Glutathione Depletion Down-regulates Tumor Necrosis Factor α-induced NF-κB Activity via IκB Kinase-dependent and -independent Mechanisms (2007)

Reduced glutathione (GSH) plays a crucial role in hepatocyte function, and GSH depletion by diethyl maleate was shown previously to inhibit expression of NF-κB target genes induced by tumor necrosis factor α (TNFα) and sensitize primary cultured mouse hepatocytes to TNF-mediated apoptotic killing. Here we demonstrate in the same system that GSH depletion down-regulates TNF-induced NF-κB transactivation via two mechanisms, depending on the extent of the depletion. With moderate GSH depletion (∼50%), the down-regulation is IκB kinase (IKK)-independent and likely acts on NF-κB transcriptional activity because TNF-induced IKK activation, IκBα phosphorylation and degradation, NF-κB nuclear translocation, NF-κB DNA binding in vitro, and NF-κB subunit RelA(p65) recruitment to κB sites of target gene promoters all appear unaltered. On the other hand, with profound GSH depletion (∼80%), the down-regulation also is IKK-dependent, and a timeline is established linking the inhibition of polyubiquitination of receptor-interacting protein 1 in TNF receptor 1 complex to partial blockage of IKK activation, IκBα phosphorylation and degradation, and NF-κB nuclear translocation. Of note, pretreatment with antioxidant trolox protects against the inhibitory effect of profound GSH depletion on IKK activation and NF-κB nuclear translocation but fails to restore expression of NF-κB target genes, revealing both IKK-dependent and -independent inhibition. These findings provide new insights into the complex effects of oxidative stress and redox perturbations on the NF-κB pathway. Reduced glutathione (GSH) plays a crucial role in hepatocyte function, and GSH depletion by diethyl maleate was shown previously to inhibit expression of NF-κB target genes induced by tumor necrosis factor α (TNFα) and sensitize primary cultured mouse hepatocytes to TNF-mediated apoptotic killing. Here we demonstrate in the same system that GSH depletion down-regulates TNF-induced NF-κB transactivation via two mechanisms, depending on the extent of the depletion. With moderate GSH depletion (∼50%), the down-regulation is IκB kinase (IKK)-independent and likely acts on NF-κB transcriptional activity because TNF-induced IKK activation, IκBα phosphorylation and degradation, NF-κB nuclear translocation, NF-κB DNA binding in vitro, and NF-κB subunit RelA(p65) recruitment to κB sites of target gene promoters all appear unaltered. On the other hand, with profound GSH depletion (∼80%), the down-regulation also is IKK-dependent, and a timeline is established linking the inhibition of polyubiquitination of receptor-interacting protein 1 in TNF receptor 1 complex to partial blockage of IKK activation, IκBα phosphorylation and degradation, and NF-κB nuclear translocation. Of note, pretreatment with antioxidant trolox protects against the inhibitory effect of profound GSH depletion on IKK activation and NF-κB nuclear translocation but fails to restore expression of NF-κB target genes, revealing both IKK-dependent and -independent inhibition. These findings provide new insights into the complex effects of oxidative stress and redox perturbations on the NF-κB pathway. TNFα 2The abbreviations used are:ActDactinomycin DBSObuthionine sulfoximineChIPchromatin immunoprecipitationDEMdiethyl maleateEMSAelectrophoretic mobility shift assayIKKIκB kinaseKAkinase assayPMHprimary cultured mouse hepatocytesRIP1receptor-interacting protein 1UbubiquitinpolyUb-RIP1polyubiquitinated RIP1ROSreactive oxygen speciesTNFtumor necrosis factorTNFR1TNF receptorTRADDTNF receptor-associated death domain proteinWCEwhole cell extractsWBWestern blottingJNKc-Jun NH2-terminal kinaseGSTglutathione S-transferasecIAP1cellular inhibitor of apoptosis protein 1iNOSinducible nitric-oxide synthase. plays an important role in liver injury in many animal models and has been implicated in the progression of human alcoholic liver disease and hepatic viral infections (1Bradham C.A. Plumpe J. Manns M.P. Brenner D.A. Trautwein C. Am. J. Physiol. 1998; 275: G387-G392PubMed Google Scholar, 2Schwabe R.F. Brenner D.A. Am. J. Physiol. Gastrointest. Liver Physiol. 2006; 290: 583-589Crossref PubMed Scopus (591) Google Scholar, 3Kaplowitz N. Nat. Rev. Drug Discov. 2005; 4: 489-499Crossref PubMed Scopus (873) Google Scholar, 4Albano E. Proc. Nutr. Soc. 2006; 65: 278-290Crossref PubMed Scopus (449) Google Scholar). TNFα exerts diverse biological effects by activation of multiple pathways promoting inflammation and both cell survival and death (5Wajant H. Pfizenmaier K. Scheurich P. Cell Death Differ. 