Focal adhesion kinase mediates TGF-beta1-induced renal tubular epithelial-to-mesenchymal transition in vitro

Abstract

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase which participates in many important cellular processes such as cell adhesion and migration. However, the role of FAK in renal tubular epithelial-to-mesenchymal transition (EMT) is still unknown. FAK was knocked down by transfection of specific small interfering RNA (siRNA) in cultured HK-2 cells, then the cells were stimulated with transforming growth factor-beta 1 (TGF-beta1). The expression of FAK, alpha-smooth muscle actin (alpha-SMA),E-cadherin, Akt, matrix metallopeptidase-9 (MMP-9),tissue inhibitor of metalloproteinase-1 (TIMP-1), and collagen IV were detected by RT-PCR, Western blot and immunofluorescence methods, respectively. Cell migration was determined by transwell assay. The results suggest that the expression of FAK was up-regulated in HK-2 cells when incubated with TGF-beta1(10 microg/l), which was accompanied by reduced expression of E-cadherin and increased expression of alpha-SMA. All these changes were restored by FAK siRNA. Akt phosphorylation was induced by the treatment with TGF-beta1, which was blocked by FAK siRNA. TGF-beta1-induced down-regulation of E-cadherin was recovered by a PI3K/Akt inhibitor, LY294002, without affecting the expression of FAK. Functionally, TGF-beta1 induced an increase in MMP-9 expression, as well as decreased expression of TIMP-1 and collagen IV, which were all restored by the FAK siRNA transfection. In addition, FAK siRNA significantly reduced TGF-beta1-induced cells migration. In conclusion, FAK may play a crucial role in mediating TGF-beta1-induced EMT through the activation of Akt pathway.