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.
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.
Department of Thoracic Medicine, National Heart and Lung Institute, Imperial College School of Medicine, London SW3 6LY, United Kingdom.
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Y
Nasuhara
I M
IM
Adcock
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Catley
P J
PJ
Barnes
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R
Newton
D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins; D007375:Interleukin-1 / Q000494:pharmacology*; D007976:Leupeptins / Q000494:pharmacology; D008025:Ligases / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D020298:U937 Cells
C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D007375:Interleukin-1; D007976:Leupeptins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D011720:Pyrazoles; D011743:Pyrimidines; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D019833:Genistein; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde