Quinones increase gamma -glutamyl transpeptidase expression by multiple mechanisms in rat lung epithelial cells

Rui-Ming Liu, Michael Ming Shi, Cecilia Giulivi, and Henry Jay Forman

Department of Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles, California 90033

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

gamma -Glutamyl transpeptidase (GGT) plays an important role in glutathione (GSH) metabolism. GGT expression is increased in oxidant-challenged cells; however, the signaling mechanisms involved are uncertain. The present study used 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a redox cycling quinone that continuously produced H2O2 in rat lung epithelial L2 cells. It was found that DMNQ increased GGT mRNA content by increasing transcription, as measured by nuclear run-on. This was accompanied by increased GGT specific activity. Cycloheximide, a protein synthesis inhibitor, blocked neither the increased GGT mRNA content nor the increased GGT transcription rate caused by DMNQ, suggesting that increased GGT transcription was a direct rather than secondary response. Previous data from this laboratory (R.-M. Liu, H. Hu, T. W. Robison, and H. J. Forman. Am. J. Respir. Cell Mol. Biol. 14: 186-191, 1996) showed that tert-butylhydroquinone (TBHQ) increased GGT mRNA content by increasing its stability. TBHQ differs markedly from DMNQ in terms of its conjugation with GSH and H2O2 generation. Together, the data suggest that quinones upregulate GGT through multiple mechanisms, increased transcription and posttranscriptional modulation, which are apparently mediated through generation of reactive oxygen species and GSH conjugate formation, respectively.

hydrogen peroxide; hydroquinone; glutathione; cycloheximide

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

GLUTATHIONE (GSH), the most abundant intracellular nonprotein thiol, participates in many important biological processes and plays an important role in the detoxification of various xenobiotic agents and oxidants. Although a few types of cells can directly take up intact GSH from the surrounding extracellular fluid, most cells depend on de novo synthesis for maintaining their intracellular GSH. Several important factors control the rate of de novo GSH synthesis. One factor is the activity of gamma -glutamylcysteine synthetase (GCS), an enzyme catalyzing the first and rate-limiting reaction in de novo GSH synthesis. Another factor is the availability of cysteine. Circulating GSH is a cysteine reservoir and serves as an important source of cysteine for the cells that express gamma -glutamyl transpeptidase (GGT) (1, 6, 16).

GGT, a membrane-bound enzyme with its catalytic site toward the outside of cells, initiates the degradation of extracellular GSH by transferring the gamma -glutamyl moiety of GSH to an amino acid-forming gamma -glutamyl amino acid and cysteinylglycine. Cysteinylglycine is then degraded by dipeptidases into cysteine and glycine, both of which can be taken up by cells and used for de novo GSH synthesis. In addition to providing cysteine for de novo GSH synthesis, GGT also participates in the GSH synthesis salvage pathway. In this pathway, GGT transfers the gamma -glutamyl moiety of extracellular GSH to cystine, the most active amino acid acceptor for the gamma -glutamyl moiety of GSH, forming gamma -glutamylcystine. gamma -Glutamylcystine is then directly taken up by the cells, where it is reduced to gamma -glutamylcysteine and cysteine (2). The gamma -glutamylcysteine is used for GSH synthesis by glutathione synthase. Utilization of extracellular cystine has been shown to be dependent on the GGT activity in human endothelial cells (8). GSH synthesis through this salvage pathway bypasses the normally rate-limiting step catalyzed by GCS. Thus, through provision of either cysteine or gamma -glutamylcysteine, GGT plays an important role in maintaining the intracellular GSH level for the majority of cells that cannot directly take up GSH from surrounding fluid.

Previous studies demonstrated that GGT was important not only for resting but also for oxidant-challenged cells to maintain their intracellular GSH concentration (5, 29, 31). NIH/3T3 fibroblasts transfected with a human placental GGT cDNA had a higher GGT specific activity and could use extracellular GSH to restore their intracellular GSH level much more efficiently than the cells transfected with only the control vector (29). These GGT cDNA-transfected cells were also more resistant to the toxicity of high concentrations of 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) than control cells when GSH was added into culture medium (31). GGT has also been shown to play an important role in protecting rat alveolar macrophages from hyperoxia toxicity (12) and bovine pulmonary artery endothelial cells from menadione toxicity (5). The mechanism by which increased GGT protects the cells from the toxicity of tert-butylhydroquinone (TBHQ) has been shown to be due to increased synthesis of intracellular GSH made possible by the increased availability of cysteine (23).

An increased expression of GGT has been found in various types of tumor cells and has been used as a marker for neoplastic formation in liver for more than 20 years. It is also known that GGT is upregulated in lung but downregulated in liver after birth (9, 17, 25). In the lung, the expression of GGT mRNAs is also switched from type I, II, and III to type III alone after birth (17). However, the signal(s) for regulating GGT expression under either pathological or physiological conditions has not been fully elucidated.

Redox cycling quinones have been used to study expression of various genes under continuous oxidative stress. The continuous generation of oxidants that can be achieved with quinones provides a model that is closer to physiological or pathological conditions to which cells may adapt than that achieved with bolus oxidants. Previously, this laboratory showed that GGT activity and GGT mRNA content were increased by oxidative stress in rat lung epithelial L2 cells using menadione, a quinone that produces reactive oxygen species by redox cycling and forms a conjugate with GSH (21). A later study showed that TBHQ, a hydroquinone that can form a conjugate with GSH but only slowly oxidizes to produce superoxide (18, 26, 27), also increased GGT activity and mRNA content (22). However, the transcription rate of the GGT gene was not changed by TBHQ, suggesting that this hydroquinone affected GGT gene expression posttranscriptionally. Because TBHQ forms a conjugate with GSH and has some capacity for generating reactive oxygen species, the signals for its effect on GGT mRNA stability remained unclear. In the present study, DMNQ, a quinone that produces superoxide and H2O2 through redox cycling but cannot form a conjugate with GSH because of the methoxy groups at the C-2 and C-3 positions (Fig. 1), has been used to test whether GGT expression can be induced by reactive oxygen species.


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Fig. 1.   Production of reactive oxygen species by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) or tert-butylhydroquinone (TBHQ) and glutathione (GSH) conjugate formation by TBHQ (based on Refs. 4 and 18). DMNQ is reduced to hydroquinones or semiquinone radicals by cellular oxidoreductases. Semiquinone radical undergoes rapid autoxidation with regeneration of parent quinone and concomitant formation of superoxide. Hydroquinone reacts rapidly with superoxide to form H2O2 and semiquinone. Superoxide dismutes to form H2O2 and oxygen. TBHQ reacts with Fe3+ to form Fe2+ and semiquinone, which then autooxidizes (18). tert-Butylquinone (TBQ) formed can be reduced in presence of microsomes (18). In addition, TBQ also reacts with GSH to form a conjugate (26, 27), whereas DMNQ cannot form a conjugate because of methoxy groups at its C-2 and C-3 positions. DT, DT-diaphorase; SG, glutathionyl moiety bound through the sulfur; Cyt, cytochrome.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Chemicals. DMNQ was kindly provided by Oxis International (Portland, OR). Cycloheximide was purchased from Sigma (St. Louis, MO). Riboprobe in vitro transcription systems were from Promega (Madison, WI). Deoxyribonuclease, proteinase K, and the RNA rapid-isolation kit were obtained from Amresco (Solon, OH). TRIzol reagent, an RNA isolation solution, was from Life Science Technologies (Grand Island, NY). All chemicals used were of at least analytic grade.

Cell culture and treatment. L2 cells derived from type II pneumocytes of adult rat lungs were obtained from the American Type Culture Collection (Rockville, MD) and grown in flasks with Ham's F-12 medium supplemented with 10% fetal bovine serum, 100 units/ml of penicillin, and 100 µg/ml of streptomycin at 5% CO2 and 37°C. DMNQ was dissolved in dimethyl sulfoxide (DMSO; final concentration of DMSO in medium was 0.1%), and cycloheximide was dissolved in the medium. L2 cells, when ~90% confluent, were treated with different compounds for various periods (see Figs. 2-6). The cells were washed once with 1× phosphate-buffered saline (PBS) buffer after treatment and harvested with a cell scraper in PBS for enzyme analysis or RNA isolation.

Enzyme activity analysis. L2 cells were suspended in 1× PBS and sonicated briefly on ice. GGT activity was measured with the fluorescent substrate gamma -glutamyl-7-amino-4-methylcoumarin as described previously (11). The specificity of this assay was confirmed using acivicin, a specific inhibitor of GGT. Protein was determined by the bicinchoninic acid method (33). The activity of GGT was expressed as picomoles of product formed per milligram protein per minute.

In vitro transcription to make GGT complementary RNA and beta -actin complementary RNA probes. A pBluescript plasmid containing a full-length human GGT coding sequence cDNA was linearized with restriction enzyme Not I and used as the template to make the GGT complementary RNA (cRNA) probe. A linearized p-TRI-beta -Actin-125 plasmid purchased from Ambion was used as the template to make the beta -actin cRNA probe. In vitro transcription was carried out using an in vitro transcription system (Promega) according to the protocol provided by the manufacturer. Briefly, 1 µg of denatured and linearized plasmid was incubated with 0.5 mM each ATP, GTP, and UTP, 12 µM CTP, 10 mM dithiothreitol, 20 units ribonuclease (RNase) inhibitor, 50 µCi of [alpha -32P]CTP (800 Ci/mmol), and 20 units of T7 RNA polymerase in a 1× transcription optimized buffer (Promega) at 37°C for 60 min. Template DNA was then digested with RNase-free deoxyribonuclease I at 37°C for 15 min. RNA was isolated after in vitro transcription by extraction with phenol-chloroform-isoamyl alcohol and was precipitated, subsequently, with ammonium acetate and ethanol.

Northern hybridization analysis of GGT mRNA. Total RNA was extracted with TRIzol reagent from L2 cells after exposure to DMNQ, cycloheximide, or combinations of these agents according to the protocol provided by the manufacturer. Ten micrograms of RNA from each sample were resolved on a 1.2% agarose gel and transferred onto a nylon membrane that was then hybridized with a full-length human GGT cRNA probe and, subsequently, with a rat beta -actin cRNA probe at 60°C for 1 h using Quikhyb (Stratagene) solution. The membrane was washed with 2× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0)-0.1% sodium dodecyl sulfate for 15 min at room temperature twice and then with 0.1× SSC-0.1% sodium dodecyl sulfate for 30 min at 55°C once. X-ray film exposure was carried out at -80°C for 1 h.

Nuclear run-on analysis of transcription rates of GGT gene. Nuclei were isolated by lysing cells in 0.5% Nonidet P-40 lysis buffer and were centrifuged at 500 g for 5 min. Isolated nuclei were incubated with 1 mM each ATP, GTP, and CTP as well as 150 µCi of [alpha -32P]UTP (600 Ci/mmol) in a reaction buffer containing 100 mM tris(hydroxymethyl)aminomethane, pH 8.0, 300 mM (NH4)2SO4, 4 mM MgCl2, 200 mM NaCl, 0.4 mM EDTA, 4 mM MnCl2, 0.1 mM phenylmethylsulfonyl fluoride, and 1.2 mM dithiothreitol at 30°C for 20 min. RNA was isolated using a rapid RNA isolation kit from Amresco according to the protocol provided by the manufacturer. Free nucleotides were removed by centrifugation through a G-50 spin column (Pharmacia Biotech, Alameda, CA) at 2,000 g for 5 min. Newly synthesized RNA was then hybridized at 60°C with the nylon membrane strips on which 5 µg each of denatured, linearized human GGT cDNA, 18S, and beta -actin cDNA had been cross-linked.

