gamma -Glutamylcysteine synthetase: mRNA stabilization and independent subunit transcription by 4-hydroxy-2-nonenal

Rui-Ming Liu, Lin Gao, Jinah Choi, 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
Materials & Methods
Results
Discussion
References

gamma -Glutamylcysteine synthetase (GCS), the rate-limiting enzyme in de novo glutathione (GSH) synthesis, is composed of one catalytic (heavy) and one regulatory (light) subunit. Although both subunits are increased at the mRNA level by oxidants, it is not clear whether they are regulated through the same mechanism. 4-Hydroxy-2-nonenal (4HNE), a lipid peroxidation product, may act as a mediator for the induction of gene expression by oxidants. In the present study, 4HNE was used to study the mechanism of induction of the two GCS subunits in rat lung epithelial L2 cells. 4HNE increased both the transcription rates and the stability of mRNA for both GCS subunits, resulting in an increased mRNA content for both subunits. Both GCS subunit proteins and enzymatic activities also increased. Emetine, a protein synthesis inhibitor, blocked the increase in GCS light subunit mRNA but not the increase in GCS heavy subunit mRNA. This suggested that although 4HNE increased transcription and stabilization of both GCS subunit mRNAs, the signaling pathways involved in the induction of the two GCS subunits differed.

messenger ribonucleic acid; oxidative stress; regulatory subunit; catalytic subunit; protein synthesis inhibitor; glutathione

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

GLUTATHIONE (GSH), the most abundant intracellular nonprotein thiol, participates in many important biological processes including the synthesis of DNA and proteins, detoxification of various xenobiotics and oxidants, and regulation of gene expression. Although a few types of cells can directly take up GSH from the surrounding fluid, most cells depend on de novo GSH synthesis to maintain their intracellular GSH content. Accumulating evidence in the literature suggests that the regulation of gamma -glutamylcysteine synthetase (GCS), the enzyme catalyzing the first and rate-limiting step in de novo GSH synthesis, is one of the major determinants of GSH homeostasis.

GCS is composed of two subunits, one heavy (GCS-HS) and one light (GCS-LS). The heavy subunit possesses all the catalytic activity of the enzyme and can be feedback inhibited by GSH. The light subunit does not have any catalytic activity; however, it has a very important regulatory function. Binding of GCS-LS to GCS-HS reduces the Michaelis-Menten constant for glutamate from 18.2 mM to a physiological concentration of 1.2 mM and increases the inhibition constant for GSH. It was suggested by Huang et al. (19) that the monomeric heavy subunit would be nonfunctional under physiological conditions because of the low glutamate and high GSH concentrations. Mulcahy et al. (36) found that cotransfection of COS cells with both GCS-HS cDNA and GCS-LS cDNA led to a greater increase in intracellular GSH content than transfection with either subunit alone when the total amount of cDNA used was equal between the cotransfection and individual transfection experiments. These data clearly suggest an important role for the light subunit in GCS activity under physiological conditions.

The regulation of GCS-HS gene expression has been extensively studied. It has been found that various agents and conditions, such as heat shock (22), heavy metals (20, 27, 61, 62), substances that deplete GSH through conjugation or by inhibiting its synthesis (2, 49, 55, 65), hormones (8, 9, 32, 42-44, 58), oxidants and antioxidants (7, 11, 16, 29, 40, 41, 47, 48, 51, 52, 55), and tumor necrosis factor (34) induce the expression of GCS-HS. Increased GCS-HS expression and GSH content have also been found in drug-resistant tumor cells, implicating a potential role for this enzyme in such resistance (4, 21, 23, 25, 35, 38, 57, 64). Regulation of the GCS-LS, on the other hand, has been less intensively studied until recently. Several compounds that induce the expression of GCS-HS have been found to induce the expression of GCS-LS as well (8, 16, 49, 55). However, some hormones such as insulin and glucocorticoid (8) or the anticancer drug cisplatin have been found to induce GCS-HS but not GCS-LS (64), indicating dissociated regulation of the two GCS subunits. A difference in the ratio of the two GCS subunit mRNA contents among various tissues further indicates that the expression of two GCS subunit genes is controlled by different mechanisms (17).

