Endotoxin-mediated nitric oxide synthesis inhibits IL-1beta gene transcription in ANA-1 murine macrophages.
On the basis of previous work demonstrating nitric oxide (NO)-mediated inhibition of nuclear factor-kappaB (NF-kappaB) DNA binding, we hypothesized that NO downregulates NF-kappaB-dependent interleukin-1beta (IL-1beta) production in an ANA-1 macrophage model of lipopolysaccharide (LPS) stimulation. In the presence of LPS (100 ng/ml), levels of IL-1beta protein and mRNA were significantly upregulated with NO synthase inhibition. Using nuclear run-on analysis and transient transfection studies, IL-1beta gene transcription and IL-1beta promoter activity were also found to be increased with inhibition of NO production. Parallel transfection studies using an NF-kappaB long terminal repeat-reporter plasmid exhibited similar findings, suggesting an NO-mediated effect on NF-kappaB activity. Gel shift studies showed that LPS-associated NF-kappaB DNA binding was increased, both in the setting of NO synthase inhibition and in a reducing environment. Repletion of NO by addition of an S-nitrosothiol restored IL-1beta protein synthesis, mRNA levels, gene transcription, promoter activity, and NF-kappaB DNA binding to levels noted in the presence of LPS alone. Our studies indicate that NO may regulate LPS-associated inflammation by downregulating IL-1beta gene transcription through S-nitrosation of NF-kappaB.
Schroeder
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RA
Department of Surgery, Georgetown University Medical Center, Washington, District of Columbia 20007, USA.
Cai
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C
Kuo
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PC
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007375:Interleukin-1 / Q000235:genetics* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000378:metabolism / Q000502:physiology*; D008623:Mercaptoethanol; D051379:Mice; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D052247:Nitric Oxide Synthase Type II; D009603:Nitroso Compounds / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D026403:S-Nitrosothiols; D014158:Transcription, Genetic / Q000502:physiology*; D015854:Up-Regulation
D004791:Enzyme Inhibitors; D007375:Interleukin-1; D008070:Lipopolysaccharides; D016328:NF-kappa B; D020030:Nitric Oxide Donors; D009603:Nitroso Compounds; D012333:RNA, Messenger; D026403:S-Nitrosothiols; D009569:Nitric Oxide; D008623:Mercaptoethanol; C032635:S-nitrosomercaptoethanol; D004247:DNA; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse