Calcium mobilization and Rac1 activation are required for VCAM-1 (vascular cell adhesion molecule-1) stimulation of NADPH oxidase activity.
VCAM-1 (vascular cell adhesion molecule-1) plays an important role in the regulation of inflammation in atherosclerosis, asthma, inflammatory bowel disease and transplantation. VCAM-1 activates endothelial cell NADPH oxidase, and this oxidase activity is required for VCAM-1-dependent lymphocyte migration. We reported previously 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 signalling 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-alpha4-integrin and mimicked by anti-VCAM-1-coated beads. VCAM-1 stimulation of calcium responses could be blocked by an inhibitor of intracellular calcium mobilization, a calcium channel inhibitor or a calcium chelator, resulting in the inhibition of NADPH oxidase activity. Addition of ionomycin overcame the calcium channel blocker suppression of 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 mobilization 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 that are required for the activation of NADPH oxidase.
Cook-Mills
Joan M
JM
Department of Pathology, University of Cincinnati, Cincinnati, OH 45267-0529, USA. joan.cook@uc.edu
Johnson
Jacob D
JD
Deem
Tracy L
TL
Ochi
Atsuo
A
Wang
Lei
L
Zheng
Yi
Y
D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism; D020013:Calcium Signaling; D002448:Cell Adhesion; D002460:Cell Line; D002465:Cell Movement; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D004789:Enzyme Activation; D008214:Lymphocytes / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D019255:NADPH Oxidases / Q000378:metabolism*; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D015220:Calcium Channels; D019010:Vascular Cell Adhesion Molecule-1; D019255:NADPH Oxidases; D020830:rac1 GTP-Binding Protein; D002118:Calcium