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[Cancer Research 64, 7780-7786, November 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

The {gamma}-Glutamylcysteine Synthetase and Glutathione Regulate Asbestos-induced Expression of Activator Protein-1 Family Members and Activity

Arti Shukla1, Trisha Flanders1, Karen M. Lounsbury2 and Brooke T. Mossman1

Departments of 1 Pathology and 2 Pharmacology, University of Vermont, Burlington, Vermont


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Asbestos fibers cause persistent increases in activator protein-1 (AP-1) family member proto-oncogenes in lung epithelial and mesothelial cells that are linked to proliferation and cell transformation. Using lung epithelial cells, the progenitor cells of lung cancers, we report that crocidolite asbestos initially depletes intracellular glutathione followed by up-regulation of both catalytic and modifier subunits of {gamma}-glutamylcysteine synthetase. In vivo asbestos inhalation experiments confirm increased protein levels of {gamma}-glutamylcysteine synthetase in mouse lungs. We also show that asbestos-induced mRNA levels of fos/jun proto-oncogenes, fra-1 transactivation, and AP-1 to DNA binding activity are glutathione-dependent. Epidermal growth factor receptor activity by asbestos is blocked by N-acetyl-L-cysteine, suggesting that it is an initial redox-activated event leading to downstream AP-1 proto-oncogene up-regulation. The overexpression of subunits of {gamma}-glutamylcysteine synthetase in combination completely blocked asbestos-induced up-regulation of AP-1 proto-oncogene expression. However, when overexpressed individually, the modifier subunit had more dramatic effects than the catalytic subunit. Our work shows that the glutathione-controlled redox status of the epithelial cell plays a pivotal role in asbestos-induced epidermal growth factor receptor and proto-oncogene activation as well as AP-1 activity.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Asbestos fibers cause oxidative stress and play an important role in the pathogenesis of many occupational lung diseases, including asbestosis, pleural effusions, pleural plaques, and malignancies such as lung cancer and pleural mesothelioma (1) . The mechanisms by which asbestos induces oxidant injury, inflammation, and cancers are poorly understood but are thought to involve the generation of reactive oxygen species after frustrated phagocytosis of long (>5 µm) asbestos fibers or from redox reactions occurring on the surface of high iron-containing fibers such as crocidolite [(Na2(Fe3+)2(Fe2+)3Si8O22(OH)2] (2) .

The delay from initial exposure to the development of asbestos-related cancers suggests that asbestos fibers act at several steps of the carcinogenic process, i.e., initiation, promotion, and progression. Our laboratory has focused on the molecular responses that occur in mesothelial and epithelial cells in vitro and in vivo after exposure to asbestos, because these cells are progenitors of asbestos-induced mesotheliomas and lung cancers, respectively. Moreover, the pulmonary epithelial cell plays a critical role in the development of pulmonary fibrosis (3) .

Activator protein-1 (AP-1), composed of members of the Fos and Jun early response proto-oncogene family, is a redox-sensitive transcription factor associated with the development of cell proliferation and tumor promotion (4 , 5) . Activation of key genes is associated with increased binding of the transcription factor, AP-1, to the DNA of the promoter region of a number of genes causally associated with cell proliferation. Crocidolite asbestos causes a persistent increase in expression of early response genes and AP-1 activity in the lung, which is preceded by phosphorylation of the epidermal growth factor receptor (EGFR) and activation of ERKs (extracellular signal-regulated kinase) 1/2 and ERK5 (6, 7, 8, 9) . Recently, we have shown that mesothelial cell transformation is accompanied by increased AP-1 binding activity and requires ERK1/2-dependent Fra-1 expression (10) .

