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Published ahead of print on September 8, 2005, doi:10.1165/rcmb.2004-0361OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 33, pp. 555-564, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0361OC

Poly(ADP-ribose)polymerase Activation Mediates Lung Epithelial Cell Death In Vitro but Is Not Essential in Hyperoxia-Induced Lung Injury

Alessandra Pagano, Claire Pitteloud, Coralie Reverdin, Isabelle Métrailler-Ruchonnet, Yves Donati and Constance Barazzone Argiroffo

Departments of Pediatrics and Pathology-Immunology, University of Geneva, Medical School, Geneva, Switzerland

Correspondence and requests for reprints should be addressed to Dr. Constance Barazzone Argiroffo, Departments of Pediatrics and Pathology, Centre Médical Universitaire, 1, rue Michel Servet, 1211, Geneva, 4, Switzerland. E-mail: constance.barazzone{at}hcuge.ch


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Hyperoxia induces extensive DNA damage and lung cell death by apoptotic and nonapoptotic pathways. We analyzed the regulation of Poly(ADP-ribose)polymerase-1 (PARP-1), a nuclear enzyme activated by DNA damage, and its relation to cell death during hyperoxia in vitro and in vivo. In lung epithelial-derived A549 cells, which are known to die by necrosis when exposed to oxygen, a minimal amount of PARP-1 was cleaved, correlating with the absence of active caspase-3. Conversely, in primary lung fibroblasts, which die mainly by apoptosis, the complete cleavage of PARP-1 was concomitant to the induction of active caspase-3, as assessed by Western blot and caspase activity. Blockade of caspase activity by Z-VAD reduced the amount of cleaved PARP-1 in fibroblasts. Hyperoxia induced PARP activity in both cell types, as revealed by poly-ADP-ribose accumulation. In A549 cells, the final outcome of necrosis was dependent on PARP activity because it was prevented by the PARP inhibitor 3-aminobenzamide. In contrast, apoptosis of lung fibroblasts was not sensitive to 3-aminobenzamide and was not affected by PARP-1 deletion. In vivo, despite evidence of PARP activation in hyperoxia-exposed mouse lungs, absence of PARP-1 did not change the extent of lung damage, arguing for redundant oxidative stress–induced cell death pathways.

Key Words: apoptosis • caspase-3 • hyperoxia • necrosis • PARP-1


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
High oxygen exposure has been used as a valuable model for acute respiratory distress syndrome (ARDS) in rodents and is characterized by extensive parenchymal cell death (1). The understanding of the molecular mechanisms and signaling pathways leading to alveolar cell death is essential for the development of efficient therapeutic treatments to protect the alveolo–capillary barrier. We previously showed that mouse alveolar cells exposed to 100% oxygen exhibit apoptotic and necrotic features in vivo. However, it is difficult to define which cell type dies by apoptosis or necrosis exclusively according to morphologic criteria (2). Numerous in vitro studies indicate that the mechanism of hyperoxia-induced cell death depends on the cell type–specific response. Indeed, human (A549) and mouse (MLE12 and MLE15) epithelial cell lines exposed to hyperoxia show several characteristics of oncotic/necrotic cell death besides the activation of an early apoptotic signaling pathway (3–6). Murine macrophages and fibroblast cell lines (Rat1) seem to undergo apoptosis because they show several apoptotic features, including DNA laddering and caspase activation (7, 8). Recently, several reports have suggested that intermediate patterns of cell death may exist (4, 5).

Hyperoxia generates reactive oxygen species, leading to massive oxidation and DNA damage (9). DNA oxidation and strand breaks have been directly detected by comet assay in cultured epithelial cell lines exposed to oxygen (10, 11) and in type II alveolar epithelial cells isolated from hyperoxia-exposed mice (12). In vivo, although free radical–mediated DNA strand breaks have not been directly shown in the lung, DNA damage has been revealed by terminal transferase nick end-labeling (TUNEL) and DNA electrophoresis (DNA laddering) in oxygen-exposed mouse lungs (2, 13).

