Protective effects of transient HO-1 overexpression on susceptibility to oxygen toxicity in lung cells

Denise M. Suttner1, Kunju Sridhar1, Christen S. Lee1, Toshiya Tomura2, Thomas N. Hansen3, and Phyllis A. Dennery1

1 Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94304; 2 Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030; and 3 Department of Pediatrics, Ohio State University, Columbus, Ohio 43210


    ABSTRACT
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Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

Rat fetal lung cells (RFL-6) were transiently transfected with a full-length rat heme oxygenase (HO)-1 cDNA construct and then exposed to hyperoxia (95% O2-5% CO2) for 48 h. Total HO activity and HO-1 protein were measured as well as cell viability, lactate dehydrogenase (LDH) release, protein oxidation, lipid peroxidation, and total glutathione to measure oxidative injury. HO-1 overexpression resulted in increased total HO activity (2-fold), increased HO-1 protein (1.5-fold), and increased cell proliferation. Immunohistochemistry revealed perinuclear HO-1 localization, followed by migration to the nucleus by day 3. Decreased cell death, protein oxidation, and lipid peroxidation but increased LDH release and glutathione depletion were seen with HO-1 overexpression. Reactive iron content could not explain the apparent loss of cell membrane integrity. With the addition of tin mesoporphyrin, total HO activity was decreased and all changes in injury parameters were normalized to control values. We conclude that moderate overexpression of HO-1 is protective against oxidative injury, but we speculate that there is a beneficial threshold of HO-1 expression.

heme oxygenase-1; antioxidant; oxidative stress; cell proliferation; nuclear protein


    INTRODUCTION
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

HEME OXYGENASE (HO) is the first and rate-limiting enzyme in the catabolism of heme. The HO-1 isoenzyme is ubiquitously expressed and is inducible by a variety of oxidant stresses (3). In its reaction, heme is degraded and bilirubin is produced. Heme, released from hemoglobin and heme proteins, has been shown to promote the formation of oxygen radicals (5, 6), which leads to cellular injury. Furthermore, there is evidence that bilirubin can protect linoleic acid from peroxyl radical-induced oxidation in vitro (31) and that hyperbilirubinemia can enhance protection against oxidative injury in vivo (11). Finally, coinduction of ferritin, associated with HO, has been demonstrated in human skin fibroblasts exposed to ultraviolet (UV) A radiation, suggesting that, through coinduction of ferritin, induction of HO-1 may lead to the sequestration of redox active iron (36). Therefore, the sum of the actions of HO may result in antioxidant effects. Indeed, there is increasing support that HO-1 is protective against both heme- and nonheme-mediated oxidant stress. Coronary endothelial cells transfected with a pRC-CMV human HO-1 cDNA construct acquired resistance to heme- and hemoglobin-induced toxicity measured by cell viability (1). Similarly, induction of HO-1 with sublethal doses of UVA radiation has been shown to protect against cell death due to UVA exposure (35). Furthermore, hamster fibroblasts made resistant to hyperoxia were found to moderately overexpress HO-1 and, when transfected with antisense oligonucleotides, developed increased susceptibility to oxygen toxicity (10). In another study (20), human pulmonary epithelial cells stably transfected with a rat HO-1 cDNA had increased viability in hyperoxia. Several models demonstrate induction of HO-1 in hyperoxia. For example, hyperoxic exposure resulted in increased HO-1 mRNA and HO-1 activity in a variety of in vitro models including fibroblasts (13), macrophages (19), and endothelial cells (17). Hyperoxia has also been shown to induce HO-1 mRNA in vivo in the lung (8).

We wanted to expand on the role of HO-1 in defense against hyperoxia specifically as it relates to the immature lung. This area of investigation is clinically important because prolonged hyperoxic exposure is known to contribute to the development of severe lung injury in immature organisms (28). If, in fact, HO serves as an endogenous antioxidant in hyperoxia, the ability to enhance its expression could have therapeutic benefits in premature infants and sick neonates.

