Ethyl pyruvate inhibits nuclear factor-kappaB-dependent signaling by directly targeting p65.
Ethyl pyruvate has been shown to have anti-inflammatory properties in numerous cell culture and animal studies. In this series of experiments, we tested the hypothesis that ethyl pyruvate inhibits signaling by the pro-inflammatory transcription factor, NF-kappaB. Ethyl pyruvate inhibited luciferase expression in lipopolysaccharide-stimulated murine macrophage-like RAW 264.7 cells transfected with an NF-kappaB-dependent luciferase reporter vector. Ethyl pyruvate also decreased NF-kappaB DNA-binding activity in lipopolysaccharide-stimulated RAW 264.7 cells and decreased lipopolysaccharide-induced expression of an NF-kappaB-dependent gene, inducible nitric oxide synthase. Ethyl pyruvate had no effect on the degradation of IkappaBalpha or IkappaBbeta in lipopolysaccharide-stimulated RAW 264.7 cells, suggesting that ethyl pyruvate acts distally to this step in the activation of NF-kappaB. In a cell-free system, binding of p50 homodimers to an NF-kappaB consensus oligonucleotide sequence was unaffected by ethyl pyruvate over a wide range of concentrations, indicating that ethyl pyruvate probably does not modify or interact with the p50 subunit of NF-kappaB. In contrast, ethyl pyruvate inhibited DNA binding by ectopically overexpressed wild-type p65 homodimers. However, ethyl pyruvate failed to inhibit the DNA-binding activity of homodimers of an overexpressed mutant form of a p65 with substitution of serine for cysteine 38. Taken together, these results suggest that ethyl pyruvate inhibits DNA-binding by covalently modifying p65 at Cys(38). We conclude that some of the beneficial anti-inflammatory effects of ethyl pyruvate may be due to modification of p65, thereby inhibiting signaling via the NF-kappaB pathway.
Han
Yusheng
Y
Department of Critical Care Medicine, 615 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15261, USA.
Englert
Joshua A
JA
Yang
Runkuan
R
Delude
Russell L
RL
Fink
Mitchell P
MP
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D020672:I-kappa B Proteins / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D011773:Pyruvates / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*; D051996:Transcription Factor RelA; D014158:Transcription, Genetic / Q000187:drug effects
C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B; C000606205:Nfkbia protein, mouse; D011773:Pyruvates; D051996:Transcription Factor RelA; C046522:ethyl pyruvate; D000072000:NF-KappaB Inhibitor alpha; D004247:DNA; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse