TNF-α enhances TGF-β-induced endothelial-to-mesenchymal transition via TGF-β signal augmentation

Abstract

The tumor microenvironment (TME) consists of various components including cancer cells, tumor vessels, cancer-associated fibroblasts (CAFs), and inflammatory cells. These components interact with each other via various cytokines, which often induce tumor progression. Thus, a greater understanding of TME networks is crucial for the development of novel cancer therapies. Many cancer types express high levels of TGF-β, which induces endothelial-to-mesenchymal transition (EndMT), leading to formation of CAFs. Although we previously reported that CAFs derived from EndMT promoted tumor formation, the molecular mechanisms underlying these interactions remain to be elucidated. Furthermore, tumor-infiltrating inflammatory cells secrete various cytokines, including TNF-α. However, the role of TNF-α in TGF-β-induced EndMT has not been fully elucidated. Therefore, this study examined the effect of TNF-α on TGF-β-induced EndMT in human endothelial cells (ECs). Various types of human ECs underwent EndMT in response to TGF-β and TNF-α, which was accompanied by increased and decreased expression of mesenchymal cell and EC markers, respectively. In addition, treatment of ECs with TGF-β and TNF-α exhibited sustained activation of Smad2/3 signals, which was presumably induced by elevated expression of TGF-β type I receptor, TGF-β2, activin A, and integrin αv, suggesting that TNF-α enhanced TGF-β-induced EndMT by augmenting TGF-β family signals. Furthermore, oral squamous cell carcinoma-derived cells underwent epithelial-to-mesenchymal transition (EMT) in response to humoral factors produced by TGF-β and TNF-α-cultured ECs. This EndMT-driven EMT was blocked by inhibiting the action of TGF-βs. Collectively, our findings suggest that TNF-α enhances TGF-β-dependent EndMT, which contributes to tumor progression.

Keywords: TGF-β; TGF-β type I receptor (ALK5); TNF-α; activin; endothelial-to-mesenchymal transition; epithelial-to-mesenchymal transition; integrin αv.

Figure 1

Effects of TGF‐β2 and TNF‐α on the expression of TGF‐β2 and TNF‐α target genes in HUAECs. HUAECs were cultured in the absence (−) or presence (+) of 1 ng/mL TGF‐β2 in combination with 10 ng/mL TNF‐α for 4 h (A, B) or 72 h (C, D), followed by qRT‐PCR analyses for TMEPAI (A, C) and ICAM1 (B, D) expression. Error bars represent standard deviation. *P < 0.05

Figure 2

Effects of TGF‐β2 and TNF‐α on endothelial and mesenchymal characteristics of multiple types of endothelial cells. HUAECs (A‐C) and HUVECs (D‐F) were cultured in the absence (−) or presence (+) of 1 ng/mL TGF‐β2 in combination with 10 ng/mL TNF‐α (HUAECs) and 15 ng/mL TNF‐α (HUVECs), respectively, for 72 h, followed by qRT‐PCR analyses for SM22α (A, D), MMP2 (B, E), and Claudin 5 (CLDN5) (C, F) expression. Error bars represent standard deviation. *P <0.05; n.s., not significant

Figure 3

Effects of TGF‐β2 and TNF‐α on mesenchymal characteristics of HUAECs. HUAECs were cultured in the absence (−) or presence (+) of 1 ng/mL TGF‐β2 in combination with 10 ng/mL TNF‐α for 72 h, followed by fluorescence immunostaining for SM22α (magenta) and nuclei (blue) (A). Scale bar, 100 μm. (B) Numbers of SM22α‐positive cells were counted in 4 fields. Data are shown as the mean ± standard deviation of 4 independent experiments. *P <0.0001.

