title: Differential effect of covalent protein modification and glutathione depletion on the transcriptional response of Nrf2 and NF-kappaB.
abstract: Liver injury associated with exposure to therapeutic agents that undergo hepatic metabolism can involve the formation of reactive metabolites. These may cause redox perturbation which can result in oxidative stress as well as protein modification leading to activation or inhibition of cellular transcriptional responses. Nevertheless, the effects of these challenges on more than one transcriptional pathway simultaneously remain unclear. We have investigated two transcription factors known to be sensitive to electrophilic stress and redox perturbation, Nrf2 and NF-kappaB, in mouse liver cells. Cellular stress was induced by the probes: N-acetyl-p-benzoquinineimine (NAPQI), the reactive metabolite of acetaminophen; dinitrochlorobenzene (DNCB), a model electrophile; and buthionine (S,R)-sulfoximine (BSO), an inhibitor of glutamate-cysteine ligase. NAPQI, DNCB and BSO can all cause glutathione (GSH) depletion; however only NAPQI and DNCB can covalently bind proteins. We also employed RNAi to manipulate Keap1 (the inhibitor of Nrf2), Nrf2 itself and NF-kappaB-p65, to understand their roles in the response to drug stress. All three chemicals induced Nrf2, but NF-kappaB binding activity was only increased after BSO treatment. In fact, NF-kappaB binding activity decreased after exposure to NAPQI and DNCB. While RNAi depletion of Keap1 led to reduced toxicity following exposure to DNCB, depletion of Nrf2 and NF-kappaB augmented toxicity. Interestingly, increased Nrf2 caused by Keap1 depletion was reversed by co-depletion of NF-kappaB. We demonstrate that Keap1/Nrf2 and NF-kappaB respond differently to electrophiles that bind proteins covalently and the redox perturbation associated with glutathione depletion, and that crosstalk may enable NF-kappaB to partly influence Nrf2 expression during cellular stress.
authors.affiliation: MRC Centre for Drug Safety Science, Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool, Merseyside, L69 3GE, UK.
authors.forename: Alvin J L
authors.initials: AJ
authors.lastname: Chia
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Goldring
authors.forename: Neil R
authors.initials: NR
authors.lastname: Kitteringham
authors.forename: Shi Quan
authors.initials: SQ
authors.lastname: Wong
authors.forename: Paul
authors.initials: P
authors.lastname: Morgan
authors.forename: B Kevin
authors.initials: BK
authors.lastname: Park
mesh_terms: D000818:Animals; D002460:Cell Line; D005978:Glutathione / Q000235:genetics / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D051379:Mice; D051267:NF-E2-Related Factor 2 / Q000235:genetics* / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics* / Q000378:metabolism*; D046188:Protein Modification, Translational / Q000502:physiology*; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology*
chemical_list: D051267:NF-E2-Related Factor 2; D016328:NF-kappa B; D011506:Proteins; D005978:Glutathione