CCAAT binding transcription factor binds and regulates human COL1A1 promoter activity in human dermal fibroblasts - Demonstration of increased binding in systemic sclerosis fibroblasts

Objective. To determine the binding factors that interact with the proximal promoter region of the human type I collagen gene, COL1A1, and to examine their involvement in its transcriptional regulation in normal and systemic sclerosis (SSc) dermal fibroblasts, Methods. Nuclear extracts from dermal fibroblasts from 4 patients with SSc and 4 age- and sex-matched control individuals were examined by electrophor esis mobility shift assays with a COL1A1 promoter fragment encompassing nuc leotides -174 to -50 bp, Supershift assays with antibodies specific to vari ous transcription factors, and competition experiments using consensus, wil d-type, or mutated oligonucleotides corresponding to their specific binding sites, were performed, The effects of specific oligonucleotides as "intrac ellular competitors"' were examined by transient transfection experiments i n SSc fibroblasts using a COL1A1 construct containing -174 bp of the promot er. Results. The findings demonstrate that the CCAAT binding transcription fact or (CBF) binds the proximal CCAAT box located at -100 to -96 bp, but not th e distal CCAAT box at -125 to -121 bp, of the human COL1A1 promoter in both SSc and normal fibroblasts, CBF binding activity was 3-5-fold higher in th e SSc fibroblasts, Moreover, the promoter activity of the -174-bp COL1A1 co nstruct was decreased by up to 50% when specific oligonucleotides were used as "intracellular competitors." In addition, Sp1 and Sp3 were other transc ription factors found to be involved in the formation of the DNA-protein co mplexes within this region of the COL1A1 promoter. Conclusion. These results indicate that the transcription factor CBF binds the human COL1A1 proximal promoter region in human dermal fibroblasts, and its binding activity is higher in SSc fibroblasts.