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J. Biol. Chem., Vol. 283, Issue 27, 18582-18590, July 4, 2008

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Hydrogen Peroxide Prolongs Nuclear Localization of NF-B in Activated Cells by Suppressing Negative Regulatory Mechanisms^*

Karine Enesa, Kazuhiro Ito, Le A. Luong, Ingvild Thorbjornsen, Chee Phua, Yasuo To, Jonathan Dean^¶, Dorian O. Haskard, Joseph Boyle, Ian Adcock, and Paul C. Evans¹

From the British Heart Foundation (BHF) Cardiovascular Sciences Unit and Respiratory Sciences, National Heart and Lung Institute, and ^¶the Kennedy Institute of Rheumatology Division, Imperial College London, W12 ONN, United Kingdom

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

Received for publication, February 19, 2008 , and in revised form, April 28, 2008.

^* This work was supported by grants from the British Heart Foundation, Kidney Research UK, and National Heart and Lung Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: BHF Cardiovascular Sciences Unit, National Heart and Lung Institute, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 ONN, UK. Tel.: 44-20-83831619; Fax: 44-20-83831640; E-mail: paul.evans{at}imperial.ac.uk.

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