4-Hydroxy-2-nonenal (4HNE) is a major lipid peroxidation metabolite formed from cellular lipids during normal metabolism and in response to xenobiotics that cause lipid peroxidation (12). It has been reported that conjugation with GSH both enzymatically and nonenzymatically is a major detoxification pathway for 4HNE (1, 24, 33, 53, 60). Toxic effects of 4HNE include inactivation of enzymes, depletion of intracellular GSH content, inhibition of cell proliferation, and impairment of the transport of glutamate (24, 33, 59, 66). Nonetheless, continuous production of 4HNE, even in cells unchallenged by oxidants, produces a steady-state concentration in the micromolar range (14). This alpha ,beta -unsaturated aldehyde can bind to the sulfhydryl, imidazole, and amino groups of proteins and cause diverse biological effects. Interestingly, the addition of exogenous micromolar 4HNE increases the expression of several genes (5, 6, 15, 26, 45, 46, 54). Such results suggest that 4HNE is involved in the regulation of gene expression even under physiological conditions. The production of 4HNE was found to be increased under several pathological conditions such as in children with autoimmune diseases (18, 31), in patients with Alzheimer's disease (18, 31), and on oxidant challenge (6, 24, 33, 45). Because the production of 4HNE is increased during oxidant challenge, it has been suggested that 4HNE might act as a mediator in the induction of gene expression by oxidants.

The present study was conducted with rat lung epithelial L2 cells in which GCS gene expression has been shown to be increased by redox cycling quinones (55). The principal purpose of the present study was to use 4HNE to further explore the mechanism of GCS induction and to determine differences and similarities in the mechanisms regulating the two subunits during oxidative stress. The significance of this study is twofold: 1) the two GCS genes are regulated by independent mechanisms in which an increase in the regulatory (but not the catalytic) subunit mRNA is dependent on de novo protein synthesis, and 2) stabilization of the mRNAs for both subunits is enhanced by 4HNE such that this mechanism contributes nearly as much as does increased transcription to increases in GCS proteins and enzymatic activity.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Chemicals. 4HNE was purchased from Cayman Chemical (Ann Arbor, MI), and the somatic cell ATP assay kit, actinomycin D, and emetine were from Sigma (St. Louis, MO). DNase, proteinase K, and an RNA rapid-isolation kit were obtained from Amresco (Solon, OH). TRIzol Reagent, an RNA isolation solution, was from Life Technologies (Grand Island, NY). All chemicals used were at least analytic grade.

Cell culture and treatment. L2 cells, originally derived from type II pneumocytes of adult rat lungs, were obtained from the American Type Culture Collection (Manassas, VA) 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 in 5% CO2 at 37°C. 4HNE was dissolved in ethanol and actinomycin D in DMSO such that the final concentrations of ethanol and DMSO in the medium were 0.1%. Emetine was dissolved in the medium. L2 cells, when ~90% confluent, were treated with different compounds as indicated in RESULTS and Figs. 1-8. The cells were washed once with 1× phosphate-buffered saline (PBS) after treatment and harvested with a cell scraper in 1× PBS for nuclei and RNA isolation and Western analysis as well as for GCS activity measurement.

Northern hybridization analysis of GCS mRNAs. GCS-HS cDNA (804 bp) and GCS-LS cDNA (1,001 bp), which were reversely transcribed from rat kidney RNA and L2 cell RNA, respectively, and amplified by PCR (52, 55), were labeled with [alpha -32P]dCTP with a random-primer DNA labeling kit from Life Technologies (Gaithersburg, MD). The GCS mRNA content was determined by Northern hybridization analysis as previously described (29). Briefly, total RNA was extracted with TRIzol Reagent (Life Technologies) according to the protocol provided by the manufacturer. Twenty micrograms of RNA from each sample were resolved on a 1.2% agarose gel and transferred onto a nylon membrane. Hybridization was carried out with GCS-LS cDNA, GCS-HS cDNA, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probes sequentially at 60°C with Quik-Hyb (Stratagene) solution. After hybridization, the membranes were washed twice with 2× sodium chloride-sodium citrate (SSC; 1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) buffer-0.1% sodium dodecyl sulfate (SDS) for 15 min at room temperature, then twice with 0.1× SSC-0.1% SDS for 15 min at 50°C. The membranes were scanned, and the radioactivity was quantitated by an Instantimager (Packard Instrument, Meriden, CT).

