Figure 2

Transforming growth factor (TGF)-β1-induced epithelial mesenchymal transition (EMT) in human bronchial epithelial cells (HBECs) is Smad-dependent. HBECs that have been pre-incubated with the activin receptor-like kinase (ALK)-5 inhibitor SB431542 (SB, 10 μM) 1 h before the addition of TGF-β1 (5 ng/ml) are unable to undergo EMT after 5 days of differentiation. By contrast, mitogen-activated protein kinase inhibition by PD98059, a MEK inhibitor (PD, 10 μM), only partially inhibits EMT. (a) Representative phase contrast images (10× magnification, scale bar = 10 μM) show that HBECs in the presence of TGF-β1 lose cell-cell contact, become more sparse and change into an elongated fibroblastoid morphology. This effect is inhibited after pre-incubation of the cells with the ALK-4,-5,-7 inhibitor SB but not by the MEK inhibitor PD. (b) Western-blot analysis shows that SB completely abolishes TGF-β1 gene regulation of E-cad and the phosho-Smad2 signal is fully inhibited in the presence of SB. An antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (c) Western-blot analysis shows that the upregulation of matrix metalloprotease (MMP)-2 by TGF-β1 is partially inhibited by the presence of bone morphogenetic proteins (BMP)4 confirming the quantitative polymerase chain reaction (qPCR) results shown in (d). Phosho-Smad1/5/8 shows active BMP signalling in the presence of BMP4 which is inhibited in the presence of TGF-β1. An antibody against GAPDH was used as loading control. (d) q(PCR) analysis shows that SB inhibits gene expression changes of collagen I α 1, MMP2, MMP-9 and E-cad induced by TGF-β1 (top 2 panels). BMP4 (50 ng/ml) induces a significant upregulation of collagen I in the presence of TGF-β1, as compared to TGF-β1 alone (top left panel) and has no significant effect on E-cad expression either alone or in combination with TGF-β1 (top right panel). In contrast, the upregulation of MMP-2 and MMP-9 expression in the presence of TGF-β1 is significantly inhibited by concomitant BMP4 treatment (bottom 2 panels). The relative expression level of each gene was normalized to GAPDH mRNA in the same sample. Statistical significance was determined by one-way ANOVA followed by Tukey test; ***P < 0.001.