Hydrogen peroxide prolongs nuclear localization of NF-kappaB in activated cells by suppressing negative regulatory mechanisms.
NF-kappaB transcription factors induce pro-inflammatory molecules (e.g. IL-8) in response to cytokines (e.g. TNFalpha, IL-1beta) or other stimuli. In the basal state, they are sequestered in the cytoplasm by inhibitory IkappaB proteins. Pro-inflammatory signaling triggers polyubiquitination of intermediaries (e.g. RIP1), which activate IkappaB kinases that trigger Ser phosphorylation and degradation of IkappaBalpha, thereby promoting nuclear translocation of NF-kappaB. A negative feedback loop exists whereby NF-kappaB drives resynthesis of IkappaBalpha, which promotes export of NF-kappaB from the nucleus to the cytoplasm. This process relies on Cezanne, a deubiquitinating cysteine protease that stabilizes resynthesized IkappaBalpha by removing polyubiquitin from modified intermediaries. H(2)O(2) is generated during inflammation. Here we examined the effects of H(2)O(2) on NF-kappaB dynamics and pro-inflammatory activation in cultured cells co-stimulated with TNFalpha or IL-1beta. Quantitative reverse transcription-PCR and enzyme-linked immunosorbent assay revealed that H(2)O(2) enhanced the induction of IL-8 by TNFalpha or IL-1beta. We demonstrated by using assays of NF-kappaB nuclear localization and by imaging of live cells expressing a fluorescent form of NF-kappaB that H(2)O(2) prolonged NF-kappaB nuclear localization in cells co-stimulated with TNFalpha or IL-1beta by suppressing its export from the nucleus. We provide evidence that H(2)O(2) suppresses NF-kappaB export by prolonging polyubiquitination of signaling intermediaries, which promotes Ser phosphorylation and destabilization of newly synthesized IkappaBalpha proteins. Finally, we observed that the catalytic activity of Cezanne and its ability to suppress RIP1 polyubiquitination and NF-kappaB transcriptional activity were inhibited by H(2)O(2). We conclude that H(2)O(2) prolongs NF-kappaB activation in co-stimulated cells by suppressing the negative regulatory functions of Cezanne and IkappaBalpha.
Enesa
Karine
K
British Heart Foundation Cardiovascular Sciences Unit, National Heart and Lung Institute, Imperial College London, W12 ONN, United Kingdom.
Ito
Kazuhiro
K
Luong
Le A
le A
Thorbjornsen
Ingvild
I
Phua
Chee
C
To
Yasuo
Y
Dean
Jonathan
J
Haskard
Dorian O
DO
Boyle
Joseph
J
Adcock
Ian
I
Evans
Paul C
PC
D021581:Active Transport, Cell Nucleus / Q000187:drug effects / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism*; D016207:Cytokines / Q000378:metabolism / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D051550:I-kappa B Kinase / Q000378:metabolism; D020672:I-kappa B Proteins / Q000378:metabolism; D018836:Inflammation Mediators / Q000378:metabolism / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D016601:RNA-Binding Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D025801:Ubiquitin / Q000378:metabolism; D054875:Ubiquitination / Q000187:drug effects / Q000502:physiology
C119724:AGFG1 protein, human; D016207:Cytokines; D020672:I-kappa B Proteins; D018836:Inflammation Mediators; D016328:NF-kappa B; D028861:Nuclear Pore Complex Proteins; D016877:Oxidants; D016601:RNA-Binding Proteins; D025801:Ubiquitin; D006861:Hydrogen Peroxide; D051550:I-kappa B Kinase; D010450:Endopeptidases; C476458:OTUD7B protein, human