GDF-11 promotes human trophoblast cell invasion by increasing ID2-mediated MMP2 expression

Growth differentiation factor-11 (GDF-11), also known as bone morphogenetic protein-11, belongs to the transforming growth factor-beta superfamily. GDF-11 was first identified as an important regulator during embryonic development. Increasing evidence has demonstrated that GDF-11 regulates the development of various organs and its aberrant expressions are associated with the risk of cardiovascular diseases and cancers. Extravillous trophoblast (EVT) cells invasion is a critical event for placenta development and needs to be finely regulated. However, to date, the biological function of GDF-11 in the human EVT cells remains unknown. HTR-8/SVneo, a human EVT cell line, and primary cultures of human EVT cells were used to examine the effect of GDF-11 on matrix metalloproteinase 2 (MMP2) expression. Matrigel-coated transwell invasion assay was used to examine cell invasiveness. A series of in vitro experiments were applied to explore the underlying mechanisms that mediate the effect of GDF-11 on MMP2 expression and cell invasion. Treatment with GDF-11 stimulates MMP2 expression, in the HTR-8/SVneo and primary human EVT cells. Using a pharmacological inhibitor and siRNA-mediated knockdown approaches, our results demonstrated that the stimulatory effect of GDF-11 on MMP2 expression was mediated by the ALK4/5-SMAD2/3 signaling pathways. In addition, the expression of inhibitor of DNA-binding protein 2 (ID2) was upregulated by GDF-11 and that was required for the GDF-11-stimulated MMP2 expression and EVT cell invasion. These findings discover a new biological function and underlying molecular mechanisms of GDF-11 in the regulation of human EVT cell invasion. Ede8tEoyGya1QPPVVUV7JY Video Abstract Video Abstract

a continuous blood supply to the placenta throughout pregnancy. Aberrant EVT cell invasion is associated with different placental diseases such as preeclampsia, intrauterine growth restriction, miscarriage, and hydatidiform mole [3,4]. To date, it is known that several members of the TGF-β superfamily are expressed in the human placenta and regulate EVT cell invasion in an autocrine and/ or paracrine manner [5][6][7].
GDF-11, also known as BMP-11, is a member of the TGF-β superfamily and plays important role in the regulation of anterior/posterior axial patterning during embryonic development [8][9][10]. Deletion of the Gdf11 gene in mice results in perinatal lethality. Although the precise cause of death remains unknown, Gdf11 -/mice died within 24 h after birth [11]. Mutations in human GDF11 and its extracellular antagonist, FST, genes are associated with the orofacial clefts [12]. Various in vivo studies in rodents have demonstrated the postnatal functions of GDF-11 in skeletal muscle, heart, brain, and bone and suggested potential therapeutic implications in those organs-related diseases [13]. In humans, the GDF-11 expression can be detected in nearly all organs including the placenta [14]. Aberrant expressions of GDF-11 are associated with the pathogenesis of cardiovascular, neurological, skeletal muscle, and age-related diseases as well as the risk of cancers [15,16].
Similar to other TGF-β superfamily members, GDF-11 activates intracellular signaling by binding to two activin type-II receptors, ActRIIA and ActRIIB, and three type-I receptors, activin receptor-like kinase 4 (ALK4), ALK5, and ALK7 that belong to serine and threonine kinase receptors. Upon ligand binding, activated receptors phosphorylate and activate the canonical SMAD2/3 or SMAD1/5/8 signaling pathways and non-canonical ERK1/2, p38, and JNK signaling pathways [13,16]. We have shown that TGF-β1 inhibits invasiveness in primary human EVT cells and an immortalized EVT cell line, HTR-8/SVneo [17][18][19]. In contrast, activins and GDF-8 stimulate human EVT cell invasion [20,21]. These studies indicate that different TGF-β superfamily members have distinct effects on the regulation of human EVT cell invasion. GDF-11 and GDF-8 share 89% sequence identity in their mature form and are believed to have similar biological functions because the same membrane receptors and intracellular signaling pathways are used and activated, respectively. However, in some contexts, GDF-11 and GDF-8 exert different biological functions [13,22]. Matrix metalloproteinase 2 (MMP2) and MMP9 mediated remodeling and degradation/activation of the extracellular matrix play an essential role in the EVT cell invasion [23]. Our recent study shows that GDF-8 stimulates human EVT cell invasion by upregulating the expression of MMP2 [21]. However, to date, the biological function of GDF-11 in human EVT cells remains unknown, and whether GDF-11 has a similar effect to GDF-8 in the regulation of EVT cell invasion is unclear. Therefore, the present study was designed to examine the effect of GDF-11 on human EVT cell invasion and to explore related underlying molecular mechanisms.

