Activation of nuclear factor-κB by depolarization and Ca2+ influx in MIN6 insulinoma cells

The purpose of the current study was to determine whether nuclear factor-kappaB (NF-kappaB) activation is a component of the depolarization/Ca2+-dependent signaling in beta-cells. MIN6 cells were transfected with a plasmid containing five tandem repeat's 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 Ca2+-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, respectivel suggesting that these pathways contribute to the activation in the depolarization response. These findings demonstrate that depolarization/Ca2+ influx 9 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 Ca2+ 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.