Molecular Mechanism of the Regulation of Glutathione Synthesis by Tumor Necrosis Factor-alpha and Dexamethasone in Human Alveolar Epithelial Cells*

Irfan RahmanDagger , Frank Antonicelli, and William MacNee

From the Rayne Laboratory, Respiratory Medicine Unit, Department of Medicine (RIE), University of Edinburgh, Medical School, Edinburgh Eh8 9AG, United Kingdom

    ABSTRACT
Top
Abstract
Introduction
References

Glutathione (GSH) is an important physiological antioxidant in lung epithelial cells and lung lining fluid. We studied the regulation of GSH synthesis in response to the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha ) and the anti-inflammatory agent dexamethasone in human alveolar epithelial cells (A549). TNF-alpha (10 ng/ml) exposure increased GSH levels, concomitant with a significant increase in gamma -glutamylcysteine synthetase (gamma -GCS) activity and the expression of gamma -GCS heavy subunit (gamma -GCS-HS) mRNA at 24 h. Treatment with TNF-alpha also increased chloramphenicol acetyltransferase (CAT) activity of a gamma -GCS-HS 5'-flanking region reporter construct, transfected into alveolar epithelial cells. Mutation of the putative proximal AP-1-binding site (-269 to -263 base pairs), abolished TNF-alpha -mediated activation of the promoter. Gel shift and supershift analysis showed that TNF-alpha increased AP-1 DNA binding which was predominantly formed by dimers of c-Jun. Dexamethasone (3 µM) produced a significant decrease in the levels of GSH, decreased gamma -GCS activity and gamma -GCS-HS mRNA expression at 24 h. The increase in GSH levels, gamma -GCS-HS mRNA, gamma -GCS-HS promoter activity, and AP-1 DNA binding produced by TNF-alpha were abrogated by co-treating the cells with dexamethasone. Thus these data demonstrate that TNF-alpha and dexamethasone modulate GSH levels and gamma -GCS-HS mRNA expression by their effects on AP-1 (c-Jun homodimer). These data have implications for the oxidant/antioxidant balance in inflammatory lung diseases.

    INTRODUCTION
Top
Abstract
Introduction
References

The tripeptide, L-gamma -glutamyl-L-cysteinylglycine, or glutathione (GSH), is a ubiquitous cellular non-protein sulfhydryl, which plays an important role in maintaining intracellular redox balance and in cellular defenses against oxidative stress (1, 2). GSH is present in high concentrations in lung epithelial lining fluid (3). It also has an important role in maintaining the integrity of the airspace epithelium, in both type II alveolar cells in vitro, and in lungs in vivo (4, 5). A physiological role for GSH as an antioxidant has been described in numerous inflammatory disorders (6). Depletion of lung epithelial lining fluid GSH has been described in conditions such as human immunodeficiency virus infection (7), idiopathic pulmonary fibrosis (8), adult respiratory distress syndrome (9), and cystic fibrosis (10). Elevated levels of GSH occur in the epithelial lining fluid of chronic smokers and in patients with lung cancer (11).

GSH is synthesized by two enzymes, gamma -glutamylcysteine synthetase (gamma -GCS,1 EC 6.3.2.2), which is the rate-limiting enzyme, and glutathione synthetase (12). The gamma -GCS holoenzyme exists as a dimer composed of heavy (gamma -GCS-HS; 73 kDa) and light (gamma -GCS-LS; 28 kDa) subunits (13). The heavy subunit possesses all of the catalytic activity (14). We have recently demonstrated that a putative AP-1 transcription factor-binding site is necessary for oxidant-mediated regulation of the catalytic gamma -GCS-HS gene promoter (15).

Inflammation results in the release of inflammatory mediators, such as cytokines, which affect the local tissue oxidant/antioxidant balance. Anti-inflammatory drugs might also influence this balance. Tumor necrosis factor-alpha (TNF-alpha ) is a ubiquitous pro-inflammatory cytokine and is recognized as an important mediator of multiple inflammatory events in the lungs. It induces chronic inflammatory changes associated with the increase in a variety of defense mechanisms, through the up-regulation of mRNA for various inflammatory mediators (16). Regulation of mRNA expression by TNF-alpha is mediated by activation of transcription factors c-Fos/c-Jun (activator protein-1, AP-1) and nuclear factor-kappa B (NF-kappa B) (17). It has been reported that prior exposure to a sublethal dose of TNF-alpha renders cells resistant to a subsequent TNF-alpha challenge. The mechanism by which this TNF-alpha -mediated cellular resistance occurs is related to the enhancement of the intracellular antioxidant capacity (18). Examples of this are increased activity of mitochondrial manganese superoxide dismutase (19), and other protective proteins, including plasminogen activator inhibitor type 2, the zinc finger protein A20, and the Bcl-2-related family member A1 (20-22). However, the molecular basis of the TNF-alpha -induced cellular tolerance is not fully understood at present. One possible mechanism may involve TNF-alpha -induced generation of reactive oxygen species (ROS) by leakage from the mitochondrial electron transport chain (23, 24). Such intracellular ROS could induce glutathione synthesis in response to this oxidant stress by a mechanism involving the up-regulation of the gamma -GCS-HS mRNA.

Corticosteriods are widely used as anti-inflammatory agents in various inflammatory lung diseases (17). Corticosteroids act to reduce inflammation and cellular damage by two different mechanisms as follows: by direct binding of glucocorticoids to their consensus glucocorticoid response element site, which activates transcription processes, and by an indirect mechanism involving nuclear protein interactions (25). These latter effects may be mediated by an interaction between the glucocorticoid-receptor complex and transcription factors such as NF-kappa B and AP-1, which regulate transcription for inflammatory mediators such as cytokines (17, 25). We have shown that AP-1 plays a critical role in enhancing gamma -GCS-HS mRNA expression, and therefore regulation of GSH synthesis (15, 26, 27). Thus corticosteroids may have an effect on intracellular GSH through a mechanism involving AP-1 (28). However, the effects of corticosteroids on GSH synthesis at the molecular level have not been studied so far.

Thus the aim of this study was to elucidate the molecular regulatory mechanisms for GSH synthesis in human alveolar epithelial cells (A549) in response to TNF-alpha and dexamethasone, important pro- and anti-inflammatory agents, respectively.

    EXPERIMENTAL PROCEDURES

Materials-- GSH, GSSG, 5,5'-dithiobis-(2-nitrobenzoic acid), sulfosalicylic acid, glutathione reductase, NADPH, triethanolamine, pyruvate kinase, lactate dehydrogenase, L-glutamate, L-alpha -aminobutyrate, phosphoenolpyruvate, NADH, dexamethasone, DL-buthionine-(SR)-sulfoximine, and ATP were obtained from the Sigma (Poole, UK) and 2-vinylpyridine from Aldrich Chemical Co., (Dorset, UK). Recombinant human TNF-alpha was purchased from R & D Systems, (Abingdon, Oxford, UK). Cell culture media/reagents and the TRIZOL reagent were obtained from Life Technologies, Inc. (Paisley, UK).

