The organization and the assembly of the fibronectin matrix is known to regulate the progression of cells through the cell cycle. As the fibronectin matrix undergoes continual remodeling in vivo, it provides a potential target for therapies (i.e., anti-metastatic or anti-angiogenic) aimed at controlling cell growth. The molecular mechanisms by which fibronectin matrix organization regulates cell growth are poorly understood. During the previous funding period we have shown that anastellin, an anti-angiogenic peptide derived from the first Type III module of fibronectin, induces remodeling of the fibronectin matrix and completely inhibits the growth of human microvessel endothelial cells. We have also shown that the remodeling of the fibronectin matrix is associated with changes in adhesion, but not growth factor, regulation of ERK pathways necessary for the expression of cyclin D and cyclin A. During the next funding period, we will evaluate the hypothesis that anastellin-induced fibronectin matrix remodeling affects integrin and LPA dependent regulation of FAK/Rho pathways necessary for ERK activity. Studies described in AIM 1 will use neutralizing antibodies, peptide agonists and antagonists, and various null cell lines to address the role of EDA+ fibronectin, the alpha4beta1 fibronectin integrin and heparan sulfate proteogiycans in the regulation of MAP kinase activity. Studies in AIM 2 will evaluate the effect of anastellin on integrin regulation of ERK. The effect of anastellin on the kinetics and extent of Rho and FAK activation will be evaluated using Western Blots and Rho pull-down assays. The ability of active forms of Rho and FAK to rescue integrin signaling to ERK in the presence of anastellin will be assessed by using adenoviral vectors expressing constitutively active forms of FAK and Rho.
In AIM 3, Western blotting, pharmacological inhibitors, and dominant/negative adenoviral constructs will be used to define the role of the Ras/Raf1 MEK1 pathway in the activation of ERK by LPA. Activation of ERK by LPA will be assessed by Western blot, nuclear localization and activation of transcription factors.
In AIM 4, the role of growth factor transactivation and Rho/Rho kinase in the activation of ERK by LPA will be assessed using pharmacological inhibitors of tyrosine kinases and adenoviral vectors containing dominant/negative Rho and Rac constructs.
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