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-α4-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.
- calcium channel
- intracellular calcium release
- NADPH oxidase
- vascular cell adhesion molecule-1 (VCAM-1)
Abbreviations used: AM, acetoxymethyl ester; BAPTA AM, bis-(o-aminophenoxy)ethane-N,N,N´,N´-tetra-acetic acid tetrakis(acetoxymethyl ester); EGFP, enhanced green fluorescent protein; DHR 123, dihydrorhodamine 123; GST, glutathione S-transferase; HEV, high endothelial venule; HUVEC, human umbilical vein endothelial cell; PAK, p21-activated kinase; ROS, reactive oxygen species; TMB-8, 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate; TNFα, tumour necrosis factor α; VCAM-1, vascular cell adhesion molecule-1; WASP, Wiskott–Aldrich syndrome protein.
- The Biochemical Society, London ©2004