Ethacrynic acid inhibits multiple steps in the NF-kappaB signaling pathway.
Ethacrynic acid has been used as a safe and effective diuretic for more than 30 years. In this study, we tested the hypothesis that ethacrynic acid is also an anti-inflammatory agent that inhibits signaling by the proinflammatory transcription factor NF-kappaB. We showed that ethacrynic acid inhibited luciferase expression in lipopolysaccharide-stimulated macrophage-like RAW 264.7 cells transfected with an NF-kappaB-dependent luciferase reporter vector and also inhibited NF-kappaB DNA binding in lipopolysaccharide-stimulated RAW 264.7 cells (electrophoretic mobility shift assay). Ethacrynic acid inhibited degradation of IkappaBalpha and IkappaBbeta in lipopolysaccharide-stimulated RAW 264.7 cells. Ethacrynic acid impaired DNA binding of wild-type p65 subunits of NF-kappaB in cells. However, DNA binding of a Cys--> Ser p65 mutant was not inhibited by ethacrynic acid, suggesting that ethacrynic acid inhibits DNA binding by alkylating p65 at Cys. In a cell-free system, binding of p50 homodimers to an NF-kappaB consensus sequence was inhibited by ethacrynic acid at concentrations from 10 to 100 microM, indicating that ethacrynic acid probably also covalently modifies the p50 subunit. These data indicate that ethacrynic acid inhibits activation of the NF-kappaB pathway at multiple points and suggest that this well-studied drug warrants further investigation as a potential therapeutic for various conditions that are associated with excessive inflammation.
Han
Yusheng
Y
Department of Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
Englert
Joshua A
JA
Delude
Russell L
RL
Fink
Mitchell P
MP
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D002474:Cell-Free System; D003545:Cysteine / Q000737:chemistry; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004976:Ethacrynic Acid / Q000378:metabolism / Q000494:pharmacology*; D017930:Genes, Reporter; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D007249:Inflammation; D008070:Lipopolysaccharides / Q000378:metabolism; D051379:Mice; D008956:Models, Chemical; D008958:Models, Molecular; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D052002:NF-kappa B p50 Subunit; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D051996:Transcription Factor RelA; D014157:Transcription Factors / Q000378:metabolism*
D000893:Anti-Inflammatory Agents; D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D011994:Recombinant Proteins; D051996:Transcription Factor RelA; D014157:Transcription Factors; D000072000:NF-KappaB Inhibitor alpha; C073701:Nfkb1 protein, mouse; D012694:Serine; D004247:DNA; D005978:Glutathione; D003545:Cysteine; D004976:Ethacrynic Acid