Establishment and characterization of scleroderma fibroblast clonal cell lines by introduction of the hTERT gene.
Lack of an adequate experimental model has hindered the ability to fully understand scleroderma (SSc) pathogenesis. Current SSc research is based on the study of cultured fibroblasts from skin biopsies. In depth characterization of the SSc fibroblast phenotype is hindered by the limited lifespan and heterogeneity of these cells. The goal of this study was to isolate high collagen-producing fibroblasts from SSc biopsies and extend their lifespan with hTERT immortalization to enable characterization of their phenotype. Fibroblasts from two pairs of closely matched normal and SSc biopsies were infected with an hTERT lentivirus. Infected colonies were isolated, cultured into clonal cell lines and analysed with respect to profibrotic gene expression. The mRNA levels of nine profibrotic genes were measured by quantitative real-time PCR. Protein levels were assessed by Western blot. The hTERT SSc clones were heterogeneous with regards to expression of the profibrotic genes measured. A subset of the SSc clones showed elevated expression levels of collagen I, connective tissue growth factor and thrombospondin 1 mRNA, while expression of other genes was not significantly changed. Elevated expression of collagen I protein and mRNA was correlative with elevated expression of connective tissue growth factor. Several hTERT clones expressed high levels of pSmad1, Smad1 and TGF-betaRI indicative of altered TGF-beta signalling. A portion of SSc clones expressed several profibrotic genes. This study demonstrates that select characteristics of the SSc phenotype are expressed in a subset of activated fibroblasts in culture. The clonal SSc cell lines may present a new and useful model to investigate the mechanisms involved in SSc fibrosis.
Division of Rheumatology, Medical University of South Carolina, Charleston, SC 29425, USA.
Bagrat
B
Kapanadze
Erin
E
Morris
Edwin
E
Smith
Maria
M
Trojanowska
D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D016922:Cellular Senescence / Q000187:drug effects; D002999:Clone Cells; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D055513:Connective Tissue Growth Factor / Q000235:genetics / Q000378:metabolism; D057075:Enzyme Assays; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D008875:Middle Aged; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012595:Scleroderma, Systemic / Q000235:genetics / Q000473:pathology*; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology
C526317:CCN2 protein, human; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D055513:Connective Tissue Growth Factor; C509186:TERT protein, human; D019098:Telomerase