Poly(ADP-ribose) polymerase-1 (PARP-1) is the most abundant nuclear enzyme of the PARP family that is activated in response to DNA damage and participates in DNA repair, genomic integrity, and cell death (14). PARP-1 binds rapidly to DNA strand breaks and adds branched poly(ADP-ribose) (PAR) polymers, using nicotinamide adenine dinucleotide (NAD+) as a substrate, on itself and other nuclear proteins (e.g., histones), thereby facilitating the action of DNA repair enzymes (15). PARP-1 has been described to be involved in the regulation of cell death (14). The presence of cleaved PARP-1 has been considered a characteristic hallmark of apoptosis. Caspases, in particular caspase-3, are known to cleave PARP-1 and therefore inhibit its activity (16, 17). On the other hand, excessive DNA damage induces massive PARP activation, leading to the depletion of cellular stores of NAD+ and ATP and consequent energy failure followed by necrotic cell death (suicide theory) (18). PARP activation has been reported in several patho-physiologic conditions characterized by oxidative stress, cell death, and inflammation, such as hemorrhagic shock, cerebral ischemia, asthma, and lipopolysaccharide-induced acute lung injury. The specific deletion of the PARP-1 gene is associated with a beneficial effect and decreased cell death in those models (19–22). NAD+ depletion and PAR accumulation have been described in total rat lungs exposed to oxygen (23), suggesting a role for PARP in response to the oxygen-mediated oxidative stress.

In this context, we hypothesized that during acute exposure to oxygen, PARP-1 might participate in directing the cell death response. We studied PARP-1 regulation in two pulmonary cell populations. We chose the A549 epithelial cell line, known to die by necrosis (3, 4), and we isolated primary fibroblasts from mouse lung that died primarily by apoptosis during hyperoxia. We also studied the role of PARP-1 in vivo by using PARP-1 –/– mice and by treating animals with the PARP inhibitor 3-aminobenzamide (3-AB) during hyperoxia. In vitro, PARP-1 was differentially regulated according to the cell type and the mode of cell death. In particular, necrosis in epithelial A549 cells but not apoptosis in lung fibroblasts was dependent on PARP activity. Despite the induction of PARP activity, which may contribute to the cellular energy failure, the absence of PARP-1 was not sufficient to prevent hyperoxia-induced cell death in vivo.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Cell Culture and Hyperoxia Exposure
Human lung adenocarcinoma A549 cell line (ATCC CCL185) were grown in F12K medium (Kaighn's modification; Gibco, Paisley, UK), supplemented with 1% penicillin-streptomycin (Gibco) and 10% fetal calf serum. Primary mouse lung fibroblasts were isolated from lung explants of Sv129 PARP-1 +/+ or Sv129 PARP-1 –/– mice as previously described (4) and grown in Dulbecco's modified Eagle's medium (glucose 1,000 mg/l; Sigma-Aldrich, Buchs, Switzerland) supplemented with 1% penicillin-streptomycin and 10% fetal calf serum. Cell plates were kept in an incubator at 37°C and monitored daily until fibroblasts reached confluence. Within 2 wk of culture initiation, cells presented similar spindle-shape morphology, as assessed by optical microscopy (24, 25). Approximately 90% of the cells were positive for the surface marker Thy1.2 and negative for the hematopoietic marker CD45 when analyzed by flow-cytometry. Lung fibroblasts were positive for vimentin, as detected by immunofluorescence (not shown). Cell phenotype was analyzed every second passage, and fibroblasts were used for the experiments between passages 6 and 12.

For each experiment, cells were plated at subconfluence (70%) and allowed to adhere 24 h before the experiment. Hyperoxic conditions were achieved by placing the cells in sealed glass chambers filled with 95% O2–5% CO2 at 37°C for up to 96 h. The oxygen concentration was checked at the beginning and end of the exposure period by an oxygen analyzer (OM 11; Beckman, Fullerton, CA) as previously described (26). Control cells were kept in air (21% O2–5% CO2) at 37°C. Medium and gases were replaced daily.

3-AB (Alexis Biochemicals, San Diego, CA) was added to cell cultures at the final concentration of 3 mM because at this dosage the pharmacologic inhibitor presented minimal toxicity and was effective in hyperoxia. The pan-caspase inhibitor Z-VAD-fmk (Caltag Laboratories, Burlingame, CA) was added at the final concentration of 100 µM as described (27).

Determination of Cell Death
Assessment of intracellular lactate dehydrogenase release. Lactate dehydrogenase (LDH) release, a marker of cell death, was measured in cell culture supernatants: 200 µl of culture medium was removed, centrifuged for 5 min at 400 g, and stored at 4°C. For intracellular LDH determination, cells were lysed by adding 200 µl of fresh medium containing 7.5% Triton X-100 (final concentration 1%). LDH extra- and intracellular content was measured using a colorimetric assay (Roche Molecular Biochemicals, Rotkreuz, Switzerland). LDH release was calculated as the ratio of extracellular to total (extracellular + intracellular) LDH content (mean of triplicates ± SD). Values were expressed as the percentage of total releasable LDH.

Trypan blue dye exclusion. Nonadherent and adherent cells were collected and stained with an equal volume of 10% Trypan blue dye solution. The extent of cell death was expressed as the percentage of blue (dead, floating + adherent) cells over total cell number.