A549 Epithelial Cells-- The human type II alveolar epithelial cell line, A549 (ECACC number 86012804), which was mycoplasma-free, was maintained in continuous culture at 37 °C, 5% CO2 in Dulbecco's modified minimum essential medium (DMEM), sodium bicarbonate, and 10% fetal bovine serum (FBS).

When required for the assays, confluent monolayers of A549 epithelial cells were washed twice with phosphate-buffered saline (PBS). Thereafter, trypsin EDTA solution was added to detach the cells. The cells were then washed with DMEM, containing 10% FBS at 250 × g for 10 min, to neutralize the trypsin, and were resuspended in DMEM with 10% FBS and maintained in 162-cm2 cell culture flasks (Corning Costar, High Wycombe, UK).

Epithelial Cell Exposure to TNF-alpha and Dexamethasone-- Monolayers of confluent A549 epithelial cells were prepared by seeding 0.8 × 106 cells/well in a 6-well or 3 × 106 cells/100-mm plates in DMEM with 10% FBS at 37 °C, 5% CO2, until 70-80% confluency was reached. Confluent monolayers were rinsed twice with DMEM and were treated with TNF-alpha (10 ng/ml) and/or dexamethasone (3 µM) for time intervals between 4 and 24 h in 2 ml or 5 ml of full media, incubated at 37 °C, 5% CO2. Thereafter, the monolayers were washed twice with cold PBS, and the cells were harvested with 0.05% trypsin/EDTA solution and resuspended in cold PBS (pH 7.4). An aliquot of cells was diluted with trypan blue, counted, and the cell viability determined. This cell suspension was thereafter used in the GSH and GSSG assays. For gamma -GCS activity, mRNA assays, and transfection experiments, monolayers were scraped using a Teflon scraper (Corning Costar, High Wycombe, UK).

GSH, GSSG, and gamma -GCS Assays-- The epithelial cells were centrifuged at 250 × g for 5 min at 4 °C, and the cell pellets were suspended in 1 ml of cold 0.6% sulfosalicylic acid, sonicated on ice, homogenized with a Teflon pestle, and vortexed vigorously. The cell homogenates were then centrifuged at 4,000 × g for 5 min at 4 °C. The supernatant was immediately used in the GSH assay by the 5,5'-dithiobis-(2-nitrobenzoic acid)-glutathione reductase recycling method described by Tietze (29). For the GSSG assay, supernatant was treated with 2-vinylpyridine and triethanolamine as described previously (30) and thereafter was used in the assay for GSH as described above.

Cell extracts for gamma -GCS activity were prepared 4 or 24 h after each treatment. The cells were washed with ice-cold PBS, scraped into PBS, and collected by centrifugation at 250 × g for 5 min at 4 °C. After two additional washes with PBS, the cells were resuspended in 100 mM potassium phosphate buffer (pH 7.4), sonicated, and homogenized using a Teflon pestle on ice with Triton X-100 to a final concentration of 0.1% (v/v). The extracts were spun at 13,000 × g for 15 min at 4 °C. The supernatants were recovered and dialyzed at 4 °C against four changes of 500 ml each of the Tris-HCl buffer (50 mM, pH 8.0) and then stored at 0 °C. The protein concentrations were determined using the bicinchoninic acid reagent assay (Pierce, Chester, UK) (31).

gamma -GCS activity was assayed by the method described by Seelig and Meister (12) using a coupled assay with pyruvate kinase and lactate dehydrogenase. The rate of decrease in absorbance at 340 nm was followed at 37 °C. Enzyme-specific activity was measured as micromoles of NADH oxidized per min/mg protein, which is equal to 1 international unit (IU). For each experimental assay, BSO (50 µM) was added to the cell homogenate and incubated for 1 h at 37 °C to check the specificity of the gamma -GCS assay.

Isolation of RNA and Reverse Transcription-- RNA was isolated from A549 cells using the TRIZOL reagent (Life Technologies, Inc., Paisley, UK). Total RNA was reverse-transcribed according to the manufacturer's instructions (Life Technologies, Inc., catalog number 8025SA). The resultant cDNA was stored at -20 °C until required.

Assessment of gamma -GCS-HS mRNA by Polymerase Chain Reaction (PCR)-- Oligonucleotide primers were chosen using the published sequence of human gamma -GCS-HS cDNA (32) and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (33) (Stratagene, Cambridge, UK). The primers for gamma -GCS-HS were synthesized by Oswel DNA Services, University of Southampton, UK (25, 34). The sequences of the primers used in the PCR were as follows: gamma -GCS-HS (sense 5'-GTG GTA CTG CTC ACC AGA GTG ATC CT-3') and (antisense 5'-TGA TCC AAG TAA CTC TGG ACA TTC ACA-3'); GAPDH (sense 5'-CC ACC CAT GGC AAA TTC CAT GGC A-3') and (antisense 5'-TC TAG ACG GCA GGT CAG GTC AAC C-3'). Five microliters of the reverse-transcribed mRNA mixture was added directly to the PCR mixture and used for the PCR reactions, which we have previously described (26, 27, 34). Bands were visualized by a UV transilluminator, and photograph negatives were scanned using a white/ultraviolet transilluminator, UVP (Orme Technologies, Cambridge, UK). The intensity of the gamma -GCS-HS mRNA (531 bp) bands were expressed as a percentage of the intensity of the GAPDH bands (600 bp). The pKS-hGCS plasmid (American Type Culture Collection, Rockville, MD, catalog number 79023) was used as a positive control for gamma -GCS-HS. Fifty femtograms was used for each experiment to check the specificity of the PCR (data not shown).

Generation of the gamma -GCS-HS Promoter Construct-- The gamma -GCS-HS promoter was isolated by PCR from human genomic DNA using the upstream oligonucleotide 5'-(-1050) GGCGACATCCAATATGAAGGCTGTG-3' and downstream oligonucleotide 5'-(+82) TTCCTACTTGTGACCAAAACCTGCG-3'. The resulting promoter fragment (-1050 to +82 bp) was sequenced and cloned into the pCRII cloning vector (Invitrogen). A HindIII and SphI fragment (1138 bp) containing the promoter was isolated and subcloned into the polylinker of the promoterless plasmid pCAT Basic Vector (Promega). This construct was denoted pCBGCS and has been described previously (15, 26, 27).

