Increased phosphorylation of transcription factor Sp1 in scleroderma fibroblasts: association with increased expression of the type I collagen gene.

Abstract

To determine the potential roles of transcription factors Sp1 and Sp3 in the increased expression of the human alpha2(I) collagen gene in scleroderma fibroblasts. Dermal fibroblasts from 7 patients with diffuse systemic sclerosis (SSc; scleroderma) of recent onset and from 7 healthy individuals were studied. The levels of expression of alpha2(I) procollagen, Sp1, and Sp3 messenger RNA (mRNA), with or without stimulation by transforming growth factor beta (TGFbeta) or oncostatin M (OSM), were evaluated by Northern blot analysis, and the respective protein levels were determined by immunoblotting. The DNA binding activity of nuclear proteins recognizing the cis-acting elements in the human alpha2(I) collagen promoter was examined by gel mobility shift assays. The levels of Sp1 phosphorylation were investigated by immunoprecipitation using an antiphosphoserine-specific antibody. SSc fibroblasts showed basal alpha2(I) collagen mRNA levels that were approximately 3 times higher than those in normal fibroblasts. TGFbeta or OSM increased human alpha2(I) collagen mRNA expression in normal dermal fibroblasts, but these cytokines failed to increase alpha2(I) collagen mRNA levels in SSc fibroblasts. There were no significant differences in the levels of expression of Sp1 or Sp3 between SSc and normal fibroblasts. However, increased Sp1 phosphorylation was detected in SSc fibroblasts compared with normal fibroblasts. Mithramycin, a specific inhibitor of Sp1 binding, abolished the increased expression of the alpha2(I) collagen gene in SSc fibroblasts, in a dose-dependent manner. These results demonstrate the involvement of Sp1 in the up-regulation of expression of the alpha2(I) collagen gene in SSc fibroblasts.