Cellular Signalling

Bone morphogenetic protein 2 induces the activation of WNT/ β-catenin signaling and human trophoblast invasion through up- regulating BAMBI

Hong-Jin Zhao, Hsun-Ming Chang, Christian Klausen, Hua Zhu, Yan Li, Peter C.K. Leung

To appear in: Cellular Signalling

Please cite this article as: H.-J. Zhao, H.-M. Chang, C. Klausen, et al., Bone morphogenetic protein 2 induces the activation of WNT/β-catenin signaling and human trophoblast invasion through up-regulating BAMBI, Cellular Signalling(2019), https://doi.org/10.1016/j.cellsig.2019.109489

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Bone morphogenetic protein 2 induces the activation of WNT/β-catenin signaling and human trophoblast invasion through up-regulating BAMBI

Hong-Jin Zhao2,3, Hsun-Ming Chang3, Christian Klausen3, Hua Zhu3, Yan Li1*, Peter C.K. Leung3*

1 School of Medicine, Shandong University, Ji’nan, P.R. China 250012; Center for Reproductive Medicine, Shandong University, Ji’nan, P.R. China
2 Shandong Provincial Hospital affiliated to Shandong University, Ji’nan, P.R. China 250021
3 Department of Obstetrics and Gynaecology, BC Children’s Hospital Research Institute, University of British Columbia, Vancouver, British Columbia, Canada V5Z 4H4 Short Title: BMP2 activates WNT/β-catenin signaling for EVT invasion

Keywords: BMP2, BAMBI, WNT/β-catenin, cyclin D1, trophoblast invasion
Address all correspondence and requests for reprints to:
Peter C.K. Leung, PhD, FCAHS, FRSC Department of Obstetrics and Gynaecology BC Children’s Hospital Research Institute University of British Columbia
Room 317, 950 West 28th Avenue
Vancouver, British Columbia, Canada, V5Z 4H4 Tel: 1-604-875-2718
Fax: 1-604-875-2717
E-mail: [email protected] OR
Yan Li, MD, PhD School of Medicine Shandong University No. 44 Wenhua Xi Road
Ji’nan, Shandong Province, China, 250012 Tel: 86-531-883-82084
Fax:86-531-883-82502
Email: [email protected]

Conflicts of Interest: The authors have nothing to disclose.Grant support: This study was supported by Foundation grant (FDN-143317) from the Canadian Institutes of Health Research to P.C.K.L. and the Fundamental Research Funds of Shandong University to Y.L.Word count: 3501 Number of figures: 6 figures+2 supplemental figures
Abstract

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.

1. Introduction

Appropriate extravillous cytotrophoblast (EVT) invasion into the uterine wall is a prerequisite for normal pregnancy. Insufficient EVT invasion leads to aberrant placenta development, contributing to several pregnancy-related complications, such as preeclampsia and intrauterine growth restriction, both of which are harmful to the health of both mothers and fetuses [1]. Thus, the research on the control of EVT invasion is of great importance. To date, a variety of signaling pathways have been linked to EVT invasion, including transforming growth factor-β (TGF-β) and WNT/β-catenin signaling pathways [2, 3].
As an important TGF-β superfamily member firstly discovered for its pivotal role in bone formation [4], BMP2 has since been shown to be expressed in many tissues including human decidual cells and primary human EVT cells [5, 6]. Moreover, BMP2 is expressed at embryonic implantation sites in mice [7]. Conditional knockout studies show that Bmp2 is essential for endometrial decidualization and fertility [8]. In vitro research has demonstrated that BMP2 promotes human trophoblast invasion through SMAD-mediated signaling [6, 9]. Like BMP2, canonical WNT/β-catenin signaling has also been reported to function in regulating bone formation and human trophoblast invasion [10, 11]. Canonical WNT proteins, including WNT1 and WNT3A, bind to seven-transmembrane frizzled receptor and a single- transmembrane co-receptor known as low density lipoprotein receptor-related protein 5/6 (LRP5/6). Receptor binding results in the phosphorylation of GSK3β, then the accumulation of active (non- phosphorylated) β-catenin in the cytoplasm and its translocation into the nucleus to cooperate with T cell factor/ lymphoid enhancer factor (TCF/LEF) in the regulation of gene expression (e.g. cyclin D1) [12, 13]. During the differentiation of rat trophoblast cells to giant cells, which are similar to human EVTs, cyclin D1 is up-regulated to allow for transition from normal mitotic cell cycle to endoreduplication cycle [14]. Similar changes in cell cycle occur during the acquisition of invasive capacity in human EVTs [15, 16], suggesting potential effects of cyclin D1 on invasive differentiation of human trophoblasts. In vitro studies with human trophoblast cell lines and/or primary EVTs show that trophoblast cell invasion is enhanced by WNT3A-induced canonical WNT signaling, but is impaired by reduced expression of cyclin D1 [11, 17, 18]. Therefore, BMP2 and WNT/β-catenin signaling play complementary roles in human

trophoblast invasion.

Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) is a transmembrane protein that is highly conserved among vertebrates [19]. It shares structural similarities with TGF-β type I receptors but lacks an intracellular serine/threonine kinase domain for signal transduction. Therefore, BAMBI often functions as an antagonist of TGF-β signaling by interfering the normal interaction between TGF-β type I and type II receptors [19]. In addition, BAMBI facilitates canonical WNT signaling during rodent myoblast cell differentiation [20]. Indeed, decreased BAMBI expression blocks the nuclear translocation of β-catenin and inhibits WNT/β-catenin signaling in gastric cancer cells and preadipocytes [21, 22]. Notably, BAMBI is overexpressed and involved in the malignancy of several cancers including osteosarcoma [23], ovarian [24] and colorectal [25] cancers. In canine kidney epithelial cells, BAMBI is up-regulated to mediate the loss of epithelial polarity induced by hypoxic-inducible factor 1 (HIF1) [26]. Intriguingly, HIF1 is also a positive regulator of HTR8/SVneo cell invasion [27], suggesting a possible link between BAMBI and HIF1-mediated human EVT invasion. BAMBI levels in human EVTs are over 2-fold higher than those in villous cytotrophoblast [28, 29], suggesting that BAMBI may be associated with trophoblast invasive differentiation. BAMBI is a target molecule of BMP2 in human granulosa cells [30], but it is unknown whether BAMBI is involved in BMP2 and/or WNT/β-catenin signaling in human trophoblast cells.
In the present study, we have examined the effect of BAMBI on the activation of canonical WNT/β- catenin signaling and human EVT cell invasion by BMP2. Our results show that BMP2 up-regulates BAMBI and activates WNT/β-catenin signaling as demonstrated by increased GSK3β phosphorylation, active β-catenin accumulation and cyclin D1 expression. Moreover, BAMBI is essential for BMP2- induced activation of WNT/β-catenin signaling as well as human trophoblast invasion.
2. Materials and Methods

