteristics. Together, this work provides a comprehensive resource that identifies strengths and limitations of current trophoblast organoid platforms., Competing Interests: Declaration of interests The authors declare that no competing interests exist., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Placental insufficiency disorders are major obstetric complications that share a common phenomenon of poor placental trophoblast cell invasion and remodeling of uterine tissues. Myostatin is a transforming growth factor (TGF)-β superfamily member well known for its important role in muscle growth control. Myostatin is also produced in the placenta and has been shown to regulate some trophoblast functions. However, its roles in placental development are still poorly understood. In this study, we tested the hypothesis that myostatin increases trophoblast cell invasion by upregulating N-cadherin via SMAD2/3-SMAD4 signaling. Primary and immortalized (HTR8/SVneo) trophoblast cells were used as study models. Matrigel-coated transwell invasion assays were used to study the effects of recombinant human myostatin on trophoblast cell invasion. Reverse transcription quantitative real-time polymerase chain reaction and Western blot were used to measure myostatin effects on N-cadherin mRNA and protein levels, respectively. Small inhibitor molecules as well as siRNA-mediated knockdown were used to block myostatin receptor and downstream signaling, respectively. Data were analyzed either by unpaired Student T test or one-way analysis of variance followed by Newman Keuls test for multiple group comparisons. Myostatin significantly increased primary and HTR8/SVneo trophoblast cell invasion. Moreover, myostatin upregulated N-cadherin mRNA and protein levels in a time-dependent manner in both study models. These effects were blocked by inhibition of TGF-β type I receptors as well as siRNA-mediated knockdown of SMAD2/3 combined or common SMAD4. Importantly, myostatin-induced trophoblast cell invasion was abolished by knockdown of N-cadherin, SMAD2/3, or SMAD4. Myostatin may increase human trophoblast cell invasion by upregulating N-cadherin via SMAD2/3-SMAD4 signaling., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The interplay between growth factors, signaling pathways and transcription factors during placental development is key to controlling trophoblast differentiation. Bone morphogenetic protein 2 (BMP2) has been implicated in trophoblast invasion and spiral artery remodeling during early placental development. However, the molecular mechanisms by which these are accomplished have not been fully elucidated, particularly for transcriptional regulation of key transcription factors. Here, we identified SOX4 as a direct target gene induced by BMP2 in first-trimester placental trophoblasts. Analysis of single-cell RNA-seq data from first-trimester placentas and decidua tissues revealed that SOX4 expression is mainly localized in extravillous trophoblast and decidual stromal cells. Moreover, gain- and loss-of-function approaches demonstrated that SOX4 exerts a pro-invasive role in human trophoblasts, and this effect contributes to BMP2-enhanced trophoblast invasion. Importantly, we found that SOX4 was required for BMP2-induced regulation of a subset of genes associated with cell migration and extracellular matrix organization. We also show that SOX4-dependent regulation of the BMP2 target SERPINE2 occurs via binding of SOX4 to regulatory elements such as enhancers, thereby promoting BMP2-induced trophoblast invasion. In conclusion, these findings uncover a novel mechanism involving SOX4 that shapes the BMP2-regulated transcriptional network during invasive trophoblast development., (© 2021 Federation of American Societies for Experimental Biology.)

Following embryo implantation, extravillous trophoblasts (EVTs) invade the maternal decidua to a certain extent during early pregnancy, which is critical for normal placentation and successful pregnancy in humans. Although sharing a similar protein structure, the transforming growth factor-β (TGF-β) superfamily members exert divergent functions in regulating EVT invasion, which contributes to a relative balance of TGF-β superfamily proteins in precisely modulating this process at the maternal-fetal interface during the first trimester of pregnancy. This review details recent advances in our understanding of the functions of TGF-β superfamily members and their corresponding receptors, signaling pathways, and downstream molecular targets in regulating human EVT invasion from studies using various in vitro or ex vivo experimental models. Also, the relevance of these discoveries about TGF-β superfamily members to adverse pregnancy outcomes is summarized. The application of 3D culture trophoblast organoids, single-cell sequencing, and microfluidic assays in EVT invasion studies will help better reveal the molecular mechanisms through which TGF-β superfamily members regulate human EVT invasion, shedding light on the development of innovative strategies for predicting, diagnosing, treating, and preventing adverse human pregnancy outcomes related to EVT invasion dysfunction., Competing Interests: Declaration of Interests We declare that none of the authors have any conflicts of interest with regard to the content of this review., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Activin A promotes human trophoblast invasion during the first trimester of pregnancy and is associated with preeclampsia and pregnancy-induced hypertension (PE/PIH) in naturally conceived pregnancies. However, whether integrin β1 mediates activin A-increased trophoblast invasion remains unknown and the evidence is limited regarding the predictive value of activin A for PE/PIH in women receiving in vitro fertilization (IVF) treatment. Here, we studied the role and underlying molecular mechanisms of integrin β1 in activin A-promoted invasion in immortalized (HTR8/SVneo) and primary human extravillous trophoblast (EVT) cells. A nest case-control study was designed to investigate the predictive/diagnostic value of activin A in IVF pregnancies. Results showed that integrin β1 expression increased after activin A treatment and knockdown of integrin β1 significantly decreased both basal and activin A-increased HTR8/SVneo cell invasion. SB431542 (TGF-β type I receptors inhibitor) abolished activin A-induced SMAD2/SMAD3 phosphorylation and integrin β1 overexpression. Activin A-upregulated integrin β1 expression was attenuated after the depletion of ALK4 or SMAD4 in both HTR8/SVneo and primary EVT cells. Furthermore, we found similar first-trimester activin A levels in IVF patients with or without subsequent PE/PIH. These results reveal that integrin β1 mediates activin A-promoted trophoblast invasion through ALK4-activated SMAD2/3-SMAD4 pathway, and the predictive/diagnostic value of first-trimester maternal serum activin A for hypertensive disorders of pregnancy might be different in IVF population., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