Primary human EVT cell isolation and culture
The study received institutional approval and was carried out in accordance with the guidelines from the Zhengzhou University Research Ethics Board (#2020-KY-140). Human EVT cells were isolated from first-trimester (6-9 weeks of gestation) placental tissue explants as previously described [18,19]. Briefly, chorionic villi were washed with a cold medium and mechanically minced into 1-2 mm fragments. Fragments of the chorionic villi were allowed to adhere for 2-3 days, after which any non-adherent material was removed. These tissue explants were further cultured for 10-14 days to allow EVT cell outgrowth, during which the culture medium was changed every 2 days. EVT cells were separated from the villous explants by brief trypsin digestion. Cells were plated in a 6-well or 12-well plate (2 × 10 4 cell/cm 2 ) without coating and cultured in a humidified atmosphere containing 5% CO 2 and 95% air at 37 °C in Dulbecco's modified Eagle's medium/nutrient mixture F-12 Ham medium (DMEM/F-12) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate. Isolated primary EVT cells were characterized by the expressions of cytokeratin-7 and HLA-G. Primary EVT cells were not passaged. Individual primary cultures were composed of cells from one individual patient. Each experiment was repeated at least three times and each time used cells derived from different patients.

Immunofluorescence staining
Cells were cultured on coverslips, fixed with cold methanol at − 20 °C, and then permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS). Cells were blocked with Dako Protein Block (Dako) for 1 h and incubated with antibodies diluted in Dako Protein Block. Alexa 488-labeled donkey anti-mouse was used as a secondary antibody. Cells were counterstained with DAPI, rinsed with PBS, mounted with Gelvatol, and examined using a Nikon Eclipse fluorescence microscope.

Western blot
Cells were lysed in cell lysis buffer (Cell Signaling Technology) supplemented with a protease inhibitor cocktail (Sigma). Equal amounts of protein were separated by SDS polyacrylamide gel electrophoresis and transferred onto PVDF membranes. After 1 h of blocking with 5% nonfat dry milk in Tris-buffered saline (TBS), the membranes were incubated overnight at 4 °C with primary antibodies diluted in 5% nonfat milk/TBS. Following primary antibody incubation, the membranes were incubated with appropriate HRP-conjugated secondary antibodies. Immunoreactive bands were detected using an enhanced chemiluminescent substrate (Bio-Rad Laboratories) and imaged with a ChemiDoc MP Imager (Bio-Rad Laboratories).

Invasion assay
Transwell cell culture inserts (8 µm pore size, 24 wells, BD Biosciences) were coated with 1 mg/mL growth factor-reduced Matrigel (BD Biosciences). Cells (1 × 10 5 cells/insert) in DMEM/F-12 medium supplemented with 0.1% FBS were incubated for 48 h against a gradient of 10% FBS. Non-invasive cells were removed with a cotton swab from the upper side of the membrane. Cells that penetrated the membrane were fixed with cold methanol, stained with crystal violet (0.5%, Sigma) for 30 min, and subsequently washed thoroughly with tap water. Each experiment was performed with triplicate inserts. In each insert, five microscopic fields were photographed under an optical microscope, and the cell number was counted manually.

Statistical analysis
The results are presented as the mean ± SEM of at least three independent experiments. All statistical analyses were analyzed by PRISM software. Multiple comparisons were analyzed using one-way ANOVA followed by Tukey's multiple comparison test. A significant difference was defined as p < 0.05. Values that are statistically different from one another (p < 0.05) are indicated by different letters. The values with any common letter are not significantly different.

