Abstract 478: GIT1 Regulates Angiogenesis By Affecting Endothelial Cell Podosome Formation And Migration
We previously showed that the G-protein coupled receptor (GPCR) kinase-interacting protein 1 (GIT1) was a key mediator for thrombin-mediated endothelial cell (EC) focal adhesion turnover and permeability. Recent data show that GIT1 traffics between three distinct cellular compartments (cytoplasm, focal adhesions and cell membrane) through interactions with diverse proteins including ARF, Rac1 and Cdc42 GTPases, p21-activated kinase (PAK), PAK-interacting exchange factor (PIX) and paxillin. Importantly, we and others showed that tyrosine kinase receptors (TKRs) such as the epidermal growth factor receptor stimulate GIT1 phosphorylation via c-Src, suggesting a role for GIT1 in TKR signaling. VEGF is the most important TKR in EC; essential for cell survival, migration and angiogenesis. Recently, actin-rich structures, whose assembly is regulated by c-Src, termed podosomes, were found to contribute to tissue invasion, matrix remodeling and cell motility. Since GIT1 is a substrate of Src, podosome formation is Src dependent, and VEGF is an essential angiogenic factor, we hypothesized that GIT1 plays an important role in angiogenesis by mediating VEGF-induced EC podosome formation and migration. Exposure of EC to VEGF (50 ng/ml) for 30 minutes stimulated GIT1 translocation and co-localization with podosomes. Mutation analysis showed that co-localization was dependent on the paxillin-binding domain of GIT1. Downregulation of GIT1 by siRNA significantly decreased VEGF-induced podosome formation. Pretreatment with the Src inhibitor PP2 or infection with dominant negative small GTPases also dramatically reduced podosome formation. Therefore, VEGF-induced EC podosome formation is dependent on Src, GIT1 and small GTPases. Finally, downregulation of GIT1 by siRNA significantly inhibited VEGF-induced EC migration and tube formation in vitro. These data suggest that GIT1 is an important mediator for VEGF-induced angiogenesis by affecting EC podosome formation and migration.