Induction of 1-cys peroxiredoxin expression by oxidative stress in lung epithelial cells

Han-Suk Kim,1 Yefim Manevich,1 Sheldon I. Feinstein,1 Jhang Ho Pak,1 Ye Shih Ho,2 and Aron B. Fisher1

1Institute for Environmental Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; and 2Institute of Chemical Toxicology, Wayne State University, Detroit, Michigan 48202

Submitted 17 March 2003 ; accepted in final form 4 April 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1-Cys peroxiredoxin (1-cysPrx), a member of the peroxiredoxin family that contains a single conserved cysteine residue, reduces a broad spectrum of hydroperoxides. We studied changes in 1-cysPrx expression in rat lungs and lung cell lines in response to oxidative stress due to hyperoxia, H2O2, or paraquat. After 60 h of hyperoxia (>95% O2), mRNA and protein levels of 1-cysPrx and peroxidase activity were significantly elevated in rat lungs by ~1.5- to 2-fold compared with the control (P < 0.05). A similar induction of 1-cysPrx was observed in mouse lungs following exposure to O2 for 63 or 72 h; enzyme induction in mouse lungs was similar for wild-type and glutathione peroxidase 1 gene-targeted mice. H2O2 and paraquat treatment induced 1-cysPrx gene expression in L2 cells. Enzyme induction was attenuated by pretreatment with Trolox or N-acetylcysteine. Actinomycin D treatment showed that stability of 1-cysPrx mRNA was not altered in the presence of H2O2 or paraquat, indicating that increased expression with oxidative stress is regulated at the transcriptional level. These data indicate that the antioxidant enzyme 1-cysPrx is induced in lung cells by oxidative stress.

antioxidant enzyme; reactive oxygen species; gene regulation; lung injury


OXIDATIVE STRESS HAS BEEN implicated in the pathogenesis of acute respiratory distress syndrome, asthma, lung fibrosis, bronchopulmonary dysplasia, and other lung pathologies (5, 15, 16). Because pulmonary epithelial cells are essential to maintain function of the alveolar-capillary barrier, their capacity to reduce reactive oxygen species (ROS) associated with oxidative stress is important to prevent the loss of cellular integrity. Previous investigators have studied the relative importance of antioxidant enzymes in the ability of lung cells to tolerate oxidative stress, focusing mainly on the classic antioxidant enzymes such as catalase, glutathione peroxidases (GPx), and superoxide dismutases (3, 4, 9, 20). However, there has been considerable uncertainty regarding the role of these enzymes in vivo so that antioxidant defenses against pulmonary oxidative stress are not fully understood.

Peroxiredoxins are a recently described superfamily of nonseleno-proteins that catalyze the thiol-dependent reduction of peroxides (21). They have been divided into two subgroups according to the number of their conserved cysteine residue(s), namely the one- and two-cysteine groups. 1-Cys peroxiredon (1-cysPrx), the only mammalian member of the one-cysteine group, is widely expressed in tissues; it is enriched in lung and especially in Clara and alveolar type II epithelial cells (14). This protein has been shown to catalyze the reduction of hydroperoxides, including phospholipid hydroperoxides, using glutathione (GSH) as an electron donor (6). We recently have shown that 1-cysPrx can reduce peroxidized membrane phospholipids in a lung epithelial cell line (17) and that antisense-mediated decrease in expression results in apoptotic cell death (18). These data indicate that 1-cysPrx can play an important role in cellular defense against oxidative stress in lung cells.

We reported previously that this protein is regulated uniquely in developing rat lung during the perinatal period (13). The present study was designed to investigate changes in expression of 1-cysPrx in rat lungs and isolated cells during oxidative stress induced by hyperoxia, H2O2, or paraquat. In addition, we investigated whether gene-targeted mice lacking cytosolic GPx (GPx1) would show enhanced expression of 1-cysPrx in response to hyperoxia. These mice are not more sensitive to hyperoxic injury than the wild type (10), raising the possibility that induction of 1-cysPrx compensates for GPx1 deficiency.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials. GSH, glutathione reductase, NADPH, tert-butyl hydroperoxide, H2O2, paraquat (PQ), and actinomycin D (Act D) were purchased from Sigma Chemical (St. Louis, MO). Diphenyl-1-pyrenylphosphine (DPPP) was from Dojindo Molecular Technology (Kumamoto, Japan), and 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA) was from Molecular Probes (Eugene, OR). Cell culture media and components were from Life Technologies (Gaithersburg, MD). Trolox, a water-soluble form of vitamin E, was obtained from Aldrich Chemical (Milwaukee, WI). 1-Palmitoyl, 2-linolenoyl-sn-glycerol-3 phosphoryl choline (PLPC) was purchased from Avanti Polar Lipids (Alabaster, AL), and the corresponding PLPC hydroperoxide (PLPCOOH) was prepared using soybean 15-lipoxygenase as described previously (6). 1-Palmitoyl-2-[9,10-3H]palmitoyl-sn-glycerol-3-phosphocholine ([3H]DPPC) was purchased from NEN (Boston, MA).