In mammals, bone morphogenetic protein 2 (BMP2) is a critical regulator of endometrial decidualization and early implantation. Insulin-like growth factor-binding protein 3 (IGFBP3) is highly expressed in the endometrium and at the maternal-fetal interface in multiple species, including humans. BMP2-induced IGFBP3 signaling has been confirmed to have a role in trophoblast cell invasion; however, the involvement of this signaling pathway in endometrial remodeling remains poorly understood. To determine the roles of BMP2 in regulating IGFBP3 expression during the transformation of endometrial stromal cells, we employed immortalized human endometrial stromal cells (HESCs) and primary human decidual stromal cells (HDSCs) as study models. We showed that BMP2 significantly increased the expression of IGFBP3 in a dose- and time-dependent manner in both HESCs and primary HDSCs. Additionally, the BMP2-induced upregulation of IGFBP3 is mediated by the inhibitor of DNA-binding 1 (ID1), and knockdown of ALK3 completely abolished BMP2-induced upregulation of ID1. Moreover, BMP2 increased the expression of matrix metalloproteinases 2 (MMP2) and promoted cell migration in HESCs and primary HDSCs. Knockdown of either IGFBP3 or ID1 significantly suppressed the basal and the BMP2-induced increase in MMP2 expression as well as the cell migration in both cell models. These data demonstrated that BMP2 upregulated the expression of ID1, which in turn induced the expression of IGFBP3, and these BMP2-induced cell activities were most likely mediated by the ALK3 type I receptor. The increased expression of IGFBP3 promoted the MMP2 expression and cell migration in both HESCs and HDSCs. These findings deepen our understanding of a newly identified mechanism by which BMP2 and IGFBP3 regulate endometrial remodeling in humans, which provides insight into potential therapies for endometrium-related diseases and pregnancy-induced complications., (© 2020 Federation of American Societies for Experimental Biology.)

Neuropeptide FF (NPFF) is well-known for its roles in the central nervous system. Despite studies demonstrating that NPFF receptor 2 (NPFFR2) mRNA is highest in placenta, nothing is known about NPFF-NPFFR2 functions in placental development. Here, we investigated the effects of NPFF-NPFFR2 on expression of syncytial [human chorionic gonadotropin (hCG) β] and fusogenic [syncytin 1, syncytin 2, and glial cells missing 1 (GCM1)] genes in first trimester primary human cytotrophoblast cells. By analyzing two publicly available microarray data sets, we found that NPFF is consistently expressed throughout gestation whereas NPFFR2 increases in first trimester and is elevated in placenta samples from women with preeclampsia. Immunohistochemistry showed that NPFFR2, syncytin 1/2, and GCM1 each displayed unique patterns of expression among different trophoblast populations in first trimester placenta. Treatment of primary human cytotrophoblast cells with NPFF increased the mRNA and protein levels of hCG β, syncytin 1, syncytin 2, and GCM1; and knockdown of NPFFR2 abolished these effects. Interestingly, GCM1 mediated NPFF-induced upregulation of syncytin 1 and syncytin 2, but not hCG β, in primary human cytotrophoblasts. Our results demonstrate that NPFF acts via NPFFR2 to enhance production of hCG β and promote GCM1-dependent expression of syncytin 1 and 2 in human cytotrophoblasts., (© 2020 Federation of American Societies for Experimental Biology.)