2003; 10: 45-65Crossref PubMed Scopus (1900) Google Scholar). Like many cell types, normal hepatocytes do not undergo apoptosis in response to TNFα because successful activation of TNF-induced NF-κB pathway blocks the TNF-induced apoptotic pathway (5Wajant H. Pfizenmaier K. Scheurich P. Cell Death Differ. 2003; 10: 45-65Crossref PubMed Scopus (1900) Google Scholar, 6Papa S. Zazzeroni F. Pham C.G. Bubici C. Franzoso G. J. Cell Sci. 2004; 117: 5197-5208Crossref PubMed Scopus (245) Google Scholar). actinomycin D buthionine sulfoximine chromatin immunoprecipitation diethyl maleate electrophoretic mobility shift assay IκB kinase kinase assay primary cultured mouse hepatocytes receptor-interacting protein 1 ubiquitin polyubiquitinated RIP1 reactive oxygen species tumor necrosis factor TNF receptor TNF receptor-associated death domain protein whole cell extracts Western blotting c-Jun NH2-terminal kinase glutathione S-transferase cellular inhibitor of apoptosis protein 1 inducible nitric-oxide synthase. NF-κB, a family of dimeric transcriptional factors, regulates the expression of a spectrum of genes involved in cell survival and anti-apoptotic functions and inflammation (7Ghosh S. Karin M. Cell. 2002; 109: 81-96Abstract Full Text Full Text PDF PubMed Scopus (3294) Google Scholar). It exists mainly as the RelA(p65) and p50 heterodimer and is retained in cytoplasm by inhibitor proteins (IκBs) in unstimulated cells (7Ghosh S. Karin M. Cell. 2002; 109: 81-96Abstract Full Text Full Text PDF PubMed Scopus (3294) Google Scholar). TNFα activates various pathways through two receptors, TNFR1 and TNFR2 (5Wajant H. Pfizenmaier K. Scheurich P. Cell Death Differ. 2003; 10: 45-65Crossref PubMed Scopus (1900) Google Scholar). The signaling from TNFα to NF-κB activation, mediated mainly through TNFR1, has been most extensively studied (5Wajant H. Pfizenmaier K. Scheurich P. Cell Death Differ. 2003; 10: 45-65Crossref PubMed Scopus (1900) Google Scholar, 8Chen Z.J. Bhoj V. Seth R.B. Cell Death Differ. 2006; 13: 687-692Crossref PubMed Scopus (102) Google Scholar, 9Hayden M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3372) Google Scholar). Upon TNFα binding, TNFR1 recruits TNF receptor-associated death domain protein (TRADD), receptor-interacting protein 1 (RIP1), and TNF receptor-associated factor 2 (TRAF2) (5Wajant H. Pfizenmaier K. Scheurich P. Cell Death Differ. 2003; 10: 45-65Crossref PubMed Scopus (1900) Google Scholar, 10Harper N. Hughes M. MacFarlane M. Cohen G.M. J. Biol. Chem. 2003; 278: 25534-25541Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). RIP1 is polyubiquitinated in TNFR1 complex (10Harper N. Hughes M. MacFarlane M. Cohen G.M. J. Biol. Chem. 2003; 278: 25534-25541Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 11Micheau O. Tschopp J. Cell. 2003; 114: 181-190Abstract Full Text Full Text PDF PubMed Scopus (2015) Google Scholar). This polyubiquitination process is promoted by TRAF2 and reversed by A20 (8Chen Z.J. Bhoj V. Seth R.B. Cell Death Differ. 2006; 13: 687-692Crossref PubMed Scopus (102) Google Scholar). TRAF2 functions as a ubiquitin ligase (E3) RIP1 through of ubiquitin that is to the J. N. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, H. M. S. P. C. 2004; PubMed Scopus Google A20 acts through two to and to RIP1 and RIP1 H. M. S. P. C. 2004; PubMed Scopus Google Scholar). RIP1 recruits IKK G. Full Text Full Text PDF PubMed Scopus Google through the of the of with in the IKK complex Nat. Cell Biol. 2006; PubMed Scopus Google Scholar, G. Z.J. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google and to activation of by phosphorylation of and in the activation (8Chen Z.J. Bhoj V. Seth R.B. Cell Death Differ. 2006; 13: 687-692Crossref PubMed Scopus (102) Google Scholar, 9Hayden M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3372) Google Scholar). IKK complex IκBα two sites E. J. PubMed Scopus Google and H. G. S. K. Karin M. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google and by (7Ghosh S. Karin M. Cell. 2002; 109: 81-96Abstract Full Text Full Text PDF PubMed Scopus (3294) Google Scholar). NF-κB the with to κB sites and on the of target genes M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3372) Google Scholar, Nat. Rev. Cell Biol. 2004; PubMed Scopus Google Scholar). It is to that in the of liver injury models disease hepatocytes to to the diverse effects of TNFα and to TNF-induced a as the most antioxidant and a of the is crucial to hepatocyte N. N. Am. J. Physiol. Gastrointest. Liver Physiol. 