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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 227-233, 2002
© 2002 American Thoracic Society

Regulation of 1-cys Peroxiredoxin Expression in Lung Epithelial Cells

Han-Suk Kim, Jhang Ho Pak, Linda W. Gonzales, Sheldon I. Feinstein and Aron B. Fisher

Institute for Environmental Medicine and Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Address correspondence to: Aron B. Fisher, M.D., Institute for Environmental Medicine, University of Pennsylvania School of Medicine, 1 John Morgan Building, 3620 Hamilton Walk, Philadelphia, PA 19104-6068. E-mail: abf{at}mail.med.upenn.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
1-cys peroxiredoxin (1-cys Prx), the only member of a Prx subfamily that contains a single conserved cysteine residue, is abundant in lung. This bifunctional protein has both glutathione peroxidase and phospholipase A2 activities compatible with a role both in protection against lung oxidant injury and also in lung phospholipid metabolism. Here we studied the developmental expression of 1-cys Prx in rat lungs and hormonal effects on protein expression in human and rat lung cells. There was little change in 1-cys Prx expression during the prenatal period, but a marked increase in expression immediately after birth. Enzymatic (peroxidase and phospholipase) activities increased gradually after birth and reached adult level at 7–14 postnatal days. Expression of the protein was induced in the presence of dexamethasone (Dex) in cultured human and rat lung epithelial cells and also was upregulated in neonatal rat lung in vivo. cAMP treatment had no effect on expression, although there was a modest synergistic effect when combined with Dex in human fetal lung epithelial cells. The increased expression of 1-cys Prx at birth may be important for surfactant phospholipid turnover related to the phopholipase A2 activity of the protein and for antioxidant defense based on its peroxidase function.

Abbreviations: phospholipase A2, aiPLA2 • dexamethasone, Dex • fetal calf serum, FCS • gestational day, GD • glutathione peroxidase, GPx • glutathione, GSH • isobutylmethylxanthine, IBMX • minimal essential medium, MEM • phosphate-buffered saline, PBS • postnatal day, PD • peroxiredoxin, Prx • type II cells, TII cells


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The peroxiredoxins (Prx), a novel family of antioxidant proteins which catalyze the reduction of peroxides using their conserved cysteine residues, can be divided into two subgroups, one containing a single conserved cysteine residue (the 1-cys Prx group), and another containing an additional conserved cysteine residue (the 2-cys Prx group) (1). To date, the 1-cys Prx group contains only one known member. This protein was first isolated from the bovine ciliary body and was shown to catalyze the reduction of H2O2 and organic hydroperoxides using glutathione (GSH) as an electron donor (2). The protein did not have GSH S-transferase activity (2, 3). Because of the absence of selenium from the protein, this enzyme was called a nonselenium glutathione peroxidase (GPx), although there remains some uncertainty as to whether GSH is the physiologic reductant (4). This protein also has been reported to have phospholipase A2 (aiPLA2) activity that is Ca2+-independent (5–7). Expression of the protein and mutagenesis studies have indicated that the bifunctional protein has distinct active sites for the peroxidase and phospholipase activities (8).

Although 1-cys Prx is not a specific lung protein, lungs exhibit especially high expression compared with other organs (6, 9). Its localization to both the cytoplasm and lysosome and its bifunctional activity have raised the possibility that it functions not only in protection against lung oxidant injury but also in the regulation of lung phospholipid metabolism. Both oxidant stress and phospholipid insufficiency are regarded as risk factors for lung diseases of the newborn (10, 11).

