Involvement of alpha V beta 5 integrin-mediated activation of latent transforming growth factor beta 1 in autocrine transforming growth factor beta signaling in systemic sclerosis fibroblasts

Objective. To confirm the involvement of alpha v beta 5 in the self-activation system in systemic sclerosis (SSc) fibroblasts. Methods. Levels of alpha v beta 5 expression were analyzed by immunoprecipitation. The promoter activity of the human alpha 2(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 (TGF beta) 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 alpha v beta 5 expression were significantly elevated in SSc fibroblasts compared with normal fibroblasts. Treatment with anti-alpha v beta 5 antibody or beta 5 antisense oligonucleotide significantly reduced human alpha 2(1) collagen gene promoter activity in SSc fibroblasts. In SSc fibroblasts pretreated with TGF beta 1 antisense oligonucleotide, the exogenous latent TGF beta 1 stimulation significantly increased human alpha 2(I) collagen gene promoter activity; this effect was significantly reduced in the presence of anti-alpha v beta 5 antibody. Phosphorylation levels and DNA binding ability of Smad3 in SSc fibroblasts were significantly reduced by treatment with beta 5 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-alpha v beta 5 antibody. Anti-alpha v beta 5 antibody reversed the myofibroblastic features of SSc fibroblasts. Conclusion. Up-regulated expression of alpha v beta 5 contributes to the establishment of autocrine TGF beta signaling in SSc fibroblasts through activation of endogenous latent TGF beta 1.