Analysis of nuclear morphology. Nuclear morphology was assessed by Hoechst 33258 (Sigma-Aldrich) staining. Cells were plated on coverslips placed in 30 mm Petri dishes. Adherent cells were incubated with Hoechst 33258 (2 µg/ml) in PBS for 5 min at 37°C, washed, and fixed with 4% buffered formalin or with 50% acetone/methanol. Slides were mounted with Mowiol 4–88 (Sigma-Aldrich) and visualized with a fluorescence microscope (Zeiss Axiophot 1 equipped with an Axiocam color CCD camera; Carl Zeiss, Oberkochen, Germany). Nuclei were scored as apoptotic if they appeared smaller and brighter, indicating nuclear condensation. Multiple high-power (40x) fields were averaged, and results were expressed as the percentage of total cells counted (around 600 cells/sample) in three independent experiments.

Quantification of apoptosis by flow cytometry. Cells were washed in PBS (1x), incubated with Annexin-V-FITC according to the manufacturer's instruction (BD Biosciences-Pharmingen, Heidelberg, Germany), and stained with propidium iodide (PI) (Sigma Aldrich). The analysis was performed with a FACScan flow-cytometer (BD Biosciences-Pharmingen).

Caspase-3 Activity
Cells (1 x 106/condition) were collected, washed in PBS (1x), and resuspended in hypotonic buffer containing 25 mM HEPES, 5 mM MgCl2, 1 mM aprotinin, 1 mM EDTA, and 1 mM Pefabloc. Protein content was measured using a BCA protein assay kit (Pierce, Rockford, IL). For the caspase activity measurement, 20–60 µg of proteins suspended in 20 µl of the buffer were distributed in a black microclear bottom 96-well plates (Greiner Bio-One GmbH; Frickenhausen, Germany). A mix (180 µl) containing 10 mM Hepes, 0.1% dimethyl-ammonio-l propanesulfonate, 1% saccharose, 5 mM DTT, and 30 µM of the specific substrate for caspase3 coupled to a fluorochrome (DEVD-AFC, stock at 12.5 mM in DMSO) was added to the sample. The accumulation of the fluorescent product (360 nm excitation/ 530 nm emission) was recorded during 3 h using a benchtop scanning fluorometer (FlexStation II; Molecular Devices, Bucher Biotec AG, Basel, Switzerland). Analysis of the data was performed using SoftMax Pro and Excel software.

Immunocytochemistry
Cells were fixed in trichloroacetic acid 10% and incubated with an anti-PAR mouse monoclonal antibody (H10, ref. no. 804–220-R100; Alexis Biochemicals) (dilution: 1/100) overnight at 4°C in PBS-Tween 0.05% as previously described (28, 29). After washing cells in PBS-Tween 0.1%, a biotinylated antimouse IgG, Fc-specific, antibody (ref. no. 115–065–071; Jackson Laboratories, San Diego, CA) (dilution: 1/500) was added and labeled with streptavidin-FITC (Caltag) (dilution: 1/500). 4,6-Diamidino-2-phenylindole (DAPI) (Roche Diagnostics, Rotkreuz, Switzerland) (dilution 1/200) was added to visualize nuclei. Slides were mounted and analyzed as described previously.

Animals and Hyperoxia Exposure
PARP-1 –/– (Sv129 mice genetic background) and their littermates (+/+) were furnished by Dr. Wang's laboratory (CIRC, Lyon, France) (30) and bred in our animal facility. Mice were identified by PCR according to the conditions described by Wang (31). PARP-1 protein deletion was verified by Western blot.

Experiments were performed with 8- to 10-wk-old mice weighing 20–25 g. PARP-1 –/– and PARP-1 +/+ mice were placed in a sealed plexiglas chamber and exposed to air or 100% O2 for 72 h as described (32). In some experiments, C57BL/6 mice were treated daily with the PARP inhibitor 3-AB (20 mg/kg/d dissolved in DMSO/PBS and given intraperitoneally) or the vehicle (DMSO/PBS) during oxygen exposure. After mice were killed, lung damage was evaluated macroscopically, and a score was given (33). Pulmonary edema was determined by measuring wet lung weight (34). The right lung was immediately frozen in liquid nitrogen and stored at –80°C for further analysis. In some experiments, the left lung was fixed and stored for histologic analysis. All study protocols were approved by the local ethics committee on animal experiments (Office Vétérinaire Cantonal of Geneva).

