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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
antioxidant enzyme; reactive oxygen species; gene regulation; lung injury
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals. Animal use was reviewed and approved by the University of
Pennsylvania Institutional Animal Care and Use Committee. Male Sprague-Dawley
rats weighing 180220 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, 62.5500 µM; PQ, 0.1100 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 60120 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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).
|
|
H2O2- and
PQ-induced 1-cysPrx mRNA expression in L2 cells. L2 cells were exposed to
PQ at concentrations of 0.1100 µM or to H2O2
at concentrations of 62.5500 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.
|
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).
|
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).
|
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).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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-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 |
---|
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 |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|