Animals. Animal use was reviewed and approved by the University of Pennsylvania Institutional Animal Care and Use Committee. Male Sprague-Dawley rats weighing 180–220 g were obtained from Charles River Breeding Labs (Kingston, NY). Male C57BL/6 mice weighing ~25 g were obtained from Jackson Labs (Bar Harbor, ME). GPx1 gene-targeted mice derived from 129/SVJ x C57BL/6 were bred at Wayne State University; the generation and characteristics of these mice have been described previously (10). Animals were maintained on a 12:12-h light/dark cycle in the animal facility of the University of Pennsylvania School of Medicine. Rats were exposed to hyperoxia for 50 h and mice for 63 or 72 h in a chamber that was continuously flushed with 100% O2 at sufficient flow to give about six volume exchanges per hour. O2 content measured in the chamber was >95%. Control groups were exposed in the same chamber to ambient air. Animals were allowed food and water ad libitum. At the end of exposure, animals were anesthetized with intraperitoneal pentobarbital (50 mg/kg body wt) and exsanguinated by incision of the abdomen and the abdominal aorta. After incision of the chest, lungs were cleared of blood by perfusion through the pulmonary artery with PBS and then removed and homogenized. In some experiments, lung alveolar type II cells were isolated from hyperoxia-exposed and control rats using collagenase plus trypsin for cellular dispersion followed by differential adherence to yield a population of ~95% purity (1). Tissues and cells were frozen and stored at -70°C until analyzed.

Cell culture. A rat lung epithelial cell line (L2) was obtained from American Type Culture Collection (Manassas, VA). These cells were chosen since they express 1-cysPrx (17) and because our studies of the role of this enzyme in oxidant stress in vivo primarily utilize rat (and mouse) models. Cells were grown in Eagle's minimum essential medium (MEM) containing 10% fetal calf serum in 60-mm-diameter culture dishes at 37°C in a humidified atmosphere of 5% CO2 in air. For exposure to oxidants, H2O2 or PQ at varying concentrations (H2O2, 62.5–500 µM; PQ, 0.1–100 µM) were added to the culture medium. In some studies, Act D (10 µg/ml) was added at the start of incubation. In other studies, cells were preincubated for 60–120 min with Trolox (0.2 mM) or N-acetylcysteine (NAC, 1 mM). The culture medium was changed every 24 h. Possible cytotoxicity of added reagents was tested by assay for lactate dehydrogenase (LDH) release into the medium.

Northern blot analysis. Total RNA was isolated from lung tissue and L2 cells using the RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Total RNA was separated by electrophoresis on a 1% agarose gel containing formaldehyde (0.66 M). The size-fractionated RNAs were then transferred onto a nylon membrane (Schleicher and Schuell, Keene, NH) by capillary action and hybridized to 32P-labeled rat 1-cysPrx cDNA probe generated by random priming using the Rad Prime DNA labeling system (GIBCO-BRL, Bethesda, MD). After a high stringency wash, the membrane was exposed to Kodak X-ray film at -80°C for 12 h. Blots were quantified by densitometric scanning of X-ray film using a FluorS MultiImager (Bio-Rad, Hercules, CA).

Western blot analysis. Tissues and cells were disrupted with lysis buffer consisting of 25 mM Tris · HCl (pH 8), 1 mM EDTA, 2 mM dithiothreitol, 10% glycerol, 1% Triton X-100, and Complete Protease Inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN). Tissues and cell extracts were homogenized by sonication and centrifuged at 10,000 g for 20 min. Protein concentration was determined using Coomassie blue (Bio-Rad). Protein samples (10 µg) were subjected to 12% SDS-PAGE and transferred to nitrocellulose membranes (Amersham, Piscataway, NJ). Western blotting was carried out using a polyclonal antibody to 1-cysPrx peptide (1:3,000 dilution) and peroxidase-conjugated secondary antibody (1:5,000 dilution) as described previously (13). The reaction was detected by chemiluminescence using an ECL kit (Amersham). The membrane then was treated with stripping solution (62.5 mM Tris · HCl, pH 6.8, 100 mM 2-mercaptoethanol, and 2% SDS) at 55°C and reprobed with a 1:500 dilution of rabbit anti-actin polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) to normalize for protein loading. Density of images on film was quantified using the FluorS MultiImager (Bio-Rad).

Enzymatic activity. GPx activity was determined by coupled NADPH/GSH reductase assay in the presence of GSH with PLPCOOH as substrate. The use of the phospholipid hydroperoxide substrate provided partial specificity for 1-cysPrx since PLPCOOH is not reduced by GPx1 (6). PLA2 activity was measured at pH 4 with a liposome-based assay using [3H]DPPC substrate and radiochemical detection of liberated [3H]palmitic acid as described previously (2).

Detection of ROS production in intact cells. Intracellular ROS generation was assessed by measurement of dichlorofluoroscein (DCF) fluorescence (25). L2 cells were treated with or without H2O2 or PQ for 1 h, rinsed twice with PBS, incubated with the membrane-permeable diacetate form of the reduced dye (H2DCF-DA, 5 µM) for 15 min, and then washed. To study antioxidant effects, we preincubated cells with Trolox or NAC for 60 min. Cell fluorescence was imaged using a Nikon Diaphot TMD epifluorescence microscope, a Hamamatsu ORCA digital camera, and MetaMorph imaging software (Universal Imaging, Downingtown, PA). The fluorescence intensity was normalized to the total number of cells by phase microscopy of the same field and expressed relative to control values.

DPPP was used as a fluorescent probe for lipid peroxidation in intact cells as described previously (17, 18). In brief, L2 cells adherent to 12 x 25-mm plastic slides were grown to confluence in 60-mm culture dishes. Slides with cells then were washed with PBS twice and placed in a standard quartz cuvette (10 x 10 mm) containing 50 µM DPPP in PBS. After incubation at 37°C for 10 min, slides were washed with PBS and analyzed in a spectrofluorometer (Photon Technology International, Bricktown, NJ). After recording the initial emission spectrum, we measured the increase in fluorescence (excitation 351 nm, emission 380 nm) continuously before and after addition of reagents (H2O2 or PQ). The effects of Trolox and NAC also were studied as described above for DCF.

Data analysis. Statistical analysis was carried out with SigmaStat (Jandel, Palo Alto, CA). Mean values and SE were calculated for each experimental group. Means of two groups were compared by the nonparametric Mann-Whitney t-test. Differences among three or more groups were evaluated by one-way analysis of variance. Values of P < 0.05 were considered statistically significant.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hyperoxia-induced 1-cysPrx expression and activities. After 50 h of hyperoxia, mRNA and protein levels of 1-cysPrx in the rat lung were significantly elevated by ~1.5- to 1.7-fold; GPx activity in lung homogenates was increased by 2.1-fold, whereas PLA2 activity was increased 1.6-fold (Fig. 1). All of these changes with hyperoxia were statistically significant compared with room air controls (P < 0.05). A similar analysis was carried out on type II alveolar epithelial cells isolated from control lungs and from lungs after rats had been exposed to hyperoxia. On comparing the effects of hyperoxia in isolated rat type II cells vs. whole rat lung, we found mRNA level and acidic, Ca2+-independent phospholipase A2 (aiPLA2) activity to show slightly greater increases, whereas increases in protein and GPx activity were similar (Fig. 1).



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Fig. 1. Effect of hyperoxia on expression of 1-cys peroxiredoxin (1-cysPrx) in rat lung and alveolar type II cells. Rats were exposed to hyperoxia (>95% O2) for 50 h; lungs were homogenized for assay or used for isolation of alveolar type II cells. A: changes in 1-cysPrx mRNA content analyzed by Northern blot. B: changes in protein content analyzed by Western blot. Representative blots are shown above each graph. Individual data were quantified as densitometry units and expressed as percentage of control values. C: effect of hyperoxia on glutathione peroxidase activity of lung homogenate with 1-palmitoyl,2-linolenoyl-sn-glycerol-3-phosphoryl choline hydroperoxide (PLPCOOH) substrate [phospholipid hydroperoxide glutathione peroxidase (GPx) activity]. D: effect of hyperoxia on acidic, Ca2+-independent phospholipase A2 (aiPLA2) activity of lung homogenate with [3H]dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) substrate in mixed unilamellar liposomes. Values are means ± SE for n = 3. *P < 0.05 compared with control.

