title: Transcriptional regulation of human alpha1(I) procollagen gene in dermal fibroblasts.
abstract: OBJECTIVE
  To clarify the fractional activity of promoters from human alpha1(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 alpha (TNFalpha) 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 alpha1(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. TNFalpha, IFNalpha, IFNbeta showed inhibitory effects on 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%.
  
  
  CONCLUSIONS
  Sequences of -2 483 to +42 bp recombined with reporter gene provide an ideal construction for transcriptional study of alpha1(I) procollagen gene. The anti-collagen capacity of TNFalpha and IFNs is associated with their transcriptional regulation. Ap-1 and Sp-1 mediate the basal transcriptional activation of human alpha1(I) procollagen gene in dermal fibroblasts. Competitors for highly active promoters might be a novel potential candidate in fibrotic blockade.
authors.affiliation: Department of Laboratory Medicine, Changzheng Hospital, 415 Fengyang Road, Shanghai 200003, China.
authors.forename: Chun-Fang
authors.initials: CF
authors.lastname: Gao
authors.forename: Hao
authors.initials: H
authors.lastname: Wang
authors.forename: Ai-Hua
authors.initials: AH
authors.lastname: Wang
authors.forename: Wei-Dong
authors.initials: WD
authors.lastname: Wan
authors.forename: Yan-Aan
authors.initials: YA
authors.lastname: Wu
authors.forename: Xian-Tao
authors.initials: XT
authors.lastname: Kong
mesh_terms: D002478:Cells, Cultured; D002675:Child, Preschool; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D020405:Dermis / Q000166:cytology*; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D008297:Male; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D024042:Collagen Type I; D016207:Cytokines; D011498:Protein Precursors; D014157:Transcription Factors