Transforming growth factor-β1 inhibits trophoblast cell invasion by inducing Snail-mediated down-regulation of vascular endothelial-cadherin protein

Abstract

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.

Keywords: Cadherins; Invasion; SMAD Transcription Factor; Snail; Transforming Growth Factor-β (TGFβ); Trophoblast; VE-cadherin.

FIGURE 1.

TGF-β1 decreases HTR-8/SVneo cell invasion. A, cells were treated with increasing concentrations of TGF-β1 (1, 5, and 10 ng/ml) and seeded onto Matrigel-coated transwell inserts. After 48 h of incubation, non-invading cells were wiped from the upper side of the filter, and the nuclei of the invading cells were stained with Hoechst 33258. The top panels show representative images of the invasion assays. The scale bar represents 200 μm. The bottom panels show the summarized quantitative results. B, cells were treated with 5 ng/ml of TGF-β1 in combination with SB431542 (10 μm). Cell invasion was examined using the Matrigel invasion assay. C, cells were transfected with 50 nm control siRNA (si-Ctrl) or TβRI siRNA (si-TβRI) for 48 h. The protein levels of TβRI were examined using Western blot analyses. D, after TβRI knockdown, cells were treated with 5 ng/ml of TGF-β1 and cell invasion was examined using the Matrigel invasion assay. E, cells were treated with 5 ng/ml of TGF-β1 every 24 h, and the number of cells was quantified using the trypan blue exclusion assay. F, cells were treated with 5 ng/ml of TGF-β1 for 24 and 48 h. The cell morphology was microscopically examined. The results of the invasion assay were expressed as the mean ± S.E. of at least three independent experiments. Values without a common letter were significantly different (p < 0.05).

FIGURE 2.

TGF-β1 down-regulates VE-cadherin in human trophoblast cells. A, HTR-8/SVneo cells were treated with increasing concentrations of TGF-β1 (1, 5, and 10 ng/ml), and the mRNA levels of VE-cadherin were examined using RT-qPCR. B, HTR-8/SVneo cells were treated with 5 ng/ml of TGF-β1, and the mRNA levels of VE-cadherin were analyzed at different time points using RT-qPCR. C, HTR-8/SVneo cells were treated with 1, 5, and 10 ng/ml of TGF-β1 for 24 and 48 h. The protein levels of VE-cadherin were examined by Western blot. D, three different primary cultures of human trophoblast cells (primary trophoblast cell (PTC); # 1, 2, and 3) were treated with 5 ng/ml of TGF-β1 for 24 h, and the protein levels of VE-cadherin were examined by Western blot. The RT-qPCR results were expressed as the mean ± S.E. of at least three independent experiments. Values without a common letter were significantly different (p < 0.05).

FIGURE 3.

TGF-β type I receptor is required for TGF-β1-induced down-regulation of VE-cadherin. A and B, HTR-8/SVneo cells were treated with 5 ng/ml of TGF-β1 in combination with SB431542 (10 μm). The mRNA (A) and protein levels (B) of VE-cadherin were examined using RT-qPCR and Western blot, respectively. C and D, HTR-8/SVneo cells were transfected with 50 nm control siRNA (si-Ctrl) or TβRI siRNA (si-TβRI) for 48 h and then treated with 5 ng/ml of TGF-β1 for 24 h. The mRNA (C) and protein (D) levels of VE-cadherin and TβRI were examined using RT-qPCR and Western blot, respectively. E, the endogenous protein levels of E-cadherin and N-cadherin in HTR-8/SVneo and two different primary human trophoblast cultures (PTC)(PTC1 and PTC2) were examined using Western blot. The human choriocarcinoma cell line JEG3 was used as a positive control to detect E-cadherin. F, HTR-8/SVneo cells were treated with 5 ng/ml of TGF-β1 for 24 h. The protein levels of E-cadherin and N-cadherin were examined using Western blot. The RT-qPCR results were expressed as the mean ± S.E. of at least three independent experiments. Values without a common letter were significantly different (p < 0.05). C indicates control, and T indicates TGF-β1.

FIGURE 4.

VE-cadherin is required to maintain the invasive capacity of human trophoblast cells. A, HTR-8/SVneo and primary trophoblast culture cells were transfected with 50 nm control siRNA (si-Ctrl) or VE-cadherin siRNA (si-VE) for 24 and 48 h. The TβRI protein levels were examined by Western blot. B, after VE-cadherin knockdown, cell invasion was examined using the Matrigel invasion assay. C, HTR-8/SVneo cells were transfected with 50 nm control siRNA (si-Ctrl) or VE-cadherin siRNA (si-VE) for 24 h and then treated with 5 ng/ml of TGF-β1 for 24 h. The VE-cadherin protein levels were examined using Western blot. D, after VE-cadherin knockdown, HTR-8/SVneo cells were treated with 5 ng/ml of TGF-β1 and cell invasion was examined using the Matrigel invasion assay. The results of the invasion assay were expressed as the mean ± S.E. of at least three independent experiments. Values without a common letter were significantly different (p < 0.05). C indicates control, and T indicates TGF-β1.

FIGURE 5.

