Activation of endothelial cell NF-κB by interleukin (IL)-1 constitutes an event critical to the progression of the innate immune response. In this context, oxidants have been associated with NF-κB activation, although the molecular source and mechanism of targeting have remained obscure. We found that RelA, essential for NF-κB activation by IL-1, was associated with the NADPH oxidase adapter protein p47phox in yeast two-hybrid, coprecipitation, and in vitro binding studies. RelA and p47-GFP also colocalized in endothelial cells in focal submembranous dorsoventral protrusions. Overexpression of p47phox synergized with IL-1β in the activation of an artificial κB-luciferase reporter and specifically augmented IL-1β-induced RelA transactivation activity. p47phox overexpression also greatly increased IL-1β-stimulated RelA phosphorylation, whereas it had no effect on I-κB degradation or on RelA nuclear translocation of κB binding. The tandem SH3 domains of p47phox were found to associate with a proline-rich mid-region of RelA (RelA-PR) located between the Rel homology and transactivation domains. The RelA-PR peptide blocked interaction of p47phox and RelA, and ectopic expression of RelA-PR abrogated IL-1β-induced transactivation of the NF-κB-dependent E-selectin promoter. Further, suppression of NADPH oxidase function through the inhibitor diphenylene iodonium, the superoxide dismutase mimetic Mn(III) tetrakis(4-benzoic acid)porphyrin (MnTBAP), or expression of a dominant interfering mutant of a separate NADPH oxidase subunit (p67(V204A)) decreased IL-1β-induced E-selectin promoter activation, suggesting that p47phox facilitates NF-κB activation through linkage with the NADPH oxidase. IL-1β rapidly increased tyrosine phosphorylation of IL-1 type I receptor-associated proteins, suggesting that oxidants may operate through inactivation of local protein-tyrosine phosphatases in the proximal IL-1β signaling pathway leading to RelA activation.
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