Involvement of the constitutive complex formation of c-Ski/SnoN with Smads in the impaired negative feedback regulation of transforming growth factor β signaling in scleroderma fibroblasts

Objective. The principal effect of transforming growth factor β1 (TGFβ1) on mesenchymal cells is its stimulation of extracellular matrix synthesis. Previous reports indicated the significance of the autocrine TGFβ loop in the pathogenesis of scleroderma. The aim of this study was to examine c-Ski and SnoN, principal molecules in the negative regulation of TGFβ signaling, to further understand the autocrine TGFβ loop in scleroderma. Methods. Levels of expression of c-Ski/SnoN on cultured normal and scleroderma fibroblasts were determined by Western blotting, Northern blotting, and immunohistochemical staining. To determine the protein-protein interaction between c-Ski/SnoN, Smads, and p300, immunoprecipitation was performed. A transient transfection assay was performed to measure promoter activity of the α2(I) collagen gene and the 3TP-Lux plasmid construct. Results. Scleroderma fibroblasts exhibited increased c-Ski/SnoN levels compared with normal fibroblasts, both in vivo and in vitro. Although c-Ski/SnoN constitutively formed a complex with Smads by immunoprecipitation, the inhibitory effect of c-Ski/SnoN on the promoter activity of human α2(I) collagen and 3TP-Lux was impaired in scleroderma fibroblasts. Immunoprecipitation analyses also revealed that overexpressed c-Ski/SnoN could not compete with p300 in these cells. Conclusion. These results indicate that impaired competition with p300 is the possible cause of dysfunction of c-Ski/SnoN in scleroderma fibroblasts and that this might contribute to maintenance of the autocrine TGFβ loop in this disease.