Enesa Karine, Ito Kazuhiro, Luong Le A, Thorbjornsen Ingvild, Phua Chee, To Yasuo, Dean Jonathan, Haskard Dorian O, Boyle Joseph, Adcock Ian, Evans Paul C
British Heart Foundation Cardiovascular Sciences Unit, National Heart and Lung Institute, Imperial College London, W12 ONN, United Kingdom.
J Biol Chem. 2008 Jul 4;283(27):18582-90. doi: 10.1074/jbc.M801312200. Epub 2008 May 12.
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.
核因子-κB转录因子在细胞因子（如肿瘤坏死因子α、白细胞介素-1β）或其他刺激下诱导促炎分子（如白细胞介素-8）。在基础状态下，它们被抑制性IκB蛋白隔离在细胞质中。促炎信号触发中间体（如RIP1）的多聚泛素化，激活IκB激酶，后者触发IκBα的丝氨酸磷酸化和降解，从而促进核因子-κB的核转位。存在一个负反馈环，即核因子-κB驱动IκBα的重新合成，这促进核因子-κB从细胞核输出到细胞质。这个过程依赖于Cezanne，一种去泛素化半胱氨酸蛋白酶，它通过从修饰的中间体上去除多聚泛素来稳定重新合成的IκBα。炎症过程中会产生过氧化氢。在这里，我们研究了过氧化氢对在肿瘤坏死因子α或白细胞介素-1β共同刺激下培养的细胞中核因子-κB动态和促炎激活的影响。定量逆转录聚合酶链反应和酶联免疫吸附测定表明，过氧化氢增强了肿瘤坏死因子α或白细胞介素-1β对白细胞介素-8的诱导。我们通过核因子-κB核定位测定以及对表达荧光形式核因子-κB的活细胞成像证明，过氧化氢通过抑制核因子-κB从细胞核输出，延长了在肿瘤坏死因子α或白细胞介素-1β共同刺激下细胞中核因子-κB的核定位。我们提供证据表明，过氧化氢通过延长信号中间体的多聚泛素化来抑制核因子-κB输出，这促进了新合成的IκBα蛋白的丝氨酸磷酸化和不稳定。最后，我们观察到过氧化氢抑制了Cezanne的催化活性及其抑制RIP1多聚泛素化和核因子-κB转录活性的能力。我们得出结论，过氧化氢通过抑制Cezanne和IκBα的负调节功能，延长了共同刺激细胞中核因子-κB的激活。
