Abstract 17504: Rassf5 Mediates Beta1 Integrin Endocytosis and Promotes Vessel Stability
Beta1-integrins are essential for vascular development and angiogenesis. Recently, we identified the small GTPase Rap1 as activator of integrins in endothelial cells (EC). RASSF5/RAPL is a downstream interaction partner of Rap1 and promotes integrin activation and adhesion in lymphocytes. Silencing of Rap1 reduced EC adhesion. Knockdown of RASSF5 significantly increased integrin-mediated adhesion on collagen, fibronectin and vitronectin indicating different roles of these molecules in integrin regulation in EC. Silencing of RASSF5 by siRNA significantly decreased angiogenic sprouting (83% inhibition) and blocked VEGF-induced cell migration (68% inhibition), while not affecting EC proliferation. Consistently, lentiviral overexpression of RASSF5 in EC induced angiogenic sprouting. Surface expression of beta1-integrins was increased after silencing of RASSF5, but total protein amount of beta1-integrin in EC remained unchanged. These data suggest that silencing of RASSF5 affect the cellular trafficking of beta1-integrin. Therefore, we investigated the role of RASSF5 in integrin endocytosis and recycling in EC. Silencing of RASSF5 reduced beta1-integrin endocytosis, but did not influence recycling to the cell surface. Furthermore, we studied the endocytic trafficking of active (matrix bound) alpha5beta1-integrins. Strikingly, knockdown of RASSF5 in EC led to a reduction in endocytosis of active matrix bound alpha5beta1-integrins. Alpha5-integrin co-immunoprecipitated with RASSF5, via its C-Terminus. Silencing of RASSF5 also enhanced actin polymerization and Rac1 activity in EC. In vivo silencing of the RASSF5 orthologue gene in the zebrafish embryo with morpholinos induced hemorrhages, suggesting that RASSF5 contributes to vessel stability. Consistently, silencing of RASSF5 in vitro led to increased EC permeability. Based on these observations, we conclude that RASSF5 interacts with alpha5beta1-integrin and mediates endocytosis of preferentially matrix bound integrins. RASSF5 regulates the activity of Rac1, thereby affecting the actin cytoskeleton reorganization and promoting EC migration and angiogenic sprouting. Our data reveal a new role of RASSF5 for alpha5beta1-integrin trafficking and vessel stability.
- © 2012 by American Heart Association, Inc.