The present study investigated developmental changes in mRNA and protein expression and activities of 1-cys Prx. We also examined the effect of a glucocorticoid on the expression of this protein using rat and human fetal cells. Glucocorticoid was chosen for study because it is well known as a regulator of other lung proteins such as surfactant proteins (12, 13) and antioxidant enzymes (14–16). Furthermore, antenatal glucocorticoid therapy has been routinely used clinically to reduce the incidence of respiratory distress syndromes and other morbidities of premature infants (17). The results provide new information related to fetal and neonatal development of antioxidant defenses and regulation of phospholipid metabolism in the lung, thereby contributing to our understanding of the pathophysiology of neonatal lung diseases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Animal use was approved by the University of Pennsylvania Institutional Animal Care and Use Committee. Adult male Sprague-Dawley rats and timed-pregnant female rats (gestational day [GD] 1 = mating day; term = GD 22) were obtained from Charles River Breeding Labs (Kingston, NY) and maintained in the animal facility of the University of Pennsylvania School of Medicine under specific pathogen–free conditions. For the developmental study, pregnant rats were delivered by Caesarean section at GD 17, 19, or 21 or were allowed to deliver naturally at GD 22. Neonatal rats were designated to be postnatal day (PD) 1 on the day of birth. Rats were studied at PD 1, 4, 7, 14, and adult (6 wk old). For the dexamethasone (Dex) study, newborn rats were injected subcutaneously once daily with Dex (0.25 µg/day) in saline or an equal volume of saline from PD 4 to PD 10. For lung harvest, rats were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg body wt) and were exsanguinated by cutting the abdominal aorta.

Cell Culture
Studies with rat cells utilized primary isolates from rat lungs and a rat lung epithelial cell line (L2 cells). A population enriched in lung alveolar type II (TII) cells was isolated from newborn (PD 4) rat lungs using collagenase plus trypsin dispersion and differential adherence (18). Cells were cultured in Waymouth's medium containing 10% fetal calf serum (FCS) for 24 h. and then were maintained for an additional 4 d in serum-free medium with or without added hormones. L2 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA) and were grown in minimal essential medium (MEM) containing 10% FCS in 60-mm-diameter culture dishes. The medium with the relevant hormone additions was changed every 24 h. The added hormones were Dex (10-8 M) with or without 8-Br-cAMP (10-4 M) plus isobutylmethylxanthine (IBMX, 10-4 M).

Human lung alveolar epithelial cells were prepared as primary isolates from human fetal lung obtained from second-trimester therapeutic abortions (19). All protocols were approved by the Committee on Human Research at Children's Hospital of Philadelphia and the Committee for Studies Involving Human Beings at the University of Pennsylvania. Distal lung parenchyma from fetuses at 19–23 wk of gestation, previously screened for the presence of maternal infection, was chopped and alveolar epithelial cells were isolated using a method similar to that described above for rat cells. After overnight culture in Waymouth medium with 10% FCS, cells were maintained for 1–5 d in serum-free medium without (control), with Dex (10-8 M) or cAMP (10-4 M) plus IBMX (10-4 M), or with all three agents. Medium with the relevant hormone additions was changed every 24 h.

Production of Antisera
A new affinity-purified rabbit polyclonal antiserum against a peptide (CEEAKQLFPKGVFTKEL) bearing an internal sequence (16 amino acids underlined) of rat 1-cys Prx plus initial cysteine for coupling was generated commercially (HTL Bio-Products, Ramon, CA) using standard protocols. This peptide corresponds to amino acids 196–211 of the deduced sequence of the rat protein. Reactivity against immunizing peptide was shown by dot blot analysis with a detection system using peroxidase-conjugated goat anti-rabbit IgG antibody (Bio-Rad, Hercules, CA) and 4-chloro-1-naphthol, and against the whole protein by Western blot using recombinant 1-cys Prx.

