1 Lung Biology Laboratory, Georgetown University Medical Center, Washington, District of Columbia 20007; and 2 Laboratory of Cell Signaling, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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Peroxiredoxin I (Prx I) and peroxiredoxin II (Prx II) are found in abundance in the cytoplasm of cells and catalyze the reduction of hydrogen peroxide with the use of electrons provided by thioredoxin. Here we examined Prx I and Prx II expression in rat lung during perinatal development and in response to hyperoxia. Prx I protein increased during late gestation and after birth fell to adult levels; conversely, Prx I mRNA increased after birth. Prx II protein concentration was unchanged in the perinatal period, but Prx II mRNA increased after birth. In response to hyperoxia begun on postnatal day 4, there was no change in Prx II expression; however, Prx I mRNA, protein, and enzymatic activity increased significantly. These data show that 1) Prx I and Prx II are developmentally regulated at the level of translational efficiency and 2) Prx I, but not Prx II, is inducible and is upregulated during the late-gestational preparation for the oxidative stress experienced by the lung at birth and during exposure to hyperoxia in the neonatal period.
antioxidant enzyme; thioredoxin peroxidase; thiol-specific antioxidant; perinatal development
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INTRODUCTION |
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THE
PEROXIREDOXIN1 (Prx)
family of proteins represents a widely distributed class of enzymes
that directly reduce hydrogen peroxide and alkyl hydroperoxides with
hydrogen derived from NAD(P)H (1-3). Prx I and Prx II
are 25-kDa proteins that exist mainly as dimers linked by disulfide
bonds between two conserved cysteines. Prx I and Prx II are frequently
referred to as 2-Cys Prx; they are found in abundance in the cytoplasm
of cells and catalyze the reduction of hydrogen peroxide with the use
of electrons provided by thioredoxin (1, 3). Thus Prx I
and Prx II are antioxidant enzymes with peroxidase activity that
eliminate peroxides generated during normal oxidative metabolism and
elevated levels of hydrogen peroxide generated during oxidant stress or
during stimulation of cell surface receptors. In addition to their
direct function as antioxidant enzymes, Prx also serve as components of
signaling cascades in which hydrogen peroxide acts as a second
messenger. Examples of this latter function include the role of Prx as
an effector of the cellular response to growth factors and tumor necrosis factor- (10), as an inhibitor of
apoptosis (13), and as a regulator of nuclear
factor-
B activation (9).
The goal of this study was to examine the protein and mRNA concentrations of Prx I and Prx II in rat lungs during perinatal development and in response to hyperoxia. These animal models were chosen for study because they represent two circumstances in which the lung is at risk for O2 toxicity, namely birth and O2 therapy of neonates during medical treatment. With the first few postnatal breaths, the lung experiences relative hyperoxia due to an abrupt change from the in utero PO2 of 20-30 Torr to 100 Torr in air breathing. Furthermore, hyperoxia is a risk factor in the development of bronchopulmonary dysplasia in premature infants who are treated with O2 and mechanical ventilation (11). During hyperoxia, there is an increase in the generation of reactive oxygen species, including hydrogen peroxide, which can interact with iron II to form the even more toxic hydroxyl radical (8). Therefore, a determination of Prx expression is important in understanding the antioxidant capacity of the lung during the perinatal period of development and under hyperoxic exposure.
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METHODS |
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Animal studies.
All animal procedures were done according to the National Research
Council's Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of Georgetown University. Timed-pregnant Sprague-Dawley rats were obtained from Taconic Farms (Germantown, NY), were maintained in the Animal Care
Facility of Georgetown University Medical Center on a 12:12-h light-dark cycle, and were allowed food and water ad libitum. For
developmental studies, pregnant rats were delivered by hysterotomy at
gestational days 18-22 or were allowed to deliver
naturally at gestational day 22. Neonatal rats were
designated to be 1 day old on the day of birth. For the postnatal
studies, male rats were raised 10/litter, and litters were adjusted to
that size within 12 h of birth. Rats were killed on
postnatal days 1-15. For hyperoxia studies, on
postnatal day 4, the rats were exposed to hyperoxia in
3.4-ft2 plastic chambers in which O2 (>95%),
CO2 (<0.1%), temperature (22-25°C), and humidity
(40-60%) were monitored. Air-breathing rats were exposed in
identical chambers in which air flowed from a compressed air generator.
To prevent O2-induced lung damage in the nursing dams, they
were alternated daily between litters of pups exposed to >95%
O2 and air. Pups were allowed free access to the dams. This
model of oxidant stress was chosen because, even though neonatal rats
are more tolerant to the damaging effects of hyperoxia than adult rats,
hyperoxic exposure during the neonatal period has untoward consequences
and thus represents physiological O2 toxicity
(7). All animals were killed by an intraperitoneal injection of xylazine (12 mg/kg) and ketamine (90 mg/kg) and then exsanguinated by cutting the abdominal aorta. The lungs were removed, frozen in liquid nitrogen, and stored at 70°C until use.
Measurement of Prx I and Prx II protein. Lung tissue was homogenized at 4°C in lysis buffer containing 25 mM Tris (pH 7.4), 40 mM KCl, 1% Triton (vol/vol), 0.1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 0.2 U/ml aprotinin. The homogenates were centrifuged at 100,000 g at 4°C for 1 h, and the resulting supernatant fractions were designated the cytosolic fraction. The protein concentration of cytosolic extracts was determined spectrophotometrically with Coomassie Plus Protein Assay Reagent (Pierce) using BSA as a standard. Lung protein extracts at 30 µg for Prx I and 10 µg for Prx II were separated by 12.5% SDS-PAGE and transferred to nitrocellulose membranes (Hybond ECL; Amersham). Membranes were blocked overnight at 4°C with 5% nonfat milk (Bio-Rad) in Tris-buffered saline-Tween [TBS-T; 0.1% Tween 20, 20 mM Tris (pH 7.6), and 137 mM NaCl]. Membranes were then incubated with polyclonal rabbit anti-Prx I or anti-Prx II (1:5,000 in TBS-T containing 0.5% nonfat milk) for 1 h at room temperature. The antibodies were generated and characterized as described (3). To visualize the immunoreactive protein, membranes were incubated with horseradish peroxidase-conjugated anti-protein G (1:3,000 in TBS-T containing 0.5% nonfat milk; Bio-Rad) for 1 h at room temperature followed by detection with an enhanced chemiluminescence kit (Amersham). The Prx-specific protein bands were quantified by laser densitometry (Molecular Dynamics) using ImageQuant Software; the data are expressed as densitometry units (DU). For each gel, pure Prx I or Prx II protein was assayed to confirm the specificity of the antibody and to ascertain that measurements of the tissue samples were done in a linear densitometric range. In addition, for the hyperoxia studies, after immunodetection of Prx, the membranes were stripped and probed with goat anti-actin antibody (Santa Cruz) as an internal standard. Actin protein was not significantly different between air- and hyperoxia-exposed rats (P > 0.05). The final data are expressed as relative DU of Prx per actin protein. We did not use this procedure for the developmental studies because the expression level of actin changes during the perinatal period.
Measurement of Prx I and Prx II mRNAs. Total RNA from rat lungs was isolated using TRI Reagent (Molecular Research Center) according to the manufacturer's instructions. RNA was quantified by absorbance at 260 nm. Total RNA (50 µg) was separated on a 1% agarose-0.66 M formaldehyde gel and transferred to a positively charged modified nylon membrane (Nytran Plus; Schleicher & Schuell, Keene, NH). Prx I and Prx II mRNAs were detected by Northern hybridization using Prx I- and Prx II-specific cDNAs prepared from pCRprx I and pCRprx II (10). The cDNAs were labeled with [32P]dCTP by the Random Primer DNA Labeling System (GIBCO BRL). Hybridization was performed as described previously (4, 5), and the autoradiographs were quantified by laser densitometry. After obtaining the Prx data, the membranes were stripped and hybridized with 18S rRNA antisense probe labeled with [32P]CTP using the T7 riboprobe system (Promega) for synthesis of high specific activity RNA. The template for the reaction was pT7 RNA 18S purchased from Ambion. 18S RNA was not significantly different between groups during any experimental condition (P > 0.05). The final Prx data are expressed as relative DU of Prx per 18S rRNA as an internal standard.
Measurement of Prx enzymatic activity. Lungs from 4-day-old rats exposed to air or >95% O2 for 72 h were perfused through the pulmonary artery with normal saline (0.9% NaCl) and then homogenized at 4°C in buffer containing 20 mM Tris (pH 7.5), 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 0.2 U/ml aprotinin. The homogenates were centrifuged at 15,000 g at 4°C for 30 min; the protein concentration of the resulting supernatant fractions was determined spectrophotometrically with Coomassie Plus Protein Assay Reagent (Pierce) using BSA as a standard. To isolate Prx I activity, 4 mg of total protein from each sample were mixed with 400 µl of Q Sepharose (Amersham Pharmacia Biotechnology) that was previously equilibrated with 20 mM Tris buffer (pH 7.5) containing 1 mM EDTA. The mixture of lung protein and Q Sepharose was incubated for 1 h with rotation at 4°C and spun down gently. The supernatant fraction containing unbound proteins was collected and used as the enzyme source. Western blot analysis was performed to ensure that only Prx I was enriched in the unbound fraction. Prx activity was measured in an assay coupled to NADPH oxidation as described previously (2); the data are expressed as a decrease of absorbance at 340 nm minus background NADPH oxidation in the absence of Prx (2).
Statistical analysis. The values for individual samples were averaged per experimental group, and the SE of the mean was calculated. The significance of the difference between two groups was obtained using a nonparametric Mann-Whitney t-test analysis. The significance of the difference between more than two groups was determined by ANOVA with Duncan's multiple range test extension.
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RESULTS |
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Perinatal expression of rat lung Prx I and Prx II.
Prx I protein expression increased during late gestation and then
decreased to adult levels (Fig.
1A). Conversely, Prx I mRNA was low during late gestation and increased during the early postnatal period (Fig. 1B). Prx II protein concentration was abundant
in rat lung but was largely unchanged during the perinatal period (Fig.
2A). By ANOVA analysis, the
only statistical difference observed was that the level of Prx II
protein at postnatal day 5 was 1.4-fold higher than that at
gestational day 20 (P < 0.05). Unlike Prx I, Prx II protein does not appear to increase in preparation for air breathing. However, analogous to Prx I, Prx II mRNA increased after birth, with the highest concentration of Prx II mRNA observed at
postnatal day 5; the level then fell to adult levels (Fig. 2B).
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Prx I and Prx II are regulated at the level of translational
efficiency.
Translational efficiency was calculated as the amount of protein
per RNA. The translational efficiencies of both Prx I and Prx II are
highest during late gestation and at postnatal day 1; the
efficiency decreases during the first postnatal week (Fig. 3).
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Prx I, but not Prx II, is upregulated by hyperoxia.
When 4-day-old rats were exposed to >95% O2, lung
Prx I protein was greater in the hyperoxia-exposed rats than in air
controls at 48 and 72 h of exposure (Fig.
4A). Prx I mRNA was increased at 24 h of hyperoxia and continued to increase at 48 and 72 h of hyperoxic exposure (Fig. 4B). Thus the increase in mRNA
preceded the rise in Prx I protein. Prx I mRNA and protein were
elevated to a similar degree (~3.4-fold at 72 h of >95%
O2). These data indicate that hyperoxia-induced Prx I is
regulated at a pretranslational level and not by a change in
translational efficiency. At 72 h of hyperoxic exposure, there was
a significant increase in Prx I enzyme activity (air 10.7 ± 0.5, hyperoxia 12.8 ± 0.6, n = 6 for each group,
P < 0.04). Prx II protein and mRNA were unchanged in
response to hyperoxia (Fig. 5,
A and B).
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DISCUSSION |
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This is the first examination of the two cytosolic 2-cysteine Prx isoforms in lung. To investigate Prx I and Prx II gene expression, we measured both mRNA and protein concentrations. The late-gestational increase in Prx I protein that we observed is likely to be part of the developmentally regulated biochemical preparation for the oxidative stress that occurs at birth when the lung experiences hyperoxia in going from a relatively anaerobic environment to an air-breathing environment. In this regard, Prx I is similar to the surfactant system and other antioxidant enzymes, the expression of which also increases during late gestation (7, 12). Data in newborn primates show that there is an O2-induced increase in thioredoxin and thioredoxin reductase at birth (6); this increase could provide reducing equivalents for Prx I activity that exclusively employ thioredoxin in the reduction of hydrogen peroxide. From the viewpoint of physiological significance, because the increase in Prx I occurs during the last 15% of gestation, a reduced level of Prx I earlier in gestation may be partly responsible for the lower antioxidant capacity of the premature lung and by inference contribute to the pathogenesis of bronchopulmonary dysplasia.
