Transforming Growth Factor-.BETA. Upregulates the Expression of Integrin and Related Proteins in MRC-5 Human Myofibroblasts

Abstract

Myofibroblasts are defined as fibroblasts that express certain features of smooth muscle differentiation. Activation of myofibroblasts plays a central role in fibrosis. Transforming growth factor-beta (TGF-beta) is a potent activator of myofibroblasts; namely, TGF-beta causes changes in myofibroblast phenotypes including morphological alterations and the expression of alpha-smooth muscle actin (alpha-SMA), a marker of myofibroblasts. Because it has been well known that humoral factors, especially, TGF-beta, and extracellular matrix components cause myofibroblast activation, we examined the expression of integrin and related proteins during activation of MRC-5 human myofibroblasts with TGF-beta. Western blot analysis revealed that TGF-beta treatment for 3 days increased the expression of alpha-SMA, which was also immunocytochemically observed as actin stress fibers. In the early phase of TGF-beta treatment, fibronectin expression was greatly increased, followed by the increased expression of integrin alphav and alpha11 and integrin beta1 and beta3. Co-immunoprecipitation assays revealed that the integrin alphav subunit was co-precipitated with integrin beta1 and beta3, and that integrin beta1 was co-precipitated with alpha11, alphav, alpha2, and alpha5. The expression of focal adhesion kinase and integrin-linked kinase proteins was also upregulated by treatment with TGF-beta. In addition, the expression of type I collagen mRNA was increased by TGF-beta, but not type III collagen mRNA, as judged by real-time PCR analysis. These results suggest the possibility that TGF-beta induces fibronectin expression in MRC-5 cells, which subsequently induces the expression of integrin receptors, alphavbeta3, alphavbeta1, and alpha11beta1. This report also shows that expression of integrin alpha11 is upregulated during the TGF-beta-mediated activation of myofibroblasts.