Measurement of H2O2 formation in L2 cells. About 90% confluent L2 cells were treated with different quinones for various periods of time in PBS containing 5 mM glucose. At each time point, duplicate samples were withdrawn from medium. To one of these duplicate samples, exogenous H2O2 was added to measure the recovery. H2O2 was determined fluorometrically using horseradish peroxidase-catalyzed p-hydroxyphenylaminoacetic acid dimerization at an excitation wavelength of 315 nm and an emission wavelength of 410 nm (14). The results were calculated based on the recovery and standards and expressed as nanomoles H2O2 per milligram protein.

Statistics. Data are expressed as means ± SE and were evaluated by one-way analysis of variance and then by Dunnett's multiple comparison analysis. P < 0.05 was considered significant.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Increase in GGT activity and mRNA content in L2 cells by DMNQ. A concentration-dependent increase in GGT-specific enzyme activity was observed after L2 cells were treated with 2.5-10 µM of DMNQ for 24 h (Fig. 2). Five or ten micromolar DMNQ significantly increased GGT activity by 151 and 172%, respectively. A previous study has shown that 10 µM DMNQ is near the highest concentration that can be used in L2 cells without clear evidence of cytotoxicity as measured by ATP content and morphology under light microscopy (32).


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Fig. 2.   Effect of DMNQ on gamma -glutamyl transpeptidase (GGT) specific activity in L2 cells. Cells were treated with different concentrations of DMNQ for 24 h. Specific activity of GGT was measured as described in EXPERIMENTAL PROCEDURES. Each bar represents a mean ± SE from 4 separate experiments. * Significant difference from DMSO control (P < 0.05).

GGT mRNA content was increased by 102, 206, and 183% when the cells were treated with 5 µM DMNQ for 6, 12, or 24 h, respectively. When the cells were treated with 10 µM DMNQ, no increase in the steady-state GGT mRNA content was observed until 12 h posttreatment. After treatment with 10 µM DMNQ for 12 and 24 h, GGT mRNA content increased by 67 and 109%, respectively, compared with DMSO control (Fig. 3). Five micromolar DMNQ caused a greater increase in GGT mRNA content than did 10 µM DMNQ at all time points tested, which may reflect a more subtle toxicity of 10 µM DMNQ than observed previously. Nonetheless, other studies showed that exposure of L2 cells to 10 µM DMNQ induced a greater increase than did exposure to 5 µM DMNQ in the mRNAs for both the catalytic and regulatory subunits of GCS, the rate-limiting enzyme for GSH synthesis (32, 35). Regardless, the results showed that this nonconjugating quinone could cause an elevation in GGT specific activity and mRNA content in rat lung epithelial L2 cells.


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Fig. 3.   Steady-state GGT mRNA content in L2 cells treated with DMNQ. Cells were treated with different concentrations of DMNQ for various time intervals. A: Northern blot hybridization was performed as described in EXPERIMENTAL PROCEDURES. B: data represent means of 2 or 3 separate experiments; error bars are SD (6-h and 12-h treatment groups, n = 3) or ranges (24-h treatment groups, n = 2).

Increased transcription rate of the GGT gene by DMNQ in L2 cells. To examine whether DMNQ affected the transcription rate of the GGT gene, nuclear run-on experiments were conducted. Five and ten micromolar DMNQ treatment for 12 h increased the transcription rate of the GGT gene by four- to sevenfold and two- to threefold, respectively, compared with solvent controls (Fig. 4). Five micromolar DMNQ caused a larger increase in the GGT gene transcription rate than did 10 µM DMNQ, which was coincident with the pattern of increase in the steady-state GGT mRNA content. These data suggested that an increased transcription rate was at least one of the mechanisms involved in the increase in GGT mRNA content in DMNQ-treated L2 cells.