Although numerous studies have suggested a central role of reactive oxygen species in asbestos fiber-induced toxicity and their contribution to asbestos-related lung diseases (2 , 11) , how the redox environment of the cell contributes to asbestos-induced alterations in proto-oncogene expression and AP-1 activity has previously been unclear. In the present investigation, we show that crocidolite asbestos at noncytolytic concentrations causes glutathione depletion of lung epithelial cells (C10 line), resulting in an imbalance of the redox status of the cell. Subsequently, the imbalance in redox status of the cell causes the initiation of cell signaling events, resulting in phosphorylation of the EGFR, overexpression of different AP-1 proto-oncogenes including fra-1, and AP-1 transactivation. We also show these events can be reversed either by chemical intervention of reduced glutathione levels or by overexpressing the plasmids for catalytic (gclc) and regulatory (modifier, gclm) subunits for {gamma}-glutamylcysteine synthetase. Our results are the first to reveal that the glutathione status of the cell governs cell signaling and AP-1 transactivation by asbestos.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
N-acetyl-L-cysteine, buthionine sulfoximine (BSO), glutathione ester, myelin basic protein, deferoxamine, and other chemicals were purchased from Sigma Chemical Company (St. Louis, MO). Catalase was from Calbiochem (La Jolla, CA). Antibody for catalytic subunit of {gamma}-glutamylcysteine synthetase was from Lab Vision Corp. (NeoMarkers, Fremont, CA). TaqMan primers and probes were synthesized from MWG Biotech, Inc. (High Point, NC). Monobromobimane was purchased from Calbiochem. In cell culture experiments crocidolite asbestos fibers, the most pathogenic asbestos type, were used (NIEHS, reference samples) and have been characterized previously for their chemical and physical features (12) . Because of the depletion of NIEHS reference samples of crocidolite fibers in quantities required for inhalation experiments, NIEHS reference standards of chrysotile (12) were used in animal studies.

Cell Culture and Exposure to Test Agents.
A contact inhibited, nontransformed murine alveolar type II epithelial cell line (C10; ref. 13 ) was propagated in Connaught Medical Research Laboratories-1066 medium containing penicillin, streptomycin, L-glutamine, and 10% fetal bovine serum (Life Technologies, Inc., Grand Island, NY). For all of the experiments, cells were grown to confluence, complete medium was removed, and medium containing 0.5% fetal bovine serum was added 24 hours before exposure to agents. Crocidolite asbestos fibers were suspended in HBSS (Life Technologies, Inc.) at 1 mg/ml, sonicated, and then titurated 10x through a 22-gauge needle to obtain a homogenous suspension, before addition directly to medium at a concentration of 5 µg/cm2 surface area of culture dish. In some experiments, cells were pre-exposed to glutathione ester for 1 hour or to N-acetyl-L-cysteine (10 mmol/L) or BSO (10 µmol/L) for 20 hours before the addition of asbestos. This protocol was necessary both to allow changes in glutathione levels to occur before exposure to asbestos, as verified by high-performance liquid chromatography, and to eliminate the possibility of an interaction between compounds and asbestos. N-acetyl-L-cysteine was dissolved in HBSS, and the pH was adjusted to 7.4 with NaOH before addition to medium. In some experiments, catalase (500 units/ml) was added to medium 1 hour before asbestos exposure, and heat-inactivated catalase was used as a control. To chelate redox-causing metals such as iron, asbestos samples were treated with 1 mmol/L deferoxamine for 24 hours, washed with HBSS, and resuspended in HBSS before addition to cells.

RNase Protection Assays.
Cells grown on 60-mm dishes were exposed to asbestos and/or different chemicals for different time periods. Total RNA was prepared and quantitated as described by Shukla et al. (14) . Steady-state mRNA levels of c-jun, junB, junD, c-fos, fra-1, fra-2, and fosB, the ribosomal probe, L32, and glyceraldehyde-3-phosphate dehydrogenase were examined with a RiboQuant multiprobe RNase protection assay system and the mfos/Jun multiprobe template set (PharMingen, San Diego, CA) according to the manufacturer’s protocol. Autoradiograms were quantitated with a Bio-Rad (Richmond, CA) phosphoimager. Results were normalized to expression of the housekeeping gene, L32.

Electrophoretic Mobility Shift Assay.
Electrophoretic gel mobility shift assays were used to assess the binding of AP-1 to DNA and the composition of AP-1 complexes. Nuclear extracts of sham or agent-exposed C10 cells were prepared as described previously (14) . The amount of protein in each sample was determined with the Bio-Rad protein assay (Bio-Rad). A 32P-end-labeled double-stranded oligodeoxynucleotide representing the specific element that contains a TPA response element consensus sequence (i.e., TGACTCA) was incubated with 3 µg of extract as described previously (14) . The components of the AP-1 complex were identified by supershift analysis with antibodies specific for Fra-1 and c-Jun (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Autoradiograms were developed and quantitated with a Bio-Rad phosphorimager.

EGFR Kinase Activity Assay.
EGFR kinase activity was determined with an immunoprecipitation kinase assay as follows: soluble protein was prepared as described elsewhere (15) ; the protein (300 µg) then was immunoprecipitated for 2 hours at 4°C with 2 µg of anti-EGFR antibody (Santa Cruz Biotechnology, Inc.); and the antigen-antibody complexes were collected by incubation with agarose protein A (BRL, Life Technologies, Inc.) for 1 hour at 4°C, then pellets were washed three times with lysis buffer and 3 times with kinase buffer (20 mmol/L HEPES, 10 mmol/L MnCl2, 10 mmol/L MgCl2, 1 mmol/L DTT, 100 µmol/L Na3VO4, and 10 µmol/L ATP) before resuspension in a reaction buffer containing 25 µL kinase buffer, myelin basic protein and 5 µCi of [{gamma}-32P]ATP (New England Nuclear, Life Science Products, Inc., Boston, MA). All of the samples were incubated for 20 minutes at 30°C. Reactions were terminated by addition of 2x SDS sample buffer, boiled, and the reaction products resolved on a 15% SDS-polyacrylamide gel. The extent of myelin basic protein phosphorylation was determined by autoradiography.

Western Blotting.
Cells grown in culture dishes were washed three times with ice-cold PBS and collected in lysis buffer [20 mmol/L Tris (pH7.6), 1% Triton X-100, 137 mmol/L NaCl, 2 mmol/L EDTA, 1 mmol/L Na3O4V, 10 mmol/L NaF, 1 mmol/L DTT, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin] before incubation on ice for 30 minutes. Cells were then centrifuged at 14,000 x g for 10 minutes at 4°C. Supernatants were collected, and protein concentrations were determined with the Bradford assay (Bio-Rad). Cell lysates (40 µg) were resolved by SDS-PAGE and transferred to nitrocellulose membranes according to standard procedures. Equal loading of protein was verified by Ponceau stain (Sigma). Membranes were washed in TBS, blocked for 30 minutes with TBS containing 5% nonfat milk, then incubated with primary antibodies at 5 µg/ml dilution in PBS containing 1% BSA overnight at 4°C. Membranes were then washed twice with TBS alone and twice with PBS containing 0.1% Tween 20 before incubation with horseradish peroxidase-conjugated secondary antibody (1:5,000 in PBS containing 0.1% Tween 20 containing 5% nonfat milk) for 1 hour at room temperature. Membranes were washed once with PBS containing 0.1% Tween 20 and three times with PBS before antibody binding was visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s protocol.

Luciferase Activity Assay.
Epithelial cells (C10) were transiently cotransfected with 2 µg reporter plasmid, fra-1 promoter-luciferase (a kind gift from Dr. Sekhar Reddy, Johns Hopkins University, Baltimore, MD), and renilla (0.5 µg) with LipofectAMINE 2000 (Invitrogen, Life Technologies, Inc., Grand Island, NY) according to the manufacturer’s instructions. After 24 hours of transfection, cells were switched to 0.5% fetal bovine serum before exposure to different agents. After exposure to asbestos (24 hours) and/or N-acetyl-L-cysteine or BSO (20 hours), total cell extracts were prepared and assayed for luciferase and renilla activity (Luciferase Assay System, Promega Corp., Madison, WI) with a luminometer (Berthold Technologies, Lumate, Germany). Luciferase activity was expressed as ratio of luciferase to renilla.