In the present report, rat fetal lung cells (RFL-6) in culture were used as an in vitro lung model. RFL-6 cells have been successfully used by others to study lung physiology. For example, RFL-6 cells were employed to investigate glucocorticoid-induced fetal lung maturation (39) and to determine the regulation of the nitric oxide receptor guanylate cyclase (29). Furthermore, RFL-6 cells were shown to have a high level of transfection efficiency compared with calf pulmonary arterial endothelial cells and rat type II alveolar epithelial cells (33). To investigate whether HO-1 is involved in cellular defense against hyperoxia, a model of HO-1 overexpression was established by transiently transfecting RFL-6 cells with the full-length rat HO-1 cDNA with calcium phosphate. A transient overexpression model was used because attempts to produce stably transfected cell lines were unsuccessful due to colonies with diminished cell growth. Control cells were sham transfected with the same vector excluding the HO-1 construct. To verify HO-1 overexpression, total HO activity and HO-1 protein were measured for 3 consecutive days after transfection. Once HO-1 overexpression was confirmed, HO-1-transfected and sham-transfected control samples were exposed to hyperoxia (95% O2-5% CO2) for 0, 24, and 48 h. Nonspecific parameters of cell injury as well as specific indicators of oxidative injury were measured. In additional experiments, HO-1-transfected cells were incubated with tin mesoporphyrin (SnMP), a competitive inhibitor of HO activity, to ensure that the observed results seen in the transfection experiments were due to enhanced HO activity. In an attempt to explain the modulation of oxidative damage with HO-1 overexpression, reactive iron was measured, and because decreased cell proliferation was found with stable transfection, proliferating cell nuclear antigen (PCNA) was detected in HO-1-transfected and control samples.


    MATERIALS AND METHODS
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

Cell Line and Culture Conditions

RFL-6 cells, derived from day 18 gestation rat fetal lungs and maintained by American Type Cell Culture (ATCC), were used in all experiments. The cells were grown in Ham's F-12K medium (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal calf serum (FCS), penicillin (100 units/ml), and streptomycin (100 µg/ml) and maintained at 37°C in a 95% room air-5% CO2 humidified chamber.

Experimental Design

Transfection with HO-1 and exposure to hyperoxia. To establish HO-1 overexpression, a pRC-CMV vector containing the full-length rat HO-1 cDNA was used. Control cells were sham transfected with the same plasmid excluding the HO-1 construct. The calcium phosphate precipitation method as described by the manufacturer (Life Technologies) was utilized as the vehicle for transfection in all experiments. Briefly, 24 h before transfection, 3 × 106 cells were plated in 75-cm2 flasks, enabling growth to 60% confluence. Thereafter, the cells were incubated for 24 h with 20 µg of either the HO-1 construct or the control plasmid precipitated in a calcium phosphate mixture. The precipitate was then replaced with fresh medium in a humidified chamber. Twenty-four hours after removal of the precipitate (day 1) and for the following 2 consecutive days (days 2 and 3), the cells were harvested for HO-1 activity, protein levels, and immunohistochemistry. On day 1 after transfection, the cultures were exposed to hyperoxia (95% O2-5% CO2) at 37°C in a humidified chamber and harvested for assays after 0, 24, and 48 h of hyperoxic exposure.

Transfection efficiency. The HO-1 plasmid (7 µg), precipitated in a calcium phosphate mixture, was added to RFL-6 cells that were plated on the previous day at 3 × 105 cells/25-cm2 flask. After 48 h, the cells were counted and replated at 103 and 102 cells/50-cm2 dish in medium containing G418 (400 µg/ml; Life Technologies). After 14 days in selection medium, colonies (>50 cells) were counted, and the transfection efficiency is expressed as a ratio of the total cells plated (16).

Additionally, transfected cells were labeled with HO-1 and propidium iodide (PI) to allow for nuclear staining. Cells showing dual labeling were counted and are expressed as a ratio of 100 cells counted per field as a measure of transfection efficiency. This was repeated in five separate experiments.

Incubation with SnMP. To verify that the changes in oxidative stress observed were due solely to the overexpression of HO-1, in additional experiments, transfected cells were incubated with SnMP (10 µM; Porphyrin Products, Logan, UT) in Ham's F-12K medium supplemented with penicillin (100 units/ml) and streptomycin (100 µg/ml), and assays in which significant differences between the HO-1-transfected and control samples were noted [i.e., HO activity, trypan blue exclusion, lactate dehydrogenase (LDH) release, protein oxidation and lipid peroxidation] were repeated. Cells were harvested after 24 h of hyperoxia because this was the time point when the most significant differences in oxidant injury parameters were observed between HO-1-transfected and sham-transfected cells in the previous experiments.

Assays

Immunohistochemical detection of PCNA. Nuclear proteins were collected as previously reported (19). Briefly, cell samples were suspended in a solution containing 0.5 M sucrose, 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 10% glycerol, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride. The samples were centrifuged at 4,000 g for 20 min at 4°C, and the pellet was resuspended in a high-salt buffer containing 20 mM HEPES (pH 7.9), 25% glycerol, 0.5 M KCl, 1.5 mM MgCl2, 0.4 mM EDTA, and 0.5 mM phenylmethylsulfonyl fluoride. After incubation on ice for 10 min, the supernatant was centrifuged at 14,000 g for 15 min. Twenty-microgram aliquots of supernatant were electrophoresed on a 12% polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membrane blots as previously described (19). The samples were incubated overnight with mouse anti-PCNA IgG (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:800 in a 1% solution of bovine serum albumin in phosphate-buffered saline with 0.05% Tween 20 (PBS-T). Blots were washed in PBS-T and incubated for 1 h at 25°C with a 1:5,000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG. Antigen-antibody complexes were visualized with the HRP chemiluminescence system according to the manufacturer's instructions (Bio-Rad, Hercules, CA).

