Tumor Necrosis Factor Increases Hepatocellular Glutathione byTranscriptional Regulation of the Heavy Subunit Chain ofg-Glutamylcysteine Synthetase*

(Received for publication, June 30, 1997, and in revised form, September 9, 1997)

Albert Morales‡§, Carmen Garcıa-Ruiz‡¶, Merce Mirandai, Montserrat Marıi, Anna Colelli,Esther Ardite**, and Jose C. Fernandez-Checa‡‡

From the Instituto de Investigaciones Biomedicas, Consejo Superior Investigaciones Cientıficas and Liver Unit andServicio de Bioquımica, Department of Medicine, Hospital Clinic i Provincial, Universidad de Barcelona,Barcelona 08036, Spain

Tumor necrosis factor (TNF) is an inflammatory cyto-kine that causes cell injury by generation of oxidativestress. Since glutathione (GSH) is a key cellular antiox-idant that detoxifies reactive oxygen species, the pur-pose of our work was to examine the regulation of cel-lular GSH, the expression of heavy subunit chain ofg-glutamylcysteine synthetase (g-GCS-HS), and controlof intracellular generation of reactive oxygen species incultured rat hepatocytes treated with TNF. Exposure ofcells to TNF (10,000 units/ml) resulted in depletion ofcellular GSH levels (50–70%) and overproduction of hy-drogen peroxide (2–3-fold) and lipid peroxidation. How-ever, cells treated with lower doses of TNF (250–500units/ml) exhibited increased levels of GSH (60–80%over control). TNF treatment increased (70–100%) thelevels of g-GCS-HS mRNA, the catalytic subunit of theregulating enzyme in GSH biosynthesis. Furthermore,intact nuclei isolated from hepatocytes treated withTNF transcribed the g-GCS-HS gene to a greater extentthan control cells, indicating that TNF regulates g-GCS-HS at the transcriptional level. The capacity to synthe-size GSH de novo determined in cell-free extracts incu-bated with GSH precursors was greater (50–70%) inhepatocytes that were treated with TNF; however, theactivity of GSH synthetase remained unaltered by TNFtreatment indicating that TNF selectively increased theactivity of g-GCS. Despite activation of nuclear fac-tor-kB (NF-kB) by TNF, this transcription factor was notrequired for TNF-induced transcription of g-GCS-HS asrevealed by deletion constructs of the g-GCS-HS pro-moter subcloned in a chloramphenicol acetyltrans-ferase reporter vector and transfected into HepG2 cells.In contrast, a construct containing AP-1 like/metal re-sponse regulatory elements increased chloramphenicolacetyltransferase activity upon exposure to TNF. Thus,

TNF increases hepatocellular GSH levels by transcrip-tional regulation of g-GCS-HS gene, probably throughAP-1/metal response element-like binding site(s) in itspromoter, which may constitute a protective mecha-nism in the control of oxidative stress induced by in-flammatory cytokines.

Tumor necrosis factor-a (TNF)1 is a polypeptide that elicits adiversity of cellular reactions, depending upon its concentra-tion and the type of cell where it acts (1–3). Thus, it has beenshown that TNF exerts an important physiological role as amodulator of immune responses by regulating specific genesneeded for the host defense against a varied repertoire ofagents. TNF appears to play a role in the control of cell cycle asDNA synthesis and cell proliferation increase in cells exposedto TNF, indicating that this cytokine acts as a mitogenic stim-uli (4). Such regulation of gene expression by TNF is mediatedby induction of early responsive genes including c-jun andtranscription factors, i.e. NF-kB (5, 6). Yet, as a proinflamma-tory cytokine, whose production is increased in a number ofstressful and pathological states, TNF promotes cell injurythrough several mechanisms including the overproduction ofROS (3, 7–10).

The ability of TNF to kill cells appears to be restricted totumor and virally infected cells since normal cells are generallyinsensitive to the toxic effects of TNF. Moreover, cells thatnormally are insensitive to TNF cytotoxicity can be sensitizedby pretreatment with inhibitors of protein and RNA synthesis(11–13). Conversely, it has been shown that sensitive cells canbe made resistant to TNF challenge by prior exposure to asublethal dose of TNF. These findings imply that TNF leads tothe induction of genes that confer protective effects on cells.Several protective genes induced by TNF have been reportedincluding plasminogen activator inhibitor type 2, the zinc fin-ger protein A20, and the Bcl-2 related family member A1(14–17).

The molecular basis of the cytotoxic action of TNF is not fullyunderstood at present; however, one of the possible mecha-nisms involved in the toxicity elicited by TNF includes thegeneration of ROS that may damage critical cellular compo-

* This work was supported in part by the National Institute onAlcohol Abuse and Alcoholism Grant AA09526, Direccion General Po-lıtica Cientıfica y Tecnica Grant PM 95-0185, Fondo InvestigacionesSanitarias Grant FISS 94-0046/01, Plan Nacional de I1D Grant SAF97-0087-C01, and Europharma. The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

‡ These authors contributed equally to this work.§ Fellow from the Fondo Investigaciones Seguridad Social.¶ Supported by a postdoctoral contract from the Ministerio de Edu-

cacion y Ciencia, Spain.i Supported by Europharma.** Supported by Madaus Cerafarma.‡‡ To whom correspondence and reprint requests should be ad-

dressed: Instituto Investigaciones Biomedicas, CSIC Liver Unit, Hos-pital Clinic i Provincial, Villarroel 170, 08036 Barcelona, Spain. Fax:34-3-451-5272.

1 The abbreviations used are: TNF, tumor necrosis factor; BSO, bu-thionine-L-sulfoximine; CAT, chloramphenicol acetyltransferase;DCFDA, 29,79-dichlorofluorescin diacetate; DCF, dichlorofluorescein;DHR, dihydrorhodamine; GST, GSH S-transferases; g-GCS-HS, g-glu-tamylcysteine synthetase heavy subunit; HPLC, high pressure liquidchromatography; Mn-SOD, manganese superoxide dismutase; MRE,metal response element; NF-kB, transcription factor kB; PCR, polym-erase chain reaction; ROS, reactive oxygen species; bp, base pair(s).

