Insulin Regulation of Nuclear Factor Kappa B Activity

工作概述：本研究的目的是研究胰岛素通过调节I{kappa}Balpha促进NF{kappa}B活化的信号通路。我们在这里表明，尽管胰岛素增加了{kappa}B依赖的报告基因表达，并增加了NF{kappa}B的p65/RelA亚基的核易位及其DNA结合，但它能够诱导磷酸化和泛素化I{kappa}Balpha的时间依赖性积累，而不会导致其蛋白水解降解。相反，细胞因子TNFalpha刺激细胞可通过I{kappa}Balpha的磷酸化、泛素化和随后的降解激活NF{kappa}B。免疫荧光研究显示，在未受刺激和胰岛素处理的细胞的细胞核中存在大量磷酸化的I{kappa}Balpha。I{kappa}B激酶α和β是I{kappa}Balpha磷酸化的核心参与者，在暴露于TNFalpha后迅速被诱导，而不是胰岛素。此外，胰岛素刺激的I{kappa}Balpha磷酸化不依赖于Ras/ERK级联的激活。Akt1或I类磷脂酰肌醇3-激酶（PI 3-激酶）显性阴性突变体的表达抑制了PI 3-激酶/Akt1信号的胰岛素刺激，而不影响I{kappa}Balpha的磷酸化。有趣的是，PI 3激酶抑制剂wortmannin和LY294002阻断胰岛素刺激的I类PI 3激酶依赖事件，其剂量远低于抑制I{kappa}Balpha磷酸化所需的剂量。这些数据表明，胰岛素通过一种独特的低亲和力wortmannin敏感途径调节I{kappa}Balpha功能。

Summary of work: The aim of this study was to examine the signaling pathways by which insulin promotes activation of NF{kappa}B through the regulation of I{kappa}Balpha. We show here that although insulin increased {kappa}B dependent reporter gene expression and augment nuclear translocation of the p65/RelA subunit of NF{kappa}B and its DNA binding, it was able to induce a time-dependent accumulation of phosphorylated and ubiquitinated I{kappa}Balpha without its proteolytic degradation. In contrast, cell stimulation with the cytokine TNFalpha allowed activation of NF{kappa}B through phosphorylation, ubiquitination and subsequent degradation of I{kappa}Balpha. Immunofluorescence studies revealed the presence of a large pool of phosphorylated I{kappa}Balpha in the nucleus of unstimulated and insulin-treated cells. I{kappa}B kinase alpha and beta, central players in the phosphorylation of I{kappa}Balpha, were rapidly induced following exposure to TNFalpha, but not insulin. Furthermore, insulin-stimulated I{kappa}Balpha phosphorylation did not depend on activation of the Ras/ERK cascade. Expression of a dominant negative mutant of Akt1 or of class I phosphatidylinositol 3-kinase (PI 3-kinase) inhibited the insulin stimulation of PI 3-kinase/Akt1 signaling without affecting phosphorylation of I{kappa}Balpha. Interestingly, the PI 3-kinase inhibitors wortmannin and LY294002 blocked insulin-stimulated class I PI 3-kinase-dependent events at much lower doses than that required to inhibit phosphorylation of I{kappa}Balpha. These data demonstrate that insulin regulates I{kappa}Balpha function through a distinct low-affinity wortmannin-sensitive pathway.