2006; PubMed Scopus Google Scholar). in that GSH depletion by TNF-induced expression of survival genes and primary cultured mouse hepatocytes to TNF-induced apoptosis H. K. G. N. 2002; PubMed Scopus Google Scholar, K. C. N. 2003; PubMed Scopus Google Scholar). The to the inhibitory effect of GSH depletion on signaling to inhibition of TNF-induced NF-κB gene assay we that GSH depletion by down-regulates TNF-induced NF-κB Western blotting and electrophoretic mobility shift assay we that GSH depletion by not inhibit NF-κB nuclear and DNA binding activity but a we that and involved and to by effect on signaling to IKK-dependent NF-κB activation and with the and of GSH depletion. the role of reactive oxygen species was by the effect of antioxidant nuclear was and recruitment to κB sites of gene promoters as an to the Cell of and cultured mouse hepatocytes used in all from of and as previously G. N. Am. J. Physiol. Gastrointest. Liver Physiol. 2002; PubMed Scopus Google Scholar). hepatocytes and in and The and in a cell with was to the TNFα the cells with by The in whole cell cells in 1 1 and inhibitor with 2 and extracts as S. O. Cell. Biol. PubMed Google Scholar). The protein of the extracts was by the of mouse TNFα was used in the of shown in and and signaling R.F. C. Proc. Sci. S. PubMed Scopus Google we used human TNFα in the of of in trolox in buthionine sulfoximine in and actinomycin D in as by the the cellular glutathione cell extracts with and to the GSH assay F. PubMed Scopus Google of GSH depletion by on TNF-induced NF-κB DNA binding activity NF-κB nuclear with TNFα TNFα extracts and by with was as in with and nuclear extracts as with a of an the of the nuclear extracts as in by with to nuclear of and p50 the also shown shown in of of GSH depletion by of GSH with by with TNFα TNFα the cellular GSH by the GSH as of GSH in the the cellular GSH was With a profound GSH depletion was in with moderate depletion and and from and from of into by as previously E. Brenner D.A. Am. J. Physiol. Gastrointest. Liver Physiol. PubMed Google Scholar). was in all as an with TNFα as and the assay system as previously Cell. Full Text PDF PubMed Scopus Google Scholar). NF-κB with by kinase by and used as a extracts with the in a binding 1 1 1 and of The on a with of and 1 was used in shift nuclear extracts with as 1 the of the against RelA(p65) and p50 used in a Western cell extracts nuclear extracts by and to The used and and RelA(p65) protein kinase protein and and Cell and from and The by with and an was in to the of The with and with protein the and by of IKK and cell extracts by normal protein by immunoprecipitation with and protein of IKK complex in with 2 and and in kinase 2 and IKK of the in an in kinase assay M. E. Karin M. PubMed Scopus Google in the kinase with 1 of glutathione S-transferase and the was and by the extent of phosphorylation of IκBα with an Cell is a inhibitor of IKK was to the in kinase in the of of the with kinase assay and kinase was from an This an IκBα of mouse of TNFR1 as O. Tschopp J. Cell. 2003; 114: 181-190Abstract Full Text Full Text PDF PubMed Scopus (2015) Google with mainly to the and of with human TNFα in the of was 2 TNFα to a of GSH depletion in all and in with and 1 cell extracts and to with and protein of TNFR1 signaling complex and in with TNFR1 RIP1 TRAF2 and and of was a previously P. N. PubMed Scopus Google from with TNFα of was an with The to of was to with and system and used in of gene in 1 of the was previously V. 2003; PubMed Scopus Google Scholar). of was to an that DNA not used the by the system the the The used in of was of two of from of with TNFα and as in of the the genes in by and the shown to of the and as of the induced by TNFα as and of and of of genes by and and in a new effect of antioxidant trolox on GSH of with trolox 1 by TNFα TNF was in a and in the of was as in with in shown IKK by as to the activity induced by cellular glutathione to the with trolox pretreatment as in as of glutathione in the the with trolox as in 1 of TNFα in the and of extracts and to to nuclear translocation of with as a with trolox as in 1 and of TNFα in the and of and The A20 1 and by as in the and as of the induced by TNFα the of moderate GSH depletion on the of and with with an inhibitor of GSH by TNF and IKK activity and protein of and IκBα and of IκBα and A20 with TNF TNF to and