Immunostaining
Immunostaining was done according to the method of Wagle and coworkers as previously described (20) with minor modification. Human fetal TII cells were fixed (2 h) with cold 4% paraformaldehyde in phosphate-buffered saline (PBS) (pH 7.4) and then washed in PBS. Cells were preincubated twice in 0.1% sodium borohydride in PBS for 5 min to reduce endogenous fluorescence. Cells were stained by sequential incubation with rabbit anti–1-cys Prx serum diluted 1:500 in PBS containing 0.3% Triton X-100, 3% bovine serum albumin, and 5% goat serum followed by incubation in Texas Red–conjugated goat anti-rabbit IgG (Organo Teknika, Durham, NC) diluted 1:1,000 in the same buffer. After extensive washes in PBS with 0.3% Triton X-100 and PBS alone, the cells were air-dried and coverslips were sealed with Mowiol (Calbiochem, San Diego, CA). As a control for nonspecific binding, rabbit preimmune serum diluted 1:500 was substituted for the primary antibody. All cells were examined by conventional fluorescence microscopy for Texas Red fluorescence. For quantitation, each TII cell was outlined and its fluorescence intensity was measured. For each condition, 5–9 cells were measured for the analysis.

Northern Blot Analysis
Total RNA was prepared from rat lungs and from rat and human cells using the RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. RNA (2 µg for rat cells, 7.5 µg for human cells) was electrophoresed on a 1% agarose gel with 0.66 M formaldehyde. The size-fractionated RNAs were transferred to a positively-charged nylon filter (Schleicher and Schuell, Keene, NH) by capillary action. 1-cys Prx–specific cDNA was labeled with 32P dCTP using Rad Prime DNA labeling system (GibcoBRL, Bethesda, MD). Hybridizations were performed with QuickHyb solution (Stratagene, LaJolla, CA). Membranes were washed and then exposed to BioMax film (Kodak, Rochester, NY) at -80°C. The bands were scanned and relative densitometric units were measured with a densitometer (Fluor-S MultiImager; Bio-Rad).

SDS-PAGE and Western Blot Analysis
Tissues and cells were homogenized with lysis buffer A (25 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 10% glycerol, 2 mM DTT, and protease inhibitor cocktail) (Boehringer Mannheim, Indianapolis, IN). Protein concentration was determined using Bio-Rad Protein assay (Bio-Rad). Extracts (~ 60 µg for human cells and ~ 20 µg for other samples) were loaded at equal protein, subjected to 12% SDS/PAGE, and then transferred to nitrocellulose membranes (Amersham Phamacia Biotech, Piscataway, NJ). After blocking by incubation with 5% skim milk in T-TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) for 1 h at room temp, the membranes were reacted for 1 h at room temp with a 1:3,000 dilution of the polyclonal antibody to 1-cys Prx, washed with T-TBS, then incubated for 1 h with 1:3,000 dilution of peroxidase-conjugated goat anti-rabbit IgG antibody. The reaction was detected by chemiluminescence using an ECL kit (Amersham Phamacia Biotech). Exposure to film and quantitation were performed as described for Northern blot. The membrane then was washed with a stripping solution (62.5 mM Tris-HCl, pH 6.8, 100 mM ß-mercaptoethanol, 2% SDS) at 55°C and reprobed with 1:500 diluted rabbit anti-actin polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) to normalize for protein loading.

Enzymatic Assays
GPx activity of tissue and cell homogenates was assayed by measuring consumption of NADPH (0.03 mM) in the presence of GSH (0.36 mM) and GSH reductase (0.23 µ/ml) with phospholipid hydroperoxide (PLPCOOH) substrate (250 µM). The substrate was prepared by treatment of sn 1-palmitoyl, 2-linolenoyl glycerophosphocholine (PLPC) with 15-lipoxygenase as previously described (4). The reaction mixture (3 ml) in addition contained 50 mM Tris HCl, 2 mM NaN3, and 0.1 mM EDTA (pH 8.0). The reaction was started by addition of protein. Fluorescence was continuously recorded at 460 nm (340 nm excitation) using a fluorescence spectrophotometer (Photon Technology Instrument, Bricktown, NJ). Activity was calculated from the linear slope as the rate of NADPH oxidation.