2.1 Culture of HTR8/SVneo human EVT cell line

The HTR8/SVneo simian virus 40 large T antigen immortalized first trimester human EVT cell line was kindly provided by Dr. P. K. Lala (Western University, Canada) [31]. Cells were cultured in DMEM (Life Technologies) supplemented with 10% (vol/vol) fetal bovine serum (FBS), 100 U/mL penicillin and
100 μg/mL streptomycin (Life Technologies). Cultures were maintained at 37°C in a humidified atmosphere with 5% CO2 in air. All vehicle or BMP2 treatments were performed in DMEM with 0.1% FBS following 18 h of starvation in DMEM with 0.1% FBS.

2.2 Primary human EVT cell isolation and culture

The study methodologies were approved by the Research Ethics Board of the University of British Columbia and all patients provided informed written consent. The study methodologies conformed to the standards set by the Declaration of Helsinki. First-trimester human placental samples (6–8 weeks) were collected from women undergoing elective termination of pregnancy. Primary human EVT cells were isolated as previously described [32] and each primary culture was comprised of cells from only one patient (passage 2-4). Briefly, the placental villi tips were finely minced and cultured for 3-4 days in flasks with DMEM (Life Technologies) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified 5% CO2/air atmosphere. After removal of non-attached fragments, attached villous tissue fragments were cultured for another 10-14 days to allow for EVT cell outgrowth. EVT cells were separated from villous fragments by trypsinization and the purity of EVT cells was confirmed by immunocytochemical staining for cytokeratin-7 and human leukocyte antigen G (HLA-G). Only cultures showing more than 99% positive staining for cytokeratin-7 and HLA-G were used in this study. Each experiment was replicated with primary EVT cells from at least 3 different placentas. All vehicle or BMP2 treatments were performed in DMEM with 0.1% FBS following 18 h of starvation in DMEM with 0.1% FBS.
2.3 Reagents and antibodies

Recombinant human BMP2 and DMH1 were obtained from R&D Systems. Mouse monoclonal anti- cytokeratin 7 (catalog no. MAB3554) and anti-HLA-G (catalog no. 11-499-C100) were obtained from Millipore and EXBIO Praha, respectively. Mouse monoclonal anti-SNAIL (catalog no. 3895) and rabbit monoclonal anti-SLUG (catalog no. 9585), monoclonal rabbit anti-phospho-GSK-3βSer9 (catalog no. 5558), monoclonal rabbit anti-GSK-3β (catalog no. 12456), rabbit monoclonal anti-non-phosphorylated- (Active)-β-catenin Ser45 (catalog no. 19807), and mouse monoclonal anti-cyclin D1 (catalog no. 2926) were purchased from Cell Signaling Technology. Mouse monoclonal anti-β-catenin (catalog no. 610153) was purchased from BD Biosciences. Mouse monoclonal anti-α-Tubulin (catalog no. sc-23948) was obtained from Santa Cruz Biotechnology. Horseradish peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were obtained from Bio-Rad Laboratories.
2.4 Matrigel-coated transwell invasion assay

Trophoblast cell invasiveness was examined using Corning Biocoat Growth Factor Reduced Matrigel Invasion Chamber (pore size, 8 μm; catalog no. 354483) as per the guidelines for use. Briefly, cells were pre-treated with vehicle or BMP2 (25 ng/mL) for 20 minutes. Then each insert was seeded with 5×104 cells suspended in 250 μL vehicle/BMP2-containing DMEM medium supplemented with 0.1% (vol/vol) FBS and 750 μL DMEM medium with 10% (vol/vol) FBS was added to the lower chamber. After 24 h incubation, cells in each insert were retreated with vehicle or BMP2 (25 ng/mL) and incubated for a further 12 h (36 h in total). At the end of the experiment, non-invading cells were removed from the upper side of the membrane and cells on the lower side were fixed with cold methanol (-20°C) and air dried. Cell nuclei were stained with Hoechst 33258 (Sigma-Aldrich) and imaged with a fluorescent microscope followed by analysis with Image-J software. Duplicate inserts were used for each individual experiment, and five random microscopic fields were counted per insert.
2.5 Reverse transcription quantitative real-time PCR (RT-qPCR)

Total RNA was extracted with TRIzol Reagent (Life Technologies) as per the manufacturer’s instructions. Reverse transcription was carried out with 2 μg RNA, random primers, and Moloney murine leukemia virus reverse transcriptase (Promega) in a final volume of 20 μL. SYBR Green or TaqMan RT- qPCR was performed on an Applied Biosystems 7300 Real-Time PCR System equipped with 96-well optical reaction plates. Each 20 μL SYBR Green RT-qPCR reaction contained 1×SYBR Green PCR Master Mix (Applied Biosystems), 20 ng cDNA, and 250 nM of each specific primer. The primers used were: BAMBI, 5′-GGCCTCAGGACAAGGAAACAG-3′ (forward) and 5′-
CGGAACCACAACTCTTTGGAAG-3′ (reverse); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-GAGTCAACGGATTTGGTCGT-3′ (forward) and 5′- GACAAGCTTCCCGTTCTCAG-3′ (reverse).
The specificity of each assay was validated by dissociation curve analysis and agarose gel electrophoresis of PCR products. Each sample was assayed in triplicate and a mean value from at least three independent experiments was used for relative quantification of mRNA levels by the comparative Cq method with GAPDH as the reference gene and using the formula 2-ΔΔCq.

