Differential IkappaB kinase activation and IkappaBalpha degradation by interleukin-1beta and tumor necrosis factor-alpha in human U937 monocytic cells. Evidence for additional regulatory steps in kappaB-dependent transcription

J Biol Chem. 1999 Jul 9;274(28):19965-72. doi: 10.1074/jbc.274.28.19965.


The IkappaB kinases (IKKs) lie downstream of the NF-kappaB-inducing kinase (NIK) and activate NF-kappaB by phosphorylation of IkappaBalpha. This leads to IkappaBalpha degradation and release of NF-kappaB. In U937 monocytic cells, interleukin (IL)-1beta (1 ng/ml) and tumor necrosis factor (TNF)-alpha; 10 ng/ml) induced kappaB-dependent transcription equally. However, IKK activity was strongly induced by TNF-alpha but not by IL-1beta. This was consistent with IkappaBalpha phosphorylation and degradation, yet TNF-alpha-induced NF-kappaB DNA binding was only 30-40% greater than for IL-1beta. This was not explained by degradation of IkappaBbeta, IkappaBepsilon, or p105 nor nuclear translocation of NF-kappaB. IkappaBalpha complexes or degradation-independent release of NF-kappaB. Dominant negative (NIK) repressed TNF-alpha and IL-1beta-induced kappaB-dependent transcription by approximately 60% and approximately 35%, respectively. These data reveal an imprecise relationship between IKK activation, IkappaBalpha degradation, and NF-kappaB DNA binding, suggesting the existence of additional mechanisms that regulate NF-kappaB activation. Finally, the lack of correlation between DNA binding and transcriptional activation plus the fact that PP1 and genistein both inhibited kappaB-dependent transcription without affecting DNA binding activity demonstrate the existence of regulatory steps downstream of NF-kappaB DNA binding. Therapeutically these data are important as inhibition of the NIK-IKK-IkappaBalpha cascade may not produce equivalent reductions in NF-kappaB-dependent gene expression.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA-Binding Proteins / metabolism*
  • Enzyme Activation
  • Gene Expression Regulation
  • Genes, Reporter
  • Genistein / pharmacology
  • Humans
  • I-kappa B Kinase
  • I-kappa B Proteins*
  • Interleukin-1 / pharmacology*
  • Leupeptins / pharmacology
  • Ligases / metabolism
  • NF-KappaB Inhibitor alpha
  • NF-kappa B / metabolism
  • Phosphorylation
  • Protein Serine-Threonine Kinases / metabolism*
  • Pyrazoles / pharmacology
  • Pyrimidines / pharmacology
  • Tumor Necrosis Factor-alpha / pharmacology*
  • U937 Cells


  • 4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine
  • DNA-Binding Proteins
  • I-kappa B Proteins
  • Interleukin-1
  • Leupeptins
  • NF-kappa B
  • NFKBIA protein, human
  • Pyrazoles
  • Pyrimidines
  • Tumor Necrosis Factor-alpha
  • NF-KappaB Inhibitor alpha
  • Genistein
  • Protein Serine-Threonine Kinases
  • CHUK protein, human
  • I-kappa B Kinase
  • IKBKB protein, human
  • IKBKE protein, human
  • Ligases
  • guanosine 3',5'-polyphosphate synthetases
  • benzyloxycarbonylleucyl-leucyl-leucine aldehyde