Phospholipase A2 activity of tissue and cell homogenates was measured at pH 4 (40 mM sodium acetate, 5 mM EDTA buffer) using [3H]-dipalmitoylphosphatidylcholine (3H-DPPC) in mixed unilamellar liposomes as substrate (8). After reaction, lipids were extracted and radio-labeled free fatty acids were separated by two-step thin layer chromatography using hexane/ether/acetic acid as a solvent system. Authentic palmitic acid was co-chromatographed as a standard. The free fatty acid spots were identified using I2 vapor, scraped from the plate, and analyzed by scintillation counting.

Data Analysis
Statistical analysis was performed with SigmaStat (Jandel Scientific, San Rafael, CA). Mean values were calculated by averaging data from each experimental group, and the standard error of the mean was calculated. Means of two groups were compared by non-parametric Mann–Whitney t test analysis. The significance of the difference between more than two groups was obtained using one-way ANOVA. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Developmental Course of 1-cys Prx Expression and Activities in Rat Lungs
Both 1-cys Prx mRNA and protein were expressed at low levels in rat lung during the intrauterine period and did not change significantly with time of gestation (Figure 1) . Protein and mRNA levels increased dramatically (~ 300%) within 24 h after birth (P < 0.05) but showed only slight change between PD 1 and adult (Figure 1). The apparent decrease in expression at PD 4 and PD 7 compared with PD 1 (Figure 1) was not statistically significant. The two enzymatic activities of the protein showed a slightly delayed pattern of postnatal expression (Figure 2) . Both aiPLA2 and GPx activities were low before birth, increased gradually after birth, and reached adult level at PD 7–14.



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  		Figure 1. Developmental course of 1-cys Prx mRNA and protein expression in rat lung. Lungs obtained from rats between gestational day (GD) 17 and post-natal day (PD) 42 were evaluated. (A) Changes in 1-cys Prx–specific mRNA content analyzed by Northern blot. (B) Changes in protein content analyzed by Western blot. Individual data were quantified as densitometry units and normalized with 18S RNA for the mRNA or actin for the protein, respectively. Representative blots are shown above each graph. Data in the graph are given as mean ± SE for three or four separate rat lungs at each age. *P < 0.05 compared with the preceding value in the graph.

 


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  		Figure 2. Developmental course of aiPLA2 and NSGPx activities of 1-cys Prx in rat lung. (A) Phospholipase A2 activity was measured in Ca2+-free buffer at pH 4 by the liberation of 3H-palmitate from [3H] DPPC in mixed unilamellar liposomes. (B) Glutathione peroxidase activity was assayed by measuring consumption of NADPH in presence of GSH and GSH reductase with PC hydroperoxide as substrate. Data are mean ± SE for three separate samples at each age. *P < 0.05 compared with preceding value in the graph.

 
Hormonal Regulation of 1-cys Prx Expression in Rat Lung Epithelial Cells
Hormonal effects on 1-cys Prx expression were investigated with alveolar type II cells isolated from newborn rat lungs (Figure 3) . This preparation contains ~ 40% TII cells with fibroblasts as the major contaminant (18). The expression of 1-cys Prx mRNA and protein in these cells was decreased after 5 d in culture without hormones. Addition of Dex to the medium increased both mRNA and protein expression compared with Day 5 in the absence of added hormone. cAMP, either alone or with Dex, had no effect on mRNA or protein expression.



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  		Figure 3. Hormonal treatment of alveolar type II (TII) cells isolated from neonatal rat lung. TII cells were isolated from postnatal day 4 rats and cultured for one day in Waymouth's medium supplemented with 10% fetal calf serum (FCS). Cells then were treated with or without dexamethasone (Dex; 10 nM) and/or cAMP (0.1 mM) in Waymouth's medium without FCS. Cells were harvested either before (Day 1) or at 4 d after (Day 5) adding hormones. (A) Cells were analyzed for mRNA by Northern blotting and for protein by Western blotting. The blots shown are representative of three separate experiments. (B) Individual data were quantified as densitometry units and normalized with 18S RNA for mRNA (solid bars) and actin for protein (shaded bars). Data are shown as percent of Day 1 control and are means ± SE for three separate experiments. *P < 0.05 compared with Day 1 control, #P < 0.05 compared with Day 5 control. C, control; D, dexamethasone; Ci, cAMP plus IBMX.