2.6 Western blot analysis

Cells were lysed in ice-cold lysis buffer (Cell Signaling Technology) with added protease inhibitor cocktail (Sigma-Aldrich). Extracts were centrifuged at 13 000 rpm for 15 minutes at 4°C and supernatant protein concentrations were determined using the DC Protein Assay (Bio-Rad Laboratories) with BSA as the standard. Equal amounts of protein were separated by standard Tris-glycine SDS-PAGE and electrotransferred to PVDF membranes. Membranes were blocked with Tris-buffered saline containing 5% (wt/vol) nonfat dry milk for 1 h and then immunoblotted overnight at 4°C with specific primary antibodies diluted in Tris-buffered saline with 5% (wt/vol) non-fat dried milk and 0.1% (vol/vol) Tween-
20. After incubation with appropriate horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature, signals were detected with enhanced chemiluminescent or SuperSignal West Femto chemiluminescent substrates (Thermo Fisher) and CL-XPosure film (Thermo Fisher). Membranes were stripped with stripping buffer (62.5mM Tris-HCl [pH 6.8], 100 mM β-mercaptoethanol, and 2% (wt/vol)

SDS) at 50°C for 30 minutes and reprobed as described above with antibodies against β-catenin. Densitometric quantification was performed using Image-Pro Plus software.
2.7 Small interfering RNA (siRNA) transfection

Cells at approximately fifty percent confluency were transfected for 48 h with 25 nM ON- TARGETplus NON-TARGETINGpool siRNA or ON-TARGETplus SMARTpool siRNA targeting human BAMBI (L-019596-00-0005; Dharmacon) using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer’s instructions. Knockdown efficiency was assessed by RT-qPCR or Western blot analysis.
2.8 Statistical analysis

Results are presented as the mean ± SEM of at least three independent experiments. Multiple group comparisons were analyzed by one-way ANOVA followed by Newman-Keuls test using PRISM software (GraphPad Software, Inc). The control in “Fold change relative to control” in the y-axis of all bar figures specifically indicates the value of the first control group in each figure. If a pair of values is significantly different (p<0.05), the values have different subscript letters (a vs. b or b vs. c) assigned to them.
3. Results

3.1 BMP2 increases BAMBI mRNA levels and the activation of WNT/β-catenin signaling in HTR8/SVneo cells
BMP2 has recently been reported to up-regulate BAMBI in human granulosa cells [30]. To examine the effects of BMP2 on BAMBI mRNA levels in human trophoblast cells, HTR8/SVneo cells were treated with 25 ng/mL BMP2 for different lengths of time (3, 6, 12 and 24 h). RT-qPCR results showed BMP2 increased BAMBI mRNA levels from 3 to 24 h (Figure 1, A). BMP2 has been shown to activate and cooperate with canonical WNT/β-catenin signaling for bone formation and pulmonary angiogenesis [33, 34]. Accordingly, we investigated the effects of BMP2 on WNT/β-catenin signaling in human

trophoblast cells. In HTR8/SVneo cells, western blot analysis showed that BMP2 treatment induced the phosphorylation of GSK3β at 6 h timepoint (Figure 1, B), increased accumulation of active β-catenin from 12 h to 24 h (Figure 1, C) and up-regulated its downstream molecule cyclin D1 protein levels from 24 h to 48 h (Figure 1, D), all of which suggest that BMP2 also facilitates canonical WNT/β-catenin signaling in HTR8/SVneo cells.
3.2 DMH1 abolishes BMP2-increased BAMBI mRNA levels and activation of WNT/β-catenin signaling in HTR8/SVneo cells
According to previous experiments, two TGF-β type I receptors, activin receptor-like kinases (ALK) 2 and/or ALK3, were generally used by BMP2 to exert its effects on human trophoblast cells [6, 9]. In this study, HTR8/SVneo cells were pre-treated with the ALK2/ALK3 inhibitor DMH1 for 1 h prior to treatment with BMP2 for a further 6 h to 24 h. As shown in Figure 2, pre-treatment with DMH1 totally blocked BMP2-induced BAMBI up-regulation (Figure 2, A), GSK3β phosphorylation (Figure 2, B and C), active β-catenin accumulation and cyclin D1 protein expression (Figure 2, D-F) in HTR8/SVneo cells, demonstrating that BMP2 increases BAMBI mRNA levels and facilitates WNT/β-catenin signaling via ALK2 and/or ALK3.
3.3 BAMBI is essential for BMP2-mediated activation of canonical WNT/β-catenin signaling in human trophoblast cells
BAMBI is a positive regulator of WNT/β-catenin signaling [20]. Thus, we used siRNA targeting BAMBI to examine its contribution to BMP2-induced WNT/β-catenin signaling. Transient transfection of HTR8/SVneo cells with siRNA targeting BAMBI for 48 h significantly suppressed BAMBI mRNA levels (Figure 3, A). Western blot results displayed that treatment of HTR8/SVneo cells with BMP2 significantly increased the levels of phosphorylated GSK3β, active β-catenin and cyclin D1, and these effects were totally abolished by BAMBI knockdown (Figure 3, B-F).

As well, we checked the effects of BMP2 on BAMBI transcription and WNT/β-catenin signaling in primary human EVT cells. Consistent with the results we observed in HTR8/SVneo cells, BMP2 treatment not only increased BAMBI mRNA levels (Figure 4, A), but also up-regulated active β-catenin accumulation and cyclin D1 protein levels (Figure 4, B and C), demonstrating the inductive effect of BMP2 on WNT/β-catenin signaling in primary human EVT cells as well. BAMBI-knockdown experiments carried out in primary EVT cells confirmed that BAMBI was essential for BMP2-induced activation of WNT/β-catenin signaling (Figure 4, D-G).
3.4 BAMBI is involved in BMP2-increased human trophoblast invasion

Next, we examined the effects of siRNA-mediated BAMBI knockdown on trophoblast invasiveness in both HTR8/SVneo and primary human EVT cells to investigate the mediation of BAMBI in BMP2- increased trophoblast invasion. Matrigel-coated transwell invasion assays showed that BMP2 treatment for 36 h significantly increased HTR8/SVneo cell invasion as we previously reported [6]. Importantly, this effect was abolished by BAMBI knockdown (Figure 5, A). Likewise, the involvement of BAMBI in BMP2-induced trophoblast invasion was further confirmed in primary human first trimester EVT cells (Figure 5, B). In summary, we demonstrated BAMBI was essential for BMP2-induced human trophoblast invasion.
4. Discussion

During placentation, a diversity of growth factors and cytokines secreted by trophoblast cells or decidual tissues cooperate with each other to tightly control human trophoblast invasion [2, 3]. Here, we show that BMP2 induces the up-regulation of BAMBI and the activation of canonical WNT/β-catenin signaling as demonstrated by the phosphorylation of GSK3β, accumulation of active β-catenin and increased cyclin D1 expression. Moreover, we demonstrate that BAMBI is essential for BMP2-induced canonical WNT/β-catenin signaling and trophoblast invasion.