GDF-11 upregulates MMP2 but not MMP9 in human EVT cells
The HTR-8/SVneo cell line was generated using human first trimester EVT cells infected with SV40 large T antigen and is the most commonly used cell model for studying the biological function of EVT cells [24]. Using this cell line makes the experiments more technically feasible, particularly for those involving gene manipulations. In women of reproductive age, the serum level of GDF-11 can reach 40 ng/mL [25]. To examine the effects of GDF-11 on MMP2 and MMP9 expressions, HTR-8/ SVneo cells were treated with 30 ng/mL recombinant human GDF-11 for different periods. RT-qPCR results showed that GDF-11 upregulated MMP2 mRNA levels in a time-dependent manner. However, the mRNA levels of MMP9 were not affected by the treatment with GDF-11 (Fig. 1A). The effects of GDF-11 on MMP2 protein levels were examined by the western blot analysis. As shown in Fig. 1B, consistent with the RT-qPCR results, GDF-11 upregulated MMP2 protein levels in HTR-8/SVneo cells. To further confirm these results, primary EVT cells isolated from first-trimester chorionic villi were used. The characteristics of EVT cells were confirmed by the expressions of cytokeratin-7 and HLA-G (Additional file 1: Fig. S1). Similarly, treatment of GDF-11 upregulated MMP2 but not MMP9 mRNA levels in primary EVT cells (Fig. 1C). The stimulatory effect of GDF-11 on MMP2 protein levels was also observed by the western blot analysis (Fig. 1D).

GDF-11 upregulates MMP2 expression through ALK4 and ALK5 in human EVT cells
In a context-dependent manner, ALK4, ALK5, and/or ALK7 are required for the biological function of GDF-11. Pretreatment of HTR-8/SVneo and primary EVT cells with SB431542, a potent ALK4/5/7 inhibitor [26], blocked the stimulatory effects of GDF-11 on the MMP2 mRNA and protein levels ( Fig. 2A, B). It has been shown that, in humans, ALK7 is mainly expressed in the adipose tissue and the expression levels of ALK7 are very low in the placenta [27]. Because SB431542 inhibits the function of ALK4 and ALK5, to further distinguish the involvement of ALK4 and ALK5 in GDF-11-induced upregulation of MMP2 expression, the siRNA-mediated knockdown approach was applied to block the function of ALK4 and ALK5 specifically in HTR-8/SVneo cells. As shown in Fig. 2C, ALK4 siRNA specifically downregulated the endogenous ALK4 mRNA levels without affecting the endogenous ALK5 mRNA levels and vice versa for ALK5 siRNA. The stimulatory effect of GDF-11 on MMP2 mRNA levels was attenuated by the knockdown of ALK4 or ALK5. Western blot analysis showed similar results that both ALK4 and ALK5 were involved in the GDF-11-induced upregulation of MMP2 protein levels in HTR-8/SVneo cells (Fig. 2D).

GDF-11 upregulates MMP2 expression by activating SMAD2/3 signaling pathways
In different cell types, GDF-11 activates SMAD2/3 and SMAD1/5/8 signaling pathways. Treatment of HTR-8/ SVneo and primary EVT cells with GDF-11 induced the phosphorylation levels of SMAD2 and SMAD3 indicating their activations. However, the SMAD1/5/8 signaling pathways were not activated by the GDF-11 in both HTR-8/SVneo and primary EVT cells. We used BMP4 as the positive control for the activation of the SMAD1/5/8 signaling pathways (Fig. 3A,B). To define the involvement of SMAD signaling pathways in GDF-11-induced upregulation of MMP2 expression, the common SMAD for the functional SMAD signaling pathways, SMAD4, was knocked down by the specific siRNA. As shown in Fig. 3C, D, the knockdown of SMAD4 attenuated the stimulatory effects of GDF-11 on both MMP2 mRNA and protein levels in HTR-8/SVneo cells. Although in most contexts, the function of SMAD2 and SMAD3 are indistinguishable, these two similar intracellular signaling proteins can exert distinct roles under some conditions [28]. To further define the individual role of SMAD2 and SMAD3 in mediating the stimulatory effect of GDF-11 on MMP2 expression, the expression of endogenous SMAD2 or SMAD3 was knocked down by the specific siRNA. Transfection of HTR-8/SVneo cells with SMAD2 siRNA specifically downregulated the endogenous SMAD2 mRNA levels without affecting the SMAD3 mRNA levels and vice versa for the SMAD3 siRNA transfection. Knockdown of SMAD2 or SMAD3 attenuated the stimulatory effect of GDF-11 on MMP2 mRNA levels (Fig. 4A). Western blot results showed similar results that both SMAD2 and SMAD3 were required for the GDF-11-induced upregulation of MMP2 expression in HTR-8/ SVneo cells (Fig. 4B).