Both bone morphogenetic protein 2 (BMP2) and WNT/β-catenin signaling promote human trophoblast cell invasion. BMP2 has been shown to up-regulate bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) in granulosa cells. Besides, studies indicate BAMBI is a positive regulator for WNT/β-catenin signaling. However, whether BMP2 can increase BAMBI expression to induce WNT/β-catenin signaling for trophoblast cell invasion is still unknown. To study the roles of BAMBI in BMP2-induced activation of WNT/β-catenin signaling and human trophoblast invasion, we used immortalized human extravillous trophoblast (EVT) cell line (HTR8/SVneo) and primary human EVT cells as study models. Messenger RNA and protein levels of target genes were checked with RT-qPCR and Western blot assay respectively. The function of target proteins was studied via small interfering RNA (siRNA)-mediated knockdown. In addition, trophoblast cell invasiveness was examined by matrigel-coated transwell assays. Our results demonstrate that BMP2 treatment increased BAMBI mRNA levels and the activation of WNT/β-catenin signaling including the raised phosphorylation of GSK3β, the up-regulation of active (non-phosphorylated) β-catenin as well as its downstream target molecule cyclin D1, all of which were totally blocked by the activin receptor-like kinases (ALK) 2/3 inhibitor DMH1 or siRNA-mediated knockdown of BAMBI in HTR8/SVneo cells. Consistently, in primary human EVT cells, BMP2 also induced the up-regulation of BAMBI and the activation of WNT/β-catenin signaling indicated by the increased levels of active β-catenin and cyclin D1, which were completely blocked by BAMBI knockdown. In conclusion, BMP2 promotes the activation of canonical WNT/β-catenin signaling and human trophoblast cell invasion by up-regulating BAMBI., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Extravillous cytotrophoblasts (EVTs) invade into the uterine wall and remodel spiral arteries for proper placentation. Studies from us and others have demonstrated that the transforming growth factor-β superfamily member bone morphogenetic protein 2 (BMP2) plays important roles in endometrial decidualization and trophoblast cell invasion. However, BMP2 has also been shown to regulate endothelial cell migration and capillary-like tube formation, as has its downstream signaling molecule inhibitor of DNA binding 1 (ID1). Interestingly, insulin-like growth factor binding protein 3 (IGFBP3) also promotes cell migration and angiogenesis in endothelial precursor cell. Moreover, Id1 has a regulatory effect on Igfbp3 expression in rat prostate epithelial cells. However, whether ID1 and IGFBP3 are integrated in BMP2 signaling and involved in the regulation of trophoblast invasive and endovascular differentiation remains unknown. The objective of our study was to examine the effects of BMP2 on ID1 and IGFBP3 expression and their roles in BMP2-regulated human trophoblast invasion and endothelial-like tube formation. Primary and immortalized (HTR8/SVneo) cultures of human trophoblast cells were employed as study models. BMP2 treatment increased ID1 and IGFBP3 mRNA and protein levels in HTR8/SVneo and primary human EVT cells. Intriguingly, ID1 was essential for BMP2-induced IGFBP3 upregulation in both study models, and BMP2-induced trophoblast invasion was attenuated by knockdown of either ID1 or IGFBP3. In addition, BMP2 significantly increased endothelial-like tube formation and knockdown of ID1 and IGFBP3 reduced basal and BMP2-induced tube formation in HTR8/SVneo cells. Similarly, BMP2 increased placenta growth factor (PlGF) production in HTR8/SVneo cells and these effects were attenuated by knockdown of ID1 or IGFBP3. Our results reveal that BMP2 promotes trophoblast cell invasion and endothelial-like tube formation by ID1-mediated IGFBP3 upregulation., (© 2020 Federation of American Societies for Experimental Biology.)

Introduction: Enhanced expression and activity of cyclooxygenase-2 (COX-2) has been reported in trophoblasts from women with preeclampsia compared with healthy pregnant women. Prostaglandin E2, the production of which is initiated by COX-2, and transforming growth factor-β1 (TGF-β1) have been shown to inhibit human trophoblast cell invasion. However, it is not known if TGF-β1 induces COX-2 in human trophoblast cells or whether COX-2 mediates the suppressive effects of TGF-β1 on trophoblast invasion., Methods: The effects of TGF-β1 on COX-2 expression were examined in the HTR-8/SVneo human trophoblast cells. The expression levels of mRNA and protein were examined by RT-qPCR and western blot, respectively. Pharmacological inhibitor and siRNA were applied to investigate the underlying molecular mechanisms. Cell invasiveness was examined by the Matrigel invasion assay., Results: Treatment with TGF-β1 induces both mRNA and protein levels of COX-2. Application of type I TGF-β receptor inhibitor SB431542 abolishes the stimulatory effects of TGF-β1 on COX-2 expression. Using an siRNA-mediated gene silencing approach, we demonstrate that activation of SMAD2/3 signaling is involved in the induction of COX-2 expression by TGF-β1. Furthermore, invasion assay shows that disruption of COX-2 expression or activity attenuates TGF-β1-inhibited cell invasion., Discussion: This study indicates that TGF-β1 upregulates COX-2 expression by activating SMAD2/3-SMAD4 signaling, and elevated COX-2 subsequently contributes to the suppression of human trophoblast cell invasion by TGF-β1. Clinically, targeting TGF-β1/COX-2 could be a useful therapeutic strategy for the treatment of placental disorders., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Bone morphogenetic protein (BMP) 2 and activin A belong to the TGF-β superfamily and are highly expressed in human endometrium and placenta. Studies have demonstrated that activin A and BMP2 play essential roles in the process of early embryo implantation by promoting human trophoblast cell invasion. However, whether activin A production can be regulated by BMP2 in human trophoblast cells remains unknown. The aim of our study was to determine the effects of BMP2 on activin A production and its role in human trophoblast invasion. Primary human extravillous trophoblast (EVT) cells were used as study models. BMP2 treatment significantly increased inhibin βA (INHBA) mRNA levels and activin A production without altering inhibin α and inhibin βB levels. BMP2-induced EVT cell invasion was attenuated by knockdown of INHBA. The increased INHBA transcription and activin A production by BMP2 were blocked by the type I receptor activin receptor (ACVR)-like kinase 2 (ALK2) and activin receptor-like kinase 3 (ALK3) inhibitor dorsomorphin homolog 1 (DMH-1). BMP2-induced INHBA upregulation was also inhibited by knockdown of type I receptor ALK3 or combined knockdown of type II receptors for BMP2 (BMPR2) and ACVR2A. Whereas BMP2 initiated both canonical SMAD1/5/8 and noncanonical SMAD2/3 signaling, only knockdown of SMAD4, but not SMAD2 and SMAD3, abolished the effects of BMP2 on INHBA. Our results show that BMP2 increases human trophoblast invasion by upregulating INHBA and activin A production via ALK3-BMPR2/ACVR2A-SMAD1/5/8-SMAD4 signaling.