 
The Dex effect was confirmed in a study of L2 cells, a rat lung epithelial cell line that constituitively expresses 1-cys Prx. Dex at a concentration of 10-6 M or 10-7 M increased 1-cys Prx mRNA and protein expression after 48 h incubation, although lower concentrations (10-8 to 10-10 M) had no effect (Figures 4A and 4B) . With 10-7 M Dex, the mRNA level was increased by 50% at 12 h and 200% at 24 h incubation compared with the control (without Dex treatment), whereas the protein level was increased by 100% at 24 h and 150% at 48 h (Figure 4C). These changes were statistically significant (P < 0.05 compared with the preceding value). No further increase in either mRNA or protein was noted beyond 48 h of incubation.



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  		Figure 4. Dexamethasone (Dex) effect on the expression of 1-cys Prx in L2 cells. Dose effect on mRNA expression (A) and protein expression (B). L2 cells were treated with or without Dex at each concentration for 48 h and analyzed by Northern blotting for mRNA, and by Western blottting for protein using 18S RNA and actin, respectively, as loading standards. The blots shown are representative of three separate experiments. C shows the time course of Dex effect on 1-cys Prx expression in L2 cells. Cells were treated with 10-7 M Dex and harvested at intervals between 6 and 96 h. Densitometry units were quantified and normalized to 18S RNA for RNA or actin for protein. Data are shown as percent of control (no treatment) and are mean ± SE for three separate experiments. *P < 0.05 compared with the preceding time. Circles, mRNA level; triangles, protein level.

 
We also studied the Dex effect on 1-cys Prx expression in rat lung in vivo (Figure 5) . Newborn rats (PD 4) were injected with Dex (0.25 µg/day) for 7 d and the whole lung was analyzed. Dex increased 1-cys Prx mRNA expression by 80% (P < 0.01); although protein expression increased by 40%, this result was not statistically significant (P = 0.056) compared with the control. The effect in TII cells isolated from those lungs was greater than for whole lung and expression of both mRNA and protein levels were increased by over 100% compared with the control (P < 0.05).



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  		Figure 5. Effect of dexamethasone (Dex) treatment on 1-cys Prx expression in neonatal rat lungs in vivo. Neonatal rats (postnatal day 4) were injected subcutaneously daily with 0.25 µg Dex in saline or an equivolume of saline between postnatal days 4 and 10. At postnatal day 10, lung homogenates and isolated alveolar type II cells were analyzed for 1-cys Prx mRNA (normalized to 18S RNA; solid bars) and protein (normalized to actin; shaded bars) by Northern and Western blots, respectively. Data are shown as percentage of control (no Dex treatment) and are mean ± SE from 3–5 rats. *P < 0.01 versus control, #P < 0.05 versus control.

 
Hormonal Regulation of 1-cys Prx Expression in Human Fetal Lung Epithelial Cells
We studied epithelial cells that were cultured after isolation from three different human lungs of gestational ages 91, 97, and 117 d and evaluated for 1-cys Prx mRNA and protein (Figure 6 and Table 1) . TII cells in this preparation are ~ 93% (19). Similar to the changes noted with rat TII cells in culture, 1-cys Prx mRNA content of human lung epithelial cells was significantly decreased after 5 d in culture without added hormones. With Dex treatment, cells at Day 5 maintained their 1-cys Prx mRNA content. cAMP treatment by itself did not prevent the decrease in mRNA expression during culture, whereas combined treatment with both Dex and cAMP showed an increased effect compared with Dex alone. The pattern of hormonal effect on protein expression was similar to that observed for mRNA. The increase in protein level with Dex + cAMP treatment was 52% compared with Day 1 control and was 107% compared with Day 5 without hormones (Table 1). GPx activity in cell homogenates using PLPCOOH as substrate was decreased by 59% in Day 5 versus Day 1 without hormones; enzymatic activity was unchanged by addition of cAMP, but was increased by 91% with Dex and 138% with Dex + cAMP compared with Day 5 control (P < 0.05) (Table 1).