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 48, Issue of November 28, pp. 30371–30379, 1997© 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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nents such as proteins, lipids, and DNA causing cell injury(7–9). Consistent with the involvement of ROS in mediating theTNF-induced injury, cellular antioxidants attenuate the dam-aging effects of free radicals, and hence, these compounds maymodulate the sensitivity of cells against TNF toxicity (18–20).For instance, previous studies indicated that the antioxidantenzyme Mn-SOD determined sensitivity to TNF-induced celldeath as TNF treatment up-regulated Mn-SOD gene expres-sion; furthermore, overexpression of Mn-SOD conferred resist-ance to TNF toxicity in a human kidney embryonal cell line(18–19). However, such a protective role of Mn-SOD may berestricted to specific cell types since studies in hepatocytesrevealed that the TNF-induced expression of Mn-SOD wasdetected only at the mRNA level without increase in the pro-tein level or enzyme activity (21).

GSH, the most abundant antioxidant in cells, plays a prom-inent role in the defense against oxidative stress-induced cellinjury. Thus, the GSH redox cycle, where reduced GSH iscofactor of GSH peroxidase, and GST downplay the conse-quences of a broad range of reactive species (22–24). GSH issynthesized from its constituent amino acids in two sequentialenzymatic reactions catalyzed by g-glutamylcysteine synthe-tase (g-GCS) and GSH synthetase. The reaction catalyzed byg-GCS is the rate-limiting step in de novo GSH synthesis;g-GCS is inhibited by GSH through a feedback mechanism (22).Previous studies have shown that manipulation of GSH levelsprior to exposure to TNF modulates the cytotoxicity of thecytokine in different cell types (7, 8). GSH depletion induced byinhibition of GSH reductase with 1,3-bis(chloroethyl)-1-nitro-sourea or by incubation with BSO, a specific inhibitor of g-GCS,prior to the exposure of cells to TNF, results in an increasedsusceptibility of hepatocytes and fibrosarcoma cells to the cy-totoxic effects of TNF (7, 8). Furthermore, its lethality can beameliorated by N-acetylcysteine, a GSH precursor, which re-plenishes cellular GSH by providing intracellular cysteine (20).

Given the importance of GSH in protecting against the oxi-dative stress elicited by TNF, the purpose of our work was toexamine the regulation of cellular GSH, expression of g-GCS-HS, and control of intracellular generation of ROS in culturedrat hepatocytes treated with TNF. Our results demonstratethat TNF increases cellular GSH levels, mediated by transcrip-tional regulation of the g-GCS-HS gene, which attenuates thegeneration of hydrogen peroxide and lipid peroxidation. Ourfindings imply that the up-regulation of cellular GSH mayrepresent an additional protective mechanism to control theconsequences of oxidative stress induced by inflammatorycytokines.

EXPERIMENTAL PROCEDURES

Materials—Recombinant human TNF (Promega, specific activity2.7 3 107 units/mg protein) was added to serum-deprived cells at a doseof 250–10,000 units/ml and maintained for 4–24 h. Actinomycin D,BSO, GSH, GSSG, cysteine, and cystine were obtained from Sigma.Monochlorobimane, 29,79-dichlorofluorescein diacetate (DCFDA), dihy-drorhodamine 123 (DHR), and cis-parinaric acid were from MolecularProbes (Eugene, OR). Trizol LS reagent was obtained from Life Tech-nologies, Inc. g-Glutamylcysteine was prepared by enzymatic hydroly-sis of GSSG and reduction with dithiothreitol prior to use as described(25).

Isolation and Culture of Rat Hepatocytes—Rat hepatocytes wereisolated by collagenase digestion; 2 3 106 cells were plated on rat tailcollagen and cultured routinely in Dulbecco’s modified Eagle’s medium/F12 medium. Cell attachment averaged 50%, and cell numbers weredetermined using a Coulter counter, model multisizer II (Coulter Elec-tronics, UK), and verified by hemocytometry. Cell viability was deter-mined by trypan blue exclusion and by measurement in the medium ofGST by conjugation of GSH with DTNB. HepG2 cells were obtainedfrom the American type culture collection (ATCC, Bethesda, MD) andseeded in Dulbecco’s modified Eagle’s medium supplemented with highglucose.

Determination of Total GSH Equivalents and Synthetic Rate ofGSH—The molecular forms of GSH equivalents, mainly GSH andGSSG, both in cells extracts and extracellular medium were determinedby HPLC (26, 27). The dynamic rate of GSH synthesis was determinedin cell-free extracts prepared by fractionation of hepatocytes. Cell ex-tracts were dialyzed overnight at 4 °C to deplete cytosol GSH content tominimize feedback inhibition of GSH on g-GCS. The GSH syntheticcapacity was determined using GSH precursors, glutamate, glycine,cysteine, and monochlorobimane as described previously in detail (28).GSH synthetase activity was assayed using glycine and g-glutamylcys-teine instead of cysteine and glutamate. The rate of GSH formation wasmonitored as the net rate of fluorescence increase of GSH-monochloro-bimane adduct catalyzed by GST over time after subtracting the BSO-inhibitable fluorescence signal (28).

Measurement of Hydrogen Peroxide and Lipid Peroxidation—Produc-tion of reactive oxygen species, mainly hydrogen peroxide and otherorganic peroxides (29, 30), and lipid peroxidation were monitored spec-trofluorometrically by DCFDA or DHR and cis-parinaric acid, respec-tively. One h prior to harvesting of cells, DCFDA or DHR (2 mg/ml) orcis-parinaric acid (5 mg/ml) was added to culture plates followed bywashing to remove excess fluorochrome, and fluorescence of theseprobes was determined as described previously (24, 29).

Preparation of Nuclear Extracts and Electrophoretic Mobility ShiftAssay of NF-kB and AP-1—Preparation of nuclear extracts and assay ofNF-kB activation using a 32P-labeled kB oligonucleotide (59-AGTT-GAGGGGACTTTCCCAGGC-39) were as described previously (29). Toconfirm binding specificity a mutated kB oligonucleotide probe (59-AGTTGAATTCACTTTCCCAGGC-39) was used as a negative control.Probes were labeled at the 59 end with T4 kinase and [g-32P]ATP (3000Ci/mmol). Proteins were separated by native 6% polyacrylamide gelelectrophoresis and visualized by autoradiography. In some cases, su-pershift assays were performed by incubating nuclear extracts withantibodies to different subunits of the NF-kB/Rel protein family gener-ously provided by Dr. N. Rice (31). Similar conditions were used for thebinding activity of AP-1 using oligonucleotide (59-CGCTTGATGAGT-CAGCCGGAA-39) that was labeled as described above for NF-kB (29).

