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.

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.

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. To examine the effects of all three activin isoforms on human trophoblast cell invasion and the involvement of N-cadherin. 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. An academic research center. 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. 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. 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.

Rationale: The identification of causal variants responsible for disease associations from genome-wide association studies (GWASs) facilitates functional understanding of the biological mechanisms by which those genetic variants influence disease susceptibility. Objective: We aim to identify causal variants in or near the FAM13A (family with sequence similarity member 13A) GWAS locus associated with chronic obstructive pulmonary disease (COPD). Methods: We used an integrated approach featuring conditional genetic analysis, massively parallel reporter assays (MPRAs), traditional reporter assays, chromatin conformation capture assays, and clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing to characterize COPD-associated regulatory variants in the FAM13A region in human bronchial epithelial cell lines. Measurements and Main Results: Conditional genetic association suggests the presence of two independent COPD association signals in FAM13A. MPRAs identified 45 regulatory variants within FAM13A, among which six variants were prioritized for further investigation. Three COPD-associated variants demonstrated significant allele-specific activity in reporter assays. One of three variants, rs2013701, was tested in the endogenous genomic context by CRISPR-based genome editing that confirmed its allele-specific effects on FAM13A expression and on cell proliferation, providing functional characterization for this COPD-associated variant. Conclusions: The human GWAS association near FAM13A may contain independent association signals. MPRAs identified multiple functional variants in this region, including rs2013701, a putative COPD-causing variant with allele-specific regulatory activity.

Bone morphogenetic protein 2 (BMP2) and activin A belong to the transforming growth factor-β (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 mRNA levels and activin A production without altering inhibin α and βB levels. BMP2-induced EVT cell invasion was attenuated by knockdown of inhibin βA. The increased inhibin βA transcription and activin A production by BMP2 were blocked by type I receptor activin receptor-like kinases 2/3 (ALK2/3) inhibitor DMH1. BMP2-induced inhibin βA up-regulation was also inhibited by knockdown of type I receptor ALK3 or combined knockdown of type II receptors BMPR2 and ACTR2A. 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 inhibin βA. Our results show that BMP2 increases human trophoblast invasion by up-regulating inhibin βA and activin A production via ALK3-BMPR2/ACVR2A-SMAD1/5/8-SMAD4 signaling.