 

The expression of 1-cysPrx mRNA and protein normalized to 18S RNA or total lung protein was similar for control (room air exposed) wild-type and GPx1(–/–) mice (Fig. 2). Significant increases in 1-cysPrx mRNA and protein expression and peroxidase activity were seen in lungs from both wild-type and gene-targeted mice exposed to hyperoxia (Fig. 2 and Table 1). Expression of 1-cysPrx in mice was similar for O2 exposure at 63 or 72 h and also was similar for the wild-type and gene-targeted mice at 72 h of O2 exposure (Table 1).



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Fig. 2. Effect of hyperoxia on 1-cysPrx expression in lungs from wild-type and GPx(–/–) mice exposed to hyperoxia for 72 h. A: 1-cysPrx mRNA expression analyzed by Northern blot with 18S RNA as a control (Con) for RNA loading. B: 1-cysPrx protein as determined by immunoblot (Western). Each lane was loaded with 10 µg of total lung protein.

 

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Table 1. Effect of hyperoxia on 1-cysPrx expression in lungs from wild-type and cytosolic glutathione peroxidase gene-targeted mice

 

H2O2- and PQ-induced 1-cysPrx mRNA expression in L2 cells. L2 cells were exposed to PQ at concentrations of 0.1–100 µM or to H2O2 at concentrations of 62.5–500 µM for 12 h. There was a concentration-dependent increase in 1-cysPrx mRNA expression with a threshold at ~10 µM PQ and 250 µM H2O2 (Fig. 3A). Concentrations of 100 µM PQ and 500 µM H2O2 were chosen for further study. These concentrations had no effect on LDH release during 24 h of incubation (data not shown). Treatment with PQ or H2O2 resulted in time-dependent induction of mRNA with a significant increase noted at 6 h of incubation and a further increase at 12 h (Fig. 3B). There was a subsequent decrease in expression when measured at 24 h of incubation.



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Fig. 3. Effect of oxidant treatment on 1-cysPrx gene expression. A: dose effects of paraquat (PQ) and H2O2. L2 cells were treated with PQ (0.1–100 µM) or H2O2 (62.5–500 µM), and 1-cysPrx mRNA expression was analyzed by Northern blot. B: time course of PQ and H2O2 effects on 1-cysPrx gene expression. L2 cells were treated with PQ (100 µM) or H2O2 (500 µM) for 3–24 h. Cells were harvested at each time point and analyzed for 1-cysPrx gene expression by Northern blot. Individual data were quantified as densitometry units and presented as percentage of the control (time 0) values. Values are means ± SE for n = 3. *P < 0.05 compared with control.

 

To evaluate the mechanism for 1-cysPrx mRNA increase, we pretreated cells with Act D as an inhibitor of transcription. Preincubation with Act D (10 µg/ml) significantly inhibited the increase of 1-cysPrx mRNA expression by 82% in PQ-treated cells and 78% in cells treated with H2O2 (Fig. 4). Act D also was used to determine whether H2O2 or PQ affected the rate of turnover of 1-cysPrx mRNA in L2 cells. Act D (10 µg/ml) was coincubated with medium ± H2O2 (500 µM) or PQ (100 µM). The decrease in 1-cysPrx mRNA with time was similar in cells under control conditions and in the presence of oxidants, indicating that stability of the mRNA was not affected by the presence of oxidants (Fig. 4). Act D treatment had no effect on LDH release by L2 cells (data not shown).



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Fig. 4. The effect of actinomycin D (Act D) on 1-cysPrx mRNA expression. A: inhibition of mRNA induction. L2 cells were treated with PQ or H2O2 along with 10 µg/ml of Act D. Cells were harvested after 12 h of incubation, and 1-cysPrx mRNA expression was analyzed by Northern blot. Individual data were quantified as densitometry units and presented as percentage increase above control (without PQ or H2O2). Values are means ± SE for n = 3. *P < 0.05 compared with control. B: 1-cysPrx mRNA stability is not affected by treatment with PQ or H2O2. L2 cells were treated with H2O2 (500 µM) or PQ (100 µM) for 12 h and then for an additional 12 h in the presence of Act D (10 µg/ml). L2 cells were harvested at each time point, and total RNA was isolated and subjected to Northern blot. Individual data were quantitated as densitometry units and presented as percentage of control (time 0) values. Values are means ± SE for n = 3.

