TGF-β1 induces phenotypic differentiation and synthesis of extracellular matrix in fibroblasts derived from urethral scar via Rho/ROCK signal pathway

Abstract

Objective To examine the effect of Rho/ROCK signal pathway on TGF-β1-induced phenotypic differentiation and synthesis of extracellular matrix in fibroblasts derived from urethral scar.Methods Fibroblasts derived from urethral scar were cultured.All experiments were performed using the cells at the fourth passage.At 80％ confluence the medium was supplemented with TGF-β1 (5,10 ng/ml).After 48 hours incubation,the productions of α-SMA were assayed by Western-blot.The productions of collagen Ⅲ and fibronectin in supernatants culture were examined using ELISA.Then the fibroblasts were stimulated with TGF-β1 (10 ng/ml) after treated with the Rho/ROCK signal pathway inhibitor Y-27632(10μmol/L).After 48 hours incubation,the productions of α-SMA,collagen Ⅲ,and fibronectin were assayed,Results TGF-β1 markedly induced α-SMA,collagen Ⅲ,and fibronectin expression in cultured fibroblasts in a dose dependent manner (P＜0.01).However,simultaneous incubation of Y-27632 significantly abrogated TGF-β1 induced α-SMA,collagen Ⅲ,and fibronectin expression (P＜0.01 ).Conclusions Rho/ROCK signaling pathway should be involved in on TGF-β1-induced phenotypic differentiation and synthesis of extraccllular matrix in fibroblasts derived from urethral scar.The findings provide a basis for prevention and treatment of urethral scar. Key words: Transforming growth factor-β; α-smooth muscle actin