Western blotting analysis of GCS proteins. The cells were sonicated and lysed in a cell lysis buffer containing 20 mM Tris · HCl, 150 mM NaCl2, 1 mM MgCl2, 1 mM CaCl2, 2.5 mM EDTA, 2.5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 µg/ml of aprotinin. After centrifugation at 105,000 g for 90 min, the supernatant (containing 100 µg of protein) was mixed with 1 volume of 2× dithiothreitol (DTT) gel loading buffer (0.125 M Tris · HCl, pH 6.5, 20% glycerol, 4% SDS, 0.0025% pyronin Y, and 15.4 mg/ml of DL-DTT) and applied to 10% SDS-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA). The membranes were blocked with 5% nonfat milk at room temperature for 30 min and incubated with rabbit polyclonal anti-rat GCS antibodies at 4°C overnight. After being washed with Tris-buffered saline containing 0.05% Tween 20, the membranes were incubated with an appropriately diluted goat anti-rabbit antibody tagged with horseradish peroxidase at room temperature for 1 h and then with an enhanced chemiluminescence (ECL Plus) reagent mixture (40:1 of reagents A and B; Amersham) for 5 min. Film exposure was carried out at room temperature with Hyperfilm ECL film (Amersham).

Measurement of GCS activity. GCS activity was measured by analyzing gamma -glutamylcysteine (gamma -GC) production by HPLC with a method described by Yan and Huxtable (63), with slight modifications. Briefly, 1.2-4 × 106 cells were harvested by scraping the cells with a rubber policeman into 1× PBS. Then, the cells were pelleted and sonicated briefly in 0.3 ml of lysis solution (0.1 M Tris · HCl, pH 8.2, 150 mM KCl, 20 mM MgCl2, and 2 mM EDTA) containing 2 µg/ml each of leupeptin and aprotinin and 50 µg/ml of PMSF. Then the sonicated samples were centrifuged at 12,000 rpm at 4°C for 20 min. To remove endogenous inhibitors, acceptors, and amino acids, the supernatant was centrifuged in Microcon-10 (Amicon, Beverly, MA) tubes for 20-30 min at 4°C at 12,000 rpm. Then 0.3 ml of the lysis solution was added, and the samples were centrifuged for another 20-30 min inside the same Microcon tubes to wash and concentrate the protein. This step was repeated once more with an additional 0.3 ml of the lysis solution. Final concentrates were tested for their protein content with a bicinchoninic acid kit supplied by Pierce (Rockford, IL). The supernatant was then frozen at -20°C until enzymatic analysis.

The GCS reaction was carried out as follows. The incubation mixture contained 20 mM L-glutamic acid, 5 mM cysteine, 5 mM DTT, 10 mM ATP, 0.1 M Tris · HCl, pH 8.2, 150 mM KCl, 20 mM MgCl2, 2 mM EDTA, and 0.04 mg/ml of acivicin to inhibit the activity of endogenous gamma -glutamyl transpeptidase. The reaction was initiated by adding protein to the incubation mixture, which had been preequilibrated at 37°C for at least 3 min. The final protein concentration in the reaction mixture was between 0.1 and 1 mg/ml. After 30 min of incubation, 100 µl of the reaction mixture were derivatized with 20 µl of 5 mM N-ethylmorpholine plus 10 µl of 5 mM monobromobimane in acetonitrile in the dark for 15 min at room temperature. The reaction was terminated by adding 20 µl of 50% sulfosalicylic acid. The resulting precipitates were removed by centrifugation, and the derivatized thiols were analyzed by HPLC after dilution in water. GCS activity was linear at protein concentrations between 0.1 and 1 mg/ml of the reaction volume for at least 30 min.