ID2 mediates GDF-11-induced upregulation of MMP2 expression
In mammals, four inhibitor of DNA-binding (ID) proteins, ID1-4, that share extensive sequence homology in the HLH motif have been identified. ID proteins can be regulated by the TGF-β superfamily [29]. ID2 has been shown to mediate the expression of MMP2 in cancer cells [30,31]. Therefore, we examined whether ID2 is involved in the GDF-11-induced upregulation of MMP2 expression. Treatment with GDF-11 induced ID2 mRNA and protein levels in both HTR-8/SVneo and primary EVT cells (Fig. 5A, B). In HTR-8/SVneo cells, knockdown of SMAD2 or SMAD3 abolished the stimulatory effect of GDF-11 on ID2 protein levels (Fig. 5C). Importantly, siRNA-mediated knockdown of ID2 attenuated the GDF-11-induced MMP2 protein levels (Fig. 5D). Collectively, these results indicate that GDF-11-induced ID2 is required for the induction of MMP2 expression in human EVT cells.

ID2-mediated MMP2 expression is required for GDF-11-stimulated EVT cell invasion
To examine the effect of GDF-11 on the invasiveness of EVT cells, the Matrigel-coated transwell invasion assay was applied. Treatment with GDF-11 stimulated invasiveness in both HTR-8/SVneo and primary EVT cells (Fig. 6A, B). To define the role of MMP2 in GDF-11-stimulated cell invasion, the function of MMP2 was blocked by the siRNA-mediated knockdown approach in HTR-8/ SVneo. As shown in Fig. 6C, both endogenous and the GDF-11 upregulated MMP2 protein levels were downregulated by the MMP2 siRNA. In addition, the stimulatory effect of GDF-11 on HTR-8/SVneo cell invasiveness was attenuated by the siRNA-mediated knockdown of MMP2 (Fig. 6D). Moreover, the knockdown of ID2 also attenuated the GDF-11-induced HTR-8/SVneo cell invasion (Fig. 6E). Taken together, these results indicate that the ID2-mediated induction of MMP2 expression is required for the GDF-11-stimulated human EVT cell invasion.