Total HO activity. Assays were conducted in subdued lighting. Twenty-microliter aliquots of cell suspension (~2 × 106 cells) were reacted with 20 µl of hemin (150 µM; Sigma, St. Louis, MO) and 20 µl of NADPH (4.5 mM; Sigma) in a septum-sealed vial at 37°C. Blanks were cell sonicates reacted with hemin only. Vials were purged with carbon monoxide (CO)-free air and allowed to incubate for 15 min. The reaction was stopped with dry ice (-78°C), and CO generation in the vial gas headspace was analyzed by gas chromatography (37). HO activity was derived by subtracting the blank value from the sample value and expressing this quantity as nanomoles of CO per milligram of protein.

Cell protein content. Sonicates were analyzed for protein content by the method of Bradford (7) and read as absorbance at 595 nm.

Antibodies. Polyclonal rabbit anti-rat HO-1 antibodies were raised against a 30-kDa soluble HO-1 protein expressed in Escherichia coli from rat liver cDNA (gift from Angela Wilks, University of California, San Francisco) by Berkeley Antibodies (Berkeley, CA) as previously described (13).

Western blot analysis for HO-1. For detection of HO-1 immunoreactive protein, 20-µg aliquots of cell sonicates (~1 × 105 cells/assay) were electrophoresed on a 12% polyacrylamide gel according to the methods of Laemmli (18). Proteins were transferred overnight to a PVDF membrane (Bio-Rad) with a Bio-Rad transblot apparatus according to the method of Towbin et al. (32). Blots were briefly washed in 1× Tris-buffered saline (TBS; 200 mM Tris and 1.5 M NaCl) and then incubated overnight at 25°C with rabbit anti-rat HO-1 IgG diluted 1:800 in blocking solution (1% nonfat milk plus 0.5% BSA in TBS-0.05% Tween 20). Blots were washed in TBS-0.05% Tween 20 and incubated for 2 h at 37°C with a 1:5,000 dilution of HRP-conjugated goat anti-rabbit IgG (Caltag Laboratories, So. San Francisco, CA). The antigen-antibody signal was visualized as previously described (10), and quantification was performed by densitometry (Molecular Analyst image-analysis software, Bio-Rad).

Immunohistochemical detection of HO-1 protein. Transfected cells grown to >80% confluence on glass slides were washed in PBS and then fixed in ice-cold 100% acetone. The cells were permeabilized in 0.3% saponin in PBS and blocked in a PBS solution containing 5% milk, 1% BSA, and 0.03% saponin. The slides were then incubated with a 1:25 dilution of rabbit anti-rat HO-1 IgG overnight in a humidified chamber. After incubation, the slides were washed twice in PBS with 0.03% saponin and 1% milk, subsequently incubated with a 1:50 dilution of fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Southern Biotechnologies, Birmingham, AL) for 2 h at 37°C, and washed twice in PBS. The slides were then mounted in phenylene diamine and viewed with an Axioskop fluorescent microscope (Zeiss) fitted with a 100-W mercury HBO100W/2 (Zeiss) lamp at excitation at 493 nm and photographed with a Nikon camera.

Verification of nuclear HO-1 protein in HO-1-transfected samples. Day 3 control and HO-1-transfected cells were released from 25-cm2 flasks by gentle scraping into PBS. The cells were fixed on ice in 0.5% paraformaldehyde for 5 min. Cell pellets were resuspended in 400 µl of a PBS solution containing 3% FCS and then permeabilized and blocked in 200 µl of a PBS solution containing 0.3% saponin and 2% FCS. The pellet was rinsed in wash solution (PBS with 0.03% saponin and 0.2% FCS) and then incubated with 200 µl of a 1:25 dilution of rabbit anti-rat HO-1 IgG overnight at room temperature. After incubation, the samples were rinsed in wash solution, and incubated with a 1:150 dilution of FITC-conjugated goat anti-rabbit IgG (Southern Biotechnologies) for 1 h at room temperature. The samples were centrifuged at 2,500 rpm for 3 min, and the supernatant was discarded between each step. After an additional wash, the cells were incubated in 100 µl of a hypotonic PI solution [0.1% Triton X-100, 0.1% sodium citrate (both from Sigma), and 50 µg/ml of PI (Molecular Probes, Eugene, OR)] at 4°C overnight. After incubation, 20 µl of the cell suspension were applied to glass slides. The slides were then mounted with Slowfade and viewed with a fluorescence microscope with a confocal laser-scanning unit (Molecular Dynamics model 2010, Sunnyvale, CA). Excitation was set at 488 nm and emission at 515-545 nm for FITC. For PI, excitation was set at 568 nm and emission at >590 nm. Side-by-side images were processed on an SGI computer system (Molecular Dynamics).