Cadherins are transmembrane proteins that mediate cell-cell adhesion by promoting the formation of adherens junctions. The regulated expression of cadherins is thought to play important roles in both normal and diseased placental development. Cadherin-11, also known as OB-cadherin, is expressed in human placenta and has been shown to be involved in regulation of trophoblast cell differentiation. We have demonstrated that transforming growth factor-beta1 (TGF-β1) promotes human trophoblast cell differentiation. In addition, cadherin-11 can be up-regulated by TGF-β1 treatment. However, the underlying molecular mechanisms that mediate TGF-β1-induced cadherin-11 expression remain unknown. In this study, we demonstrate that TGF-β1 up-regulates cadherin-11 expression in human trophoblast cells. TGF-β1 treatment activates SMAD2/3 signaling pathways. Knockdown of SMAD2 or SMAD3 attenuates the stimulatory effect of TGF-β1 on cadherin-11 expression. In addition, the transcription factors, Snail and Slug, are up-regulated by the TGF-β1 treatment. Interestingly, only knockdown of Snail abolishes the TGF-β1-induced up-regulation of cadherin-11 expression. Our results suggest that TGFβ1-SMAD2/3-Snail signaling could contribute to the human trophoblast cell differentiation by up-regulating cadherin-11 expression., (Copyright © 2017 Elsevier Inc. All rights reserved.)

BMP2 expression is spatiotemporally correlated with embryo implantation and is crucial for endometrial decidualization and fertility in mice. BMP2 has been reported to increase the mesenchymal adhesion molecule N-cadherin and enhance cell invasion in cancer cells; moreover, studies suggest that N-cadherin promotes placental trophoblast invasion. However, whether BMP2 can promote trophoblast cell invasion during placentation remains unknown. The objective of our study was to investigate the effects of BMP2 on human trophoblast cell invasion and the involvement of N-cadherin and SMAD signaling. Primary and immortalized (HTR8/SVneo) cultures of human extravillous trophoblast (EVT) cells were used as study models. Treatment with recombinant human BMP2 increased HTR8/SVneo cell transwell Matrigel invasion as well as N-cadherin mRNA and protein levels, but had no significant effect on cell proliferation. Likewise, BMP2 treatment enhanced primary human EVT cell invasion and N-cadherin production. Basal and BMP2-induced invasion were attenuated by small interfering RNA-mediated downregulation of N-cadherin in both HTR8/SVneo and primary EVT cells. Intriguingly, BMP2 induced the phosphorylation/activation of both canonical SMAD1/5/8 and non-canonical SMAD2/3 signaling in HTR8/SVneo and primary EVT cells. Knockdown of SMAD2/3 or common SMAD4 totally abolished the effects of BMP2 on N-cadherin upregulation in HTR8/SVneo cells. Upregulation of SMAD2/3 phosphorylation and N-cadherin were totally abolished by type I receptor activin receptor-like kinases 2/3 (ALK2/3) inhibitor DMH1; moreover, knockdown of ALK2 or ALK3 inhibited N-cadherin upregulation. Interestingly, activation of SMAD2/3 and upregulation of N-cadherin were partially attenuated by ALK4/5/7 inhibitor SB431542 or knockdown of ALK4, but not ALK5. Our results show that BMP2 promotes trophoblast cell invasion by upregulating N-cadherin via non-canonical ALK2/3/4-SMAD2/3-SMAD4 signaling.

Appropriate trophoblast invasion into the maternal endometrium is essential for successful human implantation and placentation. Connective tissue growth factor (CTGF), also known as CCN2, is a matricellular protein that is expressed in the placenta. Interestingly, the CTGF expression levels in the placenta and serum from patients with severe preeclampsia or fetal growth restriction are higher than those from healthy controls. However, to date, the role of CTGF in the regulation of trophoblast cell invasion remains unclear. Transforming growth factor-β1 (TGF-β1) is a potent stimulator of CTGF expression and has been shown to inhibit trophoblast cell invasiveness. However, whether CTGF mediates TGF-β1-inhibited human trophoblast cell invasion is unknown. In the present study, we show that treatment with TGF-β1 upregulates CTGF expression in a human trophoblast cell line, HTR-8/SVneo, and in primary human trophoblast cells. Our results also demonstrate that the SMAD2/3 signaling pathways are required for TGF-β1-induced upregulation of CTGF. Importantly, CTGF knockdown attenuates TGF-β1-inhibited cell invasion. Furthermore, cell invasiveness is decreased by treatment with recombinant CTGF. These results provide evidence that CTGF mediates TGF-β1-inhibited human trophoblast cell invasion., (Copyright © 2017 Endocrine Society.)