Generation of the Mutant gamma -GCS-HS Promoter Construct-- A selective mutation in the proximal AP-1-binding site (-269 to -263 bp) was introduced by PCR amplification with the mutated upstream primers. The sequences of the oligonucleotides used in the PCR were as follows: upstream 5'-ATGGTGAGTTCGTCATGTTATCAA-3' and downstream 5'-GCAGGCATGCCCAGTCTTTGCG-3' and were synthesized by MWG-BIOTECH GmbH, (Ebersberg, Germany). The mutated consensus sequence of AP-1 is denoted by the underlined letters, and the italic letters on the downstream oligonucleotide indicates additional bases from the cloning site of the pCAT Basic vector. The PCR conditions were 94 °C for 10 min and then 35 cycles at 94 °C for 30 s, 58 °C for 30 s, 72 °C for 120 s, and a final extension at 72 °C for 10 min with 1 unit of Taq DNA polymerase (Life Technologies, Inc., Paisley, UK). The resulting PCR-amplified DNA fragment (-285 to +47 bp) was confirmed by DNA sequencing. The mutated PCR fragment was isolated and subcloned into pCBGCS using HpaII and SphI restriction sites. This construct was denoted pCBmGCS.

Generation of a gamma -GCS-HS AP-1 Construct-- Multiple restriction sites within the gamma -GCS-HS promoter were utilized to create a gamma -GCS-HS construct containing a putative proximal AP-1 construct. Digestion of the gamma -GCS-HS promoter using KpnI produced a short (-1050 to -818 bp) and a large fragment (-817 to +82 bp). Further digestion of the large fragment by BalI and DraII generated a proximal fragment (-305 to +82 bp). This fragment contains a putative AP-1 at -269 to -263 bp, CAAT and TATA boxes, which was subcloned into the pCAT Basic vector (pCBGCDelta D). Restriction enzyme analyses were performed to confirm the orientation and validity of all constructs.

Transient Transfection and CAT Assay-- A549 cells (0.8 × 106 per well) were seeded into 6-well tissue culture plates and cultured at 37 °C until they were 70-80% confluent. Plasmid DNA transfections were performed using the LipofectAMINE reagent (Life Technologies, Inc.). Following treatment with TNF-alpha (10 ng/ml) and/or dexamethasone (3 µM), cell extracts were prepared and assayed for protein content using the BCA reagent (Pierce, Chester, UK) (31). Chloramphenicol acetyltransferase (CAT) activity was quantified by a CAT enzyme-linked immunosorbent assay. A beta -galactosidase expression plasmid (PSVgal, Promega) was co-transfected as an internal control to normalize transfection efficiency. In all of the transfection experiments, pCAT-Basic and pCAT-Control were used as negative and positive controls, respectively.

Preparation of Nuclear Extracts and the Electrophoretic Mobility Shift Assay (EMSA)-- Nuclear extracts were prepared by the method of Staal et al. (35). The oligonucleotides used were commercial AP-1 (5'-CGC TTG ATG AGT CAG CCG GAA-3', 3'-CGC AAC TAC TCA GTC GGC CTT-5') and NF-kappa B (5'-AGT TGA GGG GAC TTT CCC AGG C'-3, 3'-TCA ACT CCC CTG AAA GGG TCC G-5'), which were obtained from Promega. The oligonucleotides for the AP-1 site (-269 to -263 bp) present in native gamma -GCS-HS-AP-1 (5'-GAG TTC GTC ATT GAT TCA AAT AAT-3' and 3'-CTC AAG CAG TAA CTA AGT TTA TTA-5') were synthesized by MWG-BIOTECH GmbH, Ebersberg, Germany. The consensus sequence of AP-1 is indicated by bold letters. Both the oligonucleotides were end-labeled with [gamma -32P]ATP using T4 polynucleotide kinase and [gamma -32P]ATP (Promega). Binding reactions were carried out using 5 µg of nuclear extract protein for AP-1 and NF-kappa B and 25 µg for gamma -GCS-HS AP-1, 0.25 mg/ml poly(dI-dC)·poly (dI-dC) (Pharmacia Biotech, St. Albans, United Kingdom), in a 20-µl binding buffer (Promega). The protein-DNA complexes were resolved on 6% non-denaturating polyacrylamide gels at 100 V for 3-4 h. The gels were then vacuum-dried and autoradiographed overnight with an intensifying screen at -80 °C. The gel was scanned on a white/ultraviolet transilluminator UVP (Orme Technologies, Cambridge, UK).

Supershift Assay-- The nuclear extracts were preincubated with 3 µl of antiserum (1 mg/ml), at 4 °C for overnight, before analysis by EMSA as described above. Human anti-c-Jun and anti-c-Fos sera were obtained from Santa Cruz Biotechnology, Inc. These sera specifically detect the presence of the corresponding transcription factor and do not interfere with nuclear factor binding. Rabbit preimmune sera (SAPU, Edinburgh, Scotland) were incubated with the nuclear extracts as described above and used as a control.

Statistical Analysis-- Results were expressed as means ± S.E. Differences between values were compared by Duncan's multiple range test.

    RESULTS

Effect of TNF-alpha and Dexamethasone on GSH Levels in Alveolar Epithelial Cells-- TNF-alpha (10 ng/ml) significantly decreased GSH levels after 4 h treatment concomitant with an increase in GSSG levels in A549 epithelial cells. This was associated with a significant increase in GSH at 24 h, without any change in GSSG levels, compared with control values (Fig. 1 and Table I). By contrast, dexamethasone (3 µM) depleted intracellular GSH levels significantly at both 4 and 24 h. At 4 h dexamethasone produced no change in GSSG, but at 24 h GSSG was decreased compared with control levels (Fig. 1 and Table I). However, exposure to dexamethasone produced a 31% decrease in the GSH/GSSG ratio at 4 h and an 24% increase at 24 h in A549 cells, compared with control values. Co-incubation of TNF-alpha and dexamethasone produced further depletion of GSH at 24 h in epithelial cells, compared with TNF-alpha or dexamethasone alone, without any significant change in the GSH/GSSG ratio (Fig. 1 and Table I). Co-incubation of TNF-alpha and dexamethasone produced a similar decrease in GSH levels to TNF-alpha alone at 4 h, but with an 50% decrease in the GSH/GSSG ratio. Cell viability remained >95% after all of the above treatments.


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Fig. 1.   Effect of TNF-alpha (10 ng/ml) and dexamethasone (3 µM) on GSH levels at 4 and 24 h in A549 type II alveolar epithelial cells. Each histogram represents the mean and the error bars the S.E. of four experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with control values. dagger , p < 0.001, compared with TNF-alpha alone. Dex, dexamethasone.

                              
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Table I
Effects of TNF-alpha and dexamethasone on GSSG levels in alveolar epithelial cells
A549 epithelial monolayers were treated with TNF-alpha (10 ng/ml) and/or dexamethasone (Dex) (3 µM) for 4 and 24 h. Cellular GSSG levels were determined as described under "Experimental Procedures." Data are expressed as mean ± S.E. (n = 4).

Effect of TNF-alpha and Dexamethasone on gamma -GCS Activity in Alveolar Epithelial Cells-- gamma -GCS activity was not affected by TNF-alpha and/or dexamethasone treatment at 4 h, compared with control values (Fig. 2). However, TNF-alpha produced an increase in gamma -GCS activity at 24 h (Fig. 2). By contrast, dexamethasone significantly decreased gamma -GCS activity at 24 h (Fig. 2). Co-incubation of TNF-alpha with dexamethasone produced a further decrease in gamma -GCS activity at 24 h, compared with dexamethasone alone. Addition of BSO (50 µM) only inhibited 65-72% of the total gamma -GCS activity. The results were corrected for the BSO non-inhibitable enzyme activity after each treatment. The mean BSO-inhibitable gamma -GCS activity in the A549 cell homogenate was 0.065 ± 0.01 units/mg protein.


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Fig. 2.   Effect of TNF-alpha (10 ng/ml) and dexamethasone (3 µM) on gamma -GCS enzyme activity in A549 epithelial cells. A549 epithelial cells were incubated with 10 ng/ml TNF-alpha and 3 µM dexamethasone (Dex) alone or in combination for 4 h and 24 h. Each histogram represents the mean and the error bars the S.E. of three experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with control values. dagger , p < 0.001, compared with TNF-alpha alone.

Effect of TNF-alpha and Dexamethasone on gamma -GCS-HS mRNA Expression-- We investigated the mechanism of the above effects on GSH and gamma -GCS activity following TNF-alpha and dexamethasone alone or in combination. Neither TNF-alpha nor dexamethasone alone or in combination produced any significant change in the gamma -GCS-HS mRNA level after 4 h, compared with control values (Fig. 3A). However, TNF-alpha significantly increased gamma -GCS-HS mRNA expression, whereas dexamethasone treatment significantly depleted gamma -GCS-HS mRNA, after 24 h, compared with GAPDH mRNA expression (Fig. 3B). The increased gamma -GCS-HS mRNA expression produced by TNF-alpha at 24 h was completely abolished when the cells were co-treated with dexamethasone.


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Fig. 3.   Effect of TNF-alpha and dexamethasone on gamma -GCS-HS mRNA expression in A549 alveolar epithelial cells. Total RNA was isolated from control cells and cells exposed to TNF-alpha and dexamethasone (Dex) alone or in combination for 4 and 24 h. A, RNA was reversed-transcribed and used for PCR analysis of gamma -GCS-HS mRNA as described under "Experimental Procedures." B, the numeric estimates of gamma -GCS-HS mRNA levels compared with the GAPDH bands from the same sample. Each histogram represents the mean and the error bars the S.E. of the relative intensities of the gamma -GCS-HS mRNA: GAPDH band of three experiments each performed in duplicate. ***, p < 0.001 compared with control values.

Role of AP-1 in TNF-alpha and Dexamethasone-mediated Regulation of gamma -GCS-HS-- To determine if AP-1 plays an important role in TNF-alpha and dexamethasone-mediated gamma -GCS-HS gene regulation in A549 epithelial cells, we used a DNA fragment of the 5'-flanking region of the gamma -GCS-HS containing a proximal putative AP-1 site and a commercially available DNA fragment containing an AP-1 consensus in the EMSA. Nuclear proteins were isolated at 4 and 24 h after TNF-alpha and/or dexamethasone treatment and were incubated with the DNA probes containing the AP-1 site. We found that TNF-alpha exposure increased AP-1 DNA binding activities in A549 cells using both the gamma -GCS-HS AP-1 probe and commercial AP-1 probe at 4 h exposure (Figs. 4 and 5), in A549 cells compared with untreated cells. Dexamethasone treatment alone did not produce any changes in the nuclear binding of gamma -GCS-HS AP-1 probe at 4 or 24 h, compared with control values. However, dexamethasone treatment produced a significant decrease in the nuclear binding using commercial AP-1 at 24 h, without any change at 4 h, in A549 cells. TNF-alpha exposure increased gamma -GCS-HS AP-1 DNA binding activity at 24 h, without any change in AP-1 binding of the commercial probe at 24 h in A549 epithelial cells. TNF-alpha -mediated increase in AP-1 DNA binding was abolished when cells were co-treated with dexamethasone at 4 and 24 h (Figs. 4 and 5). The specificity of the binding was checked using 100-fold excess unlabeled gamma -GCS-HS AP-1 and AP-1 oligonucleotides and nonspecific oligonucleotides for NF-kappa B (data not shown).


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Fig. 4.   Effect of TNF-alpha and dexamethasone on the regulation of gamma -GCS-HS AP-1 binding activity in A549 cells. A549 cells were treated with TNF-alpha (10 ng/ml) and dexamethasone (Dex, 3 µM), alone or in combination at the times indicated. A, nuclear extracts were prepared and analyzed by EMSA using 32P-labeled synthetic double-stranded oligonucleotides containing the proximal AP-1 sequence of the gamma -GCS-HS promoter. The DNA-protein complexes formed are indicated (arrow) for gamma -GCS-HS AP-1. B, the numeric estimates of DNA binding levels were compared with the control value set at 100%. The histograms represent the means and the error bars the S.E. of the relative intensity of the bands of six experiments. **, p < 0.01; #, ***, p < 0.001 compared with control values.


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Fig. 5.   Regulation of the transcription factor AP-1 by TNF-alpha and dexamethasone. A549 epithelial cells were treated with TNF-alpha (10 ng/ml) and dexamethasone (Dex, 3 µM) at the times indicated. A, nuclear extracts were isolated and incubated in the presence of labeled AP-1 oligonucleotide. DNA binding to AP-1 was analyzed by EMSA. The unbound (free) probe in the gel is indicated at the bottom. B, densitometric quantitation of AP-1 binding was compared with the control value set at 100%. The histograms represent the mean values and the error bars the S.E. of the relative intensity of the bands of three experiments. ***, p < 0.001, compared with control values.

Involvement of c-Jun in TNF-alpha -induced AP-1 DNA Binding in A549 Epithelial Cells-- To identify which component of AP-1 (c-Fos/c-Jun) is involved in the TNF-alpha -induced AP-1 DNA binding activity in A549 cells, we used antibodies to c-Jun and c-Fos in supershift assays. By using both the commercial AP-1 probe and the gamma -GCS-HS AP-1 probe, we demonstrate that the increased AP-1 binding activity present in TNF-alpha -treated cells was related to binding of c-Jun but not of c-Fos, since the addition of the c-Jun antibody shifted the AP-1 DNA band considerably (Fig. 6). Furthermore, both oligonucleotides were equally able to bind to c-Jun-purified protein (Promega) (data not shown).


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Fig. 6.   Effect of anti-c-Jun and anti-c-Fos antibodies on the DNA binding of AP-1 induced by TNF-alpha . Nuclear extracts of A549 epithelial cells treated with TNF-alpha (10 ng/ml) for 24 h were incubated with anti-c-Jun or anti-c-Fos antibody in a 20-µl binding reaction as described under "Experimental Procedures." A supershift assay of binding activity in nuclear extracts to labeled experimental commercial AP-1 or gamma -GCS-HS AP-1-binding elements was performed. Preincubation of nuclear extract with preimmune sera is shown as a control. Incubation with anti-c-Fos, anti-c-Jun sera before the gel shift assay results in a super-retardation in band mobility (arrow with ss). Experiments were repeated three times, and a representative autograph is shown.

Role of NF-kappa B in TNF-alpha and Dexamethasone-mediated Regulation of gamma -GCS-HS-- We also determined whether the pleiotropic transcription factor NF-kappa B was also affected by these treatments. Using a commercially available NF-kappa B oligonucleotide, TNF-alpha treatment of A549 cells produced a significant increase in NF-kappa B DNA binding both at 4 and 24 h (Fig. 7). By contrast, dexamethasone produced a significant decrease in NF-kappa B DNA binding activity at 24 h, without any change at 4 h, compared with control levels. TNF-alpha -mediated increased NF-kappa B binding activity was not inhibited either at 4 or 24 h by co-treatment with dexamethasone.


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Fig. 7.   Regulation of NF-kappa B binding in response to TNF-alpha and dexamethasone. Nuclear extracts were prepared from cultured A549 epithelial cells treated with TNF-alpha (10 ng/ml) and dexamethasone (Dex, 3 µM) at the various time points as indicated. A, the electrophoretic mobility shift assay was performed using labeled NF-kappa B oligonucleotide. Positions of specific DNA-protein complexes for NF-kappa B are indicated by the arrows. B, quantification of DNA binding was performed by densitometry and compared with the control values set at 100%. The histograms represent the mean values and the error bars the S.E. of the relative intensity of the bands of three experiments. ***, p < 0.001 compared with control values.

Effect of TNF-alpha and Dexamethasone on gamma -GCS-HS Promoter Construct-derived Chloramphenicol Acetyltransferase (CAT) Activity-- To confirm that AP-1 plays a key role in TNF-alpha -induced gamma -GCS-HS gene expression, we used a CAT reporter system to explore the mechanisms by which TNF-alpha and dexamethasone regulate gamma -GCS-HS at the transcriptional level. TNF-alpha treatment of A549 cells transfected with the full gamma -GCS-HS promoter linked to the CAT reporter system (pCBGCS, Fig. 8A) produced a significant increase in CAT activity at 24 h, compared with control values (Fig. 8B). By contrast, CAT activity was significantly decreased when the cells were treated with dexamethasone at 24 h. Dexamethasone also blocked the TNF-alpha -induced CAT activity at 24 h.


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Fig. 8.   Effects of TNF-alpha and dexamethasone on the transcriptional activity of the 5'-flanking region of the gamma -GCS-HS gene in A549 alveolar epithelial cells. A, the line diagram at the top is a restriction map of the gamma -GCS-HS promoter region. The block diagram shows the relative positions of the cis-acting DNA binding elements of the promoter region cloned in the pCRII vector. The numbers in the figure represent nucleotide positions from the transcriptional start site of the gamma -GCS-HS gene which is indicated by the bent arrow. The dotted line on the left indicates an additional 50 bp from multiple cloning sites of the pCRII vector. The structure of the gamma -GCS-HS-CAT plasmid is shown below on the right. The complete 5'-flanking region (pCBGCS) and a proximal putative AP-1 construct (pCBGCSDelta D) were ligated to the CAT gene in a pCAT Basic vector (pCB) and transfected into A549 epithelial cells. Transfected A549 cells were exposed to TNF-alpha (10 ng/ml) or dexamethasone (Dex, 3 µM) alone or in combination. After 24 h incubation, the cells were harvested and assayed for CAT activity by an enzyme-linked immunosorbent assay. B, transcriptional activity was standardized by the amount of CAT activity relative to beta -galactosidase activity. The results are shown as percentages of the CAT concentration compared with that of pCBGCS. The histograms represent the means and the error bars the S.E. of the CAT-derived activities of pCBGCS and pCBGCSDelta D constructs of three transfection experiments, each performed in duplicate with the activity of pCBGCS set at 100%. **, p < 0.01; ***, p < 0.001, compared with pCBGCS. dagger , p < 0.001, compared with TNF-alpha alone.

Effect of TNF-alpha and Dexamethasone on gamma -GCS-HS AP-1 Construct-derived CAT Activity-- To establish whether the TNF-alpha -induced gamma -GCS-HS AP-1 DNA binding activity was related to activation of the gamma -GCS-HS AP-1 fragment in the CAT reporter system (pCBGCSDelta D), we transfected epithelial cells with a plasmid containing the putative AP-1 transcription factor (pCBGCSDelta D). pCBGCSDelta D displayed significant TNF-alpha -induced CAT activity in cells transfected with pCBGCSDelta D, whereas dexamethasone inhibited the CAT activity (Fig. 8B). Furthermore, TNF-alpha -induced pCBGCSDelta D CAT activity was abolished when the transfected cells were co-treated with dexamethasone.

To confirm further whether the proximal putative AP-1 site is a key regulator of TNF-alpha -induced gamma -GCS-HS promoter activation, a construct was generated from the parent pCBGCS plasmid where the wild-type proximal AP-1 site was mutated (Fig. 9A). This mutated pCBmGCS reporter construct, where the proximal AP-1 site was abrogated, showed a reduced response to TNF-alpha , compared with the wild-type gamma -GCS-HS promoter (Fig. 9B). These data imply that the proximal AP-1-binding site present in -269 to -263 bp is the major cis-regulatory element responsible for the TNF-alpha induction of the gamma -GCS-HS gene.


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Fig. 9.   Effect of a mutation in the AP-1 site at -269 to -263 bp of the gamma -GCS-HS gene on transcriptional activity. A, this shows the putative AP-1 binding element at -269 to -263 bp contained within the wild-type pCBGCS promoter construct and mutated (-269 to -263 bp) pCBmGCS promoter construct used in this experiment. The numbers in the figure represent the nucleotide positions from the transcriptional start site of the gamma -GCS-HS gene. B, basal and TNF-alpha -stimulated transcriptional activity was determined after 24 h of incubations as described in Fig. 8. ***, p < 0.001 versus control; dagger , p < 0.001 versus TNF-alpha . The histograms represent the means and the error bars the S.E. of three separate experiments performed in duplicate.