Consistent with higher mRNA levels of BAMBI in human EVTs compared with villous cytotrophoblasts [28, 29], our research shows that BAMBI facilitates BMP2-induced the activation of WNT/β-catenin signaling and human trophoblast invasion. As a pseudo-receptor of TGF-β superfamily members, BAMBI has been shown to negatively regulate TGF-β, activin and BMP signaling by interfering with the formation of functional ligand-receptor complexes [19]. However, despite belonging to the same superfamily, BMP2 and TGF-β can exert opposing functions under certain conditions. For example, BMP2 inhibits TGF-β1-induced differentiation of murine epicardial cells to smooth muscle cells [35]. Opposing effects of BMP2 and TGF-β1 also occurs in human trophoblasts; whereby BMP2 promotes and TGF-β1 inhibits human EVT cell invasion [6, 9, 36, 37]. Even for the regulation of BAMBI expression in primary EVTs, BMP2 up-regulates whereas TGF-β1 down-regulates BAMBI mRNA levels (Figure 4 A and Supplementary Figure 1). Our recent work has shown that BMP2 up-regulates BAMBI to inhibit TGF-β1 signaling and function in human granulosa cells [30]. Such inhibitory effects of BAMBI on TGF-β1 signaling may facilitate BMP2-induced human trophoblast invasion because human EVTs are known to secrete TGF-β1 [38, 39]. Possible mechanisms whereby BAMBI could inhibit TGF-β1 signaling include complexing with SMAD7, SMAD3 and TGF-β1 type I receptor (ALK5) to sequester the phosphorylation of SMAD3 and thereby disrupt TGF-β1 signaling [40]. In addition, BAMBI co- translocates with SMAD2/3 to the nucleus and alter TGF-β1-responsive gene expression in ovarian cancer cells [24]. On the other hand, knockdown of BAMBI in human preadipocytes attenuates SMAD1/5/8 phosphorylation and adipogenic effects induced by BMP4, suggesting positive roles for BAMBI in BMP signaling in this cell type [21]. Indeed, our results demonstrate that BAMBI knockdown abolishes BMP2-induced activation of WNT/β-catenin signaling and human trophoblast cell invasion.
Previous studies have shown that BMP2 can enhance canonical WNT/β-catenin signaling by increasing total β-catenin levels [33, 34]. In human trophoblast cells, BMP2 had no effects on total β-catenin but increased the accumulation of active (non-phosphorylated) β-catenin as well as the expression of its downstream target cyclin D1. Similarly, treatment of human granulosa cells with lithium chloride (LiCl), a well-known pharmacological activator of WNT/β-catenin signaling, also leads to increased levels of

active β-catenin without altering total β-catenin levels [41]. Thus, the activation of the WNT/β-catenin signaling depends more on active β-catenin accumulation compared with total β-catenin expression. In our study, BMP2-induced up-regulation in BAMBI mRNA levels (3 h) occurred prior to the increases in active β-catenin (12 h in HTR8/SVneo and 6 h in primary EVT cells), suggesting BAMBI might contribute to BMP2-induced WNT/β-catenin signaling. This was confirmed by BAMBI knockdown, which attenuated BMP2-induced the phosphorylation of GSK3β, accumulation of active β-catenin and cyclin D1, although the precise mechanisms involved are not clear. In WNT1 ligand-induced β-catenin signaling, BAMBI has been shown to strengthen the interaction between frizzled receptor and disheveled protein to inhibit β-catenin phosphorylation and subsequent degradation [42]. Intriguingly, BMP2 up- regulates several WNT ligands and their receptors [43, 44]. Moreover, the WNT pathway inhibitor Dickkopf-related protein 1 (DKK1), which blocks WNT ligands from binding to their co-receptors [45, 46], inhibits both BMP2-induced WNT/β-catenin signaling and osteoblast differentiation [33]. Thus, it is possible that BMP2 induces WNT/β-catenin signaling via canonical WNT ligand-dependent mechanisms, during which BAMBI exerts its effects as previously reported [42]. However, Vinicio et al. demonstrated that BMP2 directly induces β-catenin accumulation through BMP type II receptor-mediated GSK3β inhibition in pulmonary artery endothelial cells, in a WNT ligand-independent manner [34]. Thus, future studies examining exactly how BAMBI contributes to BMP2-induced WNT/β-catenin signaling will be of great interest.
Like BMP2, WNT/β-catenin signaling enhances human trophoblast invasion [11]. In placentas from women with severe preeclampsia, levels of WNT1, β-catenin and cyclin D1 are decreased whereas those DKK1 are increased compared to normal placentas [47], suggesting deficiencies in WNT/β-catenin signaling may contribute to preeclampsia. In contrast, abundant nuclear recruitment of β-catenin, which is a marker of hyper-activated WNT signaling, has been demonstrated in invasive EVTs from complete hydatidiform moles [11, 29]. Integration of BMP2 and WNT/β-catenin signaling has already been reported in osteoblast differentiation as well as osteogenesis, because β-catenin conditional knockout mice exhibit impaired BMP2-induced bone formation [33]. In agreement, our study shows that BMP2-