Determination of cell viability. Trypan blue exclusion was used to determine cell viability. Cells were released with 0.05% trypsin-EDTA, and 20-µl aliquots were mixed with 20 µl of 0.5% Trypan blue. The number of dead (stained) cells are expressed as a ratio of the total (stained and unstained) cells counted.

Determination of LDH. Medium from HO-1-transfected and sham-transfected control samples (100 µl) was mixed with 200 µg of NADH in 0.1 M sodium phosphate buffer (pH 7.4) containing 0.062 M Na2HPO4 and 0.038 M NaH2PO4 · H2O and allowed to incubate for 10 min in a multiwell plate. Sodium pyruvate (2.3 µmol) was then added, and samples were read at 340 nm at 2-s intervals for 2 min. LDH concentration was calculated automatically from the slope of the absorbance curve. Accuracy was determined with a standard LDH enzyme solution (Sigma enzyme control 2E).

Determination of protein oxidation. Protein oxidation was measured by detecting oxidatively generated carbonyl groups with the OxyBlot Kit (Oncor, Gaithersburg, MD). Briefly, 20-µg protein aliquots mixed with 1 volume of 12% SDS were incubated with 2 volumes of 1× 2,4-dinitrophenylhydrazine for 15 min at room temperature. The samples were then neutralized by the addition of 1.5 volumes of neutralization solution (Oncor) and electrophoresed on a 12% polyacrylamide gel. After transfer to PVDF membranes, the blots were briefly washed in blocking buffer (1% BSA in PBS-T) for 1 h and then incubated for 1 h at 25°C with rabbit anti-dinitrophenyl IgG diluted 1:150 in 1% BSA-PBS-T. the blots were washed in PBS-T and incubated for 1 h at 25°C with a 1:300 dilution of HRP-conjugated goat anti-rabbit IgG. The antigen-antibody signal was visualized by chemiluminescence with the HRP chemiluminescence system according to the manufacturer's instructions (Bio-Rad). The bands showing the most consistent signal in control and experimental samples were used for comparison between samples. Quantification was performed by densitometry (Molecular Analyst image-analysis software, Bio-Rad).

Determination of lipid peroxidation. Formation of thiobarbituric acid-reactive substances (TBARS) was measured as an estimate of membrane lipid peroxidation with the method of Wright et al. (38). Briefly, 40-µl aliquots were mixed with 0.75 ml of PBS containing 5 mM ADP and 1 mM FeCl3. After incubation at 37°C for 1 h, 0.3 ml of 10% trichloroacetic acid and 0.6 ml of 0.5% thiobarbituric acid were added. The samples were boiled for 15 min, allowed to cool, and read at 532 nm. The concentration of TBARS was calculated from the molar extinction coefficient (1.55 × 10-3 M/cm).

Determination of total glutathione. Cells were washed twice with cold (4°C) Puck's saline, scraped into cold saline, and centrifuged at 400 g. The pellets were resuspended, aliquots were assayed for protein, and 30 µl of cell extract were used to detect total glutathione content. Total glutathione was detected by the method of Anderson (2), and values are expressed as micrograms per milligram of protein as previously described (13).

Determination of reactive iron. Because the reaction of HO results in the liberation of iron from heme and iron is a known prooxidant, we examined the levels of reactive iron in HO-1-overexpressing cells to see whether this could explain some of the adverse effects noted in some instances. Iron contamination was obviated in all solutions with Chelex-100 resin (Bio-Rad). Fifty-microliter aliquots of cell lysates were incubated with a solution, pH 7.4, containing 500 µl of DNA (1 mg/ml; Sigma), 50 µl of bleomycin (1.5 U/ml; Sigma), 100 µl of MgCl2 (50 mM; Mallinckrodt Baker, Paris, KY), and 100 µl of L-ascorbic acid (75 mM; Sigma) in a 37°C shaking water bath for 1 h. The reaction was stopped with 100 µl of EDTA (0.1 M; ICN Biochemicals, Cleveland, OH). Then the samples were reacted with 500 µl of 2-thiobarbituric acid (1% wt/vol in 50 mM NaOH; Sigma) and 500 µl of hydrochloric acid (25%; Mallinckrodt Baker) at 80°C for 20 min. The absorbance at 532 nm was measured, and the iron content is expressed as micromolarity per nanogram of protein (15).