    DISCUSSION

Rapid induction of intracellular GSH synthesis occurs in response to various oxidant stresses (36-38). This may be a critical determinant of cellular tolerance to oxidant stress. In this study, we demonstrated that TNF-alpha caused a transient depletion of GSH, concomitant with an increase in GSSG levels, suggesting that TNF-alpha induced oxidative stress in epithelial cells. However, prolonged exposure (24 h) to TNF-alpha increased GSH levels, without changing GSSG levels in A549 epithelial cells. The increase in GSH was associated with increased gamma -GCS activity. The levels of GSH and gamma -GCS activity are regulated by the expression of the catalytic gamma -GCS-HS (26, 27). Recently, we and others (27, 36-43) have demonstrated that oxidants, radiation, heat shock, heavy metals, and chemotherapeutic agents can increase GSH concentrations by induction of gamma -GCS-HS expression in various cell types. In this study, we show, for the first time, that in human alveolar epithelial cells (A549), gamma -GCS-HS gene expression is induced by TNF-alpha . Similar induction of gamma -GCS-HS mRNA and elevation of GSH levels have been shown in human hepatocytes and mouse endothelial cells treated with TNF-alpha (44, 45). Elevation of glutathione levels has also been observed in cultured rat hepatocytes following treatment with TNF-alpha , which protected the cells against the cytotoxic effects of further oxidant stresses (46). However, the mechanism of this induction was not studied.

To understand the molecular mechanism of the transcriptional induction of gamma -GCS-HS in response to TNF-alpha , we cloned the 5'-flanking region of the gamma -GCS-HS into a CAT reporter system. Following transfection into A549 epithelial cells, we observed that TNF-alpha up-regulated the promoter region of the gamma -GCS-HS gene, measured as increased CAT activity. This suggests that TNF-alpha acts at the level of transcription to induce gamma -GCS-HS mRNA in epithelial cells. The signaling mechanism whereby TNF-alpha exerts its effect is currently not known. TNF-alpha is known to generate ROS, particularly the superoxide anion (Obardot 2) and H2O2, by leakage from the electron transport chain in mitochondria. This could trigger transcriptional up-regulation of gamma -GCS-HS possibly by activating signaling pathways, such as activation of the c-Jun N-terminal kinase/stress activated protein kinase (47).

We next elucidated the transcriptional regulatory mechanism by which TNF-alpha exerted its effect on the induction of gamma -GCS-HS. By using deletion and mutation studies, we have recently shown the critical role of a putative AP-1 site in oxidant-mediated regulation of the gamma -GCS-HS promoter (15). However, other investigators have suggested that an NF-kappa B-binding site in the gamma -GCS-HS promoter may be important in regulating gamma -GCS-HS gene expression (40, 42). Activation of NF-kappa B is known to be regulated by a variety of pro-inflammatory (e.g. TNF-alpha ) and anti-inflammatory agents (e.g. dexamethasone) (17). We therefore used TNF-alpha and dexamethasone to assess the role of the AP-1 and NF-kappa B transcription factors in the transcriptional up-regulation of gamma -GCS-HS. Exposure of alveolar epithelial cells to TNF-alpha produced a significant increase in the DNA binding activities of both nuclear proteins, using AP-1 and NF-kappa B as consensus probes. Thus both AP-1 and NF-kappa B transcription factors are activated by TNF-alpha . AP-1 activation by TNF-alpha was confirmed by increased CAT activity in a CAT reporter system with a cloned gamma -GCS-HS-AP-1 fragment. These data indicate that NF-kappa B binding to a consensus site in the gamma -GCS-HS promoter is not necessary for TNF-alpha -induced transcriptional activation of the gamma -GCS-HS gene and suggest the involvement of an AP-1 response element. Further confirmation of the key role of a putative proximal AP-1-binding site in the regulation of the gamma -GCS-HS gene comes from the mutational study of the putative AP-1 site (-269 to -263 bp). Mutation of the sequence 5'-TTGATTCAA-3' to 5'-TGTTATCAA-3' in the proximal AP-1 oligonucleotide (pCBmGCS) effectively eliminated TNF-alpha -induced promoter activity. This provides strong evidence that this sequence, but not NF-kappa B, is involved in the regulation of the expression of the endogenous gamma -GCS-HS subunit gene.

To identify which components of AP-1 are responsible for the up-regulation of gamma -GCS-HS, we used antibodies directed against c-Fos and c-Jun in an effort to demonstrate a supershift in the EMSA. Only an antibody that cross-reacted with c-Jun produced a supershift, supporting the view that the AP-1 EMSA data is likely to have resulted from c-Jun/c-Jun homodimer binding to gamma -GCS-HS AP-1 sites. In support of this, several investigators have recently suggested possible involvement of AP-1/Jun family members in the regulation of gamma -GCS-HS (40, 42, 43, 48).

Corticosteroids, such as dexamethasone, are known to suppress both immune responses and inflammation by activation of the glucocorticoid receptor and by interaction with various transcription factors (17). The role of intracellular GSH redox status in the regulation of transcription factors is of considerable interest (17, 49). However, the effect of dexamethasone on the regulation of GSH synthesis has not been studied. We showed that depletion of intracellular GSH by dexamethasone occurs concomitant with a decrease in gamma -GCS activity, without any change in GSSG levels in A549 alveolar epithelial cells. However, the ratio of GSH/GSSG levels was decreased by dexamethasone, suggesting that dexamethasone may impose oxidative stress in A549 epithelial cells. Depletion of liver GSH in mice and inhibition of GSH synthesis by dexamethasone have been observed in a rat hepatic cell line (50, 51). Similarly, depletion of antioxidant enzyme activities have been shown in various rat tissues by glucocorticoid supplementation, and this depletion was more pronounced when rats were challenged with oxidative stress (52). In this study, we show that the depletion of GSH, decrease in GSH/GSSG ratio, and decreased gamma -GCS activity produced by dexamethasone is also associated with a decrease in the expression of gamma -GCS-HS mRNA. However, dexamethasone had no effect on the basal level of gamma -GCS-HS gene expression at 4 h. In support of these data, dexamethasone also had no effect on basal level of manganese superoxide dismutase mRNA expression, whereas TNF-alpha -induced gene expression was completely abolished by dexamethasone treatment (53).