induced human EVT cell invasion is abolished when active β-catenin and cyclin D1 levels return to the basal following BAMBI knockdown, suggesting that BAMBI-mediated WNT/β-catenin signaling is crucial for trophoblast invasion induced by BMP2. In addition, the involvement of BAMBI in BMP2- increased expression of Snail and Slug (Supplemental Figure 2), both of which are the markers of EMT (epithelial to mesenchymal transition) during the invasive differentiation of trophoblast cells, may also account for the contribution of BAMBI to BMP2-promoted trophoblast invasion. Notably, canonical WNT/β-catenin signaling usually leads to increased cell proliferation, especially since cyclin D1 plays key roles in the transition from G1 to S phase of the cell cycle [18]. However, despite increasing canonical WNT/β-catenin signaling and cyclin D1, we have previously shown that BMP2 has no significant effects on HTR8/SVneo cell viability as assessed by MTT assay [6]. Similarly, others have shown that canonical WNT signaling has no effect on cell proliferation but promotes migration of human immortalized SGHPL-5 and primary EVT cells [48]. Importantly, cyclin D1 is involved in cell cycle transition to endoreduplication and the acquisition of polyploidy during rat trophoblast differentiation to giant cells [14], both of which occur during human trophoblast invasive differentiation [15]. Thus, in this context cyclin D1 may exert effects on trophoblast differentiation rather than proliferation.
In summary, our study demonstrates for the first time that BMP2 induces the up-regulation of BAMBI and the activation of canonical WNT/β-catenin signaling, as demonstrated by increased GSK3β phosphorylation, active β-catenin accumulation and cyclin D1 expression in human trophoblast cells. Furthermore, BAMBI up-regulation is responsible for BMP2-induced canonical WNT/β-catenin signaling activation and human EVT cell invasion (Figure 6). These findings strengthen our knowledge on placentation by highlighting a new molecular mechanism underlying the integration of BMP2 and WNT/β-catenin signaling in the regulation of human trophoblast invasion.

Author Contributions

P.C.K.L. and Y.L. designed research; H-J.Z. performed research and analyzed data; H-J.Z. and C.K. wrote the paper; H-J.Z. and H.Z. collected first trimester human placental villi; C.K. and H-M.C. revised the paper. All authors were involved in interpreting the data and approved the final article.
Acknowledgements

This study was supported by Foundation grant (FDN-143317) from the Canadian Institutes of Health Research to P.C.K.L. H-J.Z. is the recipient of a China Scholarship Council Doctoral Award. This study was also supported by “The Fundamental Research Funds of Shandong University” to Y.L. We thank the patients and staff of the CARE program at the BC Women’s Hospital & Health Centre for providing the placental samples used in this study.
References

[1] C.W. Redman, I.L. Sargent, Latest advances in understanding preeclampsia, Science 308(5728) (2005) 1592-4.
[2] J. Pollheimer, M. Knofler, Signalling pathways regulating the invasive differentiation of human trophoblasts: a review, Placenta 26 Suppl A (2005) S21-30.
[3] P.K. Lala, G.S. Hamilton, Growth factors, proteases and protease inhibitors in the maternal-fetal dialogue, Placenta 17(8) (1996) 545-55.
[4] J.M. Wozney, V. Rosen, A.J. Celeste, L.M. Mitsock, M.J. Whitters, R.W. Kriz, R.M. Hewick, E.A. Wang, Novel regulators of bone formation: molecular clones and activities, Science 242(4885) (1988) 1528-34.
[5] C.J. Stoikos, C.A. Harrison, L.A. Salamonsen, E. Dimitriadis, A distinct cohort of the TGFbeta superfamily members expressed in human endometrium regulate decidualization, Human reproduction 23(6) (2008) 1447-56.
[6] H.J. Zhao, C. Klausen, Y. Li, H. Zhu, Y.L. Wang, P.C.K. Leung, Bone morphogenetic protein 2 promotes human trophoblast cell invasion by upregulating N-cadherin via non-canonical SMAD2/3 signaling, Cell Death Dis 9(2) (2018) 174.
[7] B.C. Paria, W. Ma, J. Tan, S. Raja, S.K. Das, S.K. Dey, B.L. Hogan, Cellular and molecular responses of the uterus to embryo implantation can be elicited by locally applied growth factors, Proceedings of the National Academy of Sciences of the United States of America 98(3) (2001) 1047-52.
[8] K.Y. Lee, J.W. Jeong, J. Wang, L. Ma, J.F. Martin, S.Y. Tsai, J.P. Lydon, F.J. DeMayo, Bmp2 is critical for the murine uterine decidual response, Molecular and cellular biology 27(15) (2007) 5468-78.

