Inhibition of Endothelial Vascular Cell Adhesion Molecule-1 Expression by Nitric Oxide Involves the Induction and Nuclear Translocation of IκBα

Abstract The induction of vascular cell adhesion molecule-1 (VCAM-1) expression by tumor necrosis factor (TNF)-α requires the activation of nuclear factor-κB (NF-κB) via a process involving the phosphorylation and degradation of its cytoplasmic inhibitor, IκBα. We have shown that nitric oxide (NO) decreases VCAM-1 expression via inhibition of NF-κB activation. To determine how NO inhibits NF-κB, we studied the fate of IκBα following TNF-α stimulation in the presence of NO donorsS-nitrosoglutathione and sodium nitroprusside. Activation of NF-κB by TNF-α occurred within 15 min and coincided with rapid degradation of IκBα. Co-treatment with NO donors did not prevent IκBα phosphorylation or degradation. However, after 2 h of TNF-α stimulation, NO donors inhibited NF-κB activation and augmented IκBα resynthesis and nuclear translocation by 2.5- and 3-fold, respectively. This correlated with a 75% reduction in TNF-α-induced VCAM-1 expression. In a time-dependent manner, NO donors alone caused the nuclear translocation of IκBα. To confirm that NO donors have similar effects as endogenously derived NO, murine macrophage-like cells, RAW264.7, were co-cultured with endothelial cells. Induction of RAW264.7-derived NO inhibited lipopolysaccharide-induced endothelial VCAM-1 expression, which was reversed by the NO synthase inhibitorN ω-monomethyl-l-arginine. These findings indicate that NO inhibits NF-κB activation and VCAM-1 expression by increasing the expression and nuclear translocation of IκBα.