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  		Figure 6. Effect of dexamethasone (Dex) and cAMP on 1-cys Prx expression in type II epithelial cells of human fetus. Type II epithelial cells were isolated from a fetus of 91 d of gestation, cultured in medium without added hormones for 1 d after isolation (Day 1 cells), and cultured for an additional 4 d (Day 5 cells) with or without Dex (10 nM) and/or cAMP (0.1 mM) + IBMX (0.1 mM). Cells were analyzed by Northern blotting for mRNA (A) and Western blotting for protein (B). C, control; D, dexamethasone; Ci, cAMP + IBMX.

 

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  		TABLE 1 Hormonal effect on 1-cys Prx expression in human fetal alveolar epithelial cells

 
Expression of 1-cys Prx analyzed by immunofluorescence was low but detectable in human TII cells from the 117-d gestation that were cultured for 5 d (Figures 7A and 7B) . Treatment of cells with Dex + cAMP for 4 d significantly increased immunoreactive 1-cys Prx protein expression (+132% compared with the cells without hormones, P < 0.05; Figures 7C and 7D) as determined by fluorescence intensity.



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  		Figure 7. Effect of dexamethasone (Dex) and cAMP on 1-cys Prx immunofluorescence in human fetal alveolar epithelial cells. Isolated cells from human fetuses were treated without or with hormones (10 nM Dex plus 0.1 mM cAMP + IBMX). Cells were fixed and exposed to primary (rabbit anti-rat 1-cys Prx IgG, 1:500) and then secondary (goat anti-rabbit IgG conjugated with Texas Red) antibodies. Cells exposed to rabbit nonimmune serum instead of primary antibody were used as a negative control. (A and B) Control (no hormonal treatment) reacted with 1-cys Prx Ab. (C and D) Hormonal treatment reacted with 1-cys Prx Ab. (E and F) Hormonal treatment reacted with nonimmune serum. A, C, and E are phase contrast; B, D, and F are Texas Red fluorescence.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
1-cys Prx as a Lung Enzyme
Although 1-cys Prx is not specific to the lung, it is abundant in that organ (6) and has been estimated at almost 0.2% of soluble lung protein (9). This is the highest level of 1-cys Prx expression among all of the major organs and also the highest level of expression for any known member of the peroxiredoxin family (9). Within the lung, 1-cys Prx is localized predominantly to alveolar epithelial TII and Clara cells (6). Our previous studies have provided evidence for subcellular localization of 1-cys Prx protein in both a lysosomal fraction where the PLA2 activity could play a role in lipid degradation and in the cytosol, where the peroxidase function could be important in antioxidant defense (7). The PLA2 activity has been inferred to be important for surfactant phospholipid degradation because the presence of MJ33, an inhibitor of aiPLA2 activity, markedly decreased the degradation of internalized DPPC in rat lungs (21). The peroxidase activity may be especially important for the repair of peroxidized cell membranes because this enzyme shows high activity for GSH-dependent reduction of phosphatidylcholine hydroperoxides (4).

Developmental Course of 1-cys Prx
1-cys Prx has a unique pattern of developmental expression compared with surfactant proteins (17) and other antioxidant enzymes (22, 23). The latter, including another member of the Prx family (Prx I), increase during late gestation (24), whereas 1-cys Prx protein content was unchanged until birth. Prx I protein expression decreased gradually after birth to reach the adult level. By contrast, 1-cys Prx protein content did not show significant change between PD 1 and adult. Fujii and coworkers also studied 1-cys Prx protein expression in rat lung during prenatal and neonatal stages using Western blotting and immunohistochemical analysis (25). They reported augmented expression of this protein in lungs immediately after birth similar to results in this report, but did not report results for mRNA or enzymatic activity.