Measurement of Reduced Glutathione by High-Performance Liquid Chromatography.
Cell lysates were prepared as for Western blots. Supernatants then were mixed in a 1:1 ratio with monobromobimane solution [4 mmol/L in 50 mmol/L N-ethylmorpholine (pH 8.0); Calbiochem] and allowed to react for 5 minutes in the dark. To this final reaction mixture, 100% trichloroacetic acid was added to achieve a final trichloroacetic acid concentration of 5%. Samples were centrifuged at 3000 x g for 5 minutes to remove precipitated proteins. Supernatants were used to detect reduced glutathione by high-performance liquid chromatography with a Waters Symmetree C-18 column (150 x 4 mm). The mobile phase consisted of 10% acetonitrile and 0.25% glacial acetic acid in H2O. Reduced glutathione (GSH)-monobromobimane was detected by fluorescence (ex. 395 and em. 480 nm; ref. 16 ) and expressed per mg of protein.

Transfections.
C10 cells were transiently transfected with mammalian expression vector pCR3.1-Uni containing amino acid coding regions of the cDNAs of {gamma}-glutamylcysteine synthetase catalytic (gclc) or regulatory (modifier) subunit (gclm) of mouse or vector alone (a kind gift from Dr. Terry Kavanagh, University of Washington, Seattle, WA). The cells were transfected with LipofectAMINE 2000 according to the manufacturer’s specifications. Twenty-four hours after transfection, RNA and protein levels of both genes were substantially up-regulated as assessed by TaqMan and Western blotting, respectively (data not shown). At this time, the medium was changed to 0.5% serum-containing medium, and cells were exposed to asbestos for 8 hours. RNA was extracted from these cells with procedures described above.

TaqMan (Quantitative Reverse Transcriptase-PCR).
Total RNA was extracted from cells as described for RNase Protection Assays. Total RNA was then purified with the RNA cleaning kit from Qiagen Inc. (Valencia, CA) following the manufacturer’s instruction. After purification, the samples were treated with RNase-free DNase I (Promega) to remove contaminating genomic DNA. The RNA was then used to generate cDNA with the Reverse Transcription System (Promega), according to the manufacturer’s instruction. The Perkin-Elmer AB1 7700 prism Sequence Detection System (Applied Biosystems, Foster City, CA) was used to determine relative levels of expression of gclc and gclm. All values were normalized to the expression of hprt.

Inhalation Experiments.
Experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals (publication 85-23, 1985) following protocols approved by the University of Vermont (Institutional Animal Care and Use Committee). C57/BL6 mice (8 to 12 weeks of age) were exposed to ambient air or chrysotile asbestos (7 mg/m3 air, 6 hours/day, 5 days/week for 9 days) as described previously (17) . Briefly, mice were euthanized with pentobarbital (Abbott Laboratories, Abbot Park, IL), and lungs were instilled with PBS. Lungs (n = 4/group) were removed, snap frozen in liquid nitrogen, and stored at –80°C for Western blot analysis as described above.

Statistical Analyses.
In all of the experiments, duplicate or triplicate determinations per group per time point were done. Experiments were repeated three times or more. Results were evaluated by one-way ANOVA with the Student-Newman-Keuls procedure for adjustment of multiple pairwise comparisons between treatment groups. Differences of P = ≤0.05 were considered statistically significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Asbestos-induced Effects on Reduced Glutathione Levels in Lung Epithelial Cells.
We first examined levels of reduced glutathione as determined by high-performance liquid chromatography in C10 cells after exposure to crocidolite asbestos (5 µg/cm2) over time. Asbestos caused an initial decrease in reduced glutathione from 2 to 8 hours, which progressively increased over a 48 hours time period (Table 1)Citation . As expected, addition of N-acetyl-L-cysteine to C10 cells alone strikingly increased glutathione levels, whereas BSO caused depletion.