Figure 4

Effects of TGF‐β2 and TNF‐α on tube forming ability of HUAECs. HUAECs were cultured in the absence (−) or presence (+) of 1 ng/mL TGF‐β2 in combination with 10 ng/mL TNF‐α for 72 h, followed by tube formation assay. Cells were allowed to form tube‐like structures on a collagen gel for 8 h, followed by phase‐contrast imaging (A) and quantification (B) of tube‐like structures. Scale bars, 200 μm. Error bars represent standard deviation. *P <0.05; ND, not detected

Figure 5

Effects of TGF‐β2 and TNF‐α on expression of various TGF‐β signaling components in HUAECs. HUAECs were cultured in the absence (−) or presence (+) of 1 ng/mL TGF‐β2, 10 ng/mL TNF‐α, or a combination of both cytokines for 72 h, followed by qRT‐PCR analyses for expression of ALK5 (A), TGF‐β1 (TGFB1) (B), TGF‐β2 (TGFB2) (C), TGF‐β3 (TGFB3) (D), inhibin βA (INHBA) (E), and integrin αv (ITGAV) (F). Error bars represent standard deviation. *P <0.05; n.s., not significant

Figure 6

Effects of RelA on TGF‐β2 and TNF‐α‐induced expression of various markers in HUAECs. HUAECs transfected with negative control siRNA (NC) or siRNAs for RelA (RelA‐A and RelA‐B) were cultured in the absence (−) or presence (+) of 1 ng/mL TGF‐β2 in combination with 10 ng/mL TNF‐α for 72 h, followed by qRT‐PCR analyses for expression of RELA (A), ICAM1 (B), TMEPAI (C), SM22α (D), ALK5 (E), TGF‐β2 (TGFB2) (F), inhibin βA (INHBA) (G), and integrin αv (ITGAV) (H). Error bars represent standard deviation. *P <0.05; n.s., not significant

Figure 7

Effects of TGF‐β2 and IL‐1β on their target genes and mesenchymal cell markers in HUAECs. HUAECs were cultured in the absence (−) or presence (+) of 1 ng/mL TGF‐β2 in combination with 3 ng/mL IL‐1β for 72 h, followed by qRT‐PCR analyses for expression of ICAM1 (A), TMEPAI (B), SM22α (C), and MMP2 (D). Error bars represent standard deviation. *P <0.05; n.s., not significant

Figure 8

Quantification of Smad2/3‐activating factors in conditioned medium (CM) of HUAECs using HEK‐Blue TGF‐β reporter cells. (A) HUAECs were cultured in the absence (−) or presence (+) of 1 ng/mL TGF‐β2 in combination with 10 ng/mL TNF‐α for 60 h, followed by DMEM with 1% FBS, replacement and further culture for 16 h. (B) HEK‐Blue TGF‐β cells were cultured in the CM derived from HUAECs cultured for 16 h, followed by the measurement of absorbance at 640 nm, which represented colorimetric change of HEK‐Blue substrate by secreted alkaline phosphatase induced by Smad2/3 signal. (C, D) HEK‐Blue TGF‐β cells were cultured in the CM derived from HUAECs, in the absence (−) or presence (+) of 50 ng/mL of follistatin (C), control IgG (50 μg/mL) and anti‐TGF‐β (1D11: 50 μg/mL) (D), followed by the measurement of the absorbance at 640 nm representing colorimetric change of HEK‐Blue substrate by SEAP alkaline phosphatase induced by Smad2/3 signals. Values were normalized to the number of HUAECs responsible for secretion of Smad2/3‐activating humoral factors. Error bars represent standard deviation. *P <0.05; n.s., not significant

Figure 9

Effects of HUAEC conditioned medium (CM) on the expression of TMEPAI and mesenchymal cell markers in HSC‐4 oral cancer cells. (A‐C) HUAECs were cultured in the absence (−) or presence (+) of 1 ng/mL TGF‐β2 in combination with 10 ng/mL TNF‐α for 60 h, followed by replacement with DMEM supplemented with 1% FBS and further culture for 16 h. HSC‐4 cells were cultured in the CM derived from HUAECs for 72 h, followed by qRT‐PCR analyses for expression of TMEPAI (A), vimentin (B), and fibronectin (C). (D‐F) HSC‐4 cells were cultured in the CM derived from HUAECs, in the absence (−) or presence (+) of 50 ng/mL follistatin, control IgG (50 μg/mL), and anti‐TGF‐β neutralizing antibody (1D11: 50 μg/mL) for 72 h, followed by qRT‐PCR analysis for expression of vimentin (D, E) and fluorescence immunostaining for E‐cadherin (green), vimentin (red) and nuclei (blue) (F). Error bars represent standard deviation. *P <0.05; n.s., not significant. Scale bar, 25 μm