The transcription of human α1(I) procollagen gene (COL1A1) is suppressed by tumour necrosis factor-α through proximal short promoter elements: evidence for suppression mechanisms mediated by two nuclear-factorbinding sites

Recent studies have demonstrated that tumour necrosis factor-α (TNF-α) decreases α1(I) procollagen gene (COL1A1) expression in cultured human dermal fibroblasts. The purpose of this study was to analyse the transcriptional control of COL1A1 by TNF-α. Cultured human dermal fibroblasts were transiently transfected with plasmids containing 5´ flanking sequences of COL1A1 fused to the chloramphenicol acetyltransferase (CAT) gene, and were incubated for 48 h in medium with or without TNF-α. TNF-α inhibited the CAT activity of fibroblasts transfected with plasmids containing 2.3 kb of 5´ flanking sequences of COL1A1, whereas the activity of control plasmids containing the herpes simplex thymidine kinase promoter gene (pBLCAT) was unaltered. A series of deletion constructs or various small substitution mutations of the COL1A1 5´ flanking region fused to the CAT gene were also transfected, and CAT activity was measured after incubation with TNF-α. TNF-α suppressed COL1A1 promoter activity through proximal short promoter elements containing only 107 bp. Short substitution mutations between -101 and -97 bp or between -46 and -38 bp abolished TNF-α suppression of COL1A1 promoter activity. DNA–protein complex formation was observed involving both sites in gel retardation assays. These results suggest that TNF-α suppressed COL1A1 promoter activity through elements located between -101 and -97 bp and between -46 and -38 bp of the COL1A1 promoter, and that the suppression involved DNA-protein interactions.