title: Characterization of a DNA binding site that mediates the stimulatory effect of cyclosporin-A on type III collagen expression in renal cells.
abstract: BACKGROUND
  Previous work from our laboratory demonstrated upregulation of type III collagen by cyclosporin A (CsA) in a cellular model of renal fibroblasts 'in vitro', suggesting that a mechanism of gene transcriptional activation might be responsible for collagen accumulation in renal fibrosis resulting from chronic CsA treatment.
  
  
  METHODS
  We analysed in the same cellular model: (i) COL3A1 mRNA expression by RT-PCR; (ii) COL3A1 promoter activity by transfection of renal fibroblasts with constructs containing promoter fragments of different length fused to a reporter gene; (iii) expression of transcription factors by western blot analysis; (iv) DNA-protein binding by gel retardation assays with nuclear extracts from CsA-treated and untreated cells; and (v) site-directed mutagenesis of COL3A1 promoter to verify the role of a short DNA segment as CsA responsive element.
  
  
  RESULTS
  CsA induced a 3-5-fold increase in COL3A1 mRNA that was paralleled by a stimulation of the COL3A1 promoter. Degradation of COL3A1 mRNA was comparable in CsA-treated and -untreated cells. The target region was first limited to a 178 bp fragment from -117 to +61 (pFV1). By gel retardation, utilizing several oligonucleotides that covered the whole length of pFV1, we detected a factor able to bind the promoter DNA (oligo 31) in nuclear extracts after 3 h treatment with CsA. The binding was absent in untreated cells and it was not detected when a 10-base mutation was introduced in oligonucleotide 31. Finally, the same substitution mutation at the site of binding of this factor abolished the stimulatory effect of CsA on COL3A1 promoter. Some transcription factors, whose potential binding sites are included in the above promoter fragment, were induced by CsA treatment either soon (3 h) or late (24-72 h) after treatment and were detected by western blot analysis.
  
  
  CONCLUSIONS
  CsA induces the synthesis of type III collagen by stimulating a pathway leading to activation of COL3A1 promoter and upregulation of COL3A1 mRNA. A short promoter fragment, proximal to the transcription start site, is the target of CsA stimulation.
authors.affiliation: Nephrology Section, G. Gaslini Childrens Hospital, Department of Oncology Biology and Genetics, University of Genova, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Oleggini
authors.forename: L
authors.initials: L
authors.lastname: Musante
authors.forename: S
authors.initials: S
authors.lastname: Menoni
authors.forename: G
authors.initials: G
authors.lastname: Botti
authors.forename: M D
authors.initials: MD
authors.lastname: Duca
authors.forename: M
authors.initials: M
authors.lastname: Prudenziati
authors.forename: A
authors.initials: A
authors.lastname: Carrea
authors.forename: R
authors.initials: R
authors.lastname: Ravazzolo
authors.forename: G M
authors.initials: GM
authors.lastname: Ghiggeri
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002522:Chlorocebus aethiops; D003094:Collagen / Q000235:genetics*; D016572:Cyclosporine / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism; D005347:Fibroblasts; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007668:Kidney; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: D012333:RNA, Messenger; D011994:Recombinant Proteins; D016572:Cyclosporine; D003094:Collagen; D004247:DNA