Differential IkB Kinase Activation and IkBa Degradation by Interleukin-1b and Tumor Necrosis Factor-a in Human U937 Monocytic Cells EVIDENCE FOR ADDITIONAL REGULATORY STEPS IN kB-DEPENDENT TRANSCRIPTION*

The IkB kinases (IKKs) lie downstream of the NF-kBinducing kinase (NIK) and activate NF-kB by phosphorylation of IkBa. This leads to IkBa degradation and release of NF-kB. In U937 monocytic cells, interleukin (IL)-1b (1 ng/ml) and tumor necrosis factor (TNF)-a; 10 ng/ml) induced kB-dependent transcription equally. However, IKK activity was strongly induced by TNF-a but not by IL-1b. This was consistent with IkBa phosphorylation and degradation, yet TNF-a-induced NF-kB DNA binding was only 30–40% greater than for IL-1b. This was not explained by degradation of IkBb, IkBe, or p105 nor nuclear translocation of NF-kBzIkBa complexes or degradation-independent release of NF-kB. Dominant negative (NIK) repressed TNF-a and IL-1b-induced kBdependent transcription by ;60% and ;35%, respectively. These data reveal an imprecise relationship between IKK activation, IkBa degradation, and NF-kB DNA binding, suggesting the existence of additional mechanisms that regulate NF-kB 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-kB DNA binding. Therapeutically these data are important as inhibition of the NIK-IKK-IkBa cascade may not produce equivalent reductions in NF-kB-dependent gene expression.