NF-κB independent suppression of endothelial vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 gene expression by inhibition of flavin binding proteins and superoxide production

Oxidation-reduction (redox)-coupled mechanisms play an important role in the regulation of cell surface adhesion molecule expression. In endothelial cells membrane-bound NADH/NADPH oxidase is a significant source of intracellular superoxide (O-2(-)) production. We explored the role of flavin containing proteins such as NADH/NADPH oxidase in the induction of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) gene expression in human aortic endothelial cells (HAECs) and human dermal microvascular endothelial cells (HMECs). Treatment of HAECs by tumor necrosis factor-alpha (TNF-alpha, 100 U/ml) for 1 h induced a 31% increase in O-2(-) production within 5 min as determined by lucigenin chemiluminescence analysis of whole cells (n = 4, P<0.05). Pretreatment with the NADH/NADPH oxidase inhibitor diphenylene iodonium (DPI, 40 mu M) for 1 h inhibited O-2(-) production. DPI also inhibited TNF- and LPS-induced VCAM-1 and ICAM-1 cell surface expression and TNF-alpha-, LPS-, or IL-1 beta-induced VCAM-1 and ICAM-1 mRNA accumulation. However, DPI did not inhibit TNF-alpha-induced activation of nuclear NF-kappa B-like binding activity in HAECs and HMECs. Furthermore, DPI did not inhibit TNF-alpha-induced transactivation of NF-kappa B-driven VCAM-1 and HIV-LTR promoter gene constructs in transiently transfected HMECs. These data suggest that flavin binding proteins such as NADH/NADPH oxidase can regulate VCAM-1 gene expression independent of NF-kappa B. Furthermore, intracellular O-2(-) generation is not necessary for NF-kappa B activation or for transactivation of NF-kappa B-driven promoters. (C) 2000 Academic Press.