Samples were analyzed with a Beckman Ultrasphere ODS (250 × 4.6-mm ID, 5-mm particle size) reverse-phase stainless steel column equipped with a precolumn (Pelliquard LC-18 guard column kit with 2-cm cartridge; Supelco, Bellefonte, PA) connected to Perkin-Elmer series 410 LC pump, LC-200 fluorescence detector, and a series 200 autosampler. Solvent A consisted of 0.25% acetic acid, pH 3.9. Solvent B was 0.25% acetic acid-methanol (70:30 vol/vol), pH 3.9, and solvent C was 0.25% acetic acid-methanol (50:50 vol/vol), pH 3.9. Samples were eluted from the column at a flow rate of 1.5 ml/min with the following program: 65% solvent A and 35% solvent B were maintained for 1 min, followed by a linear gradient to 20% solvent A and 80% solvent B during the next 7 min. Then, 100% solvent B was run for 10 min, and then 100% solvent C for the next 7 min. The next sample was injected after 10 min of equilibration at 65% solvent A and 35% solvent B. The monobromobimane adduct was monitored with an excitation wavelength at 370 nm and an emission wavelength at 485 nm. Concentrations of gamma -GC were measured with standard curves generated with known amounts of gamma -GC. GCS activity is reported as nanomoles of gamma -GC per minute per milligram of protein.

Nuclear run-on analysis of transcription rates of GCS genes. Nuclei were isolated by lysing 1-5 × 107 cells in 4 ml of 0.5% Nonidet P-40 lysis buffer, which contained 10 mM Tris · HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.5% Nonidet P-40. The lysates were then centrifuged at 500 g for 5 min. Isolated nuclei were incubated with 1 mM each ATP, GTP, and CTP as well as with 150 µCi of [alpha -32P]UTP (600 Ci/mmol) in a reaction buffer containing 100 mM Tris, pH 8.0, 300 mM (NH4)2SO4, 4 mM MgCl2, 200 mM NaCl, 0.4 mM EDTA, 4 mM MnCl2, 0.1 mM PMSF, and 1.2 mM DTT at 30°C for 20 min. RNA was isolated with a rapid RNA isolation kit from Amresco according 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 GCS-HS cDNA, GCS-LS cDNA, and GAPDH cDNA probes had been cross-linked.

Measurement of intracellular GSH and GSSG contents. Intracellular contents of GSH and GSSG were determined by the well-established HPLC method of Fariss and Reed (13). Briefly, cells were acidified with 10% perchloric acid containing 2 mM EDTA and 7.5 nmol of gamma -glutamyl-glutamic acid as an internal standard. After centrifugation, 10 mM iodoacetic acid in 0.2 mM m-cresol purple was added to the supernatant, and the pH was adjusted to 8-9. The mixture was incubated in the dark at room temperature for 15 min, then 1% fluorodinitrobenzene was added, and the reaction mixture was incubated at 4°C overnight. L-Lysine was added to react with excess fluorodinitrobenzene. HPLC analysis of samples was carried out with a Perkin-Elmer HPLC system consisting of a series 410 pump, LC-90 ultraviolet spectrophotometric detector, and LCl-100 integrator after salt was removed. Elution solvents were 80% methanol (solvent D) and 0.5 M sodium acetate in 64% methanol (solvent E). After a 100-µl injection of the derivatization solution, the mobile phase was maintained at 70% solvent D and 30% solvent E for 5 min, followed by a 15-min linear gradient to 100% solvent E at a flow rate of 1.5 ml/min. The mobile phase was held at 100% solvent E for 40 min. Fluorodinitrobenzene derivatives were detected at 365 nm. Standards were run under the same conditions, and the peak areas were measured. GSH and GSSG contents were calculated based on the internal standard and GSH or GSSG standard curves.

Measurement of intracellular ATP content. Intracellular ATP content was determined as previously described (10) with a somatic cell ATP assay kit (Sigma). Briefly, cells grown in six-well plates were washed once with 1× PBS. ATP was released with 0.5 ml of somatic cell ATP releasing solution. ATP concentration was measured by the firefly luciferin-luciferase reaction with a Perkin-Elmer LS-5 fluorescence spectrophotometer in the phosphorescence mode. The ATP content was calculated based on the standard curve measured under the same conditions and normalized by protein content.