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Fig. 4.   Effect of DMNQ on GGT gene transcription rate in L2 cells. Cells were treated with DMSO (vehicle control) or 5 or 10 µM DMNQ for 12 h. Nuclei were isolated, and nuclear run-on analysis (A) was performed as described in EXPERIMENTAL PROCEDURES. B: data represent average of 2 separate experiments; error bars are ranges.

Superinduction of GGT mRNA by cycloheximide in L2 cells. To determine whether the increase in the GGT mRNA content by DMNQ required new protein (transcription factor) synthesis, the effect of cycloheximide, an inhibitor of protein synthesis, on the induction of GGT mRNA by DMNQ was studied. It was found that 1 and 10 µg/ml of cycloheximide alone increased GGT mRNA content by 40 and 114%, respectively. When the cells were treated simultaneously with 5 µM DMNQ and 1 or 10 µg/ml of cycloheximide, the steady-state GGT mRNA content in these cells was even higher than that in the cells treated with either DMNQ or cycloheximide alone (Fig. 5). These results indicated that protein synthesis was not required for an increase in GGT mRNA content in DMNQ-treated L2 cells. On the other hand, these data also indicated that the inhibition of protein synthesis itself caused and together with DMNQ enhanced the increase in GGT mRNA content.


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Fig. 5.   Superinduction of GGT mRNA by cycloheximide in L2 cells treated with DMNQ. Cells were treated with DMSO (vehicle control), 5 µM DMNQ, 1 or 10 µg/ml of cycloheximide, or DMNQ plus cycloheximide for 6 h. A: Northern hybridization was performed as described in EXPERIMENTAL PROCEDURES. B: experiment was repeated 3 times, and data are expressed as means ± SE. * Significant difference from DMSO control (P < 0.05).

Effects of cycloheximide on the GGT gene transcription rate in L2 cells. To further investigate the mechanism by which cycloheximide increased the steady-state GGT mRNA content, nuclear run-on assays were performed after the cells were treated with cycloheximide. With 10 µg/ml of cycloheximide alone, the concentration that caused an increase in steady-state GGT mRNA content, no significant change in the transcription rate of the GGT gene was observed after 6 h of treatment. When the cells were treated simultaneously with DMNQ and cycloheximide for 6 h, the transcription rate of the GGT gene was almost the same as that in the cells treated with DMNQ alone, which was two times that in DMSO control (data not shown). These results suggested that the inhibition of protein synthesis by cycloheximide had neither an effect on the transcription rate of the GGT gene itself nor an effect on the increased transcription rate of the GGT gene caused by DMNQ. Furthermore, these results also suggested that, in contrast to DMNQ, the increase in the steady-state GGT mRNA content caused by cycloheximide was not due to an increase in the transcription rate but rather to increased stability of GGT mRNA.

Production of H2O2 in L2 cells treated with quinones. To begin to understand what factors may underlie differences in the mechanisms for increasing GGT mRNA content by various quinones, the rate of H2O2 formation in L2 cells treated with these compounds was measured. It was found that the H2O2 production was increased when the cells were treated with 10 µM DMNQ or 50 µM TBHQ, the concentrations shown to increase GGT mRNA level (22) (Fig. 6). TBHQ caused a rapid but transient increase in the production of H2O2. The highest concentration of H2O2 accumulated in the incubation buffer of TBHQ-treated cells 3 min after TBHQ was added into the medium. However, the total amount of H2O2 accumulated during this period was only one-third the amount of TBHQ added. One hour after TBHQ treatment, H2O2 declined to the control level. These data suggested that TBHQ did not undergo continuous redox cycling and therefore only transiently produced reactive oxygen species within L2 cells.


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Fig. 6.   Production of H2O2 in L2 cells treated with quinones. Cells were treated with DMSO (vehicle control), 50 µM TBHQ, or 10 µM DMNQ. H2O2 was measured as described in EXPERIMENTAL PROCEDURES. Each value represents a mean of 3 samples.