Prx II protein was unchanged during late gestation, suggesting that in the lung Prx II protein is constituitively expressed, whereas Prx I protein is inducible. It was surprising to find that the mRNAs for both Prx I and Prx II were increased during the early postnatal period. The pattern of their mRNA expression is similar, suggesting that the transcription of both mRNAs may be under the control of the same developmental signal transduction pathway. However, the pattern of mRNA concentration for both Prx I and Prx II differs from the protein amount, indicating that the effective developmental expression of Prx I and Prx II is regulated at the level of translational efficiency. The data in this paper suggest that, for both Prx I and Prx II, translational efficiency is highest in late gestation and on the first postnatal day. The mechanism responsible for regulation at the level of Prx translational efficiency is not known but is at least partly specific because other antioxidant enzymes (catalase, glutathione peroxidase, and Cu,Zn superoxide dismutase) are regulated at a pretranslational level in late gestation (4, 5).
When examining the response to hyperoxia, Prx I and Prx II again demonstrated different patterns of expression. Prx I mRNA, protein, and enzymatic activity were induced by exposure to >95% O2, but Prx II mRNA and protein were unchanged. These data support our hypothesis that lung Prx II expression is constituitive, whereas Prx I expression is inducible. The hyperoxia-induced increase in Prx I expression occurs without a change in translational efficiency. At this time, we do not know the mechanism responsible for Prx I pretranslational regulation; we cannot distinguish between the possibility that Prx I gene transcription or Prx I mRNA stability is increased in response to hyperoxia. The induction of Prx I mRNA and protein was ~300%, but the increase in Prx I activity at 72 h of hyperoxia was only ~20%. These results suggest that hyperoxia causes enzyme inactivation that may then signal a need for increased Prx I expression to compensate for the oxidant-induced loss in enzyme activity.
Our findings in the developmental and hyperoxic studies suggest that, although both Prx I and Prx II are located in the cytoplasm and catalyze the same reaction, they are regulated differently and therefore may perform distinct biological activities. Prx II is present in abundance in the lung, but an increase above the steady-state level does not appear to be required to combat the elevated oxidative stress of birth or hyperoxic exposure. Prx I is inducible, and the increased expression in late gestation and during hyperoxic exposure may have a protective role against the damaging effects of reactive O2 intermediates, the concentration of which is elevated when O2 tension increases. It is difficult to assess the relative contribution of the Prx system compared with other lung peroxidases; however, we can consider their distribution, turnover, and abundance. Catalase is an abundant, high-turnover enzyme, but it is localized to the peroxisomes and is relatively inefficient at low concentrations of hydrogen peroxide. Although the turnover rate of Prx in reducing hydrogen peroxide is lower than glutathione peroxidase, Prx is efficient for the removal of hydrogen peroxide at low concentrations because of its greater abundance and low Michaelis constant (<20 µM; see Ref. 3). In various rat tissues, including lung, Prx comprises ~1-10 µg/mg of soluble protein, and the cellular concentration of glutathione peroxidase is much lower than Prx in most cells except hepatocytes (3). Thus the high abundance of Prx in the cytoplasm allows it to be an important player in the detoxification of hydrogen peroxide. It is intriguing to speculate that there are distinct subcellular pools of hydrogen peroxide subject to removal by specialized peroxidases. In this model, an inducible Prx may act not only as an antioxidant enzyme but also as a regulator of hydrogen peroxide concentration for signaling purposes.
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ACKNOWLEDGEMENTS |
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We thank Linda Knirsch for expert technical advice and Dr. Donald Massaro for critical review of this paper.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants HL-47413 and HL-20366, a Career Investigator Award of the American Lung Association to L. B. Clerch, and a grant from the Osaka Medical Research Foundation for Incurable Diseases to H.-S. Kim.
Present address of H.-S. Kim: University of Pennsylvania School of Medicine, Institute for Environmental Medicine, Philadelphia, PA 19104.
Address for reprint requests and other correspondence: L. B. Clerch, Lung Biology Laboratory, Georgetown Univ. Medical Center, Preclinical Science Bldg., GM12, 3900 Reservoir Rd. NW, Washington, DC 20007 (E-mail: clerchlb{at}georgetown.edu).
1 Prx family members were previously referred to as thioredoxin peroxidase or thiol-specific antioxidant.
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.
Received 26 May 2000; accepted in final form 22 December 2000.
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