title: Activation of nuclear factor-kappaB by depolarization and Ca(2+) influx in MIN6 insulinoma cells.
abstract: The purpose of the current study was to determine whether nuclear factor-kappaB (NF-kappaB) activation is a component of the depolarization/Ca(2+)-dependent signaling in beta-cells. MIN6 cells were transfected with a plasmid containing five tandem repeats of NF-kappaB binding sites linked to a luciferase reporter. The results of these experiments showed that KCl induced depolarization-activated NF-kappaB-dependent transcription (3.8-fold at 45 mmol/l, P < 0.01) in a concentration-dependent manner. Tumor necrosis factor-alpha (TNF-alpha), a known inducer of NF-kappaB signaling, activated this construct by 3.4-fold (P < 0.01). The response of NF-kappaB to depolarization was inhibited by the Ca(2+)-channel blocker verapamil and by the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 (70 and 62%, respectively). TNF-alpha, glucose, and KCl treatment resulted in inhibitory kappaBalpha degradation by Western blot analysis. TNF-alpha treatment and depolarization activation of NF-kappaB differed significantly in that TNF-alpha activation was not blocked by PD98059. Transfection with PKA, MEK, and MEK kinase induced NF-kappaB-dependent transcription by 20-, 90-, and 300-fold, respectively, suggesting that these pathways contribute to the activation in the depolarization response. These findings demonstrate that depolarization/Ca(2+) influx, as well as TNF-alpha treatment, can activate NF-kappaB-dependent transcription in pancreatic beta-cells, but by different signaling pathways. The current studies show that Ca(2+) signals in pancreatic beta-cells can activate transcription factors involved in the regulation of cell cycle and apoptosis. These findings now add NF-kappaB to the list of depolarization-induced transcription factors in pancreatic beta-cells.
authors.affiliation: Washington University School of Medicine, Division of Endocrinology, Diabetes and Metabolism, St. Louis, Missouri, USA. ebernal@im.wustl.edu
authors.forename: Ernesto
authors.initials: E
authors.lastname: Bernal-Mizrachi
authors.forename: Wu
authors.initials: W
authors.lastname: Wen
authors.forename: Michael
authors.initials: M
authors.lastname: Shornick
authors.forename: M Alan
authors.initials: MA
authors.lastname: Permutt
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D020672:I-kappa B Proteins / Q000378:metabolism; D007340:Insulinoma / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors; D016328:NF-kappa B / Q000502:physiology*; D011189:Potassium Chloride / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D005419:Flavonoids; D020672:I-kappa B Proteins; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D011189:Potassium Chloride; D020929:Mitogen-Activated Protein Kinase Kinases; D005947:Glucose; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D002118:Calcium