Insulin Regulation of Nuclear Factor Kappa B Activity

工作总结：这项研究的目的是研究胰岛素通过调节I{kappa}Balpha促进NF{kappa}B激活的信号通路。我们发现，尽管胰岛素增加了依赖于kappa的报告基因的表达，并增加了nf{kappa}B的p65/relA亚基的核易位及其与DNA的结合，但它能够诱导依赖于时间的磷酸化和泛素化的I{kappa}Balpha的积累，而不会发生蛋白降解。相反，细胞因子TNFpha的刺激可通过磷酸化、泛素化和随后降解I{kappa}Balpha来激活NF{kappa}B。免疫荧光研究显示，在未经刺激和胰岛素处理的细胞的细胞核中存在大量的磷酸化的Ikappa Balpha。在暴露于TNFpha后，参与Ikappa Balpha磷酸化的Ikappa BKα和Ikappa BKβ迅速被诱导，但不能被胰岛素诱导。此外，胰岛素刺激的Ikappa Balpha磷酸化并不依赖于RAS/ERK级联的激活。显性负性突变体Akt1或I类磷脂酰肌醇3-激酶(PI-3-K)的表达抑制了PI-3-K/Akt-1信号的胰岛素刺激，但不影响I{kappa}Balpha的磷酸化。有趣的是，PI 3-激酶抑制剂Wortmannin和LY294002以比抑制I{kappa}Balpha磷酸化所需的剂量低得多的剂量阻断胰岛素刺激的PI 3-K依赖事件。这些数据表明，胰岛素通过一条独特的低亲和力Wortmannin敏感途径来调节Ikappa 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.