Influence of extracellular matrix fibronectin on platelet derived growth factor signaling

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 DESCRIPTION (provided by applicant): Platelet derived growth factor (PDGF) is the major mitogen for mesenchymal cells and is used clinically to promote diabetic ulcer healing. Clinical successes using PDGF to promote wound healing have been underwhelming and inconsistent. Fibronectin is a critical ECM protein that is secreted by fibroblasts in a protomeric form and assembled into a fibrillar matrix by a cell-mediated process termed fibronectin matrix assembly. Preliminary data presented in this proposal show that cell-assembled ECM fibronectin fibrils attenuate PDGF-induced intracellular calcium release. Intracellular calcium release was measured in Fluo-4- loaded fibronectin-null (FN-/-) mouse embryonic fibroblasts (MEFs), allowing for precise control over the amount of fibronectin to which cells were exposed and the initiation of fibronectin matrix assembly. Fibronectin- mediated attenuation of PDGF-induced calcium release represents a previously undescribed mechanism by which ECM fibronectin may downregulate PDGF signaling, and thus spatially and/or temporally control PDGF activity in healing wounds. Alternatively, several diseases including pulmonary fibrosis, atherosclerosis and some types of malignancies are characterized, in part, by an upregulation of PDGF activity. Thus, a fibronectin fragment that inhibits cell responses to PDGF may be beneficial in treating these conditions. The goal of this study is to determine the mechanism and consequences of ECM fibronectin-mediated regulation of cellular responses to PDGF in fibroblasts. In order to achieve the goals of this project, the following specific aims have been developed: (1) Localize the effect of ECM fibronectin on cell responsiveness to PDGF and develop small fibronectin peptides regulate cell responsiveness to PDGF, (2) determine the mechanism by which ECM fibronectin regulates fibroblast responses PDGF and (3) determine the effect of regulation of responses to PDGF by ECM fibronectin on cell phenotype. Important tools for these studies will include calcium analysis in our Fluo-4-loaded FN-/- MEF system, as well as our laboratory's extensive library of tools used to study fibronectin matrix assembly and expertise in developing novel fibronectin-derived fusion proteins. Experimental results will provide insight into how cell responses to PDGF are regulated during normal wound healing processes, how they may be dysregulated during pathological wound healing responses, and how specific fibronectin fragments may be used therapeutically to control endogenous PDGF activity.