Inhibition of NF-kappa B by S-nitrosylation.
It is not clear if redox regulation of transcription is the consequence of direct redox-related modifications of transcription factors, or if it occurs at some other redox-sensitive step. One obstacle has been the inability to demonstrate redox-related modifications of transcription factors in vivo. The redox-sensitive transcriptional activator NF-kappaB (p50-p65) is a case in point. Its activity in vitro can be inhibited by S-nitrosylation of a critical thiol in the DNA-interacting p50 subunit, but modulation of NF-kappaB activity by nitric oxide synthase (NOS) has been attributed to other mechanisms. Herein we show that cellular NF-kappaB activity is in fact regulated by S-nitrosylation. We observed that both S-nitrosocysteine and cytokine-activated NOS2 inhibited NF-kappaB in human respiratory cells or murine macrophages. This inhibition was reversed by addition of the denitrosylating agent dithiothreitol to cellular extracts, whereas NO bioactivity did not affect the TNFalpha-induced degradation of IkappaBalpha or the nuclear translocation of p65. Recapitulation of these conditions in vitro resulted in S-nitrosylation of recombinant p50, thereby inhibiting its binding to DNA, and this effect was reversed by dithiothreitol. Further, an increase in S-nitrosylated p50 was detected in cells, and the level was modulated by TNFalpha. Taken together, these data suggest that S-nitrosylation of p50 is a physiological mechanism of NF-kappaB regulation.
Marshall
H E
HE
Department of Medicine and Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710, USA.
Stamler
J S
JS
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000818:Animals; D002460:Cell Line; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D016207:Cytokines / Q000494:pharmacology; D004247:DNA / Q000037:antagonists & inhibitors / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D020672:I-kappa B Proteins; D008623:Mercaptoethanol; D051379:Mice; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D052002:NF-kappa B p50 Subunit; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D015538:Nitrosation / Q000187:drug effects; D009603:Nitroso Compounds / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D026403:S-Nitrosothiols; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D016207:Cytokines; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D009603:Nitroso Compounds; D026403:S-Nitrosothiols; D000072000:NF-KappaB Inhibitor alpha; D008623:Mercaptoethanol; C032635:S-nitrosomercaptoethanol; D004247:DNA; C021315:S-nitrosocysteine; D019001:Nitric Oxide Synthase; D003545:Cysteine