Neutrophil adhesion and transmigration are facilitated by rapid changes in beta2-integrin activation in neutrophils, and lateral junction remodeling in endothelial cells. Recent data by our lab, and others, suggest that the small Ras-like GTPases Rap1 and Rap2 may play a role in both of these cellular responses. Rap1 has been shown to play a role in the activation of the leukocyte-specific 1132 integrins. Our preliminary data show that Rap1 and 2 (Rap 1/2) in endothelial cells are rapidly activated following stimulation with agonists known to remodel junctions, such as thrombin, histamine, and the tyrosine phosphatase inhibitor, pervanadate. Furthermore, Rap1/2 activation correlates temporally with their localization to endothelial junctions. Finally, mutagenesis studies in Drosophila link Rap1 to junctional assembly in epithelial cells. Our hypothesis is that Rap 1/2 are molecular switches for the local changes in beta2-integrin activation and junctional remodeling and thus play a critical role in neutrophil adhesion and transmigration.
The aims of this grant are to I) Assess the role of Rap1 in beta2 integrin mediated neutrophil adhesion. The importance of Rap1 in beta2 integrin activation and leukocyte adhesion in response to diverse stimuli will be evaluated. Our preliminary data and others have identified downstream effectors and regulatory proteins of Rap1. Their role in beta2-integrin activation will be assessed. II) Determine the role of Rap1 and 2 in endothelial cell function in vitro. The functional role of Rap1 and 2 in endothelial cell junction assembly, permeability and leukocyte transmigration will be assessed. Our preliminary data suggests functional interactions between Rap1 and beta-catenin, a component of endothelial cell adherens junctions and the Wnt signaling pathway. Thus we propose to examine whether stimuli that remodel junctions induce beta-catenin dependent transcription, and whether Rap1 regulates this process. III) Examine the functional consequences of Rap1 activation in endothelial cells in vivo. Genetically engineered mice with constitutively active Rap1, or with a """"""""knock-out"""""""" of this GTPase in endothelial cells will be generated: The effects of altered Rap1 expression on leukocyte migration and permeability will be examined by intravital microscopy, and in the Reverse Arthus and Shwartzman reaction, well-characterized models of inflammation. The results of these studies should help define how Rap1 integrates extracellular stimuli into coherent cellular responses required for neutrophil transmigration. This will lead to insights into the coordination of the molecular machinery underlying transmigration, a cell biological process which has consequences for endothelial pathophysiology and host defense.
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