Abstract 5282: RAPL, a New Mediator of Angiogenesis, Promotes Integrin Endocytosis in Endothelial Cells
The molecular mechanisms regulating angiogenesis are not entirely understood. Integrins are critical mediators of angiogenesis. In our previous work, we identified the small 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) in comparison to scrambled transfected EC. However, while silencing of Rap1 reduced HUVEC adhesion, silencing of RAPL unexpectedly significantly increased 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 β1-integrin as assessed by FACS, while not affecting β1-integrin mRNA and total protein levels in HUVEC. It is conceivable that the enhanced surface expression of β1-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 (internalization) of β1-integrins in HUVEC. Strikingly, silencing of RAPL blocked β1-integrin endocytosis by 43±6 % as determined by confocal microscopy. Additionally, silencing of RAPL reduced internalization of β1-integrin as assessed by a FACS-based endocytosis assay. Interestingly, RAPL co-immunoprecipitated with GEP100, a guanine nucleotide exchange factor (GEF) for Arf6, which mediates β1-integrin internalization in HeLa cells. Furthermore, silencing of GEP100 significantly impaired angiogenic sprouting of HUVEC (55 % inhibition) and VEGF-induced invasion. In conclusion, our results demonstrate for the first time that RAPL mediates, in association with GEP100, β1-integrin internalization thereby promoting angiogenic sprouting and migration. Taken together, our data identify a new angiogenic signaling pathway and shed light on the relevance of β1-integrin internalization for angiogenic functions such as sprouting and migration.