Immunohistochemistry
Left lungs were fixed by intratracheal instillation of 4% buffered formalin in phosphate buffer and embedded in paraffin. Paraffin-embedded lung tissue sections (3 µm) were stained with hematoxylin and eosin for histologic evaluation or processed for immunohistochemistry according to previously described protocols (33). Briefly, sections were deparaffinized, rehydrated, and cooked 3 x 5 min in a microwave oven to facilitate the access of the antibody. After cooling, samples were blocked with 5% BSA in Tris-buffered saline and incubated overnight with an anti-PAR monoclonal antibody (clone H10; Alexis) (dilution: 1/200). As secondary antibody, a biotinylated anti-mouse IgG, Fc-specific, antibody (Jackson) (dilution: 1/2,000) was added and labeled with streptavidin-biotin-peroxidase complex (A+B kit; Vector Laboratories, Burlingame, CA). Sections were counterstained with Hemalum. The peroxidase activity was revealed with the addition of the substrate 3,3'-diaminobenzidine tetrahydrochloride (Sigma-Aldrich). Normal mouse IgG was used instead of the primary antibody as negative control for nonspecific binding as previously described (33).

Western Blot Analysis for Cellular and Lung Extracts
Cells were washed, trypsinized, and collected by centrifugation at 400 x g. The cell pellet was lysed in total lysis buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 0.1% SDS, 1% Triton x-100, 0.5% Na-deoxycolate, proteases inhibitors). A portion of mouse frozen lung was homogenized in the same lysis buffer (40 mg/ml) in a glass Dounce homogenizer with a Teflon pestle. Homogenates were centrifuged (13,000 x g for 15 min at 4°C), and the supernatant was collected for analysis. Samples were analyzed for protein concentration with a BioRad DC Protein assay kit (ref 500–0111, Bio-Rad Laboratories, Hercules, CA). Fifty micrograms of total protein cell extracts or 100 µg of total protein lung extracts underwent electrophoresis on SDS-polyacrylamide gels and were blotted to nitrocellulose membranes (Amersham International, Amersham, UK). Membranes were blocked overnight in TBS-T buffer (Tris 0.2 M [pH 7.6], NaCl 1.5 M, 0.1% Tween-20) and 5% milk and incubated with the following antibodies: mouse monoclonal anti–PARP-1 antibody (C210, ref. no. 556362; BD Biosciences-Pharmingen; dilution 1:500), which recognizes native and cleaved forms of PARP-1; rabbit polyclonal anti-cleaved PARP-1 (Cell Signaling Technology, Beverly, MA) (dilution 1:1000); rabbit polyclonal anti-cleaved caspase-3 (9661-S; Cell Signaling Technology, Beverly, MA) (1:1,000); and rabbit polyclonal anti-actin antibody (AL-20, gift from Gabbiani laboratory) (1:2,000). Native and cleaved PARP-1 were revealed on the same membranes. Horseradish peroxidase-conjugated anti-mouse and anti-rabbit (1:3,000 dilution, Bio-Rad Laboratories) were used as secondary antibodies. Bands were visualized with a chemiluminescent substrate (ECL; Amersham International). Anti-actin antibody was used as a control for total protein loading. Quantification of signal intensity was performed on subsaturated films with the Imagequant software. Values corresponding to native and cleaved PARP-1 at the different time points during hyperoxia were expressed as fold increases over air-exposed control set as 1.

Statistical Analysis
For all parameters measured, the values for all animals in different groups were averaged, and the SD of the mean was calculated. For in vitro studies, all measurements were performed in triplicate or in duplicate when indicated. The results were expressed as mean values ± SD. The significance of differences between the values of the groups was determined with an unpaired-Student's t test. Where appropriate, two-way ANOVA with Bonferroni post-tests was used. Significance levels were set at P < 0.05.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Hyperoxia Induces a Differential Cell Death Response in Lung Epithelial Cells and Fibroblasts
To determine whether hyperoxia influences PARP expression and activity according to the mode of cell death, we first characterized cell death in pulmonary epithelial A549 cells and in primary mouse lung fibroblasts. These two cell types were exposed to hyperoxia, and their mortality was compared by measuring LDH release. In A549 cells, there was a significant increase of LDH release starting from 48 h of exposure (P < 0.001) (Figure 1A). Primary fibroblasts (Figure 1C) showed a comparable increase in LDH release. Similar results were obtained by counting Trypan blue–positive cells (A549: 60 ± 7.6% in hyperoxia-exposed cells for 72 h versus 2.3 ± 2.1% in air-exposed cells; lung fibroblasts: 22.5 ± 0.4% in hyperoxia-exposed cells for 72 h versus 1.3 ± 0.9% in air-exposed cells, n = 3, P < 0.001). We then analyzed their morphologic changes by Hoechst 33258 staining. At 72 h of oxygen exposure, A549 cells did not show any increase in fluorescence staining, whereas nuclei appeared swollen compared with air-exposed cells (Figure 1B, compare right versus left). In contrast, several nuclei of primary lung fibroblasts were shrunken and brighter, characteristic signs of apoptosis (Figure 1D, compare right versus left). Quantification of the condensed nuclei (apoptotic index) in three different experiments showed a significant increase in apoptotic fibroblasts at 72 h of hyperoxia (P < 0.001) (Figure 1E). To confirm these results, we labeled lung fibroblasts with Annexin-V-FITC, a marker of early apoptosis, and with PI, a marker of necrosis, and analyzed them by flow cytometry. At 72 h of oxygen exposure, 24.7% of the cells were single positive for Annexin (early apoptotic), and 25.7% were double positive for Annexin and PI (late apoptotic or necrotic), confirming our morphologic observations (Figure 1F).