[9] H.J. Zhao, H.M. Chang, H. Zhu, C. Klausen, Y. Li, P.C.K. Leung, Bone Morphogenetic Protein 2 Promotes Human Trophoblast Cell Invasion by Inducing Activin A Production, Endocrinology 159(7) (2018) 2815-2825.
[10] V. Krishnan, H.U. Bryant, O.A. Macdougald, Regulation of bone mass by Wnt signaling, The Journal of clinical investigation 116(5) (2006) 1202-9.
[11] J. Pollheimer, T. Loregger, S. Sonderegger, L. Saleh, S. Bauer, M. Bilban, K. Czerwenka, P. Husslein,
M. Knofler, Activation of the canonical wingless/T-cell factor signaling pathway promotes invasive differentiation of human trophoblast, The American journal of pathology 168(4) (2006) 1134-47.
[12] R.T. Moon, B. Bowerman, M. Boutros, N. Perrimon, The promise and perils of Wnt signaling through beta-catenin, Science 296(5573) (2002) 1644-6.
[13] B.T. MacDonald, X. He, Frizzled and LRP5/6 receptors for Wnt/beta-catenin signaling, Cold Spring Harbor perspectives in biology 4(12) (2012).
[14] A. MacAuley, J.C. Cross, Z. Werb, Reprogramming the cell cycle for endoreduplication in rodent trophoblast cells, Molecular biology of the cell 9(4) (1998) 795-807.
[15] T.G. Zybina, H.G. Frank, S. Biesterfeld, P. Kaufmann, Genome multiplication of extravillous trophoblast cells in human placenta in the course of differentiation and invasion into endometrium and myometrium. II. Mechanisms of polyploidization, Tsitologiia 46(7) (2004) 640-8.
[16] T.G. Zybina, P. Kaufmann, H.G. Frank, J. Freed, M. Kadyrov, S. Biesterfeld, Genome multiplication of extravillous trophoblast cells in human placenta in the course of differentiation and invasion into endometrium and myometrium. I. Dynamics of polyploidization, Tsitologiia 44(11) (2002) 1058-67.
[17] S. Sonderegger, P. Haslinger, A. Sabri, C. Leisser, J.V. Otten, C. Fiala, M. Knofler, Wingless (Wnt)-3A induces trophoblast migration and matrix metalloproteinase-2 secretion through canonical Wnt signaling and protein kinase B/AKT activation, Endocrinology 151(1) (2010) 211-20.
[18] Y. Dai, Z. Qiu, Z. Diao, L. Shen, P. Xue, H. Sun, Y. Hu, MicroRNA-155 inhibits proliferation and migration of human extravillous trophoblast derived HTR-8/SVneo cells via down-regulating cyclin D1, Placenta 33(10) (2012) 824-9.
[19] D. Onichtchouk, Y.G. Chen, R. Dosch, V. Gawantka, H. Delius, J. Massague, C. Niehrs, Silencing of TGF-beta signalling by the pseudoreceptor BAMBI, Nature 401(6752) (1999) 480-5.
[20] Q. Zhang, X.E. Shi, C. Song, S. Sun, G. Yang, X. Li, BAMBI Promotes C2C12 Myogenic Differentiation by Enhancing Wnt/beta-Catenin Signaling, International journal of molecular sciences 16(8) (2015) 17734-45.
[21] X. Luo, L.J. Hutley, J.A. Webster, Y.H. Kim, D.F. Liu, F.S. Newell, C.H. Widberg, A. Bachmann, N. Turner, C. Schmitz-Peiffer, J.B. Prins, G.S. Yang, J.P. Whitehead, Identification of BMP and activin membrane-bound inhibitor (BAMBI) as a potent negative regulator of adipogenesis and modulator of autocrine/paracrine adipogenic factors, Diabetes 61(1) (2012) 124-36.
[22] K. Liu, X. Song, H. Ma, L. Liu, X. Wen, J. Yu, L. Wang, S. Hu, Knockdown of BAMBI inhibits beta- catenin and transforming growth factor beta to suppress metastasis of gastric cancer cells, Molecular medicine reports 10(2) (2014) 874-80.
[23] L. Zhou, J. Park, K.Y. Jang, H.S. Park, S. Wagle, K.H. Yang, K.B. Lee, B.H. Park, J.R. Kim, The overexpression of BAMBI and its involvement in the growth and invasion of human osteosarcoma cells, Oncology reports 30(3) (2013) 1315-22.
[24] D. Pils, M. Wittinger, M. Petz, A. Gugerell, W. Gregor, A. Alfanz, R. Horvat, E.I. Braicu, J. Sehouli, R. Zeillinger, W. Mikulits, M. Krainer, BAMBI is overexpressed in ovarian cancer and co-translocates with Smads into the nucleus upon TGF-beta treatment, Gynecologic oncology 117(2) (2010) 189-97.
[25] J. Fritzmann, M. Morkel, D. Besser, J. Budczies, F. Kosel, F.H. Brembeck, U. Stein, I. Fichtner, P.M. Schlag, W. Birchmeier, A colorectal cancer expression profile that includes transforming growth factor beta inhibitor BAMBI predicts metastatic potential, Gastroenterology 137(1) (2009) 165-75.

[26] I. Raykhel, F. Moafi, S.M. Myllymaki, P.G. Greciano, K.S. Matlin, J.V. Moyano, A. Manninen, J. Myllyharju, BAMBI is a novel HIF1-dependent modulator of TGFbeta-mediated disruption of cell polarity during hypoxia, Journal of cell science 131(10) (2018).
[27] A.R. Highet, S.M. Khoda, S. Buckberry, S. Leemaqz, T. Bianco-Miotto, E. Harrington, C. Ricciardelli,
C.T. Roberts, Hypoxia induced HIF-1/HIF-2 activity alters trophoblast transcriptional regulation and promotes invasion, European journal of cell biology 94(12) (2015) 589-602.
[28] M. Bilban, P. Haslinger, J. Prast, F. Klinglmuller, T. Woelfel, S. Haider, A. Sachs, L.E. Otterbein, G. Desoye, U. Hiden, O. Wagner, M. Knofler, Identification of novel trophoblast invasion-related genes: heme oxygenase-1 controls motility via peroxisome proliferator-activated receptor gamma, Endocrinology 150(2) (2009) 1000-13.
[29] M. Knofler, J. Pollheimer, Human placental trophoblast invasion and differentiation: a particular focus on Wnt signaling, Frontiers in genetics 4 (2013) 190.
[30] L. Bai, H.M. Chang, J.C. Cheng, C. Klausen, G. Chu, P.C.K. Leung, G. Yang, SMAD1/5 mediates bone morphogenetic protein 2-induced up-regulation of BAMBI expression in human granulosa-lutein cells, Cellular signalling 37 (2017) 52-61.
[31] C.H. Graham, T.S. Hawley, R.G. Hawley, J.R. MacDougall, R.S. Kerbel, N. Khoo, P.K. Lala, Establishment and characterization of first trimester human trophoblast cells with extended lifespan, Experimental cell research 206(2) (1993) 204-11.
[32] J.A. Irving, J.J. Lysiak, C.H. Graham, S. Hearn, V.K. Han, P.K. Lala, Characteristics of trophoblast cells migrating from first trimester chorionic villus explants and propagated in culture, Placenta 16(5) (1995) 413-33.
[33] Y. Chen, H.C. Whetstone, A. Youn, P. Nadesan, E.C. Chow, A.C. Lin, B.A. Alman, Beta-catenin signaling pathway is crucial for bone morphogenetic protein 2 to induce new bone formation, The Journal of biological chemistry 282(1) (2007) 526-33.
[34] V.A. de Jesus Perez, T.P. Alastalo, J.C. Wu, J.D. Axelrod, J.P. Cooke, M. Amieva, M. Rabinovitch, Bone morphogenetic protein 2 induces pulmonary angiogenesis via Wnt-beta-catenin and Wnt-RhoA-Rac1 pathways, The Journal of cell biology 184(1) (2009) 83-99.
[35] C.R. Hill, N.S. Sanchez, J.D. Love, J.A. Arrieta, C.C. Hong, C.B. Brown, A.F. Austin, J.V. Barnett, BMP2 signals loss of epithelial character in epicardial cells but requires the Type III TGFbeta receptor to promote invasion, Cellular signalling 24(5) (2012) 1012-22.
[36] C.H. Graham, P.K. Lala, Mechanism of control of trophoblast invasion in situ, Journal of cellular physiology 148(2) (1991) 228-34.
[37] J.C. Cheng, H.M. Chang, P.C. Leung, Transforming growth factor-beta1 inhibits trophoblast cell invasion by inducing Snail-mediated down-regulation of vascular endothelial-cadherin protein, The Journal of biological chemistry 288(46) (2013) 33181-92.
[38] G. Xu, C. Chakraborty, P.K. Lala, Expression of TGF-beta signaling genes in the normal, premalignant, and malignant human trophoblast: loss of smad3 in choriocarcinoma cells, Biochemical and biophysical research communications 287(1) (2001) 47-55.
[39] R.L. Jones, C. Stoikos, J.K. Findlay, L.A. Salamonsen, TGF-beta superfamily expression and actions in the endometrium and placenta, Reproduction 132(2) (2006) 217-32.
[40] X. Yan, Z. Lin, F. Chen, X. Zhao, H. Chen, Y. Ning, Y.G. Chen, Human BAMBI cooperates with Smad7 to inhibit transforming growth factor-beta signaling, The Journal of biological chemistry 284(44) (2009) 30097-104.
[41] L. Bai, H.M. Chang, J.C. Cheng, G. Chu, P.C.K. Leung, G. Yang, Lithium chloride inhibits StAR and progesterone production through GSK-3beta and ERK1/2 signaling pathways in human granulosa-lutein cells, Molecular and cellular endocrinology 461 (2018) 89-99.

