The fibroproliferative response to acute and chronic lung injury results in alveolar destruction and fibrosis and significant morbidity and mortality. This response is characterized by migration of interstitial fibroblasts into a provisional alveolar matrix forming histopathologically recognizable fibroblastic foci. Cellular migration has been shown to depend on the receptor for the protease, urokinase (u-PAR), but u-PAR-related migration is independent of urokinase proteolytic activity, u-PAR lacks a transmembrane domain but is glycosylphosphotidylinositol-linked to the plasma membrane. These features constrain u-PAR to plasma membrane lipid microdomains, and suggest that interactions of u-PAR with transmembrane receptors, including integrins, play a major role in the intracellular signaling necessary for cell motility. Fibroblasts from fibrotic lungs express increased levels of u-PAR in vitro and in vivo, and u-PAR expression alters tissue fibrosis in some in vivo models. These observations have led to the hypothesis that modulation of integrin function by u-PAR affects lung fibroblast motility. Moreover, that u-PAR modulates integrin function in vitro and in vivo in a manner that is dependent on u-PAR's restricted localization within lipid rafts. These hypotheses will be tested in three specific aims. First, adhesion and motility will be assessed in human lung fibroblasts that stably overexpress u-PAR, exhibit downregulation of u-PAR, or have blocked u-PAR-integrin interactions, u-PAR-dependent motility in fibroblasts isolated from patients with fibrotic lungs will be compared with those from non-fibrotic controls. Second, adhesion and motility will be assessed in human lung fibroblasts that stably overexpress either wt u-PAR, or a non-glycosylphosphotidylinositol-linked mutant u-PAR fusion protein. The requirement for motility signaling through focal adhesion kinase will be assessed in these fibroblast clones. Third, the role of u-PAR-integrin interactions and of u-PAR's glycosylphosphotidylinositol link on fibroproliferative tissue infiltration will be determined in an in vivo murine model, u-PAR specificity is accomplished in this model using specific inhibitory peptides and gene-activated matrices. This work will provide new insight into the molecular events which govern the fibroproliferative process, and support the design of novel therapeutic agents focused on inhibiting the fibrotic response to lung injury. ? ?
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