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  		Table 1 Reduced glutathione levels in C10 cells as measured by HPLC

 
Asbestos-induced Up-regulation of AP-1 Proto-Oncogenes Is Down-regulated by N-Acetyl-L-Cysteine and Glutathione Ester and Up-regulated by BSO.
Cells were pretreated with N-acetyl-L-cysteine (10 or 20 mmol/L for 20 hours), glutathione ester (5 and 10 mmol/L for 1 hour), or BSO (5, 10, or 50 µmol/L for 20 hours) before exposing them to asbestos. Pretreatment of cells with N-acetyl-L-cysteine, the precursor of glutathione, or glutathione ester, the cell-permeable form of glutathione, caused decreases in asbestos-induced steady-state mRNA levels of c-jun, junB, c-fos, and fra-1 (Fig. 1, A and B)Citation . On the other hand, pretreatment of cells with BSO, an irreversible inhibitor of the rate-limiting enzyme of glutathione synthesis ({gamma}-glutamylcysteine synthetase), which depleted intracellular glutathione (see Table 1Citation ), increased steady-state mRNA levels of these genes (Fig. 1C)Citation . Addition of the exogenous antioxidant, catalase, and the iron chelator, deferoxamine, had no effect on asbestos-induced proto-oncongene expression (data not shown).



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  		Fig. 1. RNase protection assay showing increased steady-state mRNA levels of AP-1 family members at 8 hours after asbestos (croc) exposure and their inhibition by (A) N-acetyl-L-cysteine (10 mmol/L for 20 hours) and (B) glutathione ester (5 and 10 mmol/L for 1 hour). C, the effect of BSO (5 and 10 µmol/L for 24 hours) on asbestos-induced fos/jun family mRNA steady-state mRNA levels. D. Transcriptional activation of the fra-1 gene by asbestos (5 µg/cm2, 24 hours) and modulating effects of N-acetyl-L-cysteine and BSO. C10 cells were transiently transfected with a fra-1 promoter-luciferase reporter construct (2.0 µg) and renilla plasmid (0.5 µg). Protocols are as described in Material and Methods. *, P ≤ 0.05 in comparison to untreated control group. #, P ≤ 0.05 in comparison to asbestos-exposed groups. Bars, ±SD. (NAC, N-acetyl-L-cysteine; gapdh, glyceraldehyde-3-phosphate dehydrogenase)

 
Asbestos-induced fra-1 Transactivation, the Presence of Fra-1 in AP-1 Complexes, and Up-regulation of AP-1 DNA Binding Activity Are Inhibited by N-Acetyl-L-Cysteine and Increased by BSO.
To determine whether glutathione status affected transcriptional regulation of fra-1, a proto-oncogene associated with cell transformation (10) . We used a fra-1 promoter and luciferase reporter complex to study fra-1 transactivation by asbestos. C10 cells were transiently transfected with a promoter-reporter construct, and luciferase activity was measured as described in Materials and Methods. Asbestos exposure (5 µg/cm2) for 24 hours increased fra-1 promoter activity, which was diminished by N-acetyl-L-cysteine pretreatment and increased by BSO pretreatment (Fig. 1D)Citation .

In Fig. 2ACitation , we also show that increased AP-1 to DNA binding activity by asbestos is inhibited by N-acetyl-L-cysteine pretreatment of C10 cells (20 hours). Supershift analysis with c-Jun and Fra-1 antibodies shows markedly decreased levels of these proteins in the AP-1 complex of N-acetyl-L-cysteine pretreated samples, whereas BSO pretreatment increased fra-1 levels above those observed with asbestos alone (Fig. 2, B and C)Citation .



