Thrombin by binding to the endothelial cell surface Protease Activated Receptor-1 (PAR-1) activates heterotrimeric proteins Gq and G12/G13, which in turn induces a repertoire of signaling events that mediate increased lung vascular permeability. A primary unresolved question is the nature of the specific signaling pathways emanating from the interaction of PAR-1 with these heterotrimeric G-proteins that induce gaps between endothelial cells. Understanding these specific signals will be critical in defining the mechanisms of increased lung vascular permeability and tissue inflammation. Studies have shown a primary role of RhoA, a monomeric GTPase, in regulating endothelial barrier function. Rho activation is mediated through the tightly regulated GDP/GTP exchange cycle. Both guanine nucleotide dissociation inhibitor (GDI) and guanine nucleotide exchange factors (GEFs) are capable of regulating Rho activation. Studies have shown that the (-subunit of the heterotrimeric G protein family G12/G13 activates Rho via the intermediate p115RhoGEF, a Rho-specific GEF. Interestingly, we have also shown that in endothelial cells, thrombin induces the activation of RhoA via PKC( pathway. Since PKC( is a downstream effector of Gq, our studies suggest that in addition to classical G12/G13 pathway, Gq may play a critical role the activation of Rho. The proposed studies will address the key question that Rho is a point of confluence for PAR-1 initiated signals originating from interactions of PAR-1 with G12/G13 as well as Gq. Studies will also address the potentially important downstream action of RhoA in activating Ca2+ influx pathways in endothelial cells, and thereby in triggering the increase in endothelial permeability.
The Specific Aims are: 1. To investigate the specific roles of GDI-1 and p115RhoGEF in triggering the activation of Rho, and thereby in signaling the increase in endothelial permeability. 2. To determine the role of heterotrimeric GTP-binding protein Gq in signaling Rho activation and the loss of endothelial barrier function. 3. To determine the role of RheA in activating store-operated Ca2+ entry and signaling the increase in endothelial permeability. Studies will use genetic approaches in cultured endothelial cells and the intact mouse lung model to establish the roles of GDI-1, p115RhoGEF, and Gq in signaling Rho activation and in regulating endothelial permeability. With the information gained, it is hoped that new lines of therapy could be developed against inflammatory diseases that are characterized by """"""""leaky"""""""" pulmonary vessel.
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