Protein-protein interactions between the glucocorticoid receptor and the transcription factor AP-1 are thought to mediate a negative cross-talk between dexamethasone and TNF-alpha -induced gene regulation (17, 25). In this study, dexamethasone significantly inhibited TNF-alpha -mediated activation of gamma -GCS-HS AP-1 DNA binding activity and proximal AP-1 (pCBGCSDelta D)-derived CAT activity in alveolar epithelial cells. Dexamethasone also inhibited TNF-alpha -induced changes in GSH levels, gamma -GCS activity, gamma -GCS-HS mRNA expression, and gamma -GCS-HS promoter activity. Dexamethasone did not inhibit TNF-alpha -mediated activation of NF-kappa B but did block the increase in GSH and gamma -GCS-HS mRNA expression induced by TNF-alpha . These data support the concept that activation of NF-kappa B does not have a role in mediating the transcriptional activation of gamma -GCS-HS in response to TNF-alpha .

The results of our mutation and deletion studies confirm that the 5'-flanking proximal sequence of the gamma -GCS-HS gene, containing a putative AP-1-binding site at -269 to -263 bp, plays an important role in the transcriptional up-regulation of the gamma -GCS-HS gene in TNF-alpha -treated alveolar epithelial cells. The supershift assay showed that this AP-1 DNA-binding complex was predominantly formed by dimers of c-Jun. Furthermore, our data provide supportive evidence for negative interaction between glucocorticoid receptor and AP-1 (c-Jun) which prevents TNF-alpha -induced gamma -GCS-HS mRNA expression in A549 epithelial cells.

In conclusion, these studies show that TNF-alpha causes an increase in intracellular GSH content, gamma -GCS activity, and transcriptional activation of gamma -GCS-HS in alveolar epithelial cells, whereas dexamethasone decreased GSH levels by down-regulating the transcription of the gamma -GCS-HS gene. Electrophoretic mobility gel shift and CAT reporter assays revealed that the modulation of gamma -GCS-HS gene expression in alveolar epithelial cells by TNF-alpha and dexamethasone occurs by a mechanism involving AP-1 (c-Jun homodimer). These data may have implications for dexamethasone treatment in patients with inflammatory lung diseases, since such treatment may prevent synthesis of increased levels of the protective antioxidant GSH.

    ACKNOWLEDGEMENTS

We thank Celine Lessard and Maryline Parmentier for their technical assistance.

    FOOTNOTES

* This work was supported by the British Lung Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Rayne Laboratory, Respiratory Medicine Unit, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, UK. Tel.: 44 131 651 1523; Fax: 44 131 650 4384; E-mail: IR{at}srv1.med.ed.ac.uk.

    ABBREVIATIONS

The abbreviations used are: gamma -GCS, gamma -glutamylcysteine synthetase; gamma -GCS-HS, gamma -glutamylcysteine synthetase-heavy subunit; TNF-alpha , tumor necrosis factor-alpha ; EMSA, electrophoretic mobility shift assay; AP-1, activator protein-1; NF-kappa B, nuclear factor-kappa B; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ROS, reactive oxygen species; bp, base pair(s); CAT, chloramphenicol acetyltransferase; DMEM, Dulbecco's modified minimum essential medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; BSO, DL-buthionine-(SR)-sulfoximine.

