Abstract 17382: Rapl, a New Mediator of Angiogenesis, Promotes Integrin Endocytosis and Regulates Actin Cytoskeleton in Endothelial Cells
Integrins are critical mediators of angiogenesis. In our previous work, we identified the GTPase Rap1 as a mediator of integrin affinity in endothelial cells (EC) and angiogenesis. RAPL is an effector protein of Rap1 promoting integrin activation and adhesion in lymphocytes. However, the role of RAPL for integrin regulation in EC and angiogenesis is unclear. Silencing of RAPL with siRNA significantly decreased angiogenic sprouting of HUVEC in a 3-dimensional spheroidal system (83 % inhibition) and blocked VEGF-induced invasion (68 % inhibition), while not inhibiting EC proliferation. Surprisingly, while silencing of Rap1 reduced HUVEC adhesion, silencing of RAPL unexpectedly significantly increased integrin-dependent adhesion suggesting distinct roles for RAPL and Rap1 in integrin regulation in EC. In line with these results, silencing of RAPL significantly increased surface expression of beta1-integrin as assessed by FACS, while not affecting beta1-integrin mRNA, total protein levels and beta1-integrin activity in HUVEC. It is conceivable, that the enhanced surface expression of beta1-integrins upon silencing of RAPL is caused by a redistribution of the integrin from an intracellular pool to the cell surface. Therefore, we studied the role of RAPL for the endocytosis of beta1-integrins in HUVEC. Strikingly, silencing of RAPL blocked beta1-integrin endocytosis by 43 ± 6 % as determined by confocal microscopy. Additionally, silencing of RAPL reduced internalization of beta1-integrin subunit as assessed by FACS and of alpha5beta1-integrin as assessed by a biochemical biotinylation-based assay. Endogenous alpha5-integrin co-immunoprecipitated with overexpressed RAPL. In addition, silencing of RAPL enhanced actin stress fiber formation. In conclusion, our results demonstrate that RAPL is binding to the alpha5beta1-integrin and mediating integrin internalization and actin cytoskeleton reorganization in EC, thereby promoting angiogenic sprouting and migration. Our data identify a new angiogenic signaling pathway and shed light on the relevance of alpha5beta1-integrin endocytosis for angiogenic functions such as sprouting and migration. In ongoing experiments we are exploring the role of RAPL for angiogenesis in vivo.
- © 2010 by American Heart Association, Inc.