Interleukin-1β induction of NFκB is partially regulated by H2O2-mediated activation of NFκb-inducing kinase

Reactive oxygen species (ROS) have been demonstrated to act as second messengers in a number of signal transduction pathways, including NF kappa B. However, the mechanism(s) by which ROS regulate NF kappa B remain unclear and controversial. In the present report, we describe a mechanism whereby interleukin-1 beta(IL-1 beta) stimulation of NF kappa B is partially regulated by H2O2-mediated activation of NIK and subsequent NIK-mediated phosphorylation of IKK alpha. IL-1 beta induced H2O2 production in MCF-7 cells and clearance of this ROS through the expression of GPx-1 reduced NF kappa B transcriptional activation by inhibiting NIK-mediated phosphorylation of IKK alpha. Although IKK alpha and IKK alpha were both involved in IL-1 beta-mediated activation of NF kappa B, only the IKK alpha-dependent component was modulated by changes in H2O2 levels. Interestingly, in vitro reconstitution experiments demonstrated that NIK was activated by a very narrow range of H2O2 (1-10 mu M), whereas higher concentrations (100 mu M to 1 mM) inhibited NIK activity. Treatment of cells with the general Ser/Thr phosphatase inhibitor (okadaic acid) lead to activation of NF kappa B and enhanced NIK activity as a IKK alpha kinase, suggesting that ROS may directly regulate NIK through the inhibition of phosphatases. Recruitment of NIK to TRAF6 following IL-1 beta stimulation was inhibited by H2O2 clearance and Rac1 siRNA, suggesting that Rac-dependent NADPH oxidase may be a source of ROS required for NIK activation. In summary, our studies have demonstrated that redox regulation of NIK by H2O2 is mechanistically important in IL-1 beta induction of NF kappa B activation.