Study Hypothesis: We hypothesized that Runt-related transcription factor 2 (RUNX2), matrix metalloproteinase (MMP)2 and MMP9 are involved in basal and gonadotrophin-releasing hormone (GnRH)-induced human extravillous trophoblast (EVT) cell invasion., Study Finding: Our finding indicates that GnRH-induced RUNX2 expression enhances the invasive capacity of EVT cells by modulating the expression of MMP2 and MMP9., What Is Known Already: GnRH is expressed in first-trimester placenta and exerts pro-invasive effects on EVT cells in vitro. RUNX2 regulates MMP2 and MMP9 expression and is often associated with invasive phenotypes., Study Design, Samples/materials, Methods: First-trimester human placenta (n = 9) was obtained from women undergoing elective termination of pregnancy. The localization of RUNX2, MMP2 and MMP9 in first-trimester human placenta was examined by immunohistochemistry. Primary or immortalized (HTR-8/SVneo) EVT cells were treated alone or in combination with GnRH, GnRH antagonist Antide, MAPK kinase inhibitor PD98095, phosphatidylinositol 3-kinase inhibitor LY294002, MMP2/9 inhibitor or small interfering RNAs (siRNAs) targeting RUNX2, MMP2 and/or MMP9. Protein and mRNA levels were measured by western blot and RT-PCR, respectively. Cell invasiveness was evaluated by transwell Matrigel or collagen I invasion assays., Main Results and the Role of Chance: RUNX2, MMP2 and MMP9 were detected in the cell column regions of human first-trimester placental villi. GnRH treatment increased RUNX2 mRNA and protein levels in HTR-8/SVneo cells and primary EVTs, and these effects were attenuated by co-treatment with Antide, PD98095 or LY294002. Down-regulation of RUNX2 by siRNA reduced basal and GnRH-induced MMP2/9 expression and cell invasion. Moreover, pharmacological inhibition or siRNA-mediated knockdown of MMP2/9 reduced basal and GnRH-induced cell invasion., Limitations, Reasons for Caution: The lack of an in vivo model is the major limitation of our in vitro study., Wider Implications of the Findings: Our findings provide important insight into the functions of the GnRH - GnRH receptor system in early implantation and placentation., Large Scale Data: Not applicable., Study Funding and Competing Interests: This research was supported by Canadian Institutes of Health Research (Grant #143317) to P.C.K.L. The authors have nothing to disclose., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Context: Activin A increases matrix metalloproteinase (MMP) 2 expression and cell invasion in human trophoblasts, but whether the expression of MMP2 is essential for the proinvasive effect of activin A has yet to be determined. Moreover, the identity of the activin receptor-like kinase (ALK; TGF-β type I receptors) and downstream transcription factors (eg, SNAIL and SLUG) mediating the effects of activin on MMP2 expression and trophoblast cell invasion remains unknown., Objective: To elucidate the role of MMP2 in activin A-induced human trophoblast cell invasion as well as the involvement of ALK4 and SNAIL., Design: HTR8/SVneo immortalized human extravillous cytotrophoblast (EVT) cells and primary cultures of human first-trimester EVT cells were used as study models. Small interfering RNA (siRNA)-mediated knockdown approaches were used to investigate the molecular determinants of activin A-mediated functions., Main Outcome Measures: Levels of mRNA and protein were examined by reverse transcription-quantitative real-time PCR and Western blot, respectively. Cell invasiveness was measured by Matrigel-coated transwell assays., Results: Treatment of HTR8/SVneo cells with activin A increased the production of SNAIL, SLUG, and MMP2 without altering that of MMP9, TIMP1, TIMP2, TWIST, RUNX2, ZEB1, or ZEB2. Similarly, activin A up-regulated the mRNA and protein levels of SNAIL and MMP2 in primary EVT cells. Knockdown of SNAIL attenuated activin A-induced MMP2 up-regulation in HTR8/SVneo and primary EVT cells. In HTR8/SVneo cells, activin A-induced production of SNAIL and MMP2 was abolished by pretreatment with the TGF-β type I receptor (ALK4/5/7) inhibitor SB431542 or siRNA targeting ALK4, SMAD2/3, or common SMAD4. Likewise, knockdown of ALK4 or SMAD4 abolished the stimulatory effects of activin A on SNAIL and MMP2 expression in primary EVT cells. Importantly, activin A-induced HTR8/SVneo and primary EVT cell invasion were attenuated by siRNA-mediated depletion of ALK4 or MMP2., Conclusion: Activin A induces human trophoblast cell invasion by inducing SNAIL-mediated MMP2 expression through ALK4 in a SMAD2/3-SMAD4-dependent manner.

Remodeling of maternal spiral arteries during pregnancy requires a subpopulation of extravillous cytotrophoblasts (EVTs) to differentiate into endovascular EVTs. Activin A, which is abundantly expressed at the maternal-fetal interface, has been shown to promote trophoblast invasion, but its role in endovascular differentiation remains unknown. Vascular endothelial growth factor-A (VEGF-A) is well recognized as a key regulator in trophoblast endovascular differentiation. Whether and how activin A might regulate VEGF-A production in human trophoblasts and its relationship to endovascular differentiation have yet to be determined. In the present study, we found that activin A increased VEGF-A production in primary and immortalized (HTR8/SVneo) human EVT cells. In addition, activin A enhanced HTR8/SVneo endothelial-like tube formation, and these effects were attenuated by pretreatment with small interfering RNA targeting VEGF-A or the VEGF receptor 1/2 inhibitor SU4312. Pretreatment with the activin/TGF-β type 1 receptor (ALK4/5/7) inhibitor SB431542 abolished the stimulatory effects of activin A on phosphorylated mothers against decapentaplegic (SMAD)-2/3 phosphorylation, VEGF-A production, and endothelial-like tube formation. Moreover, small interfering RNA-mediated down-regulation of SMAD2, SMAD3, or common SMAD4 abolished the effects of activin A on VEGF-A production and endothelial-like tube formation. In conclusion, activin A may promote human trophoblast cell endothelial-like tube formation by up-regulating VEGF-A production in an SMAD2/3-SMAD4-dependent manner. These findings provide insight into the cellular and molecular events regulated by activin A during human implantation.

