Transforming growth factor beta induces fibroblast fibrillin-1 matrix formation

OBJECTIVE: Fibrillin, an extracellular matrix protein implicated in dermal fibrosis, is increased in the reticular dermis of systemic sclerosis (SSc) skin. We undertook this study to investigate the hypothesis that transforming growth factor beta (TGFbeta) or other cytokines regulate fibrillin matrix formation by normal and SSc fibroblasts. We further investigated the mechanism of TGFbeta-induced fibrillin fibrillogenesis and its relationship to myofibroblasts. METHODS: Fibrillin and fibronectin matrix deposition and alpha-smooth muscle actin expression by fibroblast cultures from normal and SSc skin treated with TGFbeta or other cytokines were analyzed by immunofluorescence. Supernatant and extracellular matrix from normal and SSc fibroblasts treated with or without TGFbeta were evaluated by Western blot and Northern blot for fibrillin protein and messenger RNA (mRNA) expression, respectively. RESULTS: Immunofluorescence demonstrated increased fibrillin matrix formation by normal and scleroderma fibroblasts after TGFbeta treatment. Other cytokines, including tumor necrosis factor alpha, interleukin-1beta (IL-1beta), IL-4, granulocyte-macrophage colony-stimulating factor, and platelet-derived growth factor, did not affect fibrillin fibrillogenesis. Fibrillin matrix formed in proximity to myofibroblasts and independently of up-regulation of fibronectin matrix or cell number. Western blot analysis of extracellular matrix confirmed increased fibrillin after TGFbeta stimulation of normal or scleroderma fibroblasts. However, TGFbeta did not alter the expression of either soluble fibrillin protein or fibrillin mRNA. CONCLUSION: Our data show that TGFbeta induces fibrillin protein incorporation into the extracellular matrix without affecting fibrillin gene expression or protein synthesis, suggesting that fibrillin matrix assembly is regulated extracellularly. TGFbeta might increase fibrillin matrix by activating myofibroblasts. Such TGFbeta-mediated effects could account for the increased fibrillin matrix observed in SSc skin.