Interleukin-1 Induction of NF B Is Partially Regulated by H2O2-mediated Activation of NF B-inducing Kinase*

Reactive oxygen species (ROS) have been demonstrated to act as second messengers in a number of signal transduction pathways, including NF B. However, the mechanism(s) by which ROS regulate NF B remain unclear and controversial. In the present report, we describe a mechanism whereby interleukin-1 (IL-1 ) stimulation of NF B is partially regulated by H2O2-mediated activation of NIK and subsequent NIK-mediated phosphorylation of IKK . IL-1 induced H2O2 production in MCF-7 cells and clearance of this ROS through the expression of GPx-1 reducedNF B transcriptional activation by inhibiting NIK-mediated phosphorylation of IKK . Although IKK and IKK were both involved in IL-1 -mediated activation of NF B, only the IKK -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 M), whereas higher concentrations (100 M to 1mM) inhibitedNIK activity. Treatment of cells with the general Ser/Thr phosphatase inhibitor (okadaic acid) lead to activation of NF B and enhanced NIK activity as a IKK kinase, suggesting that ROS may directly regulate NIK through the inhibition of phosphatases. Recruitment of NIK to TRAF6 following IL-1 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 induction of NF B activation.