Analysis of g-GCS-HS mRNA—A cDNA probe for g-GCS-HS wasgenerated by reverse transcriptase-PCR. An 804-base pair partial g-GCS-HS cDNA was prepared using rat kidney RNA as template andprimers based upon the published rat kidney g-GCS-HS sequence (32,33). A 59-primer (59-AGACACGGCATCCTCCAGTT-39 sense strand ofthe g-GCS-HS 59 sequence at positions 105–124) and a 39-primer (59-CTGACACGTAGCCTCGGTAA-39 antisense strand of the 39-sequenceat positions 890–909) were synthesized to span 804 nucleotides of theg-GCS-HS mRNA. The size of the cDNA generated was confirmed byagarose electrophoresis. The reverse transcriptase-PCR fragment wascloned into pTARGET (Promega), and several clones were sequencedusing fmol DNA sequencing system (Promega) to verify its identity;those with PCR-induced mutations were discarded. Total cellular RNA(30 mg/lane) was extracted, denatured, filtered under vacuum on nylonmembrane, and fixed with UV light. The membranes were prehybrid-ized at 65 °C, and hydridizations were performed using the 32P-labeledg-GCS-HS probe. The same blots were stripped of the hydridized g-GCS-HS and rehybridized with a cDNA fragment of 18 S rRNA used asan internal reference control to normalize for RNA loading. In somecases, total RNA was denatured in 50% formamide containing 7%formaldehyde and electrophoresed through a 1% agarose, 7% formalde-hyde gel, transferred, and fixed with UV light. Hybridization with32P-labeled g-GCS-HS probe identified a g-GCS-HS mRNA of 3.7 kilo-bases. The levels of g-GCS-HS mRNA were calculated relative to the18 S band and expressed as percentage of control. Densitometry quan-titation of autoradiographs was performed with a densitometer Prefer-ence (Seba).

Nuclear Run-on Assay of g-GCS-HS—Cultured rat hepatocytes (5 3107 cells) were treated with TNF and then washed with ice-cold phos-phate-buffered saline and nuclei prepared by lysis with 0.5% NonidetP-40 lysis buffer and differential centrifugation as described before (33).The integrity of isolated nuclei was verified by examination on a hemo-cytometer with a phase contrast microscope to ensure that cells hadbeen lysed and nuclei appeared free of cytoplasmic material. Collectednuclei were stored in 50 mM Tris, pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1mM EDTA in liquid nitrogen. Upon thawing, nuclei were resuspendedand incubated in reaction buffer (100 mM Tris, pH 8.0, 300 mM

(NH4)2SO4, 4 mM MgCl2, 200 mM NaCl, 0.4 mM EDTA, 4 mM MnCl2, 0.1mM phenylmethylsulfonyl fluoride, 1.2 mM dithiothreitol, 1 mM of ATP,GTP, and CTP, and 150 mCi of [a-32P]UTP (600 Ci/mmol, AmershamCorp.) at 30 °C for 30 min, followed by 100 units of DNase I treatmentfor 20 min at 30 °C (33). Proteinase K (100 mg) was then added and

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incubated overnight at 42 °C. Samples were then extracted with theTrizol reagent. Labeled RNA synthesized by intact nuclei was hybrid-ized with g-GCS-HS cDNA and cross-linked on strips of nylon mem-branes. Hybridization of radiolabeled RNA with 18 S cDNA was used asinternal reference control.

Generation of g-GCS-HS Promoter and Deletion Constructs—Theg-GCS-HS promoter was amplified by PCR from genomic DNA usingthe following upstream oligonucleotide 59-(1225) GGAGGCGCAGGCA-GAAGACCGA-39 and downstream oligonucleotide 59-(21088) CAGC-CAGACCTTGGGTATTCATG-39 as primers described previously (34,35). The PCR was performed for 5 min at 98 °C, 25 cycles of 30 s at94 °C, 2 min at 68 °C, and 7 min at 74 °C. Genomic DNA (150 ng) wasamplified in 20 mM Tris-HCl, pH 8.0, 50 mM KCl, 2 mM MgCl2, 0.05%detergent W-1, 200 mM dNTP, 1 unit of Taq DNA polymerase (LifeTechnologies, Inc.), and 10 pmol of each oligonucleotide to a finalvolume of 25 ml. The resulting promoter fragment (21088 to 1225) wascloned into pTARGET (Promega). Using the restriction enzymes KpnIand XhoI, two fragments of 277 and 1054 bp were subsequently ob-tained. These fragments and the total promoter (1336 bp) were sub-cloned into the polylinker of the promotorless plasmid pCAT 3 En-hancer vector (Promega). The 1054-bp fragment contained AP-1, AP-2,and MRE regulatory sites, whereas the 277-bp fragment encompassedthe NF-kB and antioxidant response element sites.

Transient Transfection and CAT Assay—HepG2 cells were seeded in6-well tissue cultures plates and cultured until cell density reached65–75% confluency. pCAT 3 Enhancer and pCAT 3 Control plasmidswere transfected using the LipofectAMINE reagent (Life Technologies,Inc.), according to the manufacturer’s instructions. Twenty four h aftertransfection, cells were exposed to TNF (500 and 10,000 units/ml)overnight. After TNF treatment, the cell extracts were isolated, and theprotein content was determined by the color change of Coomassie Bril-liant Blue G-250 dye (Bio-Rad protein assay). Chloramphenicol acetyl-transferase (CAT) activity was quantitated by the CAT enzyme-linked

immunosorbent assay (Boehringer Mannheim). b-Galactosidase ex-pression plasmid (PSVgal, Promega) was cotransfected as an internalcontrol to normalize the transfection efficiency.

Statistical Analyses—Statistical analyses for multiple comparisonsof mean values between cell preparations were made by one-way anal-ysis of variance followed by Fisher’s test.

