Insulin activates nuclear factor κB in mammalian cells through a Raf-1-mediated pathway

Abstract

We examined the effect of insulin on nuclear factor κB (NF-κB) activity in Chinese hamster ovary (CHO) cells overexpressing wild-type (CHO-R cells) or -defective insulin receptors mutated at Tyr1162 and Tyr1163 autophosphorylation sites (CHO-Y2 cells). In CHO-R cells, insulin caused a specific, time-, and concentration-dependent activation of NF-κB. The insulin-induced DNA-binding complex was identified as the p50/p65 heterodimer. Insulin activation of NF-κB: 1) was related to insulin receptor number and tyrosine kinase activity since it was markedly reduced in parental CHO cells which proved to respond to insulin growth factor-1 and phorbol 12-myristate 13-acetate (PMA) activation, and was dramatically decreased in CHO-Y2 cells; 2) persisted in the presence of cycloheximide and was blocked by pyrrolidine dithiocarbamate, aspirin and sodium salicylate, three compounds interfering with IκB degradation and/or NF-κB-IκB complex dissociation; 3) was independent of both PMA-sensitive and atypical (ζ) protein kinases C; and 4) was dependent on Raf-1 kinase activity since insulin-stimulated NF-κB DNA binding activity was inhibited by 8-bromo-cAMP, a Raf-1 kinase inhibitor. Moreover, insulin activation of NF-κB-driven luciferase reporter gene expression was blocked in CHO-R cells expressing a Raf-1 dominant negative mutant. This is the first evidence that insulin activates NF-κB in mammalian cells through a post-translational mechanism requiring both insulin receptor tyrosine kinase and Raf-1 kinase activities.