Differential I kappa B kinase activation and I kappa B alpha degradation by Interleukin-1 beta and tumor necrosis factor-alpha in human U937 monocytic cells - Evidence for additional regulatory steps in kappa B-dependent transcription

The I kappa B kinases (IKKs) lie downstream of the NF-kappa B-inducing kina se (MK) and activate NF-kappa B by phosphorylation of I kappa B alpha, This leads to I kappa B alpha degradation and release of NF-kappa B, In U937 mo nocytic cells, interleukin (IL)-1 beta (1 ng/ml) and tumor necrosis factor (TNF)-alpha; 10 ng/ml) induced kappa B-dependent transcription equally. How ever, IKK activity was strongly induced by TNF-alpha but not by IL-1 beta. This was consistent with I kappa B alpha phosphorylation and degradation, y et TNF-alpha-induced NF-KB DNA binding was only 30-40% greater than for IL- 1 beta. This was not explained by degradation of I kappa B beta, I kappa B epsilon, Or p105 nor nuclear translocation of NF-kappa B.I kappa B alpha co mplexes or degradation-independent release of NF-KB. Dominant negative (NIK ) repressed TNF-alpha and IL-1 beta-induced kappa B-ependent transcription by similar to 60% and similar to 35%, respectively. These data reveal an im precise relationship between IKK activation, I kappa B alpha degradation, a nd NF-kappa B DNA binding, suggesting the existence of additional mechanism s that regulate NF-kappa B activation. Finally, the lack of correlation bet ween DNA binding and transcriptional activation plus the fact that PP1 and genistein both inhibited KB-dependent transcription without affecting DNA b inding activity demonstrate the existence of regulatory steps downstream of NF-kappa B DNA binding. Therapeutically these data are important as inhibi tion of the NIK-IKK-I kappa B alpha cascade may not produce equivalent redu ctions in NF-kappa B-dependent gene expression.