Hydrogen peroxide prolongs nuclear localization of NF-κB in activated cells by suppressing negative regulatory mechanisms

NF-kappa B transcription factors induce pro-inflammatory molecules (e. g. IL-8) in response to cytokines (e. g. TNF alpha, IL-1 beta) or other stimuli. In the basal state, they are sequestered in the cytoplasm by inhibitory I kappa B proteins. Pro-inflammatory signaling triggers polyubiquitination of intermediaries (e. g. RIP1), which activate I kappa B kinases that trigger Ser phosphorylation and degradation of I kappa B alpha, thereby promoting nuclear translocation of NF-kappa B. A negative feedback loop exists whereby NF-kappa B drives resynthesis of I kappa B alpha, which promotes export of NF-kappa B from the nucleus to the cytoplasm. This process relies on Cezanne, a deubiquitinating cysteine protease that stabilizes resynthesized I kappa B alpha by removing polyubiquitin from modified intermediaries. H2O2 is generated during inflammation. Here we examined the effects of H2O2 on NF-kappa B dynamics and pro-inflammatory activation in cultured cells co-stimulated with TNF alpha or IL-1 beta. Quantitative reverse transcription-PCR and enzyme-linked immunosorbent assay revealed that H2O2 enhanced the induction of IL-8 by TNF alpha or IL-1 beta. We demonstrated by using assays of NF-kappa B nuclear localization and by imaging of live cells expressing a fluorescent form of NF-kappa B that H2O2 prolonged NF-kappa B nuclear localization in cells co-stimulated with TNF alpha or IL-1 beta by suppressing its export from the nucleus. We provide evidence that H2O2 suppresses NF-kappa B export by prolonging polyubiquitination of signaling intermediaries, which promotes Ser phosphorylation and destabilization of newly synthesized I kappa B alpha proteins. Finally, we observed that the catalytic activity of Cezanne and its ability to suppress RIP1 polyubiquitination and NF-kappa B transcriptional activity were inhibited by H2O2. We conclude that H2O2 prolongs NF-kappa B activation in co-stimulated cells by suppressing the negative regulatory functions of Cezanne and I kappa B alpha.