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  		Figure 1. Time-course of cell death in hyperoxia-exposed A549 cells and lung fibroblasts. Cells were exposed to air or hyperoxia for 96 h. LDH release from A549 (A) and fibroblasts (C) was measured and expressed as the percentage of the ratio of extracellular (supernatant, SPN) to total (extracellular + intracellular) LDH content. Values are expressed as mean ± SD of triplicates (one representative experiment is shown), ***P < 0.001, 48–72–96 h Hox versus air. B (A549) and D (fibroblasts) are representative fields of cells exposed to air or hyperoxia (72 h) and stained with Hoechst 33258 (magnification 40x). (E) Quantification of apoptotic nuclei of hyperoxia-exposed fibroblasts from three independent experiments. Values are expressed as mean ± SD. ***P < 0.001, 72–96 h Hox versus air. (F) Cells were exposed to air or hyperoxia for 72 h, labeled with Annexin-V-FITC and PI, and analyzed by flow cytometry.

 
We next analyzed whether the different morphologic features observed in these two cell types correlated with a differential regulation of caspase-3 activation. It is generally assumed that the morphologic changes observed during apoptosis are mainly dependent on active caspase-3 (35, 36). No cleaved (active form) caspase-3 was detected by Western blot at any time of oxygen exposure in A549 cells (Figure 2A). Conversely, active caspase-3 was present in hyperoxia-exposed primary lung fibroblasts as early as from 48 h of oxygen exposure (Figure 2B, upper panel). To confirm the presence of active caspase-3 in primary lung fibroblasts during hyperoxia, we measured caspase-3 activity in total cell lysates (Figure 2B, lower panel). Low levels of caspase-3 activity were detected in air-exposed cells and after 24 h of hyperoxia. Caspase-3 activity was strongly induced after 48 h of oxygen exposure, correlating with the detection of active caspase-3 by Western blot. These results show that lung epithelial cells respond to hyperoxia by oncosis/necrosis without caspase-3 activation, whereas lung fibroblasts undergo classical apoptosis.



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  		Figure 2. Detection of active caspase-3 in A549 cells and lung fibroblasts. Protein extracts (50 µg) from A549 (A) and mouse lung primary fibroblasts (B, upper panel) exposed to air or hyperoxia at different times were analyzed by Western blot analysis with anti-cleaved, caspase-3–specific antibody. Results represent three different experiments. Dex: Thymus extract (15 µg of protein) from a mouse treated with 250 µg of dexamethasone (intraperitoneally) and killed 6 h later was used as a positive control. Caspase-3 activity (B, lower panel) was measured in total cell lysates of lung fibroblasts exposed to air or hyperoxia at the indicated time points. Results are expressed as fluorescence emission over time, and one representative experiment out of three is shown.

 
Differential Regulation of PARP-1 Expression in Pulmonary Cells during Hyperoxia
Because the presence of cleaved PARP-1 is considered a characteristic hallmark of apoptosis, we examined the expression of native and cleaved PARP-1 in hyperoxia-exposed A549 and lung fibroblasts. In A549 cells, native PARP-1 was strongly and stably expressed during hyperoxia (Figure 3A, upper panel). Only a small amount of the cleavage product of PARP-1 (89 kD) appeared at 48 h and 72 h of oxygen exposure and was no longer detected at 96 h. Densitometric analysis of three different experiments (Figure 3A, lower panel) confirmed that, apart from some increase of cleaved PARP-1 (P < 0.05) at 48–72 h, most PARP-1 remained uncleaved. In primary lung fibroblasts (Figure 3B, upper panel), the cleaved form of PARP-1 appeared concomitantly with the disappearance of the native form, demonstrating that a complete cleavage of the protein was induced in those cells. The densitometric analysis (Figure 3B, lower panel) showed a significant reduction of the native form of PARP-1 over time of hyperoxia exposure (P < 0.001). This was accompanied with a significant increase of the cleaved form of PARP-1 (P < 0.001).