Smad-dependent signaling pathways are required for TGF-β1-induced down-regulation of VE-cadherin. A, HTR-8/SVneo cells were treated with 5 ng/ml of TGF-β1 for the indicated durations. The Smad2 and Smad3 phosphorylation levels were examined using Western blot with antibodies specific for the phosphorylated, activated forms of Smad2 (p-Smad2) and Smad3 (p-Smad3). B and C, HTR-8/SVneo cells were transfected with 50 nm control siRNA (si-Ctrl) or Smad4 siRNA (si-Smad4) for 48 h and then treated with 5 ng/ml of TGF-β1 for 24 h. The mRNA (B) and protein (C) levels of VE-cadherin and Smad4 were examined using RT-qPCR and Western blot, respectively. D and E, HTR-8/SVneo 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 5 ng/ml of TGF-β1 for 24 h. The mRNA (D) and protein (E) levels of VE-cadherin, Smad2, and Smad3 were examined using RT-qPCR and Western blot, respectively. The RT-qPCR results were expressed as the mean ± S.E. of at least three independent experiments. Values without a common letter were significantly different (p < 0.05). C indicates control, and T indicates TGF-β1.

FIGURE 6.

TGF-β1 induces Snail and Slug expression. A, HTR-8/SVneo cells were treated with 5 ng/ml of TGF-β1 for the indicated durations. The Snail, Slug, Twist, and ZEB1 mRNA levels were examined by RT-qPCR. B, HTR-8/SVneo cells were treated with increasing concentrations of TGF-β1 (1, 5, and 10 ng/ml) for 24 h. The mRNA levels of Snail and Slug were examined by RT-qPCR. C, HTR-8/SVneo cells were treated with 5 ng/ml of TGF-β1 for the indicated durations. The protein levels of Snail and Slug were examined using Western blot. D, HTR-8/SVneo cells were treated with increasing concentrations of TGF-β1 (1, 5, and 10 ng/ml) for 24 h. The protein levels of Snail and Slug were examined by Western blot. E and F, HTR-8/SVneo cells were treated with 5 ng/ml of TGF-β1 in combination with SB431542 (10 μm). The mRNA (E) and protein (F) levels of Snail and Slug were examined using RT-qPCR and Western blot, respectively. G and H, HTR-8/SVneo cells were transfected with 50 nm control siRNA (si-Ctrl) or TβRI siRNA (si-TβRI) for 48 h and then treated with 5 ng/ml of TGF-β1 for 24 h. The mRNA (G) and protein (H) levels of Snail and Slug were examined using RT-qPCR and Western blot, respectively. The RT-qPCR results were expressed as the mean ± S.E. of at least three independent experiments. Values without a common letter were significantly different (p < 0.05). C indicates control, and T indicates TGF-β1.

FIGURE 7.

Smad-dependent signaling pathways are required for the TGF-β1-induced up-regulation of Snail and Slug. A and B, HTR-8/SVneo cells were transfected with 50 nm control siRNA (si-Ctrl) or Smad4 siRNA (si-Smad4) for 48 h and then treated with 5 ng/ml of TGF-β1 for 24 h. The mRNA (A) and protein (B) levels of Snail and Slug were examined using RT-qPCR and Western blot, respectively. C and D, HTR-8/SVneo 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 5 ng/ml of TGF-β1 for 24 h. The mRNA (C) and protein (D) levels of Snail and Slug were examined using RT-qPCR and Western blot, respectively. The RT-qPCR results were expressed as the mean ± S.E. of at least three independent experiments. Values without a common letter were significantly different (p < 0.05). C indicates control, and T indicates TGF-β1.

FIGURE 8.

Snail mediates the TGF-β1-induced down-regulation of VE-cadherin. A and B, HTR-8/SVneo cells were transfected with 50 nm control siRNA (si-Ctrl), Snail siRNA (si-Snail), or Slug siRNA (si-Slug) for 48 h and then treated with 5 ng/ml of TGF-β1 for 24 h. The mRNA (A) and protein (B) levels of VE-cadherin, Snail, and Slug were examined using RT-qPCR and Western blot, respectively. C and D, HTR-8/SVneo cells were transfected with control vector (pCMV) or vector containing full-length Snail cDNA (Snail) for 24 and 48 h. The mRNA (C) and protein (D) levels of VE-cadherin and Snail were examined using RT-qPCR and Western blot, respectively. E, after knockdown and overexpression of Snail in the HTR-8/SVneo cells, cell invasion was examined using the Matrigel invasion assay. F, the Snail binding site in the human VE-cadherin promoter is highlighted by a box. The primers for the ChIP assay are underlined. G, HTR-8/SVneo cells were treated with 5 ng/ml of TGF-β1 for 24 h before being subjected to ChIP analysis. Anti-Snail or IgG antibodies were used to immunoprecipitate DNA-containing complexes. Subsequent PCR was performed with primers complementary to the VE-cadherin promoter region containing the Snail binding site. The PCR products were resolved by electrophoresis in a 1% agarose gel and visualized by ethidium bromide staining. M indicates the 100-bp DNA ladder. C indicates control, and T indicates TGF-β1.

FIGURE 9.

A schematic illustrating the signaling pathways for TGF-β1-induced down-regulation of VE-cadherin and the inhibition of cell invasion in human trophoblast cells. TGF-β1 treatment resulted in the activation of Smad2 and Smad3, which up-regulated the expression of Snail and Slug. The Smad2-dependent pathway is more involved in TGF-β1-induced up-regulation of Snail that Slug (thick versus thin arrow). In contrast, TGF-β1-induced up-regulation of Slug is primarily mediated by the Smad3-dependent pathway. Up-regulated Snail but not Slug subsequently binds to the E-box on the human VE-cadherin promoter and contributes to the TGF-β1-induced down-regulation of VE-cadherin and the inhibition of trophoblast cell invasion.