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AIM: To clarify the fractional activity of promoters fromhuman α1(I) procollagen gene,the interaction between cis-elements and consensus DNA-binding proteins responsiblefor high promoter activity,and the potential application ofpromoter competitors as well as cytokines for antifibrogenesis.METHODS: Sequence between 2 483 bp upstream of thestart of transcription and 42 bp downstream of this site wasinvestigated with serial 5’-deletion.The 5’-deleted promotersrecombined with chloramphenicol acetyltransferase (CAT)as reporter gene were transiently transfected to humandermal fibroblasts.Electrophoretic mobility shift assay wasperformed to show the DNA-protein binding capacity of thepromoter sequence.Cytokines including tumor necrosisfactor α (TNFα) and interferons (INFs) were added to theculture 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 blockthe promoter-driven CAT expression.RESULTS: Sequences of -2 483 to +42 bp and -268 to+42 bp of human α(I) procollagen gene had high activityas promoters.Binding sites for Ap-1 and Sp-1 were amongthe cis-regulatory elements recognizing consensus transcriptionfactors responsible for basal promoter activity of sequence-268 to +42 bp.TNFα,IFNα,IFNβ showed inhibitory effectson sequence -2 483 to +42 bp as promoter with activities43%,62% and 60% of control respectively.Transfection ofthe promoter competitors could reverse the promoter activityof -268 to +42 bp 40-60%.CONCLUSION: Sequences of -2 483 to +42 bp recombinedwith reporter gene provide an ideal construction fortranscriptional study of α1(I) procollagen gene.The anti-collagen capacity of TNFα and IFNs is associated with theirtranscriptional regulation.Ap-1 and Sp-1 mediate the basaltranscriptional activation of human α1(I) procollagen genein dermal fibroblasts.Competitors for highly active promotersmight be a novel potential candidate in fibrotic blockade.
AIM: To clarify the fractional activity of promoters fromhuman α1 (I) procollagen gene, the interaction between cis-elements and consensus DNA-binding proteins responsible for high promoter activity, and the potential application of promoters as well as cytokines for antifibrogenesis. METHODS: Sequence between 2 483 bp upstream of the start of transcription and 42 bp downstream of this site was investigated with serial 5’-deletion. 5’-deletion promoters recombined with chloramphenicol acetyltransferase (CAT) as reporter gene were transiently transfected to human dermal fibroblasts. Electrophoretic mobility shift assay was formed to demonstrate 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 expre ssion.RESULTS: Sequences of -2 483 to +42 bp and -268 to + 42 bp of human α (I) procollagen gene had high activityas 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 sequence -2 483 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 -2 483 to +42 bp recombined with reporter gene provide a ideal construction fortranscriptional study of α1 (procollagen gene. the anti-collagen capacity of TNFα and IFNs is associated with the transcriptional regulation. Ap-1 and Sp-1 mediate the basal transcription activation of human α1 (I) procollagen gene in dermal fibroblasts. Competitors for highly active promoters might be a novel potential candidate in fibrotic blockade .