MECHANISMS REGULATING FIBRONECTIN DEPOSITION

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Assembly and degradation of extracellular matrices are dynamic processes that are up-regulated during wound healing, embryogenesis, and metastasis. Deposition of fibronectin into the extracellular matrix is a cell-mediated process that is tightly regulated to ensured controlled matrix deposition. Although several of the early steps leading to fibronectin deposition have been identified, the mechanisms leading to the accumulation of fibronectin into disulfide-stabilized multimers are largely unknown. We have identified a disulfide isomerase activity within fibronectin and propose to determine whether this activity catalyzes the crosslinking of fibronectin into the extracellular matrix. Studies examining the deposition of fibronectin into the extracellular matrix have been regulating cell proliferation has also been difficult to assess since most adherent cells continuously produce a fibronectin matrix. We recently isolated embryonic fibronectin-null cells and have adapted them to culture under serum-free conditions. These cells provide us with the unique opportunity to selectively induce fibronectin polymerization in order to precisely determine the relationship between fibronectin polymerization and cell function. Our data indicate that fibronectin increases adhesion- dependent growth and that this effect is due to the process of fibronectin matrix assembly. These studies will provide information crucial to understanding the role of fibronectin fibronectin deposition, as occurs during atherosclerosis and fibrosis. The applicant's immediate research goals are: 1) to determine whether fibronectin deposition, as occurs during atherosclerosis and fibrosis. The applicant's immediate research goals are: 1) to determine whether fibronectin's disulfide isomerase activity catalyzes the crosslinking of fibronectin fibrils in the extracellular matrix; 2) to determine the mechanisms by which fibronectin matrix assembly positively regulates cell growth; and 3) to compare how cell growth is regulated by fibronectin matrix assembly and other extracellular matrix proteins. These studies will further my long term research goals of understanding how interactions of cells with their extracellular environment control various aspects of cell behavior including cell growth and migration, and how cells alter their extracellular environment by the regulated assembly and disassembly of extracellular matrices. This grant will provide the resources to fully exploit our newly isolated fibronectin null cell lines, to develop the technology to isolate function blocking recombinant antibodies to conserved regions of fibronectin, and to develop methods to study intracellular signaling events that are a consequence of fibronectin deposition.