Abstract
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-κB. Ethyl pyruvate inhibited luciferase expression in lipopolysaccharide-stimulated murine macrophage-like RAW 264.7 cells transfected with an NF-κB-dependent luciferase reporter vector. Ethyl pyruvate also decreased NF-κB DNA-binding activity in lipopolysaccharide-stimulated RAW 264.7 cells and decreased lipopolysaccharide-induced expression of an NF-κB-dependent gene, inducible nitric oxide synthase. Ethyl pyruvate had no effect on the degradation of IκBα or IκBβ in lipopolysaccharide-stimulated RAW 264.7 cells, suggesting that ethyl pyruvate acts distally to this step in the activation of NF-κB. In a cell-free system, binding of p50 homodimers to an NF-κB 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-κB. 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 Cys38. 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-κB pathway.
Footnotes
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This work was funded by National Institutes of Health Grant GM37631. J.A.E. is a Howard Hughes Medical Institute Medical Student Research Fellow.
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doi:10.1124/jpet.104.079707.
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ABBREVIATIONS: iNOS, inducible nitric oxide synthase; IL, interleukin; NF, nuclear factor; IKK, IκB kinase; EMSA, electrophoretic mobility shift assay; RT, reverse transcriptase; PCR, polymerase chain reaction; TBST, Tris-buffered saline/Tween 20; ROS, reactive oxygen species.
- Received October 25, 2004.
- Accepted November 1, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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