[42] Z. Lin, C. Gao, Y. Ning, X. He, W. Wu, Y.G. Chen, The pseudoreceptor BMP and activin membrane- bound inhibitor positively modulates Wnt/beta-catenin signaling, The Journal of biological chemistry 283(48) (2008) 33053-8.
[43] Q. Li, A. Kannan, W. Wang, F.J. Demayo, R.N. Taylor, M.K. Bagchi, I.C. Bagchi, Bone morphogenetic protein 2 functions via a conserved signaling pathway involving Wnt4 to regulate uterine decidualization in the mouse and the human, The Journal of biological chemistry 282(43) (2007) 31725-32.
[44] L. Yang, K. Yamasaki, Y. Shirakata, X. Dai, S. Tokumaru, Y. Yahata, M. Tohyama, Y. Hanakawa, K. Sayama, K. Hashimoto, Bone morphogenetic protein-2 modulates Wnt and frizzled expression and enhances the canonical pathway of Wnt signaling in normal keratinocytes, Journal of dermatological science 42(2) (2006) 111-9.
[45] S.L. Lewis, P.L. Khoo, R.A. De Young, K. Steiner, C. Wilcock, M. Mukhopadhyay, H. Westphal, R.V. Jamieson, L. Robb, P.P. Tam, Dkk1 and Wnt3 interact to control head morphogenesis in the mouse, Development 135(10) (2008) 1791-801.
[46] A. Glinka, W. Wu, H. Delius, A.P. Monaghan, C. Blumenstock, C. Niehrs, Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction, Nature 391(6665) (1998) 357-62.
[47] X. Wang, Z. Zhang, X. Zeng, J. Wang, L. Zhang, W. Song, Y. Shi, Wnt/beta-catenin signaling pathway in severe preeclampsia, Journal of molecular histology 49(3) (2018) 317-327.
[48] G. Meinhardt, S. Haider, P. Haslinger, K. Proestling, C. Fiala, J. Pollheimer, M. Knofler, Wnt- dependent T-cell factor-4 controls human etravillous trophoblast motility, Endocrinology 155(5) (2014) 1908-20.
Figure legends

Figure 1. BMP2 increases BAMBI mRNA levels and the activation of canonical WNT/β-catenin signaling in HTR8/SVneo cells. A-C, HTR8/SVneo cells were treated with vehicle (Ctrl) or 25 ng/mL BMP2 for different lengths of time (3, 6, 12 or 24 h). A, BAMBI mRNA levels from 3 h to 24 h time points were examined by RT-qPCR with GAPDH as the reference gene. B, The phosphorylation of GSK3β at 3 h and 6 h time points were checked via Western blot and normalized to total GSK3β. C, Active (non-phosphorylated) β-catenin protein levels from 3 h to 24 h time points were examined by Western blot and normalized to total β-catenin. D, HTR8/SVneo cells were treated with or without 25 ng/mL BMP2 every 24 h for 48 h, and cyclin D1 protein levels were analyzed by Western blot and normalized to α-tubulin. The molecular weight of p-GSK3β, GSK3β, active β-catenin, total β-catenin, cyclin D1 and α-tubulin is 46, 46, 92, 92, 36 and 55 kDa, respectively. Results are displayed as the mean
± SEM of at least three independent experiments. If a pair of values is significantly different (p<0.05), the values have different subscript letters (a vs. b or b vs. c) assigned to them.

Figure 2. DMH1 abolishes BMP2-mediated BAMBI up-regulation and activation of canonical WNT/β-catenin signaling in HTR8/SVneo cells. A-F, HTR8/SVneo cells were pre-treated with vehicle control (dimethylsulfoxide, DMSO) or 1 μM DMH1 (ALK2/3 inhibitor, 1 μM) for 1 h prior to treatment for another 3-24 h with or without 25 ng/mL BMP2. A, BAMBI mRNA levels after 12 h BMP2 treatment were measured by RT-qPCR with GAPDH as the reference gene. B-C, The phosphorylation of GSK3β after 6 h BMP2 treatment were checked via Western blot and normalized to total GSK3β. D, Representative images of Western blots for active β-catenin, total β-catenin, cyclin D1 and α-tubulin after 24 h BMP2 treatment. E-F, Quantitative results for active β-catenin (E, normalized to total β-catenin) and cyclin D1 (F, normalized to α-tubulin) are shown. Results are displayed as the mean ± SEM of at least three independent experiments. If a pair of values is significantly different (p<0.05), the values have different subscript letters (a vs. b or b vs. c) assigned to them.
Figure 3. BAMBI mediates the activation of WNT/β-catenin signaling induced by BMP2 in HTR8/SVneo cells. A-F, HTR8/SVneo cells were transfected for 48 h with 25 nM non-targeting control siRNA (siCtrl) or 25 nM siRNA targeting BAMBI (siBAMBI) prior to treatment with vehicle (Ctrl) or 25 ng/mL BMP2 for another 6 – 24 h. A, RT-PCR was used to evaluate the knockdown efficiency of BAMBI mRNA levels. B, Representative images of Western blots for phosphorylated GSK3β, total GSK3β and α- tubulin after 6 h BMP2 treatment. C, Quantitative results for phosphorylated GSK3β (normalized to total GSK3β) are shown. D, Representative images of Western blots for active β-catenin, total β-catenin, cyclin D1 and α-tubulin after 24 h BMP2 treatment. E-F, Quantitative results for active β-catenin (E, normalized to total β-catenin) and cyclin D1 (F, normalized to α-tubulin) are shown. Results are displayed as the mean ± SEM of at least three independent experiments. If a pair of values is significantly different (p<0.05), the values have different subscript letters (a vs. b or b vs. c) assigned to them.