Statistics. Data are expressed as means ± SE and were evaluated by one-way ANOVA. Significance was determined by Fisher's least significant difference test. P < 0.05 was considered significant.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

4HNE elevated the content of both GCS mRNAs. Rat lung epithelial L2 cells were treated with various concentrations of 4HNE as a function of time as indicated in Fig. 1, and Northern analyses were performed to determine relative changes in mRNAs for both GCS subunits. It was found that 4HNE caused a concentration-dependent increase in the content of both GCS-HS and -LS mRNAs. The induction reached its highest point 3 h after 4HNE treatment. At this point, the GCS-HS mRNA content was increased 1.5-, 2.5-, and 2.7-fold, whereas the GCS-LS mRNA was increased 1.5-, 4.7-, and 7.4-fold, by 5, 10, and 20 µM 4HNE, respectively. The induction of GCS mRNAs decreased after 3 h and almost reached the control level by 12 h after treatment (Fig. 1). The data indicated that 4HNE induced the expression of both GCS mRNAs at the same time, although the induction of GCS-LS was greater than that of GCS-HS.


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Fig. 1.   Induction of gamma -glutamylcysteine synthetase (GCS) catalytic heavy subunit (GCS-HS) and GCS regulatory light subunit (GCS-LS) mRNAs by 4-hydroxy-2-nonenal (4HNE). Cells were treated with 4HNE for times indicated. GCS mRNAs were measured by Northern hybridization analysis. After hybridization, membranes were scanned with an Instantimager. Top: pixel image generated by Instantimager from 1 experiment. Bottom: summary of 2-3 separate experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

4HNE caused elevation of both GCS subunit proteins. L2 cells were treated with 10 or 20 µM 4HNE or solvent vehicle for 6, 16, or 24 h. The amounts of GCS proteins were quantified by Western blotting analyses with specific rabbit anti-rat GCS antibodies. It was found that 4HNE caused a time- and concentration-dependent increase in the amount of both GCS subunit proteins (Fig. 2). The amount of GCS-LS protein increased more than that of GCS-HS protein after the cells were treated with 4HNE. These results were consistent with the results of mRNA experiments. The data indicated that 4HNE also correspondingly increased the amount of both GCS subunit proteins.


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Fig. 2.   Increase in contents of both GCS-HS and -LS proteins by 4HNE. L2 cells were treated with 4HNE or control vehicle for times indicated. Amount of GCS-HS and GCS-LS proteins was determined by Western analysis with rabbit anti-rat GCS antibodies. Top: data from 1 of 3 separate experiments. Bottom: summary of 3 separate experiments with densitometry. * Significant difference from control, P < 0.05.

4HNE increased GCS activity. GCS activity was measured to see whether the increased GCS protein content was accompanied by an increase in the activity of the enzyme. It was found that 4HNE caused a concentration- and time-dependent increase in GCS activity. Five micromolar 4HNE caused a slight but not significant increase in GCS activity at all times tested. Ten and twenty micromolar 4HNE caused a significant increase in the activity of GCS after 12 or 24 h of treatment, with a peak induction at 12 h (Fig. 3).


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Fig. 3.   4HNE increased GCS activity in L2 cells. L2 cells were treated with 4HNE or control vehicle for times indicated. GCS activity was determined by HPLC by measuring the product gamma -glutamylcysteine after derivatization with monobromobimane as described in MATERIALS AND METHODS. Basal GCS activity in L2 cells was 0.4 nmol · min-1 · mg protein-1. Each point is mean ± SE of 4-6 separate samples. * Significant difference from control, P < 0.05.

4HNE increased the transcription rates of both GCS subunit genes. Actinomycin D, an inhibitor of transcription, was used to determine whether the increased content of GCS mRNAs was due to increased transcription. It was found that actinomycin D dramatically blocked the increases in both GCS subunit mRNAs by 4HNE, suggesting that the increase in the GCS mRNA content by 4HNE was, at least in part, due to the increased transcription of the two genes (Fig. 4). To further examine whether 4HNE increased transcription of both GCS subunits, nuclear run-on analyses were performed. It was found that the transcription rates of both subunit genes were increased by 4HNE as a function of both time and concentration (Fig. 5). Increased transcription of GCS-LS mRNA occurred slightly earlier than that for GCS-HS mRNA. The data clearly indicated that 4HNE increased the transcription rates of both GCS subunit genes in L2 cells, and this increased rate of transcription was at least one of the mechanisms responsible for the increase in the content of GCS mRNAs.


