Published online May 15, 2004. doi: 10.3748/wjg.v10.i10.1447
Revised: January 5, 2004
Accepted: January 12, 2004
Published online: May 15, 2004
AIM: To clarify the fractional activity of promoters from human α1(I) procollagen gene, the interaction between cis-elements and consensus DNA-binding proteins responsible for high promoter activity, and the potential application of promoter competitors as well as cytokines for antifibrogenesis.
METHODS: Sequence between 2483 bp upstream of the start of transcription and 42 bp downstream of this site was investigated with serial 5’-deletion. The 5’-deleted promoters recombined with chloramphenicol acetyltransferase (CAT) as reporter gene were transiently transfected to human dermal fibroblasts. Electrophoretic mobility shift assay was performed to show the DNA-protein binding capacity of the promoter sequence. Cytokines including tumor necrosis factor α (TNFα) and interferons (INFs) were added to the culture medium of transiently transfected fibroblasts. Competitor DNA for the binding sites of Sp-1, Ap-1 and NF-1 was individually cotransfected transiently in order to block the promoter-driven CAT expression.
RESULTS: Sequences of -2483 to +42 bp and -268 to +42 bp of human α1(I) procollagen gene had high activity as promoters. Binding sites for Ap-1 and Sp-1 were among the cis-regulatory elements recognizing consensus transcription factors responsible for basal promoter activity of sequence -268 to +42 bp. TNFα, IFNα, IFNβ showed inhibitory effects on sequence -2483 to +42 bp as promoter with activities 43%, 62% and 60% of control respectively. Transfection of the promoter competitors could reverse the promoter activity of -268 to +42 bp 40%-60%.
CONCLUSION: Sequences of -2483 to +42 bp recombined with reporter gene provide an ideal construction for transcriptional study of α1(I) procollagen gene. The anti-collagen capacity of TNFα and IFNs is associated with their transcriptional regulation. Ap-1 and Sp-1 mediate the basal transcriptional activation of human α1(I) procollagen gene in dermal fibroblasts. Competitors for highly active promoters might be a novel potential candidate in fibrotic blockade.