Differential effects of the immunosuppressant FK-506 on human α2(I) collagen gene expression and transforming growth factor β signaling in normal and scleroderma fibroblasts

Objective. To investigate the effects of FK-506 on the expression of the human alpha 2(I) collagen gene and transforming growth factor beta (TGF beta) signaling in normal and scleroderma fibroblasts. Methods. The expression levels of type I procollagen protein and alpha 2(I) collagen messenger RNA (mRNA) were analyzed by immunoblotting and Northern blotting, respectively. The promoter activities of alpha 2(I) collagen gene and 3TP-Lux were determined by transient transfection assay. Interaction between TGF beta receptor type I and FK-506 binding protein 12 (FKBP12) was evaluated by immunoprecipitation. Results. FK-506 did not affect the basal expression of type I procollagen protein or alpha 2(I) collagen mRNA, but it significantly reduced the TGF beta 1-induced expression of type I procollagen protein and alpha 2(I) collagen mRNA in normal fibroblasts. The effect of FK-506 was regulated posttranscriptionally, but not transcriptionally. In scleroderma fibroblasts, FK-506 significantly reduced the expression of type I procollagen protein and alpha 2(I) collagen mRNA through posttranscriptional regulation, but not transcriptional regulation. FK-506 increased the basal activity of the 3TP-Lux promoter, but it did not affect the TGF beta 1-induced promoter activity in normal fibroblasts. In contrast, FK-506 did not affect the basal or the TGF beta 1-induced 3TP-Lux promoter activity in scleroderma fibroblasts. Furthermore, FKBP12, which protects TGF beta receptor type I from ligand-independent activation by TGF beta receptor type II, constitutively dissociated from TGF beta receptor type I in scleroderma fibroblasts. Conclusion. FK-506 inhibits alpha 2(I) collagen gene expression by reducing the stability of mRNA without exhibiting its activation effect on TGF beta signaling in scleroderma fibroblasts.