by normal and apoptotic and and with from with TNF the and to of and with used as and The recruitment of RelA(p65) NF-κB subunit to κB sites of NF-κB target gene promoters in hepatocytes was by a previously H. 2003; PubMed Scopus Google with to and with the cells and on in with and from hepatocytes in of nuclear and The chromatin was and with of RelA(p65) and the other the as to of the was as The complex was with protein with and on and and with the and to as and used in of gene in of was to and a κB The used in of the was from an DNA from the chromatin of recruitment of RelA(p65) to promoters as of and in and and in a new and and necrosis by and as previously H. K. G. N. 2002; PubMed Scopus Google with to to and with GSH by TNF-induced NF-κB but of of a in that GSH depletion by and TNF-induced of of genes, as and to TNF-induced apoptosis H. K. G. N. 2002; PubMed Scopus Google Scholar, K. C. N. 2003; PubMed Scopus Google Scholar). the we to κB is to the down-regulation gene a with a gene the of a κB was into by with TNFα with TNFα GSH depletion by the as as TNF-induced an inhibitory effect was not with a the κB sites that the inhibitory effect was and a inhibition was a of we the effect of GSH depletion on expression of genes by GSH depletion in with TNFα an in to a effect on the GSH depletion by gene and the depletion was moderate and in we to the effect on gene from and by of and and not TNFα of genes with the 1 IκBα and A20 and and GSH depletion by the by TNFα all genes the inhibition the was IκBα and A20 with the genes and the response is likely to to the inhibition. that GSH depletion down-regulates TNF-induced NF-κB we the down-regulation in TNF-induced NF-κB DNA binding activity NF-κB nuclear extracts from by with NF-κB DNA binding activity and by with NF-κB subunit RelA(p65) TNF as in an in NF-κB DNA binding activity as with the was κB and mainly of and heterodimer as by and GSH depletion by not TNF-induced NF-κB DNA binding activity GSH depletion by effect on the the of NF-κB DNA GSH depletion by TNF-induced NF-κB DNA binding activity a of NF-κB nuclear with the of RelA(p65) and p50 the of TNFα GSH depletion with effect on the nuclear of NF-κB, but inhibition with we that the in NF-κB DNA binding activity was to nuclear of that GSH depletion by TNF-induced NF-κB activity with NF-κB in the NF-κB DNA binding in vitro, an with the down-regulation with in both NF-κB nuclear and NF-κB DNA binding activity in vitro, of an the of the of nuclear inhibition of NF-κB GSH by and TNFα we the GSH depletion and inhibition of TNFα is to to the of GSH depletion and with inhibition of TNFα E. J. PubMed Scopus Google Scholar, G. Chem. PubMed Scopus Google is a used GSH depletion because into cells to and glutathione in by glutathione S-transferase in to the of GSH depletion by in shown in the cellular GSH was not by TNFα but of GSH depletion was in 2 with and depletion in with The depletion on the to a profound depletion of cellular GSH in to a moderate depletion in the cellular GSH in the GSH depletion is and with the depletion is and GSH with TNF-induced of IKK to of we a in to the effect of GSH depletion on TNF-induced activation of pathway. IKK activity was by kinase phosphorylation and of cellular IκBα by of whole cell and nuclear translocation of RelA(p65) and p50 was by of nuclear TNFα IKK activity and the the of in cells by a of IκBα and and a of nuclear RelA(p65) and p50 moderate GSH depletion by with effects on the activation of IKK phosphorylation and of and nuclear translocation of RelA(p65) and p50 and profound GSH depletion by in a in IKK activity as as by in IκBα and nuclear translocation of both RelA(p65) and p50 and the a in was not was of IκBα was by pretreatment of with an inhibitor of The inhibition by on IKK activation is not likely to a effect of on IKK as of of IKK complex with not of IKK activity in the assay and with On the other hand, a used protein IKK activity with we established that down-regulation of TNF-induced NF-κB activity is GSH is but also IKK-dependent GSH is against the of GSH on TNF-induced IKK and NF-κB but the used to profound cellular GSH depletion also GSH K. C. N. 2003; PubMed Scopus Google and GSH depletion is with of C. N. Google is that a role in the inhibitory effect of profound GSH depletion on TNF-induced IKK antioxidant pretreatment protects against the inhibitory a and a antioxidant M. H. M. H. H. Karin M. K. J. 2003; PubMed Scopus Google was used in the shown in GSH depletion by TNF-induced IKK with trolox the