title: 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.
abstract: 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 NFkappaB 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 NFkappaB, 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 NFkappaB 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 an 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 microns 100 microM diethyldithiocarbamate or 100 microM 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 microM) 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.
authors.affiliation: Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: G
authors.initials: G
authors.lastname: Kwon
authors.forename: J A
authors.initials: JA
authors.lastname: Corbett
authors.forename: C P
authors.initials: CP
authors.lastname: Rodi
authors.forename: P
authors.initials: P
authors.lastname: Sullivan
authors.forename: M L
authors.initials: ML
authors.lastname: McDaniel
mesh_terms: D000818:Animals; D001483:Base Sequence; D004050:Ditiocarb / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D019833:Genistein; D007340:Insulinoma; D007375:Interleukin-1 / Q000494:pharmacology*; D007515:Islets of Langerhans / Q000201:enzymology* / Q000648:ultrastructure; D007529:Isoflavones / Q000494:pharmacology; D008297:Male; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics*; D010190:Pancreatic Neoplasms; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
chemical_list: D004791:Enzyme Inhibitors; D007375:Interleukin-1; D007529:Isoflavones; D016328:NF-kappa B; D004050:Ditiocarb; D019833:Genistein; D019001:Nitric Oxide Synthase; D011505:Protein-Tyrosine Kinases