Connexin43 (Cx43)-mediated gap junctional intercellular communication (GJIC) are required for human trophoblast differentiation. To date, whether Cx43 mediates TGF-β1-induced trophoblast differentiation has not been determined. We showed that treatment with TGF-β1 increased Cx43 expression and GJIC in HTR-8/SVneo human trophoblast cells. In addition, Smad and ERK1/2 signaling pathways were involved in TGF-β1-induced up-regulation of Cx43. Moreover, TGF-β1 increased the expression of the syncytiotrophoblast marker, β-hCG. Importantly, knockdown of Cx43 abolished the TGF-β1-induced up-regulation of β-hCG. Furthermore, overexpression of Cx43 up-regulated β-hCG expression. These results provide evidence that Cx43 and GJIC activity are up-regulated by TGF-β1 in human trophoblast cells, which subsequently contributes to TGF-β1-induced trophoblast differentiation., (© 2015 Wiley Periodicals, Inc.)

GnRH is expressed in first-trimester human placenta and increases cell invasion in extravillous cytotrophoblasts (EVTs). Invasive phenotypes have been reported to be regulated by transcription factor activator protein 1 (AP-1) and mesenchymal cadherin-11. The aim of our study was to investigate the roles of AP-1 components (c-FOS/c-JUN) and cadherin-11 in GnRH-induced cell invasion in human EVT cells. Phosphorylated c-FOS and phosphorylated c-JUN were detected in the cell column regions of human first-trimester placental villi by immunohistochemistry. GnRH treatment increased c-FOS, c-JUN, and cadherin-11 mRNA and protein levels in immortalized EVT (HTR-8/SVneo) cells. Moreover, GnRH treatment induced c-FOS and c-JUN protein phosphorylation and nuclear accumulation. Pretreatment with antide, a GnRH antagonist, attenuated GnRH-induced cadherin-11 expression. Importantly, basal and GnRH-induced cadherin-11 expression and cell invasion were reduced by small interfering RNA-mediated knockdown of c-FOS, c-JUN, and cadherin-11 in HTR-8/SVneo cells. Our results suggest that GnRH induces the expression and phosphorylation of the AP-1 transcription factors c-FOS and c-JUN in trophoblast cells, which contributes to GnRH-induced elevation of cadherin-11 expression and cell invasion.

Context: GnRH and its receptor, GnRHR, were shown to promote trophoblastic cell invasion. Detection of elevated early development-related transcription factor TWIST and adhesion molecule N-cadherin in the invasive trophoblastic cells could suggest that GnRH promotes trophoblastic cell invasion through TWIST-regulated N-cadherin pathway., Objective: This study sought to investigate the regulatory effect of GnRH on TWIST and N-cadherin expression as well as invasiveness in human trophoblastic cells., Design: The expression of GnRHR, TWIST, and N-cadherin was first examined in human first-trimester chorionic villi by immunohistochemistry. Expression levels of GnRHR, TWIST, and N-cadherin were tested in primary extravillous trophoblastic (EVT) cells and an immortalized EVT cell line HTR-8/SVneo cells with incubation of GnRH and its antagonist, Antide. Small interfering RNA strategy was used to study the roles of TWIST and N-cadherin in basal- and GnRH-regulated trophoblast invasion. Matrigel-mediated transwell invasion assays were employed to assess cell invasion capacity., Results: GnRHR, TWIST, and N-cadherin were detected at the invasive site of first-trimester human placenta. GnRH treatment significantly increased TWIST and N-cadherin expression in primary EVT as well as HTR-8/SVneo cells. Pretreatment with the GnRH receptor antagonist Antide attenuated the effects of GnRH on TWIST and N-cadherin expression. Invasive capacity of primary EVT and HTR-8/SVneo cell was reduced following siRNA-mediated knockdown of either TWIST or N-cadherin. Furthermore, by knocking down endogenous TWIST, the expression level of N-cadherin was reduced as well as GnRH-induced HTR-8/SVneo cell invasion. Treatment with GnRH induces AKT phosphorylation and Phosphoinositide3-kinase inhibitor LY294002 attenuates the effects of GnRH on TWIST and N-cadherin expression and trophoblastic cell invasion., Conclusion: Our results suggest that GnRH acts via its receptor to induce AKT phosphorylation, which contributes to elevated TWIST expression. Increased levels of TWIST subsequently induce N-cadherin expression, which promotes human trophoblastic cell invasion in vitro.

Context: Inhibin β-subunits and activin receptors are expressed by human trophoblast cells. Although activin A has been shown to enhance human trophoblast cell invasion, whether two additional activin isoforms, activin B and AB, exert similar effects remains unknown. Moreover, whether the expression of mesenchymal adhesion molecule neural cadherin (N-cadherin) is essential for this proinvasive effect of activin has yet to be determined., Objective: To examine the effects of all three activin isoforms on human trophoblast cell invasion and the involvement of N-cadherin., Design: HTR8/SVneo immortalized extravillous cytotrophoblast cells and primary cultures of human extravillous cytotrophoblast cells were used as study models. Small interfering RNA-mediated knockdown approaches were used to investigate the molecular determinants of activin-mediated functions., Setting: An academic research center., Main Outcome Measures: Reverse transcription-quantitative real-time PCR and Western blot analysis were used to examine mRNA and protein levels, respectively. Cell invasiveness was assessed by Matrigel-coated transwell assays., Results: All three activin isoforms produced comparable increases in HTR8/SVneo cell invasion as well as N-cadherin expression. In addition, the up-regulatory effect of activin isoforms on N-cadherin was confirmed in primary cultures of human trophoblast cells. Interestingly, small interfering RNA-mediated down-regulation of N-cadherin attenuated basal and activin-induced invasion of both HTR8/SVneo and primary trophoblast cells. All three activin isoforms induced equivalent phosphorylation of SMAD2 and SMAD3. Importantly, activin-stimulated cell invasion, up-regulation of N-cadherin, as well as activation of SMAD2/SMAD3 were abolished by the TGF-β type I receptor inhibitor SB431542 in HTR8/SVneo cells. Furthermore, knockdown of SMAD2/3 or common SMAD4 abolished the stimulatory effects of all three activin isoforms on N-cadherin expression., Conclusion: Activin A, B, and AB produce comparable increases in human trophoblast cell invasion by up-regulating N-cadherin expression in a SMAD2/3-SMAD4-dependent manner.