Figure 4. BAMBI is crucial for the up-regulation of active β-catenin and cyclin D1 by BMP2 in primary human EVT cells. A-B, Primary EVT cells were treated with vehicle (Ctrl) or 25 ng/mL BMP2 for different lengths of time (3, 6, 12 or 24 h). A, BAMBI mRNA levels were examined by RT-qPCR with GAPDH as the reference gene. B, Active β-catenin protein levels were examined by Western blot and normalized to total β-catenin (Representative images shown in upper part and quantification results shown in lower part). C, Primary EVT cells were treated with or without 25 ng/mL BMP2 every 24 h for
48 h, and cyclin D1 protein levels were analyzed by Western blot and normalized to α-tubulin (Representative images shown on left side and quantification results shown on right side). D-G, Primary EVT cells were transfected for 48 h with 25 nM non-targeting control siRNA (siCtrl) or 25 nM siRNA targeting BAMBI (siBAMBI) prior to treatment with vehicle (Ctrl) or 25 ng/mL BMP2 for 12 h (mRNA levels) or 24 h (protein levels). D, RT-PCR was used to evaluate the knockdown efficiency of BAMBI mRNA levels. E, Representative images of Western blots for active β-catenin, total β-catenin, cyclin D1 and α-tubulin. F-G, Quantitative results for active β-catenin (F, normalized to total β-catenin) and cyclin D1 (G, normalized to α-tubulin) are shown. Results are displayed as the mean ± SEM of at least three independent experiments. If a pair of values is significantly different (p<0.05), the values have different subscript letters (a vs. b or b vs. c) assigned to them.
Figure 5. BAMBI is involved in BMP2-induced human trophoblast cell invasion. HTR8/SVneo (A) or primary human EVT cells (B) were transfected for 48 h with 25 nM non-targeting control siRNA (siCtrl) or 25 nM siRNA targeting BAMBI (siBAMBI) prior to treatment with vehicle (Ctrl) or 25 ng/mL BMP2 for a further 36 h. Matrigel-coated transwell invasion assays were used to examine the effects of BAMBI knockdown on BMP2-induced invasion in HTR8/SVneo and primary EVT cells (scale bar 100 μm). Corresponding summarized quantitative results are displayed as the mean ± SEM of at least three independent experiments. If a pair of values is significantly different (p<0.05), the values have different subscript letters (a vs. b or b vs. c) assigned to them.

Figure 6. Proposed model of the signaling pathway mediating BMP2-induced BAMBI up- regulation, canonical WNT/β-catenin signaling activation and increased human trophoblast cell invasion. The binding of BMP2 to type I receptors ALK2 and/or ALK3 leads to up-regulation of BAMBI, which in turn activates WNT/β-catenin signaling, as indicated by the increase of GSK3β phosphorylation, the accumulation of active β-catenin and the up-regulation of the downstream target gene cyclin D1. BAMBI also accounts for BMP2-increased expression of Snail and Slug, both of which is involved in the invasive differentiation of human trophoblasts. The activation of WNT/β-catenin signaling and the up- regulation of SNAIL and SLUG may contribute to BAMBI-mediated human trophoblast invasion induced by BMP2.
Supplementary Figure Legends

Supplementary Figure 1. TGF-β1 reduces BAMBI mRNA levels in primary human EVT cells. Primary EVT cells were treated with vehicle (Ctrl) or 5 ng/mL TGF-β1 for different lengths of time (3, 6, 12 or 24 h), and BAMBI mRNA levels were examined by RT-qPCR with GAPDH as the reference gene. Results are displayed as the mean ± SEM of at least three independent experiments. If a pair of values is significantly different (p<0.05), the values have different subscript letters (a vs. b or b vs. c) assigned to them.

Supplementary Figure 2. BAMBI knockdown attenuates BMP2-induced upregulation of Snail and Slug. A, HTR8/SVneo cells were treated with vehicle (Ctrl) or 25 ng/mL BMP2 for different lengths of time (3, 6, 12 or 24 h). Snail, Slug and α-tubulin protein levels were examined via Western blot. B, HTR8/SVneo cells were transfected for 48 h with 25 nM non-targeting control siRNA (siCtrl) or 25 nM siRNA targeting BAMBI (siBAMBI) prior to treatment with vehicle (Ctrl) or 25 ng/mL BMP2 for another 6 h. Representative images of Western blots for Snail, Slug and α-tubulin were shown upside and quantitative results for SLUG and SNAIL (normalized to α-tubulin) are shown downside. The molecular weight of SNAIL, SLUG and α-tubulin is 29, 30 and 55 kDa, respectively. Results are displayed as the

mean ± SEM of at least three independent experiments. If a pair of values is significantly different (p<0.05), the values have different subscript letters (a vs. b or b vs. c) assigned to them.

BMP2 increased BAMBI mRNA levels in HTR8/SVneo and primary human extravillous trophoblast cells.
BMP2 activated canonical WNT/β-catenin signaling which promotes human trophoblast invasion.
DMH1 blocked BMP2-induced up-regulation of BAMBI and activation of WNT/β- catenin signaling.
BAMBI mediated BMP2-induced activation DMH1 of WNT/β-catenin signaling and trophoblast invasion.