    REFERENCES
Top
Abstract
Introduction
References
  1. Rahman, I., Li, X. Y., Donaldson, K., Harrison, D. J., and MacNee, W. (1995) Am. J. Physiol. 269, L285-L292[Abstract/Free Full Text]
  2. Meister, A., and Anderson, M. E. (1983) Annu. Rev. Biochem. 52, 711-760[CrossRef][Medline] [Order article via Infotrieve]
  3. Cantin, A. M., North, S. L., Hubbard, R. C., and Crystal, R. G. (1987) J. Appl. Physiol. 63, 152-157[Abstract/Free Full Text]
  4. Li, X. Y., Donaldson, K., Rahman, I., and MacNee, W. (1994) Am. J. Respir. Crit. Care Med. 149, 1518-1525[Abstract]
  5. Lannan, S., Donaldson, K., Brown, D., and MacNee, W. (1994) Am. J. Physiol. 266, L92-L100[Abstract/Free Full Text]
  6. Morris, P. E., and Bernard, G. R. (1994) Am. J. Med. Sci. 307, 119-127[Medline] [Order article via Infotrieve]
  7. Staal, F. J. T., Ela, S. W., Roederer, M., Anderson, M. T., and Herzenberg, L, A. (1992) Lancet 339, 909-912[CrossRef][Medline] [Order article via Infotrieve]
  8. Cantin, A. M., Hubbard, R. C., and Crystal, R. G. (1989) Am. Rev. Respir. Dis. 139, 370-372[Medline] [Order article via Infotrieve]
  9. Bunnel, E., and Pacht, E. R. (1993) Am. Rev. Respir. Dis. 148, 1174-1178[Medline] [Order article via Infotrieve]
  10. Roum, J. H., Behld, R., McElvancy, N. G., Borok, Z., and Crystal, R. G. (1993) J. Appl. Physiol. 75, 2419-2424[Abstract]
  11. Melloni, B., Lefebvre, M.-A., Bonnaud, F., Vergnenegre, A., Grossin, L., Rigaud, M., and Cantin, A. (1996) Am. J. Respir. Crit. Care Med. 154, 1706-1711[Abstract]
  12. Seelig, G. F., and Meister, A. (1984) J. Biol. Chem. 259, 3534-3538[Abstract/Free Full Text]
  13. Seelig, G. F., Simondsen, R. P., and Meister, A. (1984) J. Biol. Chem. 259, 9345-9347[Abstract/Free Full Text]
  14. Huang, C. S., Chang, L. S., Anderson, M. E., and Meister, A. (1993) J. Biol. Chem. 268, 19675-19680[Abstract/Free Full Text]
  15. Rahman, I., Smith, C. A. D., Antonicelli, F., and MacNee, W. (1998) FEBS Lett. 427, 129-133[CrossRef][Medline] [Order article via Infotrieve]
  16. Arai, K. I., Lee, F., Miyajima, A., Miyatake, S., Arai, N., and Yokota, T. (1990) Annu Rev. Biochem. 59, 783-836[CrossRef][Medline] [Order article via Infotrieve]
  17. Rahman, I., and MacNee, W. (1998) Thorax 53, 601-612[Free Full Text]
  18. Zimmerman, R. J., Marafino, B. J., Jr., Chan, A., Landre, P., and Winkelhake, J. L. (1989) J. Immunol. 142, 1405-1409[Abstract/Free Full Text]
  19. Wong, G. H., and Goeddel, D, V. (1988) Science 242, 941-944[Medline] [Order article via Infotrieve]
  20. Dickison, J. L., Bates, E. J., Ferrante, A., and Antalis, T. M. (1995) J. Biol. Chem. 270, 27894-27904[Abstract/Free Full Text]
  21. Opipari, A. W., Jr., Hu, H. M., Yabkowitz, R., and Dixit, V. M. (1992) J. Biol. Chem. 267, 12424-12427[Abstract/Free Full Text]
  22. Karsan, A., Yee, E., and Harlan, J. M. (1996) J. Biol. Chem. 271, 27201-27204[Abstract/Free Full Text]
  23. Schulze-Osthoff, K., Bakker, A. C., Vanhaesebroeck, B., Beyaert, R., Jacob, W. A., and Fiers, W. (1996) J. Biol. Chem. 267, 5317-5323[Abstract/Free Full Text]
  24. Richter, C., Cogvadze, V., Laffranchi, R., Schlapbach, R., Schweizer, M., and Suter, M. (1995) Biochim. Biophys. Acta 127, 67-74
  25. Tsai, M. J., and O'Malley, B. W. (1994) Annu. Rev. Biochem. 63, 451-486[CrossRef][Medline] [Order article via Infotrieve]
  26. Rahman, I., Smith, C. A. D., Lawson, M. F., Harrison, D. J., and MacNee, W. (1996) FEBS Lett. 396, 21-25[CrossRef][Medline] [Order article via Infotrieve]
  27. Rahman, I., Bel, A., Mulier, B., Lawson, M. F., Harrison, D. J., MacNee, W., and Smith, C. A. D. (1996) Biochem. Biophys. Res. Commun. 229, 832-837[CrossRef][Medline] [Order article via Infotrieve]
  28. Rahman, I., Bel, A., Mulier, B., Donaldson, K., and MacNee, W. (1998) Am. J. Physiol. 275, L80-L86[Abstract/Free Full Text]
  29. Tietze, F. (1969) Anal. Biochem. 27, 502-522[Medline] [Order article via Infotrieve]
  30. Griffith, O. W. (1980) Anal. Biochem. 106, 207-212[Medline] [Order article via Infotrieve]
  31. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Anal. Biochem. 150, 76-85[Medline] [Order article via Infotrieve]
  32. Gipp, J. J., Chang, C., and Mulcahy, R. T. (1992) Biochem. Biophys. Res. Commun. 185, 29-35[Medline] [Order article via Infotrieve]
  33. Maier, J. A. M., Voulalas, P., Roeder, D., and Maciag, T. (1990) Science 249, 1570-1573[Medline] [Order article via Infotrieve]
  34. Rahman, I., Smith, C. A. D., Lawson, M. F., Harrison, D. J., and MacNee, W. (1997) FEBS Lett. 411, 393-395[CrossRef]
  35. Staal, F. J. T., Roederer, M., Herzenberg, L. A., and Herzenberg, L. A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9943-9947[Abstract]
  36. Shi, M. M., Kugelman, A., Iwamoto, T., Tian, L., and Forman, H. J. (1994) J. Biol. Chem. 269, 26512-26517[Abstract/Free Full Text]
  37. Morales, A., Miranda, M., Sanchez-Reyes, A., Colell, A., Biete, A., and Fernandez-Checa, J. C. (1998) FEBS Lett. 427, 15-20[CrossRef][Medline] [Order article via Infotrieve]
  38. Kondo, T., Yoshida, K., Urata, Y, Goto, S., Gasa, S., and Taniguchi, N. (1993) J. Biol. Chem. 268, 20366-20372[Abstract/Free Full Text]
  39. Ishikawa, T., Bao, J. J., Yamane, Y., Akimaru, K., Frindrich, K., Wright, C. D., and Kuo, M. T. (1996) J. Biol. Chem. 271, 14981-14988[Abstract/Free Full Text]
  40. Yao, K. S., Godwin, A. K., Johnson, S. W., Ozols, R. F., O'Dwyer, P. J., and Hamilton, T. C. (1995) Cancer Res. 55, 4367-4374[Abstract]
  41. Mulcahy, R. T., Wartman, M. A., Bailey, H. H., and Gipp, J. J. (1997) J. Biol. Chem. 272, 7445-7454[Abstract/Free Full Text]
  42. Tomonari, A., Nishio, K., Kurokawa, H., Fukumoto, H., Fukuoka, K., Iwamoto, Y., Usuda, J., Suzuki, T., Itakura, M., and Saijo, N. (1997) Biochem. Biophys. Res. Commun. 236, 616-621[CrossRef][Medline] [Order article via Infotrieve]
  43. Sekhar, K. R., Meredith, M. J., Kerr, L. D., Soltaninassab, S. R., Spitz, D. R., Xu, Z. Q., and Freeman, M. L. (1997) Biochem. Biophys. Res. Commun. 234, 488-593
  44. Morales, A., Garcia-Ruiz, C., Miranda, M., Mari, M., Collell, A., Ardite, E., and Fernandez-Checa, J. C. (1997) J. Biol. Chem. 272, 30371-30379[Abstract/Free Full Text]
  45. Urata, Y., Yamamoto, H., Goto, S., Tsushima, H., Akazawa, S., Yamashita, S., Nagataki, S., and Kondo, T. (1996) J. Biol. Chem. 271, 15146-15152[Abstract/Free Full Text]
  46. Imanishi, H., Scales, W. E., and Campbell, D. A., Jr. (1997) Biochem. Biophys. Res. Commun. 230, 120-124[CrossRef][Medline] [Order article via Infotrieve]
  47. Lo, Y. Y. C., Wong, J. M. S., and Cruz, T. F. (1996) J. Biol. Chem. 271, 15703-15707[Abstract/Free Full Text]
  48. Tu, Z., and Anders, M. W. (1998) Biochem. Biophys. Res. Commun. 244, 801-805[CrossRef][Medline] [Order article via Infotrieve]
  49. Sen, S. K., and Packer, L. (1996) FASEB J. 10, 709-720[Abstract/Free Full Text]
  50. Madhu, C., Maziasc, T., and Klaassen, C. D. (1992) Toxicol. Appl. Pharmacol. 115, 191-198[Medline] [Order article via Infotrieve]
  51. Lu, S. C., Kuhlenkamp, J., Garcia-Ruiz, C., and Kaplowitz, N. (1991) J. Clin. Invest. 88, 260-269[Medline] [Order article via Infotrieve]
  52. McIntosh, L. J., Hong, K. E., and Sopolsky, R. M. (1998) Brain Res. 791, 209-214[CrossRef][Medline] [Order article via Infotrieve]
  53. Antras-Ferry, J., Maheo, K., Morel, F., Guillouze, A., Cillard, P., and Cillard, J. (1997) FEBS Lett. 403, 100-104[CrossRef][Medline] [Order article via Infotrieve]


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