Human trophoblast cells express transforming growth factor-β (TGF-β) and TGF-β receptors. It has been shown that TGF-β1 treatment decreases the invasiveness of trophoblast cells. However, the molecular mechanisms underlying TGF-β1-decreased trophoblast invasion are still not fully understood. In the current study, we demonstrated that treatment of HTR-8/SVneo human trophoblast cells with TGF-β1 decreased cell invasion and down-regulated the expression of vascular endothelial cadherin (VE-cadherin). In addition, the inhibitory effect of TGF-β1 on VE-cadherin was confirmed in primary cultures of human trophoblast cells. Moreover, knockdown of VE-cadherin using siRNA decreased the invasiveness of HTR-8/SVneo cells and primary cultures of trophoblast cells. Treatment with TGF-β1 induced the activation of Smad-dependent signaling pathways and the expression of Snail and Slug. Knockdown of Smads attenuated TGF-β1-induced up-regulation of Snail and Slug and down-regulation of VE-cadherin. Interestingly, depletion of Snail, but not Slug, attenuated TGF-β1-induced down-regulation of VE-cadherin. Furthermore, overexpression of Snail suppressed VE-cadherin expression. Chromatin immunoprecipitation analyses showed the direct binding of Snail to the VE-cadherin promoter. These results provide evidence that Snail mediates TGF-β1-induced down-regulation of VE-cadherin, which subsequently contributed to TGF-β1-decreased trophoblast cell invasion.

The invasion of extravillous cytotrophoblasts (EVT) into the underlying maternal tissues and vasculature is a key step in human placentation. The molecular mechanisms involved in the development of the invasive phenotype of EVT include many that were first discovered for their role in cancer cell metastasis. Previous studies have demonstrated that N-cadherin and its regulatory transcription factor Twist play important roles in the onset and progression of cancers, but their roles in human trophoblastic cell invasion is not clear. The goal of the study was to examine the role of Twist and N-cadherin in human trophoblastic cell invasion. Twist and N-cadherin mRNA and protein levels were determined by RT-PCR and Western blotting in human placental tissues, highly invasive EVT, and poorly invasive JEG-3 and BeWo cells. Whether IL-1β and TGF-β1 regulate Twist mRNA and protein levels in the EVT was also examined. A small interfering RNA strategy was employed to determine the role of Twist and N-cadherin in HTR-8/SVneo cell invasion. Matrigel assays were used to assess cell invasion. Twist and N-cadherin were highly expressed in EVT but were poorly expressed in JEG-3 and BeWo cells. IL-1β and TGF-β1 differentially regulated Twist expression in EVT in a time- and concentration-dependent manner. Small interfering RNA specific for Twist decreased N-cadherin and reduced invasion of HTR-8/SVneo cells. Similarly, a reduction in N-cadherin decreased the invasive capacity of HTR-8/SVneo cells. Twist is an upstream regulator of N-cadherin-mediated invasion of human trophoblastic cells.

Context: The formation of the multinucleated syncytiotrophoblast of the human placenta by the terminal differentiation and fusion of mononucleate cytotrophoblasts is a critical step in pregnancy. Previous studies have demonstrated that this cellular event is dependent on a progressive decrease in the levels of the cell-adhesion molecule, E-cadherin., Objective: The aim of the study was to examine the role of Twist, a transcription factor identified as a key repressor of E-cadherin expression, in the differentiation of human trophoblastic cells., Design: The expression of Twist or E-cadherin were first examined in first-trimester chorionic villi by immunohistochemistry. Gain- or loss-of-function studies on Twist were then performed in BeWo choriocarcinoma cells. The presence or absence of multinucleated syncytium was confirmed by indirect immunofluorescence using antibodies directed against Twist, E-cadherin, or desmoplakin, a cellular marker of mononucleate cytotrophoblasts., Results: The formation of multinucleated syncytium was associated with increased Twist and a decreased E-cadherin expression. Similarly, exogenous expression of Twist resulted in a continuous and progressive decrease in E-cadherin expression and the subsequent formation of syncytium in BeWo cells maintained under normal culture conditions. In contrast, small interfering RNA specific for Twist inhibited 8-Br-cAMP (8-bromoadenosine 3',5'-cyclic monophosphate)-mediated differentiation and fusion of over time in culture., Conclusions: Twist is an upstream regulator of the E-cadherin-mediated terminal differentiation and fusion in a human trophoblastic cell line in vitro.

