Differential IκB kinase activation and IκBα degradation by Interleukin-1β and tumor necrosis factor-α in human U937 monocytic cells -: Evidence for additional regulatory steps in κB-dependent transcription

The I kappa B kinases (IKKs) lie downstream of the NF-kappa B-inducing kinase (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 monocytic cells, interleukin (IL)-1 beta (1 ng/ml) and tumor necrosis factor (TNF)-alpha; 10 ng/ml) induced kappa B-dependent transcription equally. However, 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, yet 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 complexes 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 imprecise relationship between IKK activation, I kappa B alpha degradation, and NF-kappa B DNA binding, suggesting the existence of additional mechanisms that regulate NF-kappa B activation. Finally, the lack of correlation between DNA binding and transcriptional activation plus the fact that PP1 and genistein both inhibited KB-dependent transcription without affecting DNA binding activity demonstrate the existence of regulatory steps downstream of NF-kappa B DNA binding. Therapeutically these data are important as inhibition of the NIK-IKK-I kappa B alpha cascade may not produce equivalent reductions in NF-kappa B-dependent gene expression.