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  		Fig. 2. Electrophoretic mobility shift assay showing AP-1 binding to DNA at 8 hours after addition of asbestos fibers. A. Effects of N-acetyl-L-cysteine and BSO on asbestos-induced AP-1 binding to DNA. B. Supershift analysis with (B) c-Jun and (C) Fra-1 antibodies in N-acetyl-L-cysteine and BSO pretreated samples. (NAC, N-acetyl-L-cysteine)

 
EGFR Kinase Activity Induced by Asbestos Is Inhibited by N-Acetyl-L-Cysteine.
As asbestos is known to phosphorylate EGFR, which is linked causally to ERK 1/2 activation and AP-1 proto-oncogene expression (8) , we studied the time course of asbestos-induced EGFR activity and the role of antioxidants in an EGFR kinase activity assay. Asbestos caused progressive increases in EGFR activity from 1 to 8 hours (Fig. 3A)Citation , but levels returned to control activity at 48 hours. Pretreatment of cells with N-acetyl-L-cysteine (10 mmol/L for 20 hours) before asbestos exposure for 8 hours decreased phosphorylation of myelin basic protein by EGFR, indicating its inhibition of asbestos-induced EGFR activity (Fig. 3B)Citation . However, catalase (Fig. 3C)Citation and deferoxamine (Fig. 3D)Citation had no effect on asbestos-induced EGFR activation.



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  		Fig. 3. Kinase activity assay showing (A) time frame of increased EGFR activity by asbestos (5 µg/cm2). B, effects of N-acetyl-L-cysteine. C, catalase. D, deferoxamine. Epidermal growth factor (5 ng/ml) and H2O2 (300 µmol/L) were added for 1 hour as positive controls. Arrows show phosphorylated myelin basic protein, which was used as a substrate. (MBP, myelin basic protein; EGF, epidermal growth factor; NAC, N-acetyl-L-cysteine; Cat, catalase; Def, deferoxamine)

 
Asbestos Exposure Causes Increased Protein and mRNA Levels of {gamma}-Glutamylcysteine Synthetase.
Protracted asbestos exposure resulted in time-dependent increases in mRNA and protein levels of both catalytic and modifier subunits of {gamma}-glutamylcysteine synthetase as determined by TaqMan and Western blotting, respectively (Fig. 4, A–C)Citation . Significant increases in mRNA levels of both catalytic and modifier subunits were observed at 8 hours, persisting for 96 hours after initial exposures to asbestos (Fig. 4, A and B)Citation . Western blot analysis with an antibody against the catalytic subunit showed moderate increases in protein levels at 24 and 48 hours (Fig. 4C)Citation . Asbestos inhalation for 9 days also caused increased protein levels of the catalytic subunit of {gamma}-glutamylcysteine synthetase in whole lung samples from mice (Fig. 4D)Citation .



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  		Fig. 4. A and B. Quantitative-PCR (TaqMan) data showing increases in mRNA levels of subunits of {gamma}-glutamylcysteine synthetase after asbestos exposures at different time points. *, P ≤ 0.05 in comparison to 0 hours group. C. Increase in protein levels of {gamma}-glutamylcysteine synthetase catalytic subunit after asbestos exposure as analyzed by Western blotting. Kidney was used as a positive control for protein expression. D. Increase in protein levels of {gamma}-glutamylcysteine synthetase catalytic subunit in murine lung tissues after 9 days of inhalation of asbestos. Bars, ±SD. ({gamma}GCS, {gamma}-glutamylcysteine)

 
Transfection with Subunits of {gamma}-Glutamylcysteine Synthetase Inhibits Asbestos-induced AP-1 Proto-Oncogene Up-regulation and Depletion of Reduced Glutathione.
C10 cells were transiently transfected with either catalytic (gclc) or modifier (gclm) subunits alone or together. Twenty-four hours after transfection, expression of both subunits was confirmed by TaqMan, indicating a several-fold increase in levels of these genes (data not shown). Individual subunit transfection experiments with asbestos showed more dramatic inhibition of asbestos-induced proto-oncogene expression by the modifier subunit as compared with the catalytic subunit (Fig. 5A)Citation . However, cells transfected with both subunits of {gamma}-glutamylcysteine synthetase showed complete inhibition of asbestos-induced mRNA levels (Fig. 5B)Citation . Table 1BCitation verifies that C10 cells transfected with {gamma}-glutamylcysteine synthetase subunits exhibited elevated levels of reduced glutathione, although additive effects were not observed in cotransfection groups. However, cotransfection of both subunits was more effective than transfection of individual subunits in boosting glutathione levels that were reduced by asbestos.



