Cyclic Stretch-induced TGF-β1 and Fibronectin Expression is Mediated by β1-integrin Through C-Src- and STAT3-dependent Pathways in Renal Epithelial Cells

Extracellular matrix (ECM) proteins, including fibronectin, may contribute to the early development and progression of renal interstitial fibrosis associated with chronic renal disease. Recent studies showed that β1-integrin is associated with the development of renal fibrosis in a murine model of unilateral ureteral obstruction (UUO). However, the molecular events responsible for β1-integrin-mediated signaling, following UUO, have yet to be determined. In this study, we investigated the mechanism by which mechanical stretch, an in vitro model for chronic obstructive nephropathy, regulates fibronectin and transforming growth factor-β1 (TGF-β1) expression in cultured human proximal tubular epithelium (HK-2) cells. Mechanical stretch upregulated fibronectin and TGF-β1 expression and activated signal transducer and transcription factor 3 (STAT3) in a time-dependent manner. Stretch-induced fibronectin and TGF-β1 were suppressed by a STAT3 inhibitor, S3I-201, and by small interfering RNA (siRNA) targeting human STAT3 (STAT3 siRNA). Similarly, fibronectin and TGF-β1 expression and STAT3 activation induced by mechanical stretch were suppressed by the Src family kinase inhibitor PP2 and by transfection of HK-2 cells with a dominant-negative mutant of c-Src (DN-Src), whereas PP3, an inactive analog of PP2, had no significant effect. Furthermore, mechanical stretch resulted in increased β1-integrin mRNA and protein levels in HK-2 cells. Furthermore, neutralizing antibody against β1-integrin and silencing of β1-integrin expression with siRNAs resulted in decreased c-Src and STAT3 activation and TGF-β1 and fibronectin expression evoked by mechanical stretch. This work demonstrates, for the first time, a role for β1-integrin in stretch-induced renal fibrosis through the activation of c-Src and STAT3 signaling pathways.