In contrast with TBHQ, DMNQ caused a constant increase in H2O2 production. One hour after the cells were treated with DMNQ, the accumulated H2O2 in the incubation buffer of DMNQ-treated cells reached 80 µM, which was eight times the amount of DMNQ added, suggesting that DMNQ underwent significant redox cycling and therefore continuously produced superoxide and H2O2.

H2O2 production was measured after the medium was changed to a PBS buffer that contained 5 mM glucose. This was done to avoid quenching of H2O2 by components present in the complete medium. It is, however, not possible to culture L2 cells for longer than 4 h in the absence of serum. Although this is not exactly the same condition as that used for the GGT induction study, these measurements provide information about the relative amount of H2O2 production in these cells after treatment with different quinones.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Reactive oxygen species have been proposed as signals for the activation of various genes. The present studies showed that DMNQ, a quinone that cannot form a conjugate with GSH but produces H2O2 continuously, increased GGT specific activity and mRNA content by increasing the GGT gene transcription rate. These results suggested that reactive oxygen species were at least one of signals involved in increasing GGT transcription by this quinone. This conclusion is supported by the observations made by Knickelbein et al. (19) and Takahashi et al. (34). Knickelbein et al. (19) found that hyperoxia increased steady-state GGT mRNA content in rat lung and suggested that an oxygen-dependent mechanism might be involved in the regulation of GGT gene expression during fetal development. More recently, Takahashi et al. (34) reported that nitrogen dioxide inhalation also increased GGT protein and mRNA contents in rat lung. Although neither group of authors showed a direct increase in the transcription rate of the GGT gene, their data clearly indicated that reactive oxygen species could increase the expression of GGT.

In contrast with DMNQ, TBHQ increased steady-state GGT mRNA content but not the transcription rate of the GGT gene (22). This suggested that a posttranscriptional mechanism was responsible for the increase in GGT mRNA content by this hydroquinone. To explore the potential difference of signals produced by these two compounds, the rate of H2O2 generation was measured in L2 cells treated with TBHQ or DMNQ. Although Kahl et al. (18) have suggested that TBHQ can redox cycle in the presence of microsomal preparations, NADPH, GSH, and oxygen, we found that TBHQ only transiently produced a low level of H2O2 in L2 cells. The amount of H2O2 produced in TBHQ-treated cells was much less than that in DMNQ-treated cells. Most importantly, it appears that TBHQ may not undergo continuous redox cycling in these cells, since the concentration of H2O2 detected in the medium of TBHQ-treated cells reached its maximum within 3 min after TBHQ was added into the medium and then declined to the control level rapidly within 1 h. tert-Butylquinone (TBQ), the oxidation product of TBHQ, rapidly conjugates nonenzymatically with GSH (26, 27). Apparently, this GSH-TBQ conjugate did not undergo further redox cycling in L2 cells. On the other hand, DMNQ underwent significant redox cycling and constantly produced a larger amount of H2O2 in L2 cells. Indeed, after 1 h, the amount of H2O2 in the medium was eight times the amount of DMNQ added. The differences in the biochemistry of TBHQ and DMNQ resulted in marked differences in the mechanisms by which these two quinones modulate the expression of the GGT gene. The results suggest that formation of a GSH-TBQ conjugate may be responsible for the enhanced GGT mRNA stability, while the continuous generation of reactive oxygen species through DMNQ redox cycling increases the transcription rate of the GGT gene. It should be noted that neither 10 µM DMNQ nor 50 µM TBHQ is cytotoxic to L2 cells during prolonged (>16 h) incubation in complete medium (7, 32).

The DMNQ model provides a significant difference from models using bolus addition of H2O2 or other agents that are consumed by cellular metabolism. The latter often require concentrations that may significantly impair cellular systems. DMNQ provides a model that may more closely mimic the physiological oxidative stress underlying chronic pathological processes in which adaptation may occur. As illustrated by the current results, a significant difference may also be reflected in the mechanisms of gene expression.