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Fig. 4.   Effect of actinomycin D on induction of GCS mRNAs by 4HNE. L2 cells were treated with 4HNE with and without actinomycin D for 6 h. GCS mRNA contents were measured by Northern analysis as described in MATERIALS AND METHODS.


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Fig. 5.   Increased rates of transcription for both GCS mRNAs by 4HNE. Cells were treated with 4HNE or control vehicle for 1.5 or 3 h. Transcriptional rates of GCS genes were measured by nuclear run-on analyses as described in MATERIALS AND METHODS. Data were quantified by an Instantimager. Each point is mean ± SE; nos. in bars, no. of separate experiments. * Significant difference from control, P < 0.05.

4HNE increased the stability of both GCS subunit mRNAs. Because actinomycin D did not completely block the induction of both GCS subunit mRNAs by 4HNE (Fig. 4), it was suggested that, in addition to the increased transcription rates of both GCS genes, increased stability of GCS mRNAs might also contribute to the increased content of GCS mRNAs by 4HNE. Therefore, the effect of 4HNE on the degradation rate of GCS mRNAs was investigated to further explore the mechanisms involved in 4HNE-induced increases in the content of both GCS mRNAs. Northern analyses were performed to quantify the GCS mRNA content at different time intervals after the cells were treated with actinomycin D (to block transcription) plus 10 µM 4HNE or actinomycin D plus control vehicle. The ratio of GCS mRNA to GAPDH mRNA was used to calculate the degradation rates of GCS mRNAs. It was found that the half-lives of both subunit mRNAs were almost doubled by 4HNE treatment (Fig. 6), confirming that 4HNE also posttranscriptionally modulated the expression of both GCS genes by stabilizing their mRNAs.


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Fig. 6.   Increased stability of GCS mRNAs by 4HNE. L2 cells were treated with 10 µM 4HNE (+4HNE) + 0.05 µg/ml of actinomycin D (AD) or solvent (-4HNE) + 0.05 µg/ml of AD for times indicated. RNA was isolated at different times after treatment. Northern hybridizations were performed, and radioactivity was quantified by Instantimager. Top: data from 1 experiment. Bottom: summary of 3 separate experiments. Data are means ± SE. * Significant difference from control, P < 0.05.

Difference in the mechanism of induction of two GCS subunits by 4HNE. To determine whether the increased content of both GCS subunit mRNAs was due to direct or indirect effects of 4HNE, emetine, a protein synthesis inhibitor, was used. It was found that emetine almost completely eliminated the increase in GCS-LS mRNA but had no effect on the increase in GCS-HS mRNA by 4HNE (Fig. 7), indicating that the induction of GCS-LS but not of GCS-HS required new protein synthesis. The data suggested that although the two subunits were upregulated at both the transcriptional and posttranscriptional levels, the signaling mechanisms differed.


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Fig. 7.   Effect of emetine on induction of GCS mRNAs by 4HNE. L2 cells were treated with 4HNE with or without emetine for 6 h. GCS mRNA contents were measured by Northern hybridization analysis as described in MATERIALS AND METHODS. Top: data from 1 experiment. Bottom: means (±range) of data from 2 separate experiments.

4HNE elevated the intracellular GSH content. The effect of 4HNE on the intracellular GSH content was also examined. As expected from its nonenzymatic and enzyme-catalyzed reactions with GSH, 4HNE caused an initial depletion in GSH content in a concentration-dependent manner. Nevertheless, there was a subsequent marked increase in the intracellular GSH content that reached 1.3, 1.7, and 2.0 times the control value 8 h after the cells were treated with 5, 10, and 20 µM 4HNE, respectively. GSH content then decreased with time. However, by 24 h posttreatment, the intracellular GSH content in 10 and 20 µM 4HNE-treated cells was still significantly higher than that of the control group (Fig. 8A). The data suggested that 4HNE increased the synthesis of GSH in rat lung epithelial L2 cells.