 

Oxidative stress and induction of 1-cysPrx mRNA expression. To confirm that oxidative stress is responsible for the induction of 1-cysPrx gene expression, we preincubated L2 cells with antioxidants (Trolox or NAC), then exposed them to H2O2 or PQ. Oxidant-mediated induction of 1-cysPrx mRNA expression was blocked by addition of either of the antioxidants (Fig. 5). Trolox inhibited the effect of H2O2 on gene expression by 65% and the effect of PQ by 55% (Fig. 5). NAC also markedly attenuated 1-cysPrx induction by treatment with H2O2 or PQ (Fig. 5).



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Fig. 5. The effect of antioxidants on 1-cysPrx gene induction by PQ and H2O2 as determined by Northern blot. L2 cells were pretreated with Trolox (0.2 mM) and N-acetylcysteine (NAC; 1 mM) for 2 h and then incubated with PQ (100 µM) or H2O2 (500 µM). Individual data were quantified as densitometry units and presented as percentage increase above control (without antioxidants). Values are means ± SE for n = 3.

 

We assessed the generation of intracellular ROS and lipid peroxidation during incubation with H2O2 and PQ by fluorescence microscopy using the fluorescent probes H2DCF-DA and DPPP. Both H2O2 and PQ treatment resulted in increased DCF fluorescence, indicating oxidation of the fluorophore; both Trolox and NAC significantly inhibited the increase of DCF fluorescence (Fig. 6A). DPPP fluorescence measured in cellular lipid extracts was increased about five- to six-fold after treatment with either PQ or H2O2 (Fig. 6B). Trolox inhibited this increase almost completely, whereas NAC was slightly less effective (Fig. 6B).



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Fig. 6. Oxidative stress in L2 cells treated with H2O2 or PQ. A: the effect of antioxidants on 2,7-dichlorofluorescein (DCF) fluorescence. L2 cells were treated with H2O2 (panels 1–6) or PQ (panels 7–12), and DCF fluorescence was measured without added antioxidants (Con; panels 2 and 8) or in the presence of Trolox (Tro; panels 4 and 10) or NAC (panels 6 and 12). DCF fluorescence is presented in pseudocolor using MetaMorph imaging software to show the relative fluorescence intensity (the fluorescence scale is presented at bottom). Odd numbered panels are phase-contrast images, each corresponding to the subsequent fluorescent image. B: the effect of antioxidants on diphenyl-1-pyrenylphosphine (DPPP) fluorescence. L2 cells adherent to plastic slides were incubated at 37°C for 10 min with DPPP (50 µM), washed twice, and placed in a 10 x 10-mm quartz spectrofluorometer cuvette. After recording the initial emission spectrum, we measured the increase in fluorescence at 380 nm continuously before and after addition of H2O2 or PQ ± antioxidants (AO). Values are means ± SE for n = 3. *P < 0.05 vs. respective control.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxiredoxins, a family of 20- to 30-kDa peroxidases, are represented in organisms from all kingdoms (7, 21). The family comprises multiple isoforms, including six isoforms revealed in the recently available human genome. These isoforms have unique properties and tissue and organellar distribution. Prx I–V each contain two conserved cysteine residues and utilize thioredoxin as the redox cofactor (7, 21, 23). By contrast, 1-cysPrx (also known as Prx VI) contains only a single conserved cysteine (11, 14) and utilizes GSH as the redox cofactor (2, 6). In the rat, 1-cysPrx is expressed at relatively high levels in the lung and brain (14). In addition to its peroxidase activity, this bifunctional enzyme has PLA2 activity that is Ca2+ independent and shows maximal activity at an acidic pH; site-directed mutagenesis has indicated distinct active sites for the peroxidase and phospholipase activities (2).

