title: Characterization of the phenotype of high collagen-producing fibroblast clones in systemic sclerosis, using a new modified limiting-dilution method.
abstract: BACKGROUND
  Overproduction of type I collagen in fibroblasts of systemic sclerosis (SSc) is the hallmark of fibrosis. Establishment and characterization of the phenotype of SSc fibroblasts has been hindered by the heterogeneity between fibroblasts and the lack of adequate cloning methods.
  
  
  OBJECTIVE
  To establish and investigate the characteristics of the SSc high collagen-producing fibroblast phenotype.
  
  
  METHODS
  Primary cultured fibroblasts from skin biopsies of patients with SSc and normal controls were cloned by a new modified limiting-dilution method. All clones were divided into different subpopulations based on their α1(I) procollagen (COL1A1) mRNA level detected by real-time reverse transcriptase PCR assay. In the different subpopulations, cell growth and cycle distribution were analysed by MTT and flow cytometry, COL1A1 promoter activity was examined by transient transfection, and the binding activity of Sp1 to the COL1A1 proximal promoter was investigated by quantitative chromatin immunoprecipitation.
  
  
  RESULTS
  The clonogenicities of SSc and normal control fibroblasts were similar, but the mean COL1A1 mRNA level of clones and the percentage of the subpopulation with a high COL1A1 mRNA level were significantly higher in SSc fibroblasts than in controls. There was no significant difference on cell growth and cycle between different subpopulations of SSc and control fibroblasts. The COL1A1 proximal promoter activity and its binding activity to Sp1 in the clones were strongly correlated with their COL1A1 mRNA level.
  
  
  CONCLUSIONS
  Overproduction of collagen in an SSc fibroblast subpopulation seems to result mainly from the abnormally activated transcription of COL1A1 rather than from overproliferation of fibroblasts. The new modified limiting-dilution method provides a useful means for characterizing cells with heterogeneous phenotypes.
authors.affiliation: Department of Dermatology, Zhongshan Hospital, Fudan University, Shanghai, China.
authors.forename: L
authors.initials: L
authors.lastname: Zhu
authors.forename: D
authors.initials: D
authors.lastname: Gao
authors.forename: J
authors.initials: J
authors.lastname: Yang
authors.forename: M
authors.initials: M
authors.lastname: Li
mesh_terms: D000293:Adolescent; D000328:Adult; D001706:Biopsy; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D002648:Child; D024042:Collagen Type I / Q000096:biosynthesis*; D000090602:Collagen Type I, alpha 1 Chain; D005260:Female; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D005434:Flow Cytometry; D006801:Humans; D008297:Male; D008875:Middle Aged; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012595:Scleroderma, Systemic / Q000473:pathology*; D055815:Young Adult
chemical_list: D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D012333:RNA, Messenger