INTERLEUKIN-1-BETA-INDUCED NITRIC-OXIDE SYNTHASE EXPRESSION BY RAT PANCREATIC BETA-CELLS - EVIDENCE FOR THE INVOLVEMENT OF NUCLEAR FACTOR KAPPA-B IN THE SIGNALING MECHANISM

Recent evidence indicates that overproduction of nitric oxide mediates cytokine-induced inhibition of insulin secretion by pancreatic islets. The current studies were designed to-characterize signaling events involving the transcriptional factor NF kappa B in interleukin-1 (IL-1)-induced expression of inducible nitric oxide synthase (iNOS) by primary and transformed rat pancreatic beta-cells. Due to limitations of cell numbers of purified primary beta-cells, biochemical and molecular studies were performed primarily using the insulinoma cell Line, RINm5F. Inhibitors of NF kappa B, diethyldithiocarbamate, pyrrolidine dithiocarbamate, and N-acetyl cysteine prevent IL-1-induced iNOS expression at the level of messenger RNA, protein, and nitrite generation. IL-1 induces a time-dependent translocation of NF kappa B from cytosol to nucleus, with maximal translocation observed approximately 15-30 min after IL-1 treatment, as determined by electrophoretic mobility shift assays. The specificity of the band containing the NF kappa B DNA-protein complex was shown by competition with a 150-fold excess of nonradiolabeled NF kappa B oligonucleotide. Supershift assays using immunoglobulins G against NF kappa B subunits p50 and p65 indicate that the protein complex contains a heterodimer of p50 and p65. IL-1-induced translocation of NF kappa B was blocked by 100 mu M diethyldithiocarbamate or 100 mu M pyrrolidine dithiocarbamate, further establishing a critical role for NF kappa B in the induction of iNOS by IL-1 in rat pancreatic beta-cells. Activation of tyrosine kinase appears to precede NF kappa B activation, as the tyrosine kinase inhibitor genistein (100 mu M) blocks IL-1-induced translocation of NF kappa B. An understanding of the signal transduction pathway of cytokine-induced nitric oxide generation by beta-cells will provide strategies of intervention to further evaluate the role of nitric oxide in mediating beta-cell dysfunction.