Characterization of the phenotype of high collagen-producing fibroblast clones in systemic sclerosis, using a new modified limiting-dilution method

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. Aim. 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. Conclusion. 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.