Previous studies have shown that the peroxidase activity of 1-cysPrx has an important antioxidant role (17, 18). Enzyme induction was observed in rat lungs in the perinatal period compatible with the response to oxidant stress associated with elevation of lung O2 concentration at birth (13). Overexpression of 1-cysPrx in a lung epithelial cell line (H441) that does not normally express the enzyme augmented the H2O2-degrading activity of the cells and inhibited cellular lipid peroxidation (17). Treatment of L2 cells with an antisense oligonucleotide for 1-cysPrx resulted in accumulation of phosphatidylcholine hydroperoxides in cellular membranes and apoptotic cell death (18). Thus decreased 1-cysPrx resulted in injury due to endogenously produced oxidants, whereas overexpression of the enzyme was protective against oxidative stress. Exposure to hyperoxia causes ROS-mediated pulmonary damage characterized by inflammation and death of lung cells. Previous studies have demonstrated that rats generally die at ~72 h of exposure to 1 ATA O2, whereas mice are somewhat more resistant, with a 50% mortality at ~96 h (10). Hence, we exposed rats for 50 h and mice for 63 or 72 h when there was minimal overt evidence of pulmonary injury. At the chosen time points, all animals were alive in the present study. As demonstrated previously (10), GPx(–/–) mice did not show greater sensitivity to the toxic effects of O2, although the present study did not examine this in detail.

The present study demonstrated induction of 1-cysPrx expression with hyperoxia in both rat and mouse lungs. Induction of 1-cysPrx was indicated by increases in mRNA, protein content, and enzyme activities. Similarity of response in the wild-type and gene-targeted mice indicates that induction of 1-cysPrx is not necessary to compensate for the loss of GPx activity. Because mice with GPx knockout are not more sensitive to hyperoxia (10), the results suggest that 1-cysPrx may play a significant role in protection against hyperoxic stress, although that possibility will require additional studies utilizing a 1-cysPrx knockout mouse model. Because 1-cysPrx in the lung is enriched in alveolar type II epithelial cells (14), we also examined expression of the enzyme in these cells isolated from lungs of O2-exposed rats. The increase in 1-cysPrx mRNA expression and aiPLA2 activity in type II cells was similar or slightly higher than for whole lung. Hyperoxia has been shown previously to induce Prx I in neonatal rat lungs, whereas Prx II was unaffected (12).

To study further the regulation of 1-cysPrx expression by oxidative stress, we utilized a rat lung epithelial (L2) cell line that expresses endogenous 1-cysPrx. H2O2 and PQ were used to induce oxidative stress, which was confirmed by use of fluorescent probes DCF and DPPP. Reduced DCF is localized to the cytosol and reacts with intracellular oxidants to become fluorescent (22). DPPP is targeted predominantly to cellular membranes, and increased fluorescence is an index of lipid peroxidation (24). Fluorescence of both DCF and DPPP was increased in cells treated with H2O2 or PQ and was blocked by pretreatment with Trolox or NAC. Both of the oxidants, when used at concentrations below the level of cellular lethality, induced expression of 1-cysPrx mRNA in L2 cells. Treatment with Act D, a transcriptional inhibitor, inhibited the increased mRNA expression to oxidative stress but had no effect on RNA half-life, indicating regulation of the gene at the transcriptional level. Attenuation of oxidant-mediated induction of 1-cysPrx mRNA by antioxidant treatment of cells confirmed that ROS were involved in gene induction.

The molecular mechanism of gene regulation in response to oxidative stress is still unclear. However, previous studies have indicated that redox-sensitive transcription factors such as NF-{kappa}B and activator protein-1 may be important in regulating the expression of some antioxidant enzymes (8, 19). The promoter region of the murine 1-cysPrx gene has sequences that are compatible with recognition sites for these transcription factors (15), although there have not yet been definitive studies to confirm their role.

Our previous studies using lung epithelial cell lines have shown that overexpressing 1-cysPrx protects against oxidative stress associated with Cu2+-ascorbate treatment (17), whereas antisense treatment results in oxidative stress and increased apoptotic cell death (18). In the present study, we have shown induction of 1-cysPrx expression by oxidative stress. These results provide evidence that 1-cysPrx can function as an antioxidant enzyme and may play a central role in protection of lung cell membranes against oxidative stress.


    ACKNOWLEDGMENTS
 
We thank Chandra Dodia, Jin Wen Chen, and Majed Nounou for assistance in the early phases of this study.

This work was presented in preliminary form at Experimental Biology 1999 (Washington, DC), Experimental Biology 2000 (San Diego, CA), and Experimental Biology 2002 (New Orleans, LA).

Present address for H.-S. Kim: Dept. of Pediatrics, Osaka Medical College, Osaka, Japan.

DISCLOSURES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-65543 and HL-19737.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. B. Fisher, Inst. for Environmental Medicine, Univ. of Pennsylvania School of Medicine, 1 John Morgan Bldg., 3620 Hamilton Walk, Philadelphia, PA 19104-6068 (E-mail: abf{at}mail.med.upenn.edu).

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


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 DISCUSSION
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