Discussion
Previous immunohistochemical analysis results have shown that GDF-11 is expressed in the placenta and the secretory phase of the human endometrium which suggests the role of GDF-11 in the regulation of implantation [32]. However, thus far, there is no study directly investigating the biological role of GDF-11 in the human Fig. 4 Both SMAD2 and SMAD3 participate in the GDF-11-induced MMP2 expression in HTR-8/SVneo cells. A and B, Cells were transfected with 50 nM control siRNA (si-Ctrl), SMAD2 siRNA (si-SMAD2), or SMAD3 siRNA (si-SMAD3) for 48 h, and then treated with 30 ng/mL GDF-11 (G11) for 24 h. The mRNA (A) and protein (B) levels of MMP2, SMAD2, and SMAD3 were examined by RT-qPCR and western blot, respectively. The results are expressed as the mean ± SEM of at least three independent experiments. Multiple comparisons were analyzed using one-way ANOVA followed by Tukey's multiple comparison test. Values that are statistically different from one another (p < 0.05) are indicated by different letters. The values with any common letter are not significantly different placenta or endometrium. In the present study, we revealed the novel biological role of GDF-11 in stimulating the human placental EVT cell invasion. The stimulatory effect of GDF-11 on invasiveness was mediated by the induction of MMP2. Our results also delineated that ALK4 and ALK5-mediated SMAD2/3 signaling pathways were involved in the GDF-11-induced MMP2 expression. In addition, we showed that the transcriptional regulator ID2 mediated the MMP2 expression induced by the GDF-11. This study suggests that GDF-11 may play an important role in the implantation by regulating EVT cell invasion.
To date, only a handful of studies have investigated the roles of GDF-11 on cell migration/invasion. However, most studies are done on cancer cells. In oral cancer cells, treatment with GDF-11 stimulates cell  [33]. In contrast, GDF-11 suppresses invasiveness in human liver, pancreatic, and triple-negative breast cancer cells [34][35][36]. These results suggest the effect of GDF-11 on cell migration/invasion is in a cell-type-dependent manner. GDF-11 has remarkable sequence similarity with GDF-8 [13]. Our recent study shows that GDF-8 stimulates HTR-8/SVneo cell invasion by upregulating MMP2 but not MMP9 expression [21]. In the present study, we observed that GDF-11 stimulated MMP2 but not MMP9 expressions which are the same as the results obtained from GDF-8. It is known that the biological function of GDF-8 in myogenic cells is mainly mediated by ALK4, while ALK5 mediates GDF-8 function in non-myogenic cells [37]. Agreeing with this, our previous study shows that the stimulatory effect of GDF-8 on MMP2 expression in HTR-8/SVneo cells is mediated by ALK5 but not ALK4 [21]. Interestingly, in the same cells, here we revealed that both ALK4 and ALK5 were involved in the GDF-11-induced upregulation of MMP2. Collectively, these results indicate that GDF-11 and GDF-8 utilize different receptors to exert the same pro-invasive role in human EVT cells.
It has been shown that GDF-11 and GDF-8 predominantly utilize ALK4 or ALK5 to elicit signal The results are expressed as the mean ± SEM of at least three independent experiments. Multiple comparisons were analyzed using one-way ANOVA followed by Tukey's multiple comparison test. Values that are statistically different from one another (p < 0.05) are indicated by different letters. The values with any common letter are not significantly different transduction via SMAD2 and SMAD3 [22]. Using the siRNA-mediated knockdown approach, we defined that both SMAD2 and SMAD3 were involved in GDF-11-induced MMP2 expression in HTR-8/SVneo cells. GDF-11 is also known as BMP-11. Like other BMP proteins, one study has demonstrated that GDF-11 can activate SMAD1/5/8 signaling pathways in human umbilical vein endothelial cells [38,39]. Using BMP4 as a positive control, we found that SMAD1/5/8 signaling pathways were not activated by the GDF-11 in both HTR-8/SVneo and primary EVT cells. It is interesting to note that inhibiting SMAD signaling pathways did not fully block the stimulatory effect of GDF-11 on MMP2 expression. These results suggest the involvement of other SMAD-independent non-canonical signaling pathways in GDF-11-stimulated MMP2 expression. Thus, further investigation is warranted to explore whether other signaling pathways contribute to GDF-11-induced MMP2 expression in human EVT cells.
Because lacking the DNA-binding domain, ID proteins act as transcriptional modulators by heterodimerizing with bHLH transcription factors to inhibit their DNA binding activity [40,41]. The expression of MMP2 is regulated by the ID2 and ID2 promotes cell migration and invasion in different types of human cancer cells [30,31,42]. Consistent with those previous studies, using ID2 siRNA, we showed that induction of ID2 was required for the GDF-11-stimulated MMP2 expression. In addition, the knockdown of ID2 attenuated the GDF-11-induced invasiveness in HTR-8/SVneo cells. In the human placenta, after differentiation, both mRNA and protein levels of ID2 are downregulated in cytotrophoblast cells. Overexpression of ID2 inhibits cytotrophoblast cell invasion but dramatically promotes cell migration [43]. The cause of the opposite effects on cell migration and invasion by ID2 overexpression in human cytotrophoblast cells remains unknown. However, the anti-invasive effect of ID2 in human cytotrophoblast cells is in contrast to our results in HTR-8/ SVneo cells. The major factor that leads to this distinct effect may be the supra-physiological overexpression which can commonly induce off-target effects. In addition, the HTR-8/SVneo cell line was generated using first-trimester EVT cells infected with SV40 large T antigen [24]. The immortalization process and the different natures of cytotrophoblast and EVT cells could also result in the opposite effects of ID2 in trophoblast cell invasion.

Conclusions
In summary, in the present study, we report a novel function of GDF-11 in the human placenta which GDF-11 stimulates human EVT cell invasion by upregulating MMP2 expression. Mechanically, we delineate that ALK4/5-mediated SMAD2/3 signaling pathways are involved in the stimulatory effect of GDF-11 on MMP2 expression. In addition, we show that ID2 protein is upregulated by GDF-11 and that is required for GDF-11-stimulated MMP2 expression and EVT cell invasion. Our study not only discovers the function of GDF-11 but also provides important insights into the regulation of MMP2 expression in the human placenta.