RESULTS AND DISCUSSION

Regulation of Hepatocellular GSH Levels by TNF and Con-trol of ROS—One of the consequences of the oxidative stressinduced by TNF is the generation of ROS that mediates theinjury of cells exposed to TNF (7, 8, 20). Since many of thecellular effects elicited by TNF appear to be dependent on theconcentration of the cytokine and in view of the observationthat TNF exerts an important physiological role in variouscellular functions, we investigated the effect of TNF on theregulation of total cellular GSH equivalents (GSH 1 GSSG)and generation of hydrogen peroxide as indicator of ROS incultured rat hepatocytes exposed to a wide range of TNF con-centrations (250–10,000 units/ml; 9–370 ng/ml). Hydrogenperoxide was monitored by quantitation of fluorescence of DCF,a highly fluorescent probe sensitive to peroxides, which isformed from DCFDA (29, 30). As seen in Fig. 1, cells labeledwith DCFDA displayed a significant increase in DCF fluores-cence when incubated with a high dose of TNF (10,000 units/ml) (Fig. 1B). To confirm further the burst of ROS induced byTNF, cells were labeled with DHR, another probe that assessesgeneration of peroxides. Compared with control hepatocytes,TNF (10,000 units/ml) led to a significant increase in DHR

FIG. 1. Dose-dependent effect of TNF on the oxidative stress response of hepatocytes. Cultured rat hepatocytes were incubated for 20 hwith various concentrations of TNF as indicated. A, total cellular GSH equivalents (GSH 1 GSSG) were determined by HPLC. B, hydrogenperoxide measurement following fluorescence of DCF by staining of cells with DCFDA was determined as described under “ExperimentalProcedures.” C, the estimation of lipid peroxidation of cells treated with 10,000 units/ml TNF was performed by labeling cells with cis-parinaricacid followed by determination of fluorescence as described (23). D, parallel cultures were labeled with DHR followed by washing to remove excessprobe to determine the effect of treatment of TNF at 500 units/ml (panel b) and 10,000 units/ml (panel c) compared with control cells (panel a).Magnification 200 3. Parameters were determined in duplicate plates, and the means 6 S.D. of six different experiments are shown. *, p , 0.05versus control; #, p , 0.05 versus 2500units/ml and 10,000 units/ml TNF; ¶, p , 0.05 versus 2500 units/ml TNF.

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fluorescence indicating generation of hydrogen peroxide (Fig.1D, panel c). Since the levels of hydrogen peroxide increased asresult of TNF exposure, it was important to determine the totalcellular GSH equivalents under these circumstances. A deple-tion of cellular GSH was observed at the highest concentrationof TNF, an effect that was significant compared with controlcells or cells incubated with lower doses of TNF (Fig. 1A andTable I). This effect was reflected mainly as a decrease inreduced GSH, which was translated into a lower GSH/GSSGratio, suggesting that TNF induced an oxidative stress in hepa-tocytes. One of the characteristic effects of oxidative stress isperoxidation of membrane lipids. We labeled hepatocytes withcis-parinaric acid, a fluorescent fatty acid analogue, to deter-mine the extent of lipid peroxidation. Cells treated with TNF(10,000 units/ml) displayed a greater loss of cis-parinaric acidfluorescence (Fig. 1C). Despite the degree of lipid peroxidationseen in hepatocytes treated with such a level of TNF, cytoxicitywas minimal as viability of TNF-treated hepatocytes was sim-ilar to control cells (85–90% after 20 h of treatment).

To gain a better understanding of the regulation of cellularGSH by TNF and its consequences on ROS generation, weexamined the effect of lower doses of the cytokine on theseparameters. In contrast to the results seen with 10,000units/ml TNF, TNF at 250–500 units/ml did not increase thelevel of hydrogen peroxide determined as fluorescence of DCFor DHR (Fig. 1, B and D). Interestingly, however, total GSHequivalents increased (60–80%) above control levels, mainlyreflected as an increase of reduced GSH levels (Fig. 1 and TableI). TNF at 2,500 units/ml did not influence the level of eitherROS or GSH, probably reflecting an equilibrium between ROSgeneration and their detoxification by GSH. Kinetic analyses ofthe effects of TNF on GSH levels revealed that the increase ofreduced cellular GSH required 3–5 h of incubation with TNF.During this course of incubation, TNF did not affect the levelsof reduced GSH nor GSSG in cells or medium.

The steady-state concentration of cellular GSH is regulatedby its transport through specific plasma membrane GSH car-riers (36). Recent observations indicated that apoptosis of hu-man Jurkat T lymphocytes induced with anti-FAS/APO-1 an-tibody was preceded by a rapid and specific efflux of reducedGSH (37). Such a stimulated efflux process led to a quantitativeloss of cellular reduced GSH that was recovered in the extra-cellular medium without change in the levels of GSSG. Incontrast to these observations, our data indicated that thedepletion of hepatocellular GSH induced by a high dose of TNFwas not accounted for by an increased efflux of GSH into theextracellular medium (Table I). Our results imply that TNFleads to an overproduction of ROS that exhausts cellular GSHstores, probably by formation of disulfides with proteins fa-vored by the oxidized environment. Although the proportion ofGSSG released into the medium was greater in cells treatedwith TNF (10,000 units/ml), the magnitude of this increase wasinsufficient to account for the observed cellular depletion of

GSH levels induced by TNF (Table I). It is noteworthy that inaddition to GSH and GSSG found in the medium, anothermolecular form, a cysteine-GSH mixed disulfide, formed bytranspeptidation of GSH in the presence of cystine, was de-tected in the extracellular medium, although its magnitudewas similar for control and TNF-treated hepatocytes (Table I).

Regardless of the mechanism, a lower availability of reducedGSH may be a critical factor that sensitizes cells to the chain ofreactions leading to cell death induced by either Fas ligand orTNF (7, 37). In the case of the hepatotoxicity induced by TNF,previous studies revealed that pretreatment of murine hepato-cytes with 1,3-bis(chloroethyl)-1-nitrosourea, which inhibitsthe GSH reductase and disrupts the GSH redox cycle, depleteshepatocellular GSH resulting in cytotoxicity (7). In our case, inagreement with these findings, we observed that depletion ofGSH levels by pretreatment with diethyl maleate or BSO sen-sitized cultured rat hepatocytes to TNF or acidic sphingomy-elinase treatment.2 Taken together, these results regarding theregulation of cell GSH and ROS reflect a divergent response ofhepatocytes to TNF exposure, which seems to be dependent onthe dose of TNF. The up-regulation of cellular GSH levelsevoked by a low dose of TNF (250–500 units/ml) attenuated thegeneration of ROS induced by TNF. However, at higher levels(10,000 units/ml), TNF resulted in depletion of reduced GSH,an effect that was accompanied by an increase of ROS, indicat-ing that the former was overwhelmed by the increased gener-ation of reactive species.

