Transforming growth factor-␤1 (TGF-␤1)-induced epithelial-to-mesenchymal transition (EMT) contributes to the pathophysiological development of kidney fibrosis. Although it was reported that TGF-␤1 enhances ␤ 1 integrin levels in NMuMG cells , the detailed molecular mechanisms underlying TGF-␤1-induced ␤ 1 integrin gene expression and the role of ␤ 1 integrin during EMT in the renal system are still unclear. In this study , we examined the role of ␤ 1 integrin in TGF-␤1-induced EMT both in vitro and in vivo. TGF-␤1-induced augmentation of ␤ 1 integrin expression was required for EMT in several epithelial cell lines, and knockdown of Smad3 inhibited TGF-␤1-induced augmentation of ␤ 1 integrin. TGF-␤1 triggered ␤ 1 integrin gene promoter activity as assessed by luciferase activity assay. Both knockdown of Smad3 and mutation of the Smad-binding element to block binding to the ␤ 1 integrin promoter markedly reduced TGF-␤1-induced ␤ 1 integrin promoter activity. Chromatin immunoprecipitation assay showed that TGF-␤1 enhanced Smad3 binding to the ␤ 1 integrin promoter. Furthermore, induction of unilateral ureteral obstruction triggered increases of ␤ 1 integrin in both renal epithelial and interstitial cells. In human kidney with chronic tubulointerstitial fibrosis, we also found a concomitant increase of ␤ 1 integrin and ␣-smooth muscle actin in tubule epithelia. Blockade of ␤ 1 integrin signaling dampened the progression of fibrosis. Taken together, ␤ 1 integrin mediates EMT and subsequent tubulointerstitutial fibrosis, suggesting that inhibition of ␤ 1 integrin is a possible therapeutic target for prevention of renal fibrosis.

We previously demonstrated that α3β1 integrins are essential to hepatocyte growth factor (HGF)-independent branching tubulogenesis in Mardin-Darby Canine Kidney (MDCK) cells. However, the involvement of integrin downstream signaling molecules remains unclear. In the present study, we successfully isolated cell lines possessing different tubulogenic potentials from the MDCK cells; cyst clones (CA4, CA6) forming cystic structures when cultured in 0.3% type I collagen gel and mass clones (M610, M611, M612) forming aggregated masses. Cyst clones maintained cystic structure in 0.1% collagen gel, whereas mass clones spontaneously developed into tubules. Both clones exhibited various morphologies when cultured on a dish: cyst clones formed aggregated islands, while mass clones were more scattered and exhibited higher migration capacity. Among several focal adhesion machinery proteins examined, only the expression and phosphorylation level of focal adhesion kinase (FAK) in mass clones was higher than in cyst clones, while other proteins showed no obvious differences. However, overexpression of wild type FAK in CA6 cells did not facilitate branching tubule formation in 0.1% collagen gel. Targeted decrease in the expression level of FAK in M610 cells with the application of antisense cDNA resulted in a marked reduction of branching tubule formation in 0.1% collagen gel and showed a down-regulation of fibronectin assembly, which is known to promote tubulogenesis. In contrast, overexpression of wild type FAK in CA6 cells had no effect on fibronectin assembly. Taken together, our data demonstrates that FAK is required, but not sufficient for HGF-independent branching tubulogenesis in MDCK cells.

Previous study demonstrated that low substratum rigidity down-regulates focal adhesion proteins. In this study we found that cells cultured on collagen gel exhibited higher migration capacity than those cultured on collagen gel-coated dishes. Low rigidity of collagen gel induced delayed but persistent phosphorylation of ERK1/2. Inhibition of collagen gel-induced ERK1/2 phosphorylation by MEK inhibitors and ERK2 kinase mutant induced a rounding up of the cells and prevented collagen gel-induced cell migration. Interestingly, phosphorylated ERK1/2 induced by low rigidity was present in focal adhesion sites and the lipid raft. MbetaCD (Methyl-beta-cyclodextrin), a lipid raft inhibitor, inhibited collagen gel-induced ERK1/2 phosphorylation, and cell migration. Overexpression of FAK C-terminal fragment (FRNK) in MDCK cells triggered ERK phosphorylation. Meanwhile, low substratum rigidity induced degradation of FAK into a 35 kDa C-terminal fragment. A calpain inhibitor that partially rescued FAK degradation also prevented low rigidity-induced ERK phosphorylation. However, MbetaCD did not prevent low rigidity-induced FAK degradation. Taken together, we demonstrate that the degradation product of FAK induced by collagen gel triggers activation of ERK1/2, which in turn facilitates cell spreading and migration through the lipid raft.