Improvement of the mitochondrial antioxidant defense status prevents cytokine-induced nuclear factor-κB activation in insulin-producing cells
Improvement of the mitochondrial antioxidant defense status prevents cytokine-induced nuclear factor-κB activation in insulin-producing cells
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DOI：
10.2337/diabetes.52.1.93
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发表时间：
2003-01-01
期刊：
影响因子：
7.7
通讯作者：
Tiedge, M
中科院分区：
文献类型：
--
作者：
Azevedo-Martins, AK;Lortz, S;Tiedge, M
促炎细胞因子（白细胞介素-1β [IL-1β]、肿瘤坏死因子-α [TNF-α] 和γ-干扰素 [IFN-γ]）在胰腺 β 细胞中启动多种信号级联，影响参与破坏和保护 β 细胞的基因的表达水平。通过诱导型一氧化氮合酶 (iNOS) 和氧自由基生成一氧化氮 (NO) 在细胞因子介导的 β 细胞破坏中发挥着关键作用。在这些信号级联中，转录因子核因子-kappaB (NF-kappaB) 的激活至关重要，许多细胞因子敏感基因在其启动子区域含有该转录因子的结合位点。本研究的目的是表征产生胰岛素的 RINm5F 细胞中细胞因子介导的 NF-κB 激活以及随后 iNOS 蛋白的表达，通过细胞保护酶过氧化氢酶 (Cat)、谷胱甘肽过氧化物酶 (Gpx) 和细胞质 Cu/Zn 超氧化物歧化酶 (Cu/ZnSOD) 的过表达来改善抗氧化防御状态。具有不同线粒体抗氧化防御状态的 RINm5F 细胞是通过稳定过表达有义（MnSOD 有义）和反义（MnSOD 反义）方向的 MnSOD 构建体而产生的。通过 MnSOD 的过表达，RINm5F 细胞中细胞因子诱导的（IL-1β 或由 IL-1β + TNF-α + IFN-γ 组成的细胞因子混合物）NF-κB 激活减少了 >80%。在细胞因子刺激的 MnSOD 感知细胞中，iNOS 启动子的活性保持在基础水平。相反，与对照细胞相比，细胞因子刺激的 MnSOD 反义细胞中 MnSOD 基因表达的抑制导致 NF-κB 的激活增加三倍，iNOS 启动子的激活增加两倍。 MnSOD 感知细胞细胞因子孵育 6 和 8 小时后，iNOS 蛋白表达显着降低。 RINm5F MnSOD 反义细胞中 MnSOD 的低活性水平增加了 iNOS 蛋白的表达，特别是在细胞因子介导的毒性的早期阶段。 Cat、Gpx 和细胞质 Cu/ZnSOD 不影响 NF-kappaB 和 iNOS 启动子的激活。总之，MnSOD 的过度表达可以特异性地灭活线粒体来源的氧自由基，从而显着降低胰岛素生成细胞中 NF-κB 的激活。由于早期细胞因子信号通路中的这种保护作用，iNOS（β细胞破坏过程中的一个重要事件）的诱导也显着减少。结果证明线粒体衍生的活性氧 (ROS) 在细胞因子敏感转录因子 NF-kappaB 的激活中发挥着关键作用。过度表达。因此，MnSOD 可能通过抑制氧自由基形成、防止 NF-κB 激活和 iNOS 表达而有益于 β 细胞存活。
Proinflammatory cytokines (interleukin-1beta [IL-1beta], tumor necrosis factor-alpha [TNF-alpha], and gamma-interferon [IFN-gamma]) initiate a variety of signal cascades in pancreatic beta-cells that affect the expression level of genes involved in both the destruction and the protection of the beta-cell. The generation of nitric oxide (NO) via the inducible NO synthase (iNOS) and oxygen free radicals play a key role in cytokine-mediated beta-cell destruction. Within these signal cascades, the activation of the transcription factor nuclear factor-kappaB (NF-kappaB) is crucial, and many cytokine-sensitive genes contain binding sites for this transcription factor in their promoter regions. The aim of this study was to characterize the cytokine-mediated activation of NF-kappaB and the subsequent expression of iNOS protein in insulitin-producing RINm5F cells with an improved antioxidant defense status by overexpression of the cytoprotective enzymes catalase (Cat), glutathione peroxidase (Gpx), and the cytoplasmic Cu/Zn superoxide dismutase (Cu/ZnSOD). RINm5F cells with diverse mitochondrial antioxidative defense status were generated by stable overexpression of MnSOD constructs in sense (MnSOD sense) and antisense orientation (MnSOD antisense). Cytokine-induced (IL-1beta or cytokine mix consisting of IL-1beta + TNF-alpha + IFN-gamma) activation of NF-kappaB in RINm5F cells was reduced by >80% through overexpression of MnSOD. The activity of the iNOS promoter remained at basal levels in cytokine-stimulated MnSOD sense cells. In contrast, the suppression of MnSOD gene expression in cytokine-stimulated MnSOD antisense cells resulted in a threefold higher activation of NF-kappaB and a twofold higher activation of the iNOS promoter as compared with control cells. The iNOS protein expression was significantly reduced after a 6- and 8-h cytokine incubation of MnSOD sense cells. The low activity level of MnSOD in RINm5F MnSOD antisense cells increased the iNOS protein expression in particular during the early phase of cytokine-mediated toxicity. Cat, Gpx, and the cytoplasmic Cu/ZnSOD did not affect the activation of NF-kappaB and the iNOS promoter. In conclusion, the overexpression of MnSOD, which inactivates specifically mitochondrially derived oxygen free radicals, significantly reduced the activation of NF-kappaB in insulin-producing cells. As a consequence of this protective effect in the early cytokine signaling pathways, the induction of iNOS, an important event in the beta-cell destruction process, was also significantly reduced. The results provide evidence that mitochondrially derived reactive oxygen species (ROS) play a critical role in the activation of the cytokine-sensitive transcription factor NF-kappaB. Overexpression. of MnSOD may thus be beneficial for beta-cell survival through suppression of oxygen free radical formation, prevention of NF-kappaB activation, and iNOS expression.