A large duplication in the fibrillin-1 gene (Tsk fibrillin-1) is responsible for the phenotype of the Tsk mouse, a model for skin disease in human systemic sclerosis (SSc). The best defined function of fibrillin is in supporting elastic fibers and we have recently found that Tsk mice show markedly increased hypodermal elastic fibers associated with striking changes in fibulin-2 and fibulin-5, proteins that regulate formation of elastic fibers. We are investigating the hypotheses that altered hypodermal deposition of fibulins causes skin tethering in Tsk mice and that altered dermal fibulin deposition increases elastic fibers in human SSc. We will study the effect of Tsk fibrillin-1 on fibulin-2 and -5 matrices by staining cells conditionally expressing Tsk-fibrillin-1. We will test the importance of fibulin-2 on Tsk skin tethering and altered elastogenesis by breeding Tsk mice to fibulin-2 deleted mice, we will also study the effect of fibulin-2 on elastogenesis in fibroblasts overexpressing or deleted of fibulin-2 in cell culture. Fibrillin associates with several other matrix proteins, including MAGP-2. We have found markedly increased MAGP-2 matrix in the skin of Tsk mice and human SSc. Upon expression of Tsk fibrillin-1 in cell lines, we find increased MAGP-2 and collagen matrices, suggesting that Tsk fibrillin-1 might augment collagen deposition through increased MAGP-2. The effect of increased MAGP-2 expression on collagen, fibulin and elastic fiber matrices will be studied in cell lines and transgenic mice. TGF-beta may interact indirectly with fibrillin-1 through latent TGF-beta binding proteins and TGF-beta regulates fibrillin-1 and MAGP-2 matrices in vitro. Since these are features of Tsk dermal pathology, the importance of TGF-beta on the Tsk phenotype and altered elastogenesis will be tested by examining MAGP-2 and fibulin-2 expresssion in Tsk mice bred to smadS deleted or fibroblast-targeted TGF-beta Rll deleted mice. Fibroblast cultures from these mice will also be examined for alterations in TGF-beta activity, and MAGP-2 and fibulin-2 mRNA expression. LTBP-1 or LTBP-4 will be examined as possible mediators of Tsk fibrillin-1 activation of TGF-beta signaling. Together these studies should help clarify the pathogenesis of altered elastogenesis and fibrosis associated with Tsk fibrillin-1. These findings will be correlated with studies on human SSc skin. ? ?
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