Increased phosphorylation of transcription factor Sp1 in scleroderma fibroblasts - Association with increased expression of the type I collagen gene

Objective. To determine the potential roles of transcription factors Sp1 an d Sp3 in the increased expression of the human alpha 2(I) collagen gene in scleroderma fibroblasts. Methods, Dermal fibroblasts from 7 patients with diffuse systemic sclerosis (SSc; scleroderma) of recent onset and from 7 healthy individuals were stu died, The levels of expression of alpha 2(I) procollagen, Sp1, and Sp3 mess enger RNA (mRNA), with or without stimulation by transforming growth factor beta (TGF beta) or oncostatin M (OSM), were evaluated by Northern blot ana lysis, and the respective protein levels were determined by immunoblotting. The DNA binding activity of nuclear proteins recognizing the cis-acting el ements in the human alpha 2(I) collagen promoter was examined by gel mobili ty shift assays. The levels of Sp1 phosphorylation were investigated by imm unoprecipitation using an antiphosphoserine-specific antibody. Results. SSc fibroblasts showed basal alpha 2(I) collagen mRNA levels that were similar to 3 times higher than those in normal fibroblasts, TGF beta o r OSM increased human alpha 2(I) collagen mRNA expression in normal dermal fibroblasts, but these cytokines failed to increase alpha 2(I) collagen mRN A levels in SSc fibroblasts. There were no significant differences in the l evels of expression of Sp1 or Sp3 between SSc and normal fibroblasts. Howev er, increased Sp1 phosphorylation was detected in SSc fibroblasts compared with normal fibroblasts, Mithramycin, a specific inhibitor of Spl binding, abolished the increased expression of the alpha 2(I) collagen gene in SSc f ibroblasts, in a dose-dependent manner. Conclusion. These results demonstrate the involvement of Sp1 in the up-regu lation of expression of the alpha 2(I) collagen gene in SSc fibroblasts.