inhibition was plays a role in the inhibition we cellular glutathione and that with trolox the cellular glutathione the depletion of GSH by the inhibition of TNF-induced NF-κB nuclear translocation was by trolox pretreatment and but the was not to inhibition of TNF-induced expression of NF-κB target genes, as A20 and the of nuclear inhibition of NF-κB the inhibition of TNF-induced expression of IκBα and A20 by to is likely the of IKK-dependent inhibition of NF-κB nuclear translocation. GSH with TNF-induced of RIP1 in TNFR1 and of the IKK activation not the activation was most by profound GSH depletion and the inhibition by antioxidant we that the signaling is likely of we the effect of profound GSH depletion on TNFR1 complex TNFα signaling a previously O. Tschopp J. Cell. 2003; 114: 181-190Abstract Full Text Full Text PDF PubMed Scopus (2015) Google Scholar). unstimulated with human TNFα in the of and cell extracts 2 and TNFR1 was by with a and by of TNFR1, and TRAF2 in the TNFα to of TNFR1 and the recruitment of and TRAF2 to the signaling complex as as 2 that TNFα induced an process of polyubiquitination of RIP1 in TNFR1 polyubiquitinated RIP1 2 as a of with RIP1 by the the by the of RIP1 and These in RIP1 not in cell extracts polyubiquitination of TRAF2 was not of but a TRAF2 that the of TRAF2 was profound GSH depletion inhibitory effect 2 on receptor the recruitment of and TRAF2 in but the of TNF-induced was and the in was with a of RIP1 2 but was the of polyubiquitination of RIP1 of both 2 and and of RIP1 The TRAF2 was also by profound GSH depletion the is we cell extracts by and that with profound GSH TNFα not protein of TNFR1, and TRAF2 in the in in TNFR1 complex was likely to in polyubiquitination process the recruitment of RIP1 is an TNF-induced activation, and shown a crucial role of in the that the role of in TNFα signaling in a in TNFR1 in signaling as phosphorylation of TNFα IKK activity by phosphorylation of and in the activation of in by a shown in TNFα induced a activation of IKK by phosphorylation on and to This phosphorylation was both and the of profound GSH with the IKK activity in signaling both and pathways of we activation by with and response to phosphorylation of and and phosphorylation of both was with profound GSH depletion. TNF-induced phosphorylation of c-Jun by was established a linking profound GSH depletion by to the polyubiquitination of RIP1 in signaling complex and to the in pathway. TNF-induced activation of and signaling pathways to by profound GSH the is most likely by the and GSH by TNF-induced of and with TNF-induced of to of NF-κB of transcriptional we to TNF-induced phosphorylation of and as as of by and a that in was in response to TNF but not and not The phosphorylation TNF but in with effect on the process The inhibitory effect of with is likely to nuclear translocation of as both by trolox pretreatment and On the other hand, we that nuclear of was 1 TNF with with not inhibit the with with not NF-κB DNA binding activity in cells by and used from and with an against the of The recruitment of to κB sites of and promoters was by with TNFα in recruitment of to promoters in a the response genes IκBα and the of recruitment and with the of the same two the recruitment response gene the of with not inhibit TNF-induced recruitment of all genes that transcriptional activity of not DNA binding activity of is by moderate GSH depletion. that moderate GSH depletion not TNF-induced nuclear and TNF-induced binding of to κB sites of NF-κB target gene the inhibition of NF-κB transcriptional activity in is to the of phosphorylation and of and and with and but is of GSH to TNF-induced GSH depletion is pretreatment of with an inhibitor of GSH moderate GSH depletion was by pretreatment with cell pretreatment effect on TNF-induced IKK activation phosphorylation and of IκBα but TNF-induced expression of genes as IκBα and A20 Of note, TNFα a of GSH a in with we that moderate GSH depletion in TNF-induced NF-κB activity of pathway. GSH depletion by necrosis and in the of the death of is a of apoptosis and necrosis with activation H. K. G. N. 2002; PubMed Scopus Google Scholar). moderate GSH depletion by activation not an in death as with the by to TNF-induced apoptosis with of and as pretreatment with and not These as moderate GSH depletion TNF-induced NF-κB transactivation the expression of anti-apoptotic and survival genes, as and and the activation by was and that by TNFα the cellular of an inhibitor of as activation H. G. S. K. Karin M. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). that the apoptotic death was and moderate by and by by as with inhibitor of new apoptosis a The activation of was not the two but the in the activation the of the has not been but is that inhibition of the NF-κB pathway and and activation with in response to in that GSH depletion by of genes, to apoptosis H. K. G. N. 2002; PubMed Scopus Google Scholar, K. C. N. 2003; PubMed Scopus Google Scholar). This has in to the role of TNFα in liver with GSH depletion oxidative redox perturbations and a of to the effect of to the we the the same we demonstrate that GSH depletion down-regulates NF-κB transactivation via and that cell GSH by expression of NF-κB gene in a κB and expression of anti-apoptotic genes and to the of to TNF-induced apoptosis and moderate GSH depletion was via an inhibition of GSH by pretreatment and The down-regulation is of the as not TNF-induced IKK activation, IκBα degradation, and NF-κB nuclear translocation and not with NF-κB DNA binding activity in binding in of NF-κB transcriptional activity of NF-κB by as phosphorylation and in many binding of transcriptional and the of target genes M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3372) Google Scholar, Nat. Rev. Cell Biol. 2004; PubMed Scopus Google Scholar). we TNF-induced nuclear phosphorylation and and the two not by with and and not is to the of phosphorylation and of and the effect of moderate GSH depletion. redox of NF-κB on a has been redox and DNA binding N. M. M. S. H. K. H. H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google of phosphorylation and transcriptional activity C. E. J. PubMed Scopus Google the phosphorylation and the is a of family with transcriptional a with and is in to moderate GSH depletion with of induced by On the other hand, also demonstrate that cell GSH by expression of genes K. C. N. 2003; PubMed Scopus Google through TNF-induced nuclear translocation of NF-κB and in an and not as a inhibitor of IKK activity the is likely the of in TNFR1 signaling as a in and the in phosphorylation of IKK and in IKK IκBα and and It is that RIP1 is an of as and C. P. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, N. N. Biol. 2006; PubMed Scopus Google to as M. E. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google been to the pathway. is likely to recruitment of RIP1 to TNFR1 as of RIP1 in both receptor complex and cell by pretreatment M. E. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). pretreatment cellular RIP1 and is to IKK activation C. P. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). the of profound GSH depletion in system not recruitment and cellular of RIP1 TNFα the of in recruitment of IKK complex to TNFR1 signaling the in in the receptor complex in is likely to to the inhibition of TNFα signaling the pathway. This is by that TNF-induced activation of and pathways was by profound GSH depletion both of of RIP1 in TNFα TRAF2 is the ubiquitin ligase polyubiquitination of RIP1 and A20 is the that the effects of GSH depletion on the activity of TRAF2 and the recruitment and activity of A20 to in we that a role in the inhibition of TNFα signaling in as pretreatment with antioxidant trolox against the effects of profound GSH depletion by on TNF-induced IKK activation GSH The and species to the effect of NF-κB nuclear translocation as is not to the inhibition of expression of NF-κB target genes revealing that profound GSH depletion not with NF-κB nuclear translocation but also nuclear NF-κB GSH depletion inhibit TNF-induced NF-κB transactivation via IKK-dependent and -independent with profound GSH depletion both in moderate GSH depletion the inhibition of IKK pathway. It is of that a and of is depletion of GSH K. C. N. 2003; PubMed Scopus Google that the of oxidative inhibition of IKK GSH GSH K. C. N. 2003; PubMed Scopus Google is not to oxidative findings redox the and activity of the NF-κB transcriptional moderate GSH depletion is by glutathione the that the redox is to GSH a in effects on redox on the activation in was not in with TNFα This is likely to inhibition of NF-κB activity by as with and and the to TNF-induced apoptosis with we demonstrate in that GSH depletion down-regulates TNF-induced NF-κB transactivation via IKK-dependent and -independent is to the and species of and of with profound GSH depletion and of inhibition of by moderate GSH the findings the oxidative stress and redox perturbations to cell survival pathways in response to by the Cell of the of Liver by the of