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  		Fig. 5. RNase protection assay showing effect of overexpression of catalytic or regulatory subunit (A) or both subunits (B) on asbestos-induced up-regulation of AP-1 proto-oncogene steady-state mRNA levels. C10 cells were transiently transfected with plasmids containing cDNA for catalytic or regulatory subunit or both. *, P ≤ 0.05 in comparison with respective control group.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The redox environment of a cell is composed of a number of factors that are classified as pro-oxidants or antioxidants. A balance between pro-oxidants and antioxidant status generally occurs in homeostatic, but exposure to fibers at high concentrations can tilt this balance, eliciting oxidative stress (2 , 11 , 18 , 19) . Asbestos fibers also can induce antioxidant enzymes such as superoxide dismutases and catalase in a number of cell types in an attempt to curb oxidative stress (2 , 20 , 21) .

Glutathione is considered to be the major antioxidant in the lung and is present in large quantities in the epithelial lining fluid, presumably because of release from type II epithelial cells (22) . In this study, we show that asbestos-induced up-regulation of fra-1 and other AP-1 family member genes (c-jun, junB, and c-fos) is dependent on intracellular glutathione levels. Exposure of C10 cells to asbestos initially causes a decrease in reduced glutathione levels, which later increase at 24 and 48 hours, corresponding to peaks of increased {gamma}-glutamylcysteine synthetase protein levels. Although not much is known about maintenance of intracellular pools of glutathione, the release of glutathione has been documented from rat alveolar macrophages after exposure in vitro to toxic particulates, such as silica and asbestos (23) . A report by Brown et al. (24) also shows that fibers can deplete both GSH and ascorbate from lung lining fluid. The reactive oxygen species generation by asbestos may account for the lowering of cellular GSH. The capability of asbestos fibers to generate active oxygen species after phagocytosis by the cells (18) and their ability to act as Fenton catalysts after mobilization of iron from fibers (19) may explain the importance of redox control in the cascade of cell signaling events initiated by asbestos.

In the present investigation, pretreatment of cells with catalase or deferoxamine (an iron chelator) had no effects on asbestos-induced proto-oncogene response, ruling out the role of iron catalysis and other extracellularly generated free radicals and suggesting the importance of intracellular oxidant generation by fibers. Increases in mRNA and protein levels of {gamma}-glutamylcysteine synthetase at later time points after asbestos exposure may account for compensation of initially depleted intracellular glutathione and suggests an adaptive response of the epithelial cell against asbestos-induced oxidative stress.

Other reports indicate a role of thiols in the activation of AP-1-dependent genes by a variety of different environmental stresses and toxicants (25 , 26) . For example, certain chemical oxidant stresses may activate signaling pathways leading to c-fos or c-jun induction through a common mechanism involving redox control of the AP-1 complex (27, 28, 29) . A recent report reveals that p53-dependent apoptosis, a phenomenon associated with a number of environmental carcinogens, is reduced by N-acetyl-L-cysteine (30) . Studies here with the fra-1 promoter show that levels of glutathione affect proto-oncogene gene expression at the transcriptional level and not through stabilization of steady-state mRNA levels.

Crocidolite asbestos also phosphorylates EGFR (7) , and this event leads to Src-dependent ERK1/2 activation and cell proliferation (9) . New data here show that asbestos-induced EGFR phosphorylation involves redox regulation because it was inhibited by N-acetyl-L-cysteine pretreatment. Our results support various other reports that indicate redox regulation of EGFR in different cell types. For example, N-acetyl-L-cysteine markedly suppresses EGF-induced dimerization and activation of EGFR in HEK293, A431, and NIH3T3 cells (31) . Also, EGFR transactivation by tumor necrosis factor {alpha} is regulated in a redox-dependent manner in A431 cells and can be suppressed by N-acetyl-L-cysteine or the endogenous reducing molecule, thioredoxin (32) . GSH depletion is also a potential mechanism for initiating the activation of the EGFR by alkylating agents in rat liver epithelial cells.3

