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

Endocrinology. 1995 Nov;136(11):4790-5. doi: 10.1210/endo.136.11.7588208.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Ditiocarb / pharmacology
  • Enzyme Activation
  • Enzyme Inhibitors / pharmacology
  • Gene Expression*
  • Genistein
  • Insulinoma
  • Interleukin-1 / pharmacology*
  • Islets of Langerhans / enzymology*
  • Islets of Langerhans / ultrastructure
  • Isoflavones / pharmacology
  • Male
  • Molecular Sequence Data
  • NF-kappa B / antagonists & inhibitors
  • NF-kappa B / metabolism*
  • Nitric Oxide Synthase / genetics*
  • Pancreatic Neoplasms
  • Protein-Tyrosine Kinases / antagonists & inhibitors
  • Protein-Tyrosine Kinases / metabolism
  • Rats
  • Rats, Sprague-Dawley
  • Signal Transduction*
  • Tumor Cells, Cultured

Substances

  • Enzyme Inhibitors
  • Interleukin-1
  • Isoflavones
  • NF-kappa B
  • Ditiocarb
  • Genistein
  • Nitric Oxide Synthase
  • Protein-Tyrosine Kinases