Proinflammatory cytokines (interleukin-1β [IL-1β], tumor necrosis factor-α [TNF-α], and γ-interferon [IFN-γ]) initiate a variety of signal cascades in pancreatic β-cells that affect the expression level of genes involved in both the destruction and the protection of the β-cell. The generation of nitric oxide (NO) via the inducible NO synthase (iNOS) and oxygen free radicals play a key role in cytokine-mediated β-cell destruction. Within these signal cascades, the activation of the transcription factor nuclear factor-κB (NF-κB) 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-κB 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-1β or cytokine mix consisting of IL-1β + TNF-α + IFN-γ) activation of NF-κB 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-κB 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-κB and the iNOS promoter. In conclusion, the overexpression of MnSOD, which inactivates specifically mitochondrially derived oxygen free radicals, significantly reduced the activation of NF-κB 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 β-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-κB. Overexpression of MnSOD may thus be beneficial for β-cell survival through suppression of oxygen free radical formation, prevention of NF-κB activation, and iNOS expression.
Address correspondence and reprint requests to Dr. Markus Tiedge, Institute of Clinical Biochemistry, Hannover Medical School, D-30623 Hannover, Germany. E-mail:.
Received for publication 12 June 2002 and accepted in revised form 30 September 2002.
Cat, catalase; EMSA, electrophoretic mobility shift assay; Gpx, glutathione peroxidase; IFN-γ, γ-interferon; IL-1β, interleukin-1β; iNOS, inducible nitric oxide synthase; NF-κB, nuclear factor-κB; ROS, reactive oxygen species; SEAP, secreted alkaline phosphatase; SOD, superoxide dismutase; TNF-α, tumor necrosis factor-α.