Calcium mobilization and RAC1 activation are required for VCAM-1-stimulated NADPH oxidase activity

The cell adhesion molecule VCAM-1 plays an important role in regulation of inflammation in atherosclerosis, asthma, inflammatory bowel disease, or transplantation. VCAM-1 activates endothelial cell NADPH oxidase and this oxidase activity is required for VCAM-1-dependent lymphocyte migration. We previously reported that a mouse microvascular endothelial cell line promotes lymphocyte migration that is dependent on VCAM-1 but not on other known adhesion molecules. Here we have investigated the signaling mechanisms underlying VCAM-1 function. Lymphocyte binding to VCAM-1 on the endothelial cell surface activated an endothelial cell calcium flux that could be inhibited with anti-alpha 4-integrin and mimicked by anti-VCAM-1-coated beads. VCAM-1 stimulation of calcium responses could be blocked by an intracellular calcium mobilization inhibitor, a calcium channel inhibitor, or a calcium chelator, resulting in the inhibition of NADPH oxidase activity. Addition of ionomycin overcame the calcium channel blocker-suppressed, VCAM-1-stimulated NADPH oxidase activity, but could not reverse the inhibitory effect imposed by intracellular calcium blockage, indicating that both intracellular and extracellular calcium mobilizations are required for VCAM-1-mediated activation of NADPH oxidase. Furthermore, VCAM-1 specifically activated the Rho family GTPase Rac1 and VCAM-1 activation of NADPH oxidase was blocked by a dominant negative Rac1. Thus, VCAM-1 stimulates the mobilization of intracellular and extracellular calcium and Rac1 activity which are required for the activation of NADPH oxidase.