| Leukocyte transendothelial migration (TEM) is a critical regulated step in inflammation and is therefore a potential target in the treatment of inflammatory diseases. During TEM, leukocyte beta-integrins bind to and cluster intracellular adhesion molecule-1 (ICAM1) on the surface of the endothelium. This has been shown to initiate signaling in endothelial cells that facilitate leukocyte transmigration. In the course of studying the role of ICAM1 in leukocyte TEM, we have discovered two signaling pathways that are required for TEM. First, we found that crosslinking of ICAM1 activates Src and Pyk2 tyrosine kinases, which results in tyrosine phosphorylation of VE-cadherin on Y658 and Y731. These phosphorylation events require both Src and Pyk2. Accordingly, inhibition of endothelial Src or Pyk2 reduced transmigration of leukocytes. Importantly, expression of Y658F or Y731F mutants of VE-cadherin, which cannot be phosphorylated on these residues, also inhibits leukocyte TEM demonstrating that VE-cadherin tyrosine phosphorylation is required for TEM. A second set of studies focused on the means by which ICAM1 might regulate formation of transmigratory cups around adhering leukocytes. We found that exogenously expressed ICAM1 colocalized in dorsal ruffles with the small GTPase RhoG and a RhoG-specific exchange factor, SGEF. Additionally, a proline-rich region in the ICAM1 cytoplasmic tail directly interacts with the SH3 domain of SGEF. Crosslinking of ICAM1 was observed to activate RhoG in endothelial cells. Finally, we found that siRNA-mediated knockdown of SGEF or RhoG inhibited formation of transmigratory cups, as well as leukocyte TEM. In this thesis we describe two ICAM1-dependent signaling pathways; one that contributes to leukocyte-mediated disassembly of endothelial adherens junctions via VE-cadherin phosphorylation, and one that mediates formation of transmigratory cups via activation of RhoG. These signals are required for efficient leukocyte TEM and may therefore be suitable targets for anti-inflammatory therapies. |