Metastatic carcinoma cells exploit the same molecular machinery that allows human placental cytotrophoblasts to develop an invasive phenotype. As altered expression levels of ADAMTS (ADisintegrin And Metalloproteinase with ThromboSpondin repeats) subtypes have been associated with cancer progression, we have examined the function and regulation of members of this gene family in epithelial cell invasion using cultures of highly invasive extravillous cytotrophoblasts and the poorly invasive JEG-3 cytotrophoblast cell line as model systems. Of the multiple ADAMTS subtypes identified in first trimester human placenta and these two trophoblastic cell types, only ADAMTS-12 was preferentially expressed by extravillous cytotrophoblasts. Transforming growth factor-β1 and interleukin-1β, two cytokines that promote and restrain cytotrophoblast invasion in vitro, were also found to differentially regulate trophoblastic ADAMTS-12 mRNA levels. Loss- or gain-of-function studies confirmed that ADAMTS-12, independent of its proteolytic activity, plays a specific, non-redundant role in trophoblast invasion. Furthermore, we demonstrated that ADAMTS-12 regulated cell-extracellular matrix adhesion and invasion through a mechanism involving the αvβ3 integrin heterodimer. This study identifies a novel biological role for ADAMTS-12, and highlights the importance and complexity of its non-proteolytic domain(s) pertaining to its function.

The potential roles of GnRH I and GnRH II have been assigned in promoting the invasive capacity of human trophoblasts by regulating matrix metalloproteinases-2 and -9, type I tissue inhibitor of matrix metalloproteinase, and urokinase plasminogen activator/plasminogen activator inhibitor protease systems during human placentation, and GnRH II has been shown to be more potent than GnRH I. However, the mechanisms for the differential effects of these two hormones remain unclear. In this study, we examined the invasion-promoting effects and the signaling pathways of GnRH I and GnRH II in human trophoblasts. The data revealed that both GnRH I and GnRH II were key autocrine and/or paracrine regulators in facilitating trophoblast invasion. The GnRH receptor antagonist (Antide) and specific small interfering RNA for GnRH receptor inhibited the regulatory effects of GnRH I, but not GnRH II, on trophoblast invasion. Both GnRH I and II activated protein kinase C, ERK1/2, and c-Jun N-terminal kinase to mediate their effects on trophoblast invasion, whereas only GnRH II elicited invasion-promoting action through transactivating the tyrosine kinase activity of epidermal growth factor receptor in trophoblasts. Our observations elucidate a ligand-dependent selective cross-communication between GnRH receptor and epidermal growth factor receptor signaling systems in human trophoblastic cell, and this would further our understanding on the differentially biological significance of these two forms of GnRH in extrapituitary tissues.

An intricate balance between the production of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs), modulates the overall proteolytic activity of trophoblasts during human implantation. In these studies we have examined the ability of classical GnRH I and the second form of this hormone (GnRH II) to regulate MMP-2, MMP-9, and TIMP-1 mRNA and protein levels in extravillous cytotrophoblasts propagated from explants of first trimester chorionic villi. GnRH I and GnRH II were found to increase MMP-2 and MMP-9 mRNA and protein levels in these primary cell cultures in a dose- and time-dependent manner using quantitative competitive-PCR and ELISA. In contrast, these two hormones decreased trophoblastic TIMP-1 mRNA and protein levels. Cetrorelix, a GnRH receptor antagonist, inhibited the regulatory effects of GnRH I, but not GnRH II, on MMP-2, MMP-9, and TIMP-1 expression in these cells. Collectively, these observations suggest that GnRH I and GnRH II differentially regulate MMP-2, MMP-9, and TIMP-1 expression in human trophoblasts, possibly via distinct receptor-mediated intracellular signaling pathways.

Activin is known to play an important regulatory role in reproduction, including pregnancy. To further examine the role and signaling mechanism of activin in regulating placental function, the steady-state level of activin type I receptor (ActRI) mRNA in immortalized extravillous trophoblasts (IEVT) cells was measured using competitive PCR (cPCR). An internal standard of ActRI cDNA for cPCR was constructed for the quantification of ActRI mRNA levels in IEVT cells. ActRI mRNA levels were increased in a dose-dependent manner by activin-A with the maximal effect observed at the dose of 10 ng/ml. Time course studies revealed that activin-A had maximal effects on ActRI mRNA levels at 6 hours after treatment. The effects of activin-A on ActRI mRNA levels was blocked by follistatin, an activin binding protein, in a dose-dependent manner. In addition, inhibin-A inhibited basal, as well as activin-A-induced ActRI mRNA levels. These findings provide evidence, for the first time, that activin-A modulates ActRI mRNA levels in human trophoblast cells.

The regulated expression of the urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor (PAI-1) is believed to modulate the invasive capacity of human trophoblastic cells in vitro and in vivo. To date, the factors capable of regulating the expression of uPA and PAI-1 in these cells remain poorly characterized. In these studies, we have examined the ability of the classical mammalian GnRH I and the second form of GnRH (GnRH II) to regulate uPA and PAI-1 mRNA and protein expression levels in primary cultures of human extravillous cytotrophoblasts using quantitative competitive PCR and ELISA, respectively. Both GnRH I and II increased uPA and concomitantly decreased PAI-1 mRNA and protein expression levels in our extravillous cytotrophoblast cultures in a dose- and time-dependent manner. Cetrorelix, a peptide GnRH antagonist specific for the GnRH I receptor, was capable of inhibiting the regulatory effects of GnRH I, but not GnRH II on uPA and PAI-1 expression levels in primary cell cultures. Taken together, these observations suggest that GnRH I and GnRH II may facilitate trophoblast invasion by increasing the ratio of uPA/PAI-1 expression via interactions with two distinct GnRH receptors.