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  		Figure 3. Western blot analysis for PARP-1 in A549 cells and lung fibroblasts. A549 cells (A) and mouse primary lung fibroblasts (B) were exposed to air or hyperoxia for the indicated times, and cell lysates (50 µg) were analyzed by Western blot. An anti–PARP-1 specific antibody was used to recognize the native form (116 kD). The same membranes were re-blotted with a specific anti-cleaved PARP-1 antibody that recognizes the 89-kD form. An anti-actin antibody was used for control of protein loading. Lower panels in A and B represent the densitometric analysis of three independent experiments, and the values ± SD are expressed as fold increases over air-exposed cells set as 1. White columns: native form of PARP-1. Gray column: cleaved form of PARP-1 (cleaved PARP-1: #P < 0.01, ###P < 0.001, 72 h Hox versus air; native PARP-1: ***P < 0.001, 48–72–96 h Hox versus air).

 
To confirm caspase-dependent PARP-1 cleavage in those cells, we exposed fibroblasts to hyperoxia (72 h) in the presence or absence of the caspase inhibitor Z-VAD-fmk. This treatment abolished caspase-3 activity (Figure 4A) and decreased PARP-1 cleavage (Figure 4B), indicating that PARP-1 cleavage in lung fibroblasts exposed to hyperoxia is a caspase-dependent phenomenon, probably mediated by caspase-3.



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  		Figure 4. Inhibition of caspase-3 activity and PARP-1 cleavage in primary lung fibroblasts. (A) Caspase activity was measured in total cell lysates of lung fibroblasts exposed to air (open circles) or hyperoxia for 72 h in the presence (open triangles) or absence (filled triangles) of Z-VAD (100 µM). Values are expressed as fluorescence emission over time. (B) Protein extracts (50 µg) from mouse lung primary fibroblasts exposed to air or hyperoxia for 72 h in the presence or absence of Z-VAD (100 µM) were blotted with anti-cleaved PARP-1 antibody and anti-actin antibody as control loading.

 
These results suggest that hyperoxia differentially regulates the rate of PARP-1 cleavage depending on the cell type.

Inhibition of PARP Activity Prevents Necrosis in A549 but Not Apoptosis in Lung Fibroblasts
Strong DNA damage induces PARP activation, which can be indirectly revealed by the accumulation of its product, PAR (18). We stained A549 cells with an anti-PAR antibody (Figure 5A). Air-exposed cells did not show intranuclear specific staining (a). After 48 and 72 h of oxygen exposure, intranuclear PAR accumulation was detectable (b and c). This staining was specifically due to PARP activity because it was abolished by the treatment with the PARP inhibitor, 3-AB (d). To assess whether cell death during hyperoxia was dependent on PARP activity, we measured LDH release in the absence or presence of 3-AB. The treatment with 3-AB significantly prevented LDH release in hyperoxia-exposed A549 cells (P < 0.001) (Figure 5B). Moreover, 3-AB did not affect nuclear morphology of hyperoxia-exposed A549 cells, as revealed by DAPI nuclear staining (Figure 5A; compare c [untreated cells] versus d [3-AB–treated cells]), confirming that PARP-inhibitor did not induce apoptosis in these cells. These results suggest that hyperoxia-induced necrotic death of epithelial cells is dependent on PARP activity.



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  		Figure 5. PAR accumulation and inhibition of PARP activity in hyperoxia-exposed A549 cells. (A) A549 were exposed to air (a) or to hyperoxia for 48 h (b) and 72 h in the absence (c) or presence (d) of 3 mM 3-AB (72 h hyperoxia). Cells were labeled with an anti-PAR monoclonal antibody (green fluorescence, a, b, c, d, left panel). Nuclei were visualized by DAPI staining (blue staining, a, b, c, d, right panels). Magnification x40. (B) LDH release was measured in cells treated with 0.3% DMSO (control) or 3-AB (3 mM in 0.3% DMSO) and exposed to air (72 h) or hyperoxia (72 h). Values are expressed as mean ± SD (n = 3, ***P < 0.001, 72 h Hox versus air).