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Fig. 8.   Effect of 4HNE on intracellular GSH and GSSG contents. L2 cells were treated with different concentrations of 4HNE for times indicated. Intracellular GSH (A) and GSSG (expressed as ratio of GSSG to total GSH; B) contents were measured by HPLC method. Each point is mean ± SE of 4-6 separate experiments. * Significant difference from control, P < 0.05.

Intracellular GSSG content was also measured, and the ratio of GSSG to total GSH was calculated. It was found that 10 and 20 µM 4HNE caused a significant increase in the ratio of GSSG to total GSH after 1 h of treatment (Fig. 8B). By 2 h, the ratio was still significantly higher in 20 µM 4HNE-treated cells compared with control cells. However, no differences in the ratios were observed after 4 h of treatment between the 4HNE treatment groups and the control group. The data suggested that 4HNE caused a transient oxidative stress to the cells under these experimental conditions.

4HNE did not cause cytotoxicity under these conditions. To examine whether the concentrations of 4HNE used in this study were cytotoxic, the effects of 4HNE on the cell morphology and intracellular ATP content were studied. It was found that 4HNE had no significant effect on the morphology of the cells (data not shown) or on the intracellular ATP content after the cells were treated with 5, 10, and 20 µM 4HNE for 1, 8, and 24 h compared with the control group (data not shown). The data suggested that the concentrations of 4HNE used in present study were not toxic to the cells.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Oxidative stress has been reported to be one of the circumstances in which the expression of both GCS subunit genes was increased. The present investigation demonstrated that 4HNE, a lipid peroxidation product, caused an increase in the transcription, mRNA stability, and protein content of both GCS subunits along with an increase in GCS activity and GSH synthesis in rat lung epithelial L2 cells, without cytotoxicity. Although the increase in transcription of both subunit mRNAs has also been shown with a variety of xenobiotic agents, this study also demonstrated an increase in GCS mRNA stability after exposure to a slightly elevated concentration of this physiological cellular component. Moreover, this increase in mRNA stability contributed to approximately the same extent as did the increase in transcription that has been observed with 4HNE or any of the other agents.

The present study also demonstrated that although 4HNE increased the transcription of both GCS subunit genes, the signaling pathways involved in the activation of these two GCS genes might be different. The results from experiments employing the protein synthesis inhibitor emetine indicated that the induction of GCS-LS, but not of GCS-HS, required new protein synthesis. Therefore, the increased transcription of GCS-LS mRNA was considered an indirect response to 4HNE. In contrast, the proteins required for the increased transcription of GCS-HS mRNA were already present in sufficient quantity in the cells. Because the transcription of GCS-HS mRNA was increased without new protein synthesis, it was considered a direct response to 4HNE. Previous investigations (48, 52) have shown that the induction of GCS-HS by other oxidants is a direct response. Shi et al. (52) have shown that the induction of GCS-HS by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) was not affected by cycloheximide, another protein synthesis inhibitor. Rahman et al. (48) reported that cycloheximide had no effect on the induction of GCS-HS by cigarette smoke condensate. To our knowledge, however, no prior evidence has been published regarding whether the induction of GCS-LS was a direct or an indirect response to any agent. Although several other reports (39, 49, 55) have described coordinate regulation of the two GCS subunit mRNAs by various agents, this study demonstrated for the first time that the mechanism of induction of the two GCS subunit mRNAs differed even when they were both upregulated by 4HNE.

Several putative transcription factor binding sites have been found in the 5' promoter region of GCS-HS, including several antioxidant responsive elements, activator protein (AP)-1, AP-2, an AP-1-like or metal response element, SP-1, and nuclear factor-kappa B (8, 16, 34, 37, 39, 47, 48, 50, 56, 64). However, which cis element is involved in the induction of GCS-HS is not clear. Several laboratories have reached conflicting conclusions, partially due to differences in the cell lines or compounds used. Most of the same cis elements, with the notable exception of nuclear factor-kappa B, are also present in the 5' promoter region of GCS-LS. The present study did not address which cis element(s) is involved in the induction of either GCS mRNA by 4HNE. Nonetheless, the results of the experiments with the protein synthesis inhibitor emetine, which indicated that protein synthesis is required for the activation of transcription of GCS-LS but not of GCS-HS, demonstrate a difference in the regulation of the two subunits. There are putative elements in common in the promoter regions of both GCS subunit genes (including AP-1 and antioxidant responsive elements). Although these elements could be involved in 4HNE-induced transcriptional activation of both subunits, at least one other protein must be synthesized before transcription of GCS-LS can occur.