Transcriptional Regulation of g-GCS-HS Gene by TNF—Inview of these results, we investigated the mechanism account-ing for the up-regulation of cellular GSH levels. GSH is syn-thesized by the sequential action of two enzymes, g-GCS andGSH synthetase. g-GCS is the regulatory and rate-limitingenzyme that is composed of two subunits, heavy and lightchains, that are discoordinately synthesized. The catalytic ac-tivity resides in the heavy subunit, whereas the light chainlowers the Km for glutamate and increases feedback inhibitionby GSH on the heavy subunit (32). Thus, we determined theeffect of TNF on the level of g-GCS-HS mRNA. As seen in Fig.2, TNF increased the g-GCS-HS mRNA levels compared withcontrol hepatocytes. Northern blot analyses confirmed thatTNF treatment increased the 3.7-kilobase size of g-GCS-HSmRNA (not shown). Interestingly, the effect of TNF on thesteady-state mRNA level of g-GCS-HS was significant at bothlow (500 units/ml) and high (10,000 units/ml) concentrations ofTNF, although the level of induction by the low dose wasgreater than the induction seen by the high dose (Fig. 2A). Suchan increase in the level of mRNA of g-GCS-HS may reflecteither a stabilization of its mRNA or an increased transcriptionrate of g-GCS-HS gene. To discern between these possibilities,we isolated intact nuclei from cells treated with TNF to exam-

2 C. Garcıa-Ruiz, M. Marı, E. Ardite, M. Miranda, A. Colell, A.Morales, and J. C. Fernandez-Checa, manuscript in preparation.

TABLE ICellular and medium GSH molecular forms of cultured rat hepatocytes exposed to TNF

Cultured hepatocytes were treated with TNF at the dose indicated for 20 h. Thereafter, medium was removed and cells scraped and treated with10% trichloroacetic acid. The molecular forms of GSH equivalents from cells and medium were determined by HPLC as described under“Experimental Procedures.” Results are expressed as mean 6 S.D. of n 5 7 cell preparations.

Cell Medium

GSH GSSG GSH GSSG Cys-GS

nmol/106 cells nmol/106 cells

Control 32.4 6 7.2 3.8 6 0.7 7.9 6 1.3 1.9 6 0.9 4.9 6 1.2TNF (500 units/ml) 56.5 6 10.7a 5.1 6 1.2 8.8 6 1.5 2.8 6 0.8 5.4 6 0.6TNF (10,000 units/ml) 15.7 6 4.3a,b 2.1 6 0.8a,b 6.1 6 0.9 4.5 6 0.7a 3.9 6 0.8

a p , 0.05 versus control.b p , 0.05 versus TNF at 500 units/ml.

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ine the transcription rate of g-GCS-HS gene by nuclear run-onassay. Intact nuclei isolated from cells treated with TNF re-vealed an increased capacity to transcribe the g-GCS-HS gene(Fig. 2B).

The next question we examined was if the greater levels ofg-GCS-HS mRNA were paralleled by higher enzymatic activ-ity. We determined the capacity to synthesize GSH de novo inthe presence of unlimited GSH precursors in cell-free extractsisolated from hepatocytes that were pretreated with TNF. Thedynamic synthetic rate of GSH was determined using mono-chlorobimane, a probe that forms a highly fluorescent adductwith GSH catalyzed by GST (28). GSH synthesis in cytosolextracts incubated with GSH substrates (glutamate, glycine,and ATP) and cysteine as the sulfur amino acid reflects theactivities of both g-GCS and GSH synthetase. The GSH syn-thetic rate increased significantly (40–60%) in cell extractsisolated from hepatocytes treated with TNF (500 units/ml)compared with control cells (Fig. 3A). Similar results wereobtained with higher doses of TNF (10,000 units/ml) (data notshown). g-Glutamylcysteine, the product of g-GCS, is the sub-strate for GSH synthetase to which glycine is added formingthe final product, GSH. When g-glutamylcysteine was used in

the in vitro synthetic assay instead of cysteine, the formation ofGSH reflected the activity of GSH synthetase. In contrast tothe results obtained using cysteine as sulfur amino acid GSHprecursor, the formation of GSH from g-glutamylcysteine wassimilar in hepatocytes with or without TNF treatment, demon-strating that the cytokine did not affect the specific activity ofGSH synthetase (Fig. 3B). These findings indicate that theincreased capacity of hepatocytes to synthesize GSH conferredby TNF was due to a greater activity of g-GCS that paralleledthe increase in mRNA levels induced by TNF exposure. Similarfindings were recently reported where interleukin-6 treatmentof tumor-bearing mice displayed a significant increase in thehepatic g-GCS activity that was associated with a decrease inthe hepatocellular sulfate level and lower glutamine to gluta-mate ratio (38).

To examine further the role of g-GCS activity on the home-ostasis of cellular GSH and to decipher its impact on TNF-induced generation of ROS, these parameters were determinedin conditions where g-GCS activity was inhibited by BSO. BSOtreatment for 4 h did not significantly affect the GSH levels, yetit led to a significant depletion of cellular GSH after 20 h ofBSO exposure (Fig. 4, A and B); however, this effect was not

FIG. 2. Regulation of g-GCS-HSmRNA by TNF. Cultured hepatocyteswere incubated in the presence of TNF at500 and 10,000 units/ml for 20 h. A, totalRNA extracted from cells was hybridizedwith cDNA for g-GCS-HS or 18 S rRNA,which was used as reference control andanalyzed by slot blot as described under“Experimental Procedures.” The magni-tude of increase of g-GCS-HS mRNA wascalculated relative to the levels of 18 Sand expressed as means 6 S.D. of fourdifferent cell preparations. B, intact nu-clei of cultured cells incubated with TNF(500 units/ml) were isolated, and thetranscription of the g-GCS-HS gene wasdetermined as described under “Experi-mental Procedures” relative to 18 S. Per-centage increase over control was deter-mined in four different cell preparations.*, p , 0.05 versus control; #, p , 0.05versus 10,000 units/ml TNF.