 
We detected PAR accumulation in hyperoxia-exposed apoptotic lung fibroblast nuclei (Figure 6A, 72 h). However, the treatment with 3-AB did not prevent fibroblast cell death (Figure 6B, left panel) and did not change the number of apoptotic cells at 72 h of oxygen exposure (Figure 6B, right panel). Fibroblast cell death was not affected by PARP inhibition at earlier or later time points (data not shown). We also exposed primary lung fibroblasts isolated from PARP-1 +/+ and PARP-1 –/– mice to the same conditions and assessed cell death and apoptosis as described previously. No significant difference in LDH release (23.5 ± 1.5% in 72 h hyperoxia-exposed PARP-1 +/+ versus 28.7 ± 0.1% in 72 h hyperoxia-exposed PARP-1 –/– cells, not significant [NS]) or in the apoptotic index (11.3 ± 6.8% in 72 h hyperoxia-exposed PARP-1 +/+ versus 12.03 ± 6.6% in 72 h hyperoxia-exposed PARP-1 –/–, NS) was found between PARP-1 +/+ and PARP-1 –/– fibroblasts. In conclusion, our data suggest that necrotic cell death in hyperoxia-exposed lung epithelial cells is dependent on PARP activity, whereas apoptosis of fibroblasts is not.



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  		Figure 6. PAR accumulation and inhibition of PARP activity in primary lung fibroblasts. (A) Cells were exposed to air (a) or to hyperoxia for 72 h (b) and labeled with an anti-PAR monoclonal antibody (green fluorescence, left panel). Nuclei were visualized by DAPI staining (blue staining, right panel). White arrowheads indicate staining of apoptotic nuclei. Magnification x40. (B) LDH release was measured in lung fibroblasts (left panel) treated with 0.3% DMSO (control) or 3-AB (3 mM in 0.3% DMSO) and exposed to air (72 h) or hyperoxia (72 h). Apoptotic nuclei (right panel) were also counted in lung fibroblasts exposed to the same conditions. Values are expressed as mean ± SD (n = 3, NS).

 
PARP Activity Is Induced by Hyperoxia In Vivo but Is Not Mandatory for Alveolar Cell Death
To study the role of PARP-1 within the lung during hyperoxia in vivo, we analyzed the expression of PARP-1 in lung protein extracts from Sv129 wild-type mice exposed to air or hyperoxia for 24, 48, and 72 h. The expression of native PARP-1 did not change over time, and no cleaved PARP-1 was detected in total lung extracts of hyperoxia-exposed mice (Figure 7A). We then examined lungs of hyperoxia-exposed mice for the presence of PARP activity by immunohistochemistry. A strong intranuclear accumulation of PAR was detected in alveolar cells of hyperoxia-exposed lungs (Figure 7B, compare a, air versus b, 72 h hyperoxia), indicating PARP activation in vivo. A weak signal was also detected at earlier time points (not shown). Some nuclear staining for PAR was present in lungs from PARP-1 –/– mice exposed to the same conditions but was strongly reduced compared with PARP-1 +/+ mice (Figure 7B, compare b versus c).



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  		Figure 7. In vivo analysis of PARP-1 expression and activity in hyperoxia-induced lung injury. (A) Total protein extracts from lungs (100 µg) of air-breathing and oxygen-exposed mice (hyperoxia 24, 48, and 72 h) were analyzed by Western blot. An anti-PARP-1 specific antibody was used to recognize the native form (116 kD). The same membranes were re-blotted with an anti-cleaved PARP-1 antibody, which recognizes the 89-kD form. An anti-actin antibody was used for control of protein loading. Dex: thymus extract (15 µg of protein) from a mouse treated with 250 µg of dexamethasone (intraperitoneally) and killed 6 h later. No cleaved PARP-1 could be detected in lung extracts. (B) Paraffin-embedded tissues sections from air-breathing PARP-1 +/+ (a), hyperoxia-exposed (72 h) PARP-1 +/+ (b), and hyperoxia-exposed (72 h) PARP-1 –/– (c) were stained with anti-PAR mouse monoclonal antibody. Micrographs of representative fields are shown. Magnification x63. (C) Right lung wet weight of PARP-1 +/+ and PARP-1 –/– mice exposed to air and 72 h of hyperoxia. Results are expressed as mean ± SD (n = 23 for hyperoxia-exposed groups, n = 3 for air-exposed groups, NS).