Statistical Analysis

For comparison between treatment groups, the null hypothesis that there was no difference between treatment means was tested by a single-factor analysis of variance (ANOVA) for multiple groups or unpaired t-test for two groups (Statview 4.02, Abacus Concepts, Berkeley, CA). Significance (P < 0.05) between groups was determined by means of the Fisher method of multiple comparisons.


    RESULTS
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Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

Transfection Efficiency

The efficiency of the HO-1 transfection was 42.8 ± 6.3%. This was determined from eight separate experiments. With dual labeling of the cells with HO-1 and PI, 39.9 ± 7.2% of all cells in a given field were seen to express HO-1, corroborating the level of transfection efficiency seen. We controlled for native HO-1 expression in untransfected cells by subtracting the percentage of dual-labeled cells seen when averaging three fields in sham-transfected cultures from the total seen in transfected cells.

Detection of PCNA

The PCNA p36 protein is expressed at high levels in proliferating cells and is a known marker for cells in the early G1 and S phases of the cell cycle (25). This immunoreactive PCNA was expressed at higher levels in the HO-1-transfected cells on day 2 than in the sham-transfected control cells (Fig. 1). Densitometric analysis revealed a 2.5 ± 0.3-fold increase in PCNA protein compared with that in control cells (P < 0.05).


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Fig. 1.   Representative Western analysis for proliferating cell nuclear antigen (PCNA) detection in RFL-6 cells transfected with heme oxygenase (HO)-1 on day 2 (lane T) and sham-transfected control cells (lane S). Two examples for each group are shown.

Total HO Activity and HO-1 Protein

The HO-1-transfected cells showed a twofold increase in total HO activity on days 1 and 2 compared with that in sham-transfected control cells. By day 3, HO activity had returned to control values (Fig. 2). Immunoreactive HO-1 protein was higher on day 2 in the HO-1-transfected cells compared with that in the sham-transfected control cells (Fig. 3). Similarly, detection of HO-1 protein by immunohistochemistry in the HO-1-transfected cells showed an increased HO-1 signal in the perinuclear region compared with that in sham-transfected control cells on day 2. On day 3, HO-1 had migrated to the nuclear region (Fig. 4). In fact, this was verified by labeling HO-1 in isolated nuclei, thus confirming the migration of HO-1 protein to the nuclear region (Fig. 4, inset).


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Fig. 2.   Total HO activity in transfected RFL-6 cells. HO activity was derived from CO content of samples treated with hemin and NADPH. S, sham-transfected control cells after 1-3 days; T1, T2, and T3, HO-1-transfected cells 1-3 days, respectively, after transfection. Values are expressed per mg protein normalized to sham-transfected control cell values and are means ± SE of 4 experiments. * P < 0.05 vs. sham-transfected control cells.


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Fig. 3.   A: representative of 4 Western analyses for HO-1-immunoreactive protein content in transfected RFL-6 cells. Lane C, control derived from liver from CoCl2-treated adult rat; lanes S1, S2, and S3, sham-transfected control cells after 1-3 days, respectively; lanes T1, T2, and T3, HO-1-transfected cells after 1-3 days, respectively. B: HO-1-immunoreactive protein content in transfected RFL-6 cells in A quantified by densitometry. Mean signal intensity in HO-1-transfected cells was normalized to mean signal in sham-transfected control cells for each time point after transfection. Values are means ± SE of 4 experiments. * P < 0.05 vs. sham-transfected control cells.


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Fig. 4.   Immunohistochemical detection of HO-1 protein in transfected RFL-6 cells. Cells grown and fixed on slides were assayed for immunoreactive protein as described in MATERIALS AND METHODS and examined with a Zeiss fluorescent microscope at ×100 magnification. Fluorescent signal represents HO-1 labeled with FITC. 1A: HO-1-transfected cells incubated only with secondary antibody show no fluorescence (negative control). 1, B-D: HO-1-transfected cells on days 1-3, respectively, after transfection. 2A: sham-transfected cells incubated only with secondary antibody show no fluorescence (negative control). 2, B-D: sham-transfected cells on days 1-3, respectively, after transfection. Note that fluorescent signal intensity is visibly higher in a subpopulation of RFL-6 cells transfected with HO-1 on day 2 (1C, arrow) compared with that in sham-transfected control cells. By day 3 (1D), fluorescent signal in HO-1-transfected cells had migrated to nuclear region (arrowhead). Inset: nuclear localization of HO-1 on day 3. Some (~39%) of isolated nuclei stained with propidium iodide (PI; red signal) also demonstrate FITC-labeled HO-1 (green signal). Side-by-side images of the same field are shown.

