Involvement of alphavbeta5 integrin-mediated activation of latent transforming growth factor beta1 in autocrine transforming growth factor beta signaling in systemic sclerosis fibroblasts

Abstract

Objective: To confirm the involvement of alphavbeta5 in the self-activation system in systemic sclerosis (SSc) fibroblasts.

Methods: Levels of alphavbeta5 expression were analyzed by immunoprecipitation. The promoter activity of the human alpha2(I) collagen gene was determined by transient transfection assay. Phosphorylation levels and DNA binding ability of Smad3 were investigated by immunoprecipitation and DNA affinity precipitation, respectively. The localization of active transforming growth factor beta (TGFbeta) was determined by coculture assay using TMLC cells (mink lung epithelial reporter cells that stably express a portion of the plasminogen activator inhibitor 1 promoter). The morphologic features of cells were determined by immunofluorescence analysis.

Results: Levels of alphavbeta5 expression were significantly elevated in SSc fibroblasts compared with normal fibroblasts. Treatment with anti-alphavbeta5 antibody or beta5 antisense oligonucleotide significantly reduced human alpha2(I) collagen gene promoter activity in SSc fibroblasts. In SSc fibroblasts pretreated with TGFbeta1 antisense oligonucleotide, the exogenous latent TGFbeta1 stimulation significantly increased human alpha2(I) collagen gene promoter activity; this effect was significantly reduced in the presence of anti-alphavbeta5 antibody. Phosphorylation levels and DNA binding ability of Smad3 in SSc fibroblasts were significantly reduced by treatment with beta5 antisense oligonucleotide. The luciferase activity of TMLC cells cocultured with SSc fibroblasts was significantly elevated compared with that of TMLC cells cocultured with normal fibroblasts and was significantly reduced in the presence of anti-alphavbeta5 antibody. Anti-alphavbeta5 antibody reversed the myofibroblastic features of SSc fibroblasts.

Conclusion: Up-regulated expression of alphavbeta5 contributes to the establishment of autocrine TGFbeta signaling in SSc fibroblasts through activation of endogenous latent TGFbeta1.