It is well known that antioxidant response elements are present in the promoter regions of several genes involved in xenobiotic metabolism and antioxidant defense, such as glutathione S-transferase and NAD(P)H:quinone reductase. This cis-acting element is involved in the induction of these genes by H2O2 (3, 10). Although a putative antioxidant response element sequence has also been found in the promoter region of the GGT gene (17, 34), whether this element is involved in the activation of GGT transcription by DMNQ remains to be resolved.

Further investigation of the mechanism of induction of GGT by DMNQ focused on the question of whether the increase in GGT transcription was a primary or secondary response to the production of reactive oxygen species. It was found that cycloheximide did not block but rather enhanced the induction of GGT mRNA by DMNQ. Nuclear run-on study showed that cycloheximide had no effect on the increased GGT gene transcription rate caused by DMNQ. These results suggest that the DMNQ-induced increase in the GGT gene transcription rate does not require new protein synthesis and is a direct rather than secondary response of these cells to oxidative stress.

In the same experiments, it was also found that cycloheximide alone increased steady-state GGT mRNA content. Although induction and superinduction of mRNA by protein synthesis inhibitors have been well documented (13, 15, 20, 24, 30, 37), the precise molecular mechanisms underlying this phenomenon have not been extensively characterized. One current hypothesis is that the protein synthesis inhibitor increases the stability of mRNA by inhibiting the synthesis of either an mRNA destabilization protein or RNase (15, 28). Another hypothesis is that the protein synthesis inhibitor inhibits the synthesis of a labile trans-acting protein that acts as a transcription repressor (30, 36, 37). In the latter case, the protein synthesis inhibitor would be expected to increase the gene transcription rate. In some cases, induction or superinduction may be due to a combination of both mechanisms. To sort out the mechanism by which cycloheximide increased the steady-state GGT mRNA level and caused a superinduction of GGT mRNA in the presence of DMNQ, nuclear run-on experiments were performed. The result showed that cycloheximide, at the concentration that induced or superinduced GGT mRNA, had no effect on the transcription rate of the GGT gene in the presence or absence of DMNQ. This suggests that the increase in GGT mRNA content by cycloheximide is not due to derepression of transcription but rather to an increased stability of GGT mRNA. The same mechanism has been proposed for the regulation of several genes such as monocyte chemoattractant protein-1 (15), CYP1A1 (30), and creatine phosphokinase (28). We conclude that the superinduction of GGT by cycloheximide in the presence of DMNQ is due to a combination of transcriptional activation of the GGT gene by DMNQ and increased stability of GGT mRNA by cycloheximide. The induction and superinduction of GGT mRNA by cycloheximide also confirms that posttranscriptional regulation is an important mechanism involved in the regulation of GGT gene expression.

In summary, reactive oxygen species appear to be at least one of the signals responsible for the transcriptional activation of the GGT gene by quinones. Nonetheless, both transcriptional and posttranscriptional mechanisms play roles in the regulation of GGT gene expression in rat lung epithelial L2 cells. As new protein synthesis is not required for transcriptional activation of the GGT gene by DMNQ, an increase in GGT transcription appears to be a primary response to oxidative stress. Aside from the obvious oxidative stress imposed by redox cycling quinones, hyperoxia, or nitrogen dioxide exposure, other pathological processes may also produce an increase in reactive oxygen species. For example, reactive oxygen species have been implicated in carcinogenesis and may be responsible for increased GGT expression in tumors.

    ACKNOWLEDGEMENTS

We thank Dr. Enrique Cadenas, Dr. David Ann, and Jinah Choi for helpful comments.

    FOOTNOTES

This work was supported by National Institute of Environmental Health Sciences Grant ES-05511.

Present address of M. M. Shi: Dept. of Environmental Health, Harvard School of Public Health, Boston, MA 02115.

Address for reprint requests: R.-M. Liu, Dept. of Molecular Pharmacology and Toxicology, Univ. of Southern California, 1985 Zonal Ave., Los Angeles, CA 90033.

Received 7 July 1997; accepted in final form 3 December 1997.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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