Cell Viability

No differences in cell viability were observed before hyperoxic exposure; however, when the HO-1-transfected and sham-transfected control samples were exposed to hyperoxia for 24 and 48 h, the HO-1-transfected cells had 75 and 65% less cell death, respectively, compared with that in control samples (Fig. 5).


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Fig. 5.   Relative cell death in transfected RFL-6 cells exposed to hyperoxia. Trypan blue exclusion was used to determine number of dead RFL-6 cells exposed to hyperoxia. Values are expressed as a ratio to sham-transfected control cell values at each time point of hyperoxic exposure and are means ± SE of 4 experiments. S, sham-transfected control cells at 0, 24, and 48 h of hyperoxia; T1, T2, and T3, 0, 24, and 48 h, respectively, of hyperoxia in HO-1-transfected cells. * P < 0.05 vs. sham-transfected control cells.

LDH Release

No differences in LDH release were observed before hyperoxic exposure, but HO-1-transfected cells had a 2.5-fold increase in LDH release after 24 h of hyperoxic exposure compared with that in sham-transfected control cells (Fig. 6). By 48 h of hyperoxia, LDH levels were again not different between groups.


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Fig. 6.   Relative lactate dehydrogenase (LDH) release in transfected RFL-6 cells exposed to hyperoxia. LDH release was detected spectrophotometrically with a commercially available kit. Values are expressed as a ratio to sham-transfected control cell values at each time point of hyperoxic exposure and are means ± SE of 4 experiments. * P < 0.05 vs. sham-transfected control cells.

Protein Oxidation

There was no significant difference in protein oxidation between the HO-1-transfected and sham-transfected control samples at 0 h of hyperoxia. After 24 h of hyperoxic exposure, protein oxidation was significantly decreased in the HO-1-transfected cells compared with that in the sham-transfected control cells (Fig. 7). By 48 h of hyperoxia, no differences were again observed.


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Fig. 7.   A: representative of 4 Western analyses of protein carbonyl groups in transfected RFL-6 cells exposed to hyperoxia. Samples were treated with 2,4-dinitrophenylhydrazine, neutralized, and then subjected to Western analysis with anti-dinitrophenyl antibodies. Lane U, unoxidized control; lane O, oxidized control; lanes S1, S2, and S3, sham-transfected control cells after 0, 24, and 48 h, respectively, of hyperoxia; lanes T1, T2, and T3, HO-1-transfected cells after 0, 24, and 48 h, respectively, of hyperoxia. B: relative protein carbonyl content in transfected RFL-6 cells exposed to hyperoxia in A quantified by densitometry. Mean protein carbonyl signal intensity in HO-1-transfected cells was normalized to mean signal in sham-transfected control cells at 24 h of hyperoxia. Values are means ± SE of 4 experiments. * P < 0.05 vs. sham-transfected control cells.

Lipid Peroxidation

After 24 h of hyperoxic exposure, TBARS, measured as an index of lipid peroxidation, were decreased twofold compared with those in control cells (Fig. 8). There was no difference in the amount of TBARS detected between groups at any other time point measured.


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Fig. 8.   Relative thiobarbituric acid-reactive substances (TBARS) in transfected RFL-6 cells exposed to hyperoxia. TBARS were detected spectrophotometrically. Values are expressed as a ratio to sham-transfected control values for each time point of hyperoxic exposure and are means ± SE of 4 experiments. * P < 0.05 vs. sham-transfected control cells.

Glutathione Content

The HO-1-transfected cells had a 2.5- and 1.5-fold decrease in total glutathione compared with that in sham-transfected control cells at 0 and 24 h of hyperoxia, respectively. A 25% decrease in glutathione was observed in the HO-1-transfected cells after 48 h of hyperoxic exposure, although this was not significant (Fig. 9).


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Fig. 9.   Relative glutathione depletion in transfected RFL-6 cells exposed to hyperoxia. Total glutathione levels were detected spectrophotometrically. Values are expressed as a ratio to sham-transfected control values at each time point of hyperoxic exposure and are means ± SE of 4 experiments. * P < 0.05 vs. sham-transfected control cells.

Iron Content

Because the HO reaction leads to the release of iron, we evaluated the differences in reactive iron between HO-1-transfected and sham-transfected cells. No differences in reactive iron content could be observed with HO-1 transfection at any point during hyperoxic exposure.

Incubation With SnMP

With the addition of SnMP to the HO-1-transfected cells and subsequent exposure to 24 h of hyperoxia, total HO activity returned to the level in the sham-transfected control cells (a 55% decrease in HO activity compared with that in HO-1-transfected samples not treated with SnMP; Fig. 10A). Cell viability also decreased to control values when the HO-1-transfected cells were treated with SnMP (Fig. 10B). Additionally, LDH levels, previously increased in the HO-1-transfected samples, decreased toward sham-transfected control levels when the HO-1-transfected cells were treated with SnMP (Fig. 10C). Furthermore, SnMP treatment resulted in TBARS levels similar to those in control cells; namely, a 1.7-fold increase was observed in the SnMP-treated cells compared with that in untreated HO-1-transfected cells (Fig. 10D). Similarly, the amount of protein oxidation increased threefold, approaching sham-transfected control values, when the HO-1-transfected samples were treated with SnMP (Fig. 11).


