Fibronectin (FN) is an indispensable link between cells and their microenvironment. In vivo, FN matrix acts as a structural framework to organize cells and control their behaviors. 2 major types of FN matrix are the fibrin-FN provisional matrix that forms at sites of tissue injury and the cell-assembled fibrillar FN matrix deposited in most tissues. We have developed in vitro three-dimensional (3-D) matrix models that resemble these two FN networks and allow us to define the effects of matrix structure and composition on cell behavior. In this proposal, these model systems will be used to study the role of FN in adhesion modulation, FN matrix assembly, and fibroblast differentiation. Using the fibrin-FN matrix, we have shown dramatic effects on cell morphology and signaling by tenascin contraction, and suppression of Rho GTPase and focal adhesion kinase (FAK) signaling. Whether this is a general mechanism of adhesion modulation will be determined by testing cell responses to the presence of other adhesion modulatory proteins, thrombospondin-1, vitronectin, or fibulin-1, in the fibrin-FN matrix. Regulatory effects of FN and tenascin-C on differentiation of wound fibroblasts into highly contractile myofibroblasts will also be addressed with this system. Our other model is a 3-D FN matrix prepared from cultures of highly confluent fibroblasts. Fibroblasts growing in this 3-D matrix assemble significantly increased amounts of FN matrix. The mechanism by which the 3-D FN matrix stimulates matrix assembly will be determined by analyzing its effects on FN conformation and integrin activity. The ability of this 3-D FN matrix to modulate the effects of anti-tumor agents on cancer cells will also be examined. Analyses of FN matrix assembly will extend to the molecular level with a focus on the first 2 type III modules, III 1-2, which play a regulatory role in FN fibril formation. Matrix assembly by FN mutants will complement structural studies on HIi-2 fragments. The proposed studies will provide novel information about FN matrix functions and regulation of FN matrix assembly and are highly relevant to many biological processes.
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