Bernal-Mizrachi Ernesto, Wen Wu, Shornick Michael, Permutt M Alan
Washington University School of Medicine, Division of Endocrinology, Diabetes and Metabolism, St. Louis, Missouri, USA.
Diabetes. 2002 Dec;51 Suppl 3:S484-8. doi: 10.2337/diabetes.51.2007.s484.
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.
本研究的目的是确定核因子-κB（NF-κB）激活是否是β细胞去极化/钙（Ca2+）依赖性信号传导的一个组成部分。将含有与荧光素酶报告基因相连的五个串联重复NF-κB结合位点的质粒转染到MIN6细胞中。这些实验结果表明，氯化钾（KCl）以浓度依赖性方式诱导去极化激活的NF-κB依赖性转录（45 mmol/L时为3.8倍，P<0.01）。肿瘤坏死因子-α（TNF-α）是一种已知的NF-κB信号诱导剂，可使该构建体激活3.4倍（P<0.01）。NF-κB对去极化的反应受到钙通道阻滞剂维拉帕米和丝裂原活化蛋白激酶激酶（MEK）抑制剂PD98059的抑制（分别为70%和62%）。通过蛋白质印迹分析，TNF-α、葡萄糖和KCl处理导致抑制性κBα降解。TNF-α处理和NF-κB的去极化激活存在显著差异，因为TNF-α激活不受PD98059的阻断。用蛋白激酶A（PKA）、MEK和MEK激酶转染分别诱导NF-κB依赖性转录20倍、90倍和300倍，表明这些途径有助于去极化反应中的激活。这些发现表明，去极化/Ca2+内流以及TNF-α处理均可激活胰腺β细胞中NF-κB依赖性转录，但通过不同的信号通路。目前的研究表明，胰腺β细胞中的Ca2+信号可激活参与细胞周期和细胞凋亡调节的转录因子。这些发现现在将NF-κB添加到胰腺β细胞中去极化诱导的转录因子列表中。