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Fig. 10.   A: Total HO activity in transfected RFL-6 cells incubated with 10 µM tin mesoporphyrin (SnMP) and exposed to hyperoxia for 24 h. B: relative cell death in transfected RFL-6 cells incubated with SnMP and exposed to hyperoxia for 24 h. C: relative LDH release in transfected RFL-6 cells incubated with SnMP and exposed to hyperoxia for 24 h. D: relative lipid peroxidation in HO-1-transfected cells treated with SnMP and exposed to hyperoxia for 24 h. T, HO-1-transfected cells without SnMP treatment; M, HO-1-transfected cells with SnMP treatment. Values are expressed as a ratio to sham-transfected control values and are means ± SE of 4 experiments. * P < 0.05 vs. sham-transfected control cells. dagger  P < 0.05 vs. HO-1-transfected cells without SnMP treatment.


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Fig. 11.   A: representative of 3 Western analyses of protein carbonyl groups in transfected RFL-6 cells incubated with SnMP and exposed to hyperoxia for 24 h. B: relative protein carbonyl content in transfected RFL-6 cells exposed to hyperoxia in A subjected to Western analysis, visualized by chemiluminescence, and quantified by densitometry. Mean protein carbonyl signal intensity in HO-1-transfected cells was normalized to mean signal in sham-transfected control cells at 24 h of hyperoxia. Values are means ± SE of 3 experiments. * P < 0.05 vs. sham-transfected control cells. dagger  P < 0.05 vs. HO-1-transfected cells without SnMP treatment.


    DISCUSSION
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

HO-1 is inducible by a variety of agents that facilitate the generation of reactive oxygen species. Additionally, in the reaction of HO, heme, a prooxidant (5, 6), is degraded; bilirubin, an antioxidant (11, 31), is produced; and ferritin may be coinduced (36), thus leading to the potential sequestration of redox active iron. Therefore, the sum of the reactions of HO may result in antioxidant effects. The present study was undertaken to investigate whether rat fetal lung fibroblasts in culture (RFL-6) acquired enhanced protection against oxygen toxicity with HO-1 overexpression. With the calcium phosphate precipitation method, a transient twofold overexpression of HO-1 was successfully established. Attempts at obtaining a stably transfected cell line failed due to lack of proliferation of cell colonies. The lack of proliferation is in agreement with a previous report (20) of decreased proliferation in HO-1-transfected cells exposed to hyperoxia. However, we could not document decreased proliferation in the transient transfection model. Perhaps this was due to the overall lower level of HO-1 expression (2-fold) than what could be achieved in a stable line (3- to 5-fold). Recent observations (9, 14) corroborate that a 2- to 2.5-fold overexpression of HO results in increased cell proliferation in several cell lines. This effect is felt to be mediated by CO or heme availability. Perhaps at higher levels of HO expression, with loss of heme and excess CO, opposite effects are observed as previously suggested (24). This remains to be systematically explored.

We noted increased HO expression on day 2 after transfection and observed HO-1-immunoreactive protein in the cytoplasm as expected. However, on day 3, there was migration of immunoreactive HO-1 signal into the nucleus. This was substantiated in isolated nuclear preparations. This phenomenon has not previously been reported and may indicate that, as with other stress proteins such as heat shock protein 70 (21, 30), HO-1 can serve either as a chaperone or a nuclear messenger. Interestingly, when HO-1 was localized in the nucleus, the level of HO activity was lowered, yet there was still improved cell survival in hyperoxia. It is not clear whether this suggests that HO-1 protein itself could serve a protective role in the nucleus. It may also be that nuclear HO-1 inhibits HO-1 expression because others (27) report refractoriness of HO-1 to further induction and possible negative feedback mechanisms. This area of investigation requires further exploration.

Overexpression of HO-1 was associated with enhanced protection against oxygen toxicity as evidenced by increased cell viability. This is in agreement with data showing that rabbit coronary endothelial cells transfected with human HO-1 acquired increased resistance to heme and hemoglobin, measured by cell viability, compared with sham-transfected control endothelial cells (1). Additionally, Lee et al. (20) demonstrated increased cell viability in hyperoxia when human lung epithelial cells were transfected with rat HO-1. The aforementioned studies used cell viability as an index of oxidative injury in the investigation of the role of HO-1 in antioxidant defense. In the present study, we expanded on these observations by examining specific markers of oxidative injury, namely, protein oxidation, lipid peroxidation, and glutathione depletion. When RFL-6 cells were exposed to hyperoxia, both protein oxidation and lipid peroxidation were significantly decreased in the HO-1-transfected fibroblasts compared with those in the sham-transfected control cells. These results, in combination with the observed increased cell viability in hyperoxia seen with HO-1 overexpression, lend further support for a protective role of HO-1 against oxygen toxicity in the lung.

