Local tissue invasion and subsequent metastasis of tumors results from a tightly controlled dynamic between pericellular proteolysis and cell migration. Vitronectin is a plasma protein also found in the extracellular matrix, which supports integrin dependent cell adhesion and regulates the function of key components of the plasminogen activator system. Vitronectin synthesis and accumulation in the matrix is upregulated in a variety of tumors where it functions as a template on which cells coordinate migration as well as proteolytic remodeling of the extracellular matrix. During the previous funding period we have demonstrated that the heparin binding domain of vitronectin can down-regulate the polymerization of the fibronectin matrix. Our hypothesis is that the binding of vitronectin's hepann binding domain to cell surfaces decreases fibronectin polymerization by affecting a5B1 integrin function and cellular contractility. Our preliminary data suggest that both the plasminogen activator receptor as well as cell surface proteoglycans are part of the molecular mechanism by which matrix vitronectin affects fibronectin matrix assembly.
In Aim 1 we will use recombinant vitronectin proteins and peptides to map the sequences in vitronectin's heparin binding domain which regulate fibronectin matrix assembly.
In Aim 2, we will use solid phase binding assays, chemical crosslinking and cell adhesion assays to determine whether uPAR and svndecans are receptors for vitronectin's heparin binding domain. The role of uPAR in the down regulation of matrix assembly by vitronectin will be detennined using uPAR null cell lines. In the third Aim fibronectin binding assays. cell adhesion assa s and immunolocalization will be used to determine whether vitronectin's heparin binding domain can regulate affinity/avidity of the a5B1 integrin. Association of a5f31 with known integrin modulatory proteins will be evaluated using co-immunoprecipitation, affinity chromatography and immunolocalization. In the fourth aim we will evaluate the effect of vitronectin's heparin binding domain on the levels of active rho. We will also assess the effect of vitronectin on cell contractility by measuring phosphorylation of myosin light chain and collagen gel contraction. As the levels of flbronectin in the matrix can affect cell growth, tumor metastasis and angiogenesis, understanding the mechanisms by which vitronectin regulates polymerization of fibronectin will provide new insights into how the extracellular matrix may be a useful target for therapies directed at tumor metastases.
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