Both nonenzymatic and enzyme-catalyzed conjugation with GSH contribute to detoxification of 4HNE (1, 53). Thus the increase in GCS, which contributes to the elevation of GSH in response to 4HNE exposure, appears to be an adaptive response. Interestingly, aldose reductase, another enzyme involved in the metabolic detoxification metabolism of 4HNE, has also been shown to be induced by 4HNE (54).

4HNE has been found to induce the expression of several other genes that respond to oxidative stress. Because oxidant challenge increases the production of this unsaturated aldehyde, it has been suggested that 4HNE might act as a mediator for the induction of gene expression by oxidants. A previous study (55) from this laboratory showed that the redox cycling quinone DMNQ increased transcription of both GCS subunit mRNAs in rat lung epithelial L2 cells. The induction of GCS gene expression occurred earlier in 4HNE-treated cells than in DMNQ-treated cells, which was consistent with the hypothesis that 4HNE acts as a downstream signal for the induction of GCS by DMNQ. On the other hand, the effect of DMNQ lasted longer than that of 4HNE, which was also expected with a bolus addition of 4HNE with its short half-life. DMNQ can redox cycle and thereby continuously generates hydrogen peroxide (30, 51) and, potentially, 4HNE.

Although the signals responsible for the induction of GCS gene expression by 4HNE are unknown, the reactions of 4HNE implicated in its induction of other proteins may be revealing. 4HNE can form covalent cross-links with proteins via a Michael addition to lysine, cysteine, and histidine residues. As an aldehyde, 4HNE can also form Schiff bases with amino groups on proteins and small molecules. It has been reported that covalent binding of 4HNE to protein by a Michael addition might act as a signal for the activation of genes such as transforming growth factor-beta 1 or procollagen type I (45, 46, 54). The corresponding saturated aldehyde (nonanal) could not induce the expression of these genes, even though this compound could also bind to amino groups by Schiff bases formation. Therefore, it was suggested that the Michael reaction with thiol groups was more important for the induction of gene expression.

4HNE can also conjugate with GSH, which is considered a detoxification reaction. Conjugation with GSH will lead to a decrease in GSH content, which may cause oxidative stress due to decreased ability to reduce endogenously generated hydroperoxides. The ratio of GSSG to total glutathione has been suggested to be a good indicator of cell oxidative status. In the present study, there was a significant but transient elevation of the GSSG-to-(GSH+GSSG) ratio during the early phase of 4HNE exposure (Fig. 8B). Although this change in ratio actually reflected a decrease in GSH rather than an increase in GSSG, it persisted for several hours, suggesting that an oxidative stress was occurring. Nonetheless, as GSH synthesis began to increase, GSSG content also increased but remained proportional to GSH content as reflected in the steady ratio of GSSG to (GSH+GSSG) from 4 to 24 h. Thus, because this increase in the GSSG content occurred after the induction of GCS gene expression, it was not a contributing factor in the induction of GCS.

Iodoacetamide is an alkylating agent that, like 4HNE, conjugates with GSH. Activation of heat shock factor by iodoacetamide has been suggested to result from the oxidation of protein thiols due to the depletion of GSH (28). On the other hand, 4HNE directly activates heat shock factor through formation of a hemiacetal adduct (3). Thus, in an analogous manner, the question of whether 4HNE acts directly on the transcriptional machinery for GCS or indirectly through promoting a greater oxidative state (increased hydroperoxide steady state) or through both pathways will probably require determining exactly which signaling proteins are involved. Such speculation provides a rationale for further investigation of the chemical species that provide the signals for the adaptive response of GCS induction.

    ACKNOWLEDGEMENTS

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

    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests: H. J. Forman, Dept. of Molecular Pharmacology and Toxicology, School of Pharmacy, PSC 516, Univ. of Southern California, 1985 Zonal Ave, Los Angeles, CA 90033.

Received 28 May 1998; accepted in final form 16 July 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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