Despite evidence for increased protection against hyperoxia with HO-1 overexpression, HO-1-transfected RFL-6 cells showed decreased cell membrane integrity as evidenced by increased LDH release. In contrast, induction of HO-1 by preirradiation in human skin fibroblasts resulted in a fourfold increase in HO activity and less LDH release during subsequent exposure to UVA radiation compared with cells that had not been preirradiated (35). In a transient overexpression model, a significant number of cells within the sample were not transfected, and, therefore, some of the transfected cells may be expressing HO-1 well beyond the twofold average detected by Western blotting. Perhaps the increased LDH release in the HO-1-transfected samples was due to injury in the cells that are overexpressing HO-1 beyond a critical threshold. Existence of a beneficial threshold of HO-1 overexpression is supported by the observation that oxygen-resistant hamster fibroblasts (O2R95) overexpressing HO-1 cDNA were no more resistant to oxygen toxicity. In fact, these cells demonstrated some degree of increased oxygen toxicity when transfected with HO-1 cDNA, thereby achieving a fourfold overexpression of HO-1 compared with that in parent cells (13). As with HO-1, a twofold overexpression of superoxide dismutase, a known important antioxidant enzyme, resulted in a reduction in both microbicidal and fungicidal activities in intraperitoneal macrophages (23), suggesting a beneficial threshold of other antioxidants as well.

Similar to LDH release, glutathione depletion was noted with HO-1 overexpression. This is contradictory to other observations that suggested that inhibition of HO activity rather than overexpression is associated with glutathione depletion. For example, exposure of HO-1 antisense-transfected O2R95 cells to hyperoxia (10) and inhibition of HO in rabbit corneal epithelial cells (26) both resulted in lower levels of total glutathione. The explanation for the finding in this report is not clear. Because the sham-transfected cells did not display similar glutathione depletion, this phenomenon is unlikely to be related to the transfection procedure itself.

To ensure that the protection against oxygen toxicity seen in the transfection experiments was due to differences in HO-1 expression between the HO-1-transfected and sham-transfected cells, we incubated the HO-1-transfected cells with SnMP and evaluated whether inhibition on the day when HO activity was highest would reverse the effects noted with transfection. This metalloporphyrin has been used clinically as a competitive inhibitor of HO (34), and preliminary data (12) suggest that SnMP does not result in the paradoxical induction of HO-1 seen with other metalloporphyrins. Furthermore, SnMP has not been shown to alter other heme-dependent enzymes, such as nitric oxide synthase, as do other metalloporphyrins (22). With the addition of SnMP, HO activity was inhibited to 60% of the activity seen in the HO-1-transfected samples and cytoprotection associated with HO-1 overexpression was significantly diminished. Specifically, there was increased cell death, increased protein oxidation, and increased lipid peroxidation after incubation with SnMP. Interestingly, elevated LDH levels previously observed with HO-1 overexpression returned to baseline, further supporting that the decreased cell integrity is also attributable to HO-1 overexpression in this model.

Because iron is released from the HO reaction and overexpression could allow for increases in the level of this prooxidant, we measured reactive iron to understand whether increased release of iron could explain some of the detrimental effects of HO-1 overexpression. No differences were observed between the groups. It remains to be seen whether other factors such as CO or cGMP could contribute to the detrimental effects of HO-1 overexpression as reported by others (4).

In conclusion, moderate overexpression of HO-1 improves resistance to oxygen toxicity in RFL-6 cells as demonstrated by increased cell viability and decreased protein oxidation and lipid peroxidation. However, there is some cytotoxicity associated with HO-1 overexpression, suggesting a duality of effects. Establishing an overexpression model of HO-1 with predictable ranges of HO activity could potentially lead to a better understanding of the conditions under which HO is beneficial and by which mechanisms. Furthermore, if the data presented here reflect the in vivo situation, therapeutic strategies to enhance HO-1 should be undertaken with a complete understanding of the potential duality of this endogenous antioxidant.


    FOOTNOTES

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

Address for reprint requests: P. A. Dennery, Dept. of Pediatrics, Stanford Univ. School of Medicine, 750 Welch Rd. #315, Palo Alto, CA 94304.

Received 17 July 1998; accepted in final form 28 October 1998.


    REFERENCES
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

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