Cotransfection with both catalytic and modifier subunits of {gamma}-glutamylcysteine synthetase resulted in complete inhibition of asbestos-induced up-regulation of fra-1, c-jun, junB, and c-fos steady-state mRNA levels. However, in epithelial cells transfected with these subunits individually, the modifier subunit was found to be more effective than the catalytic subunit in protecting cells from asbestos-induced up-regulation of proto-oncogenes. One possibility is that coexpression of the multidrug resistance protein occurs with the catalytic subunit and not with the modifier subunit as has been shown in other models (33 , 34) . Multidrug resistance protein coexports GSH, glucuronoids, and sulfate-conjugated drugs, thereby lowering intracellular glutathione levels. Although the catalytic subunit of the enzyme, which we show here is increased in asbestos-exposed lung tissue, seems to be more important in our transfection experiments, reports show that the modifier unit of {gamma}-glutamylcysteine synthetase may also be involved in conferring drug resistance (35) or age-associated decreases in GSH in the brain (36) . The {gamma}-glutamylcysteine synthetase modifier subunit knockout mice represent a model of chronic GSH depletion that may aid in future work to determine the critical role of {gamma}-glutamylcysteine synthetase status in asbestos-induced lung disease (37) .

We conclude that intracellular glutathione may play an essential role in protecting intact cells against asbestos fiber-induced up-regulation of AP-1 proto-oncogenes and transformation. Our studies show for the first time that up-regulation of fra-1 (and other AP-1-dependent genes) is glutathione dependent and can be prevented either by chemical manipulation or by overexpressing {gamma}-glutamylcysteine synthetase, a rate-limiting enzyme for glutathione biosynthesis. These approaches may also be valuable in altering asbestos-induced EGFR activation and signaling pathways leading to both proto-oncogene up-regulation and functional changes, for example, cell injury, proliferation, and inflammation contributing to asbestos-associated cancers and fibroproliferative lung diseases. Our findings suggest that the mechanisms of GSH in modulating AP-1 are similar in different cell types, for example, lung epithelial and mesothelial cells (6) , that are targets of asbestos carcinogenicity. Fig. 6Citation shows a hypothetical schema whereby oxidants elaborated by asbestos cause decreases in gluthathione and altered redox status of these cells. These events are associated with AP-1 up-regulation, a key pathway influencing cell proliferation and transformation. The molecular mechanisms that links redox potential to AP-1 activation are undoubtedly complex and are under investigation in a number of laboratories.



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  		Fig. 6. Diagram showing relationship of our results to the development of asbestos-related lung cancers.

 


   ACKNOWLEDGMENTS
 
Dr. Albert van der Vliet (Department of Pathology, University of Vermont, Burlington, VT) provided valuable assistance with high-performance liquid chromatography analyses. We acknowledge Dr. Terry Kavanaugh (University of Washington) for {gamma}-glutamylcysteine synthetase constructs and Dr. Sekhar Reddy (Johns Hopkins University, Baltimore, MD) for the fra-1 promoter constructs. We also wish to acknowledge Mary Lou Shane and Tim Hunter from the Vermont Cancer Center DNA Analysis Facility at the University of Vermont for providing support for quantitative reverse transcriptase-PCR studies. Laurie Sabens, Christine Germano, and Masha Stern (Cell Imaging and Analysis Core) provided valuable assistance in the manuscript preparation. Dr. Pamela Vacek (Department of Medical Biostatistics, University of Vermont, Burlington, VT) aided in statistical analyses.


   FOOTNOTES
 
Grant support: Program Project Grant PO1 HL67004 from the National Heart Lung and Blood Institute.

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

Requests for reprints: Brooke T. Mossman, Department of Pathology, 215 Health Science Research Facility, University of Vermont College of Medicine, Burlington, VT 05405. Phone: (802) 656-0382; E-mail: brooke.mossman{at}uvm.edu

3 K. Abdelmohsen, L-O. Klotz, and H. Sies, unpublished data. Back

Received 4/23/04. Revised 7/16/04. Accepted 8/20/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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