At birth, lung cells experience marked environmental changes that include exposure to stress hormones (e.g., corticosteroids and catecholamines), to mechanical stretch, and to an increased alveolar O2 concentration. A rapid increase of 1-cys Prx as demonstrated in these lungs immediately after birth might be an important mechanism for adaptation to these stresses. The changes in expression of 1-cys Prx protein and in RNA during the perinatal period were similar, indicating that 1-cys Prx expression is regulated mainly at the transcriptional level rather than by translational efficiency. This differs from the mechanism for control of expression for Prx I and II which are regulated at the level of translation (24). In contrast to the findings for mRNA and protein expression, increases in the two enzymatic activities of 1-cys Prx were delayed and occurred gradually during the postnatal period, only reaching adult levels at PD 7–14. Although the mechanisms responsible for regulation of the activities are not known, the results suggest that posttranslational modifications might be important.

Hormonal Regulation of 1-cys Prx Expression
Dexamethasone has been shown to regulate functional development of the lung, including the expression of surfactant proteins (12, 13) and some antioxidant enzymes (e.g., SOD, GPx, and catalase) (14–16). Both neonatal rat and human fetal TII epithelial cells in primary culture lose surfactant-related differentiated functions, but recent studies have shown that these can be restored by the addition of Dex along with cAMP (18, 19). 1-cys Prx expression also was decreased during primary cell culture and its level of expression was maintained by Dex in both differentiated (newborn rat lung) and undifferentiated (human fetal lung) cells. Although the sequence of human or rat 1-cys Prx genomic DNA has not been reported, Lee and coworkers identified several motifs in the noncoding region of exon 1 of the mouse gene encoding 1-cys Prx as putative binding sites for steroid hormone receptor (26). Unlike the effect on surfactant protein expression, cAMP by itself had no effect on 1-cys Prx expression, although there was a modest synergistic effect with Dex when evaluated in the immature type II cells from human fetal lung. The synergistic effect of cAMP shown in human fetal type II cells could be caused either by an intermediate factor synthesized in the presence of cAMP, or through alterations of gene transcription, although there was no evidence for a cAMP binding site in mouse 1-cys Prx (26). The discrepancy between human fetal cells and rat newborn cells in response to cAMP could result from a different state of cell differentiation or from a difference in promoter elements between the human and murine genes. L2 cells were used to study the dose response to Dex. An effect on expression of 1-cys Prx was seen only at relatively high Dex concentrations (> 10-7 M) and the rate of response was relatively slow. These results suggest a relatively low density of corticosteroid receptors in L2 cells.

We found that Dex also increased 1-cys Prx expression in rat lung in vivo. Although Dex may affect morphology of neonatal rat lung, including an increase in TII cell number, that would not account for the effect because 1-cys Prx protein and mRNA were normalized to actin or 18S rRNA, respectively. Furthermore, analysis of isolated type II cells showed an even greater increase of 1-cys Prx protein expression than that seen for the whole lung.

In summary, the bifunctional enzyme 1-cys Prx is developmentally regulated in the rat lung. There is little change during the prenatal period and a marked increase in expression at birth that subsequently changes little during postnatal development. Enzymatic activities also increased at birth, with a further gradual increase during the postnatal period to reach adult levels at 7–14 d. Expression of the protein is induced in the presence of dexamethasone with possibly a synergistic effect of cAMP. The increased expression at birth may be important for surfactant phospholipid turnover related to the PLA2 activity of the protein and for antioxidant defense based on its peroxidase function.


   Acknowledgments
 
The authors thank Dr. Phillip Ballard for helpful suggestions, Dr. Yefim Manevich for providing the phospholipid hydroperoxide substrate, and Jain-Qin Tao, Kris DeBolt, and Kathy Notarfrancesco for technical assistance. This work was supported by grants HL 19737 and HL 65543.

Received in original form December 18, 2001

Received in final form March 5, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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