title: Improvement of the mitochondrial antioxidant defense status prevents cytokine-induced nuclear factor-kappaB activation in insulin-producing cells.
abstract: 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 insulin-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.
authors.affiliation: Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany.
authors.forename: Anna Karenina
authors.initials: AK
authors.lastname: Azevedo-Martins
authors.forename: Stephan
authors.initials: S
authors.lastname: Lortz
authors.forename: Sigurd
authors.initials: S
authors.lastname: Lenzen
authors.forename: Rui
authors.initials: R
authors.lastname: Curi
authors.forename: Decio L
authors.initials: DL
authors.lastname: Eizirik
authors.forename: Markus
authors.initials: M
authors.lastname: Tiedge
mesh_terms: D000818:Animals; D002374:Catalase / Q000378:metabolism; D016207:Cytokines / Q000494:pharmacology / Q000502:physiology*; D004338:Drug Combinations; D005979:Glutathione Peroxidase / Q000378:metabolism; D007328:Insulin / Q000096:biosynthesis*; D007340:Insulinoma / Q000378:metabolism*; D007375:Interleukin-1 / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology*; D016328:NF-kappa B / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D010088:Oxidoreductases / Q000378:metabolism*; D010190:Pancreatic Neoplasms / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000502:physiology; D013482:Superoxide Dismutase / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D016207:Cytokines; D004338:Drug Combinations; D007328:Insulin; D007375:Interleukin-1; D016328:NF-kappa B; D010088:Oxidoreductases; D002374:Catalase; D005979:Glutathione Peroxidase; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D013482:Superoxide Dismutase