Cytokine-induced activation of nuclear factor-κB is inhibited by hydrogen peroxide through oxidative inactivation of IκB kinase

Rapid activation of the I kappaB kinase (IKK) complex is considered an obligatory step in the activation of nuclear factor-kappaB (NF-kappaB) in response to diverse stimuli. Since oxidants have been implicated in the regulation of NF-kappaB, the focus of the present study was the activation of IKK by tumor necrosis factor alpha (TNF alpha) in the presence or absence of hydrogen peroxide (H2O2). Exposure of mouse alveolar epithelial cells to H2O2 was not sufficient to activate IKK degrade I kappaB alpha, or activate NF-kappaB. In contrast, TNF alpha induced IKK activity rapidly and transiently resulting in I kappaB alpha degradation and NF-kappaB activation. Importantly, in the presence of H2O2 the ability of TNF alpha to induce IKK activity was markedly decreased and resulted in prevention of I kappaB alpha degradation and NF-kappaB activation. Neither tyrosine kinases nor phosphatidylinositol 3-kinases, known regulators of NF-kappaB by oxidants, were involved in IKK inhibition by H2O2 Direct addition of H2O2 to the immunoprecipitated IKK complex inhibited enzyme activity. Inhibition of IKK activity by H2O2 was associated with direct oxidation of cysteine residues present in the IKK complex and occurred only in enzymatically active IKK. In contrast to previously published observations, our findings demonstrate that the oxidant H2O2 reduces NF-kappaB activation by inhibiting activated IKK activity.