FIG. 3. Measurement of GSH synthetic rate in cell-free extracts of hepatocytes. A, cytosol fractions from control or TNF (500units/ml)-treated cells (20 h) were isolated and dialyzed at 4 °C to minimize feedback inhibition by GSH. The GSH synthetic rate was determinedby the formation of GSH-bimane fluorescent adduct using substrates and precursors (glutamate, glycine, ATP, and cysteine) for GSH. B, activityof GSH synthetase was determined using g-glutamylcysteine and glycine as GSH precursors and GSH-bimane fluorescent adduct followed overtime. Results are the means 6 S.D. of four different experiments. *, p , 0.05 versus control.

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accompanied by an increased fluorescence of DCF (Fig. 4),indicating that depletion of GSH is insufficient for ROS over-production. Furthermore, addition of TNF (500 units/ml) tocells pretreated with BSO resulted in a further depletion ofcellular GSH compared with control or TNF-treated cells thatwere associated with a significant increase in DCF fluores-cence, indicating overproduction of hydrogen peroxide. Similarresults were obtained when gene transcription was blockedwith actinomycin D. The effect of the transcription blocker oncellular GSH was modest as GSH levels did not decrease com-pared with control cells (Fig. 5). However, as in the case withBSO treatment, TNF, at the dose that efficiently increasedcellular GSH levels, led to a significant depletion of GSH levelswith respect to either control or TNF-treated cells, an effectthat was accompanied by a significant increase of hydrogenperoxide (Fig. 5). Although blocking of transcription generallysensitizes cells against TNF, at the dose of TNF used (500units/ml), hepatocytes remained viable (80–90%). However,exposure of cells to a greater dose of TNF (10,000 units/ml)resulted in apoptosis as revealed by Hoechst-labeled hepato-cytes, a fluorochrome that monitors chromatin condensationand fragmentation (data not shown). These findings suggestthat the up-regulation of cellular GSH induced by TNF can beviewed as an adaptive mechanism to control the extent of ROSgeneration. Preventing the increase of GSH levels by inhibitionof the rate-limiting enzyme of GSH synthesis, g-GCS, or block-ing its transcription unmasks the full capacity of TNF to over-produce ROS.

Conversely, it can be inferred that if the molecular mecha-nisms by which TNF leads to ROS generation were disrupted,cells exposed to a high dose of TNF could display increasedGSH levels. Since mitochondria play a critical role in the TNF-induced overproduction of ROS (8, 23, 24), we examined theeffect of rotenone and thenoyltrifluoroacetone, blockers of elec-tron transfer at complex I and II of respiration, respectively, onthe effects of high doses of TNF (10,000 units/ml) on ROS andGSH levels. As seen in Fig. 6, blocking electron transfer attheses sites resulted in attenuation of TNF-induced ROS gen-eration, which was translated in greater cell GSH levels com-pared with control or TNF-treated cells. These results areconsistent with recent findings in isolated rat liver mitochon-dria incubated with ceramide, one the mediators of the biolog-ical effects of TNF, which highlight mitochondria as criticalplayers in the generation of ROS induced by TNF (24). Similarfindings were observed when hepatocytes were incubated withdesferrioxamine prior to TNF exposure.3 Desferrioxamine pre-

vented the TNF-induced ROS generation as DCF fluorescencewas similar for control and TNF-treated hepatocytes; conse-quently, the levels of total reduced GSH of TNF-exposed cellswere greater than control cells. The increased GSH levels ob-served under these circumstances are consistent with the find-ings shown above that TNF leads to increased levels of g-GCS-HS mRNA. Therefore, these findings support the viewthat TNF initiates signaling pathways that culminate in over-production of ROS and induction of cell GSH levels. The pre-dominance of these cellular responses depends on the level atwhich TNF acts.

Our results have provided evidence that TNF leads to sus-tained elevation of hepatocellular GSH, an effect that may beconsidered as a protective mechanism to withstand the TNF-induced oxidative stress. This adaptive mechanism of GSHinduction has also been observed in cells exposed to sublethalconcentrations of quinones and Michael reaction acceptors (33,39). The mechanism whereby structurally unrelated sub-stances, such as TNF and compounds with electrophilic orelectron-deficient centers, lead to induction of g-GCS-HSmRNA remains to be characterized. A common feature of theseagents is their ability to generate ROS, and consequently oxi-dative stress, which could mediate the induction of g-GCS-HSgene by increasing its transcriptional rate. Oxidative stress caninfluence gene transcription by activation of transcription fac-

3 A. Morales, C. Garcıa-Ruiz, M. Miranda, M. Marı, A. Colell, E.Ardite, and J. C. Fernandez-Checa, unpublished observations.

FIG. 4. Inhibition of g-GCS by BSOand consequences on the cellularGSH and ROS induced by TNF. Hepa-tocytes were pretreated with BSO (1 mM)for 15 min before addition of TNF (500units/ml). Following incubation of cellswith TNF for 4 h (A) or 20 h (B), cells weredeproteinized for determination of totalGSH levels by HPLC (open bars). Parallelplates were labeled with DCFDA to mon-itor generation of hydrogen peroxide byfluorescence of DCF (closed bars) as de-scribed under “Experimental Proce-dures.” Results are the means 6 S.D. ofn 5 4–5 individual cell preparations. *,p , 0.05 versus control; #, p , 0.05 versusTNF alone.

FIG. 5. Effect of actinomycin D on the cellular GSH and ROSlevels induced by TNF exposure. Hepatocytes were incubated withTNF (500 units/ml) in the presence or absence of actinomycin D (Act D)(0.8 mg/ml) for 20 h. Cells were scraped from plates and total GSHequivalents determined by HPLC (open bars). Simultaneously in par-allel plates hydrogen peroxide formation (striped bars) was determinedby fluorescence of DCF. Results are the mean 6 S.D. of n 5 4–5 cellpreparations. *, p , 0.05 versus control; #, p , 0.05 versus TNF alone.

