Nitric oxide represses inhibitory kappaB kinase through S-nitrosylation.
Nitric oxide (NO) possesses antiinflammatory effects, which may be exerted via its ability to inhibit the transcription factor, NF-kappaB. A commonly proposed mode of action for inhibition of NF-kappaBbyNO involves interference with NF-kappaB binding to DNA. Because activation of inhibitory kappaB kinase (IKK), the prerequisite enzyme complex necessary to induce NF-kappaB, is subject to redox regulation, we assessed whether IKK could present a more proximal target for NO to inhibit NF-kappaB activation. We demonstrate here that S-nitrosothiols (SNO) caused a dose-dependent inhibition of the enzymatic activity of IKK, in lung epithelial cells and in Jurkat T cells, which was associated with S-nitrosylation of the IKK complex. Using biotin derivatization of SNO, we revealed that IKKbeta, the catalytic subunit required for NF-kappaB activation, was a direct target for S-nitrosylation. A mutant version of IKKbeta containing a Cys-179-to-Ala mutation was refractory to inhibition by SNO or to increases in S-nitrosylation, in contrast to wild-type IKKbeta, demonstrating that Cys-179 is the main target for attack by SNO. Importantly, inhibition of NO synthase activity in Jurkat T cells resulted in activation of IKK, in association with its denitrosylation. Moreover, NO synthase inhibition enhanced the ability of tumor necrosis factor alpha to activate IKK, illustrating the importance of endogenous NO in regulating the extent of NF-kappaB activation by cytokines. Collectively, our findings demonstrate that IKKbeta is an important target for the redox regulation of NF-kappaB by endogenous or exogenous NO, providing an additional mechanism for its antiinflammatory properties.
Reynaert
Niki L
NL
Department of Pathology, University of Vermont, Burlington, VT 05405, USA.
Ckless
Karina
K
Korn
Solange H
SH
Vos
Nanda
N
Guala
Amy S
AS
Wouters
Emiel F M
EF
van der Vliet
Albert
A
Janssen-Heininger
Yvonne M W
YM
D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases; D019169:Jurkat Cells; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009569:Nitric Oxide / Q000494:pharmacology / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D010396:Penicillamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011650:Pulmonary Alveoli / Q000166:cytology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D026403:S-Nitrosothiols / Q000378:metabolism / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D019323:omega-N-Methylarginine / Q000494:pharmacology
D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D020030:Nitric Oxide Donors; D011994:Recombinant Proteins; D026403:S-Nitrosothiols; C110881:S-nitro-N-acetylpenicillamine; D014409:Tumor Necrosis Factor-alpha; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; C496564:Chuk protein, mouse; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; C496562:Ikbkb protein, mouse; C496566:Ikbke protein, mouse; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D010396:Penicillamine; D003545:Cysteine