 
To determine if PARP-1 plays a role in pulmonary cell death, we exposed PARP-1 +/+ and PARP-1 –/– mice to 100% oxygen for 72 h and analyzed the extent of lung injury as described previously (33). The alveolar damage, as assessed by macroscopic examination (macroscopic score: 2.75 ± 1.94 in PARP-1 –/– versus 1.95 ± 1.53 in PARP-1 +/+ mice; P = NS, n = 23) and measured by lung weight (Figure 7C), was similar in both groups. Absence of PARP-1 did not interfere with the amount of apoptosis in vivo because lung extracts from PARP-1 +/+ and PARP-1 –/– mice presented the same extent of DNA fragmentation, as assessed by DNA electrophoresis (not shown). In accord with these results, the administration of the PARP inhibitor 3-AB did not change lung injury and mouse survival (not shown). These data demonstrate that the absence of PARP-1 or the treatment with a PARP inhibitor are not able to prevent hyperoxia-induced cell death and lung injury and suggest that PARP-1 is not mandatory for alveolar cell death in vivo.


   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Our study provides evidence that in vitro hyperoxia induces specific cell death responses, according to the cell type, by mechanisms involving differential PARP regulation and activation. We showed that A549 cells, derived from lung alveolar type II epithelial cells, die by necrosis when exposed to hyperoxia. We did not detect the presence of the active form of the effector caspase-3, in agreement with our previous data where no cleaved caspase-3 was detected in hyperoxic mouse lungs (33). These results confirm previous reports showing that in vitro hyperoxia promotes necrosis in A549, MLE15, and MLE12 lung epithelial cells lines and in bronchial primary epithelial cells, as assessed by analysis of nuclear morphology, electron-microscopy, DNA electrophoresis, and flow-cytometry analysis (Annexin-V, PI staining) (3–6, 37). In these previous studies, the morphology of these cells was similar to that observed in vivo when mice were exposed to hyperoxia, supporting the concept that alveolar epithelial cells are more prone to necrosis (2). Nevertheless, A549 cells are able to respond to other forms of oxidative stress (e.g., H2O2 treatment) or to specific apoptotic stimuli (e.g., ceramide-mediated signaling and etoposide treatment) by apoptosis and caspase activation (3, 38, 39). Moreover, in these cells hyperoxia can induce the activation of the Bid/caspase-8 pathway, cytochrome c release, and activation of caspase-9 in spite of a final outcome of necrosis (4). Therefore, we can conclude that the apoptotic signaling machine is intact in those cells and that necrosis is specific to the cell death stimulus (high oxygen tension). For these reasons, A549 cells are considered a good model to study cell signaling and specific cell death pathway of lung epithelial cells in response to hyperoxia.

Primary lung fibroblasts exposed to hyperoxia exhibited features of apoptosis, according to the nuclear morphology, flow-cytometry analysis, and the presence of caspase-3 activity. The difference observed between the number of apoptotic cells detected by Hoechst staining and FACS analysis and the higher percentage of LDH release can be explained by the accumulation over time of dying cells or by some heterogeneity of the primary cell response to hyperoxia. These data support previous reports showing that hyperoxia is able to induce apoptosis in fibroblasts, endothelial cells, and macrophages (7, 8, 40).

No potential mechanisms have been identified to explain cell-type dependent differential death responses. Because caspase-9 was shown to be activated in A549 and fibroblastic cells during hyperoxia (4, 8) in vitro, whereas active caspase-3, which is downstream to caspase-9 (41), was present only in fibroblasts, we suggest that apoptosis in lung fibroblasts is dependent on the activation of caspase-3. However, recently, active caspase-3 was found in isolated type II cells from hyperoxic rats. These cells did not present any apoptotic aspect, suggesting that the activation of caspase-3 may not always lead to apoptosis (42). In that case, we can speculate that the apoptosis/necrosis shift may be regulated downstream of the signaling cascade.

Cleavage of PARP-1 is mainly dependent on the presence of active caspase-3 (43, 44). We found that native PARP-1 was only partially cleaved in A549 cells during hyperoxia. This correlated with the absence of active caspase-3 in these cells. In contrast, in lung fibroblasts, in which activated caspase-3 was detected during hyperoxia, all native form of PARP-1 was cleaved. Treatment with the caspase inhibitor Z-VAD-fmk reduced PARP-1 cleavage in hyperoxia-exposed A549 cells (45). In our experiments, the same inhibitor prevented caspase-3 activity and reduced the amount of cleaved PARP-1 in lung fibroblasts, suggesting that PARP-1 cleavage requires caspase-3 in these cells. Because it is known that hyperoxia induces caspase-8 activation in A549 cells, it is likely that, in the absence of active caspase-3, caspase-8 may be responsible for residual caspase proteolytic activity (4, 44).

Intranuclear PAR accumulation was detected in both cell types, indicating that PARP activity increased during hyperoxia. In A549 cells, we found positive cells at 48 and 72 h of hyperoxia. We could not detect any PAR accumulation before this time (6–24 