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tors such as NF-kB that senses the redox environment of thecell.

Role of NF-kB in the TNF-induced Transcription of g-GCS-HS: Functional Analyses of g-GCS-HS Promoter—We next ex-plored the mechanism by which TNF regulates g-GCS-HS atthe transcriptional level. Activation of the pleiotropic factorNF-kB is a well-described event initiated by a variety of stimuliincluding TNF (23, 40). Such a transcription factor becomeactivated upon release and subsequent degradation of the in-hibitor moiety IkB-a that sequesters NF-kB in an inactive formin cytosol of resting cells. Upon activation, NF-kB translocatesto the nuclei where it binds to specific binding sites in thepromoter of responding genes. Recent evidence revealed ananti-apoptotic functional role of NF-kB in determining the fateof cells in response to TNF (41–43). Thus transgenic micelacking the p65/RelA gene die during development as the resultof massive apoptosis in the liver (41). In addition, cells express-

ing a repressor of IkB-a or its dominant negative mutant dis-played an increased susceptibility to apoptosis induced by TNF.The mechanisms whereby NF-kB protects cells against TNF-induced cell death is currently unknown. Recent studies havepartially characterized the 59-flanking region of g-GCS-HS pro-moter identifying several regulatory cis-acting elements in-cluding kB binding site(s) (34, 35, 44). These observations,together with studies showing the involvement of NF-kB in theinduction of detoxification enzymes such as NAD(P)H:quinoneoxidoreductase (DT-diaphorase) (45), prompted us to examinewhether transcription factor NF-kB mediates the transcrip-tional activation of g-GCS-HS induced by TNF.

We first verified if TNF efficiently activated NF-kB by deter-mining the pattern and dose-dependent activation of this tran-scription factor in response to TNF. Nuclear extracts of hepa-tocytes incubated with TNF and analyzed by gel retardationassay revealed the activation of NF-kB in a dose-dependentfashion, being significant at 250 units/ml and maximal at10,000 units/ml (Fig. 7, A and C). Such activation was specificfor NF-kB as the retarded band was displaced by competitionwith a fold excess of unlabeled probe, in contrast to the lack ofdisplacement by a mutated kB oligonucleotide (not shown).Several members of the NF-kB/Rel family of transcription fac-tors have been identified that can form dimers in several homoor hetero combinations, the p65(RelA)/p50 heterodimer beingone of the best characterized examples (40). To identify themembers of NF-kB activated by TNF, we performed super shiftassays where nuclear extracts were incubated with specificantibodies to different NF-kB/Rel members before addition oflabeled kB probe. TNF resulted in a predominant activation ofthe heterodimer p65/p50 with minimal activation of ho-modimer p50 (Fig. 7B). The magnitude of NF-kB activation atthe highest dose of TNF used (10,000 units/ml) was 2–3-foldgreater than the activation seen with 500 units/ml TNF; as thelevels of ROS constitute one of the major triggers of NF-kBactivation, these results reflect the effect of oxidative stressinduced by TNF on NF-kB activation (Fig. 7) (9, 29). In con-trast, the dose of TNF that resulted in maximal induction ofg-GCS-HS mRNA was 500 units/ml (Fig. 2), suggesting thatactivation of NF-kB and induction of g-GCS-HS mRNA in re-sponse to TNF correlated inversely.

To determine the functional role of kB in the transcriptionalregulation of g-GCS-HS and to assess the contributory role ofother factors and cis-regulatory elements contained in the 59-flanking region of g-GCS-HS, we partially characterized thepromoter of g-GCS-HS gene prepared by PCR from genomic

FIG. 6. Effect of rotenone/thenoyltrifluoroacetone on the TNF-induced GSH depletion and generation of ROS. Hepatocytes wereincubated with rotenone/thenoyltrifluoroacetone (Rot/TTFA), 20 and 15mM, respectively, before the addition of TNF (10,000 units/ml) andincubated for 20 h. Following this incubation, cells were harvested forGSH determination by HPLC (open bars), and DCF fluorescence toassess the levels of hydrogen peroxide (stripped bars) was determinedas described under “Experimental Procedures.” Results are themeans 6 S.D. of n 5 3 individual preparations. *, p , 0.05 versuscontrol; #, p , 0.05 versus TNF alone.

FIG. 7. Activation of transcriptionfactor NF-kB by TNF. Hepatocyteswere treated with TNF at the dose indi-cated. A, nuclear extracts were isolatedand incubated in the presence of labeledkB oligonucleotide as described under“Experimental Procedures.” As control,nuclear extracts were isolated from hepa-tocytes in the absence of TNF. DNA bind-ing to kB was analyzed by gel retardation.Specificity of binding to kB was verifiedby displacement with excess (100-fold)unlabeled kB oligonucleotide (not shown).B, parallel aliquots of nuclear extractswere incubated with antibodies to differ-ent members of kB/Rel family before ad-dition of labeled kB oligonucleotide. Con-trol (Ctrl) in this panel refers to nuclearextracts isolated from TNF-treated hepa-tocytes in the absence of antibodies. C,densitometric quantitation of het-erodimer p65/p50 activated in A. *, p ,0.05 versus control; #, p , 0.05 versusTNF (250–2000 units/ml).

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DNA of hepatocytes. Subsequently, hepatocytes were tran-siently transfected with plasmids containing the full-length ordeletion constructs of the 59-flanking region of the g-GCS pro-moter linked to the CAT reporter gene (34, 35, 44) (Fig. 8A).Cells that were transfected with the full-length construct,21088/1225, displayed a significant TNF-stimulated CAT ac-tivity. However, the shorter construct (227 bp), containing NF-kB-like and xenobiotic response/antioxidant response-like ele-ments, resulted in minimal CAT expression following TNFtreatment compared with the full-length plasmid. High levelsof transcriptional activation of g-GCS-HS promoter were in-duced by TNF when cells were transfected with the deletionplasmid, 2756/1210, which contained AP-1/AP-like transcrip-tion factor and MRE, as the magnitude of CAT activity inducedby TNF was similar to that seen with the full-length plasmid(Fig. 8B). Futhermore, to establish whether TNF treatmentinduced DNA binding to the g-GCS-HS AP-1 site, we performedelectrophoretic mobility shift assays with nuclear extracts pre-pared from hepatocytes that were treated with TNF and with aradiolabeled oligonucleotide encompassing the AP-1 bindingsite. Although hepatocytes displayed a constitutive AP-1 acti-vation, after an initial decline in AP-1 activation, TNF led to afurther activation of AP-1 being significant at 4 h post-treat-ment (Fig. 9).

These findings indicate a dispensable role of NF-kB bindingsite for the TNF-induced transcriptional activation of g-GCS-

HS gene and suggest the involvement of AP-1-like responseelements. These findings are in agreement with previous re-ports where c-jun/c-jun homodimers (AP-1) mediate the regu-lation of g-GCS-HS gene induced by cisplatin and cadmium (34,46). Recent observations also indicated an induction of g-GCS-HS by cigarette smoking in human alveolar epithelial cells thatwas associated with AP-1 response element (47). At present wecannot completely discard the involvement of other cis-regula-tory elements located further upstream in the 59-flanking re-gion of g-GCS-HS gene (44). Additional work will be needed tofully characterize the cis-regulatory elements responsible forinduction of g-GCS-HS by TNF.

Concluding Remarks—The ability of mammalian cells tomaintain cellular functions during oxidative stress depends onthe rapid induction of protective antioxidant systems. Overpro-duction of TNF in certain pathological states results in cyto-toxicity mediated by a variety of mechanisms including activa-tion of phospholipase A2, increased generation of lipidintermediates such as ceramide, and formation of ROS leadingto oxidative stress (3, 7, 8, 23, 24). Our findings providedevidence for the first time that TNF up-regulates the levels ofreduced GSH as an adaptive control mechanism that attenu-ates the extent of ROS generation implying that cellular GSHdepletion prior to exposure to TNF would determine a greatervulnerability to the cytotoxicity of TNF in a variety of cells2 (7,8). In particular, as mitochondria stand as the source of the

FIG. 8. TNF induction of g-GCS-HS-CAT promoter constructs transfectedin HepG2 cells. A, the 59-flanking regionof g-GCS-HS was generated by PCR usingspecific primers described under “Experi-mental Procedures.” The PCR fragmentwas isolated from agarose gels and di-gested with restriction enzymes to gener-ate distinct fragments of the indicatedsize. These fragments were subcloned inthe pCAT reporter vector as described un-der “Experimental Procedures.” ARE, an-tioxidant response element; XRE, xenobi-otic response element. B, deletionconstructs were transiently transfected inHepG2 cells. Following transfection, TNF(500 units/ml) was added and incubatedfor 20 h. Subsequently, cells were har-vested, and extracts were isolated andused for CAT activity that was deter-mined by enzyme-linked immunosorbentassay. The pCAT reporter vector did notresult in measurable CAT activity in cellextracts. CAT activity was normalized forprotein content. Results are means 6 S.D.of n 5 3 individual experiments. *, p ,0.05 versus control; #, p , 0.05 versuscells transfected with 227 pb plasmid.

FIG. 9. AP-1 binding in nuclear ex-tracts of hepatocytes treated withTNF. Nuclear extracts were preparedfrom cultured rat hepatocytes treatedwith TNF (500 units/ml) for various peri-ods as indicated. Electrophoretic mobilityshift assay was performed by incubatingnuclear extracts with AP-1 oligonucleo-tide as described under “ExperimentalProcedures.” Quantification of DNA bind-ing was performed by densitometryshown in the right panel. Results are themean 6 SD of n 5 3 different experi-ments. *, p , 0.05 versus control.

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burst of ROS induced by TNF, depletion of GSH in this or-ganelle would favor a greater generation of ROS contributing tothe TNF-induced cytotoxicity (24). Our findings are consistentwith recent observations that nitric oxide protected culturedrat hepatocytes from TNF-induced apoptosis by a mechanismthat required expression of heat shock protein 70 (48). Al-though these studies did not examine the regulation of hepa-tocellular GSH, it is possible that the protective role of heatshock protein 70 may have been mediated by an increase in thehepatocellular levels of GSH. In this regard, Mehlen et al. (49)have provided evidence that overexpression of small heat shockproteins protected murine fibrosarcoma cells against TNF-in-duced cell death by a mechanism that required up-regulation ofcellular levels of GSH. Although these authors did not studythe regulation of g-GCS, it is likely that the increased cellularGSH levels seen after overexpression of heat shock protein 27may have been mediated by increased activation of g-GCS, asthe protection afforded by heat shock proteins was abrogatedby BSO pretreatment (49). The possibility that heat shockproteins may play an important role in mediating the TNF-induced transcriptional regulation of g-GCS deserves furtherwork, which is currently under investigation. In this regard,previous observations have demonstrated that TNF induces arapid phosphorylation of heat shock proteins (50).

Our findings indicate a dispensable role of NF-kB in medi-ating the transcriptional activation of g-GCS. Despite activa-tion of this transcription factor by TNF that mediates theexpression of a repertoire of genes in response to TNF, othertranscription factors that are also activated by TNF may par-ticipate in the gene regulation of this cytokine. In line with this,recent advances in the identification of TNF intermediateshave linked the binding of TNF to its receptor 55-kDa TNFreceptor subtype with the activation of mitogen-activated pro-tein kinase cascade leading to activation of transcription factorAP-1 (51, 52). Futhermore, since TNF elicits independent sig-naling cascades to transmit its action to the cell interior (53),future studies will be required to determine which signalingroute mediates the TNF regulation of the g-GCS gene. Eluci-dation of the signaling mechanisms involved in the increasedtranscription of g-GCS may be helpful in designing strategiesto control the oxidative stress induced by inflammatory cyto-kines in pathological situations, including tumor-induced ca-chexia, as GSH levels would control the extent and conse-quences of overproduction of ROS.

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Regulation of Cellular GSH by TNF 30379

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Esther Ardite and José C. Fernández-ChecaAlbert Morales, Carmen Garci?a-Ruiz, Merce Miranda, Montserrat Mari?, Anna Colell,

-Glutamylcysteine SynthetaseγRegulation of the Heavy Subunit Chain of Tumor Necrosis Factor Increases Hepatocellular Glutathione by Transcriptional

doi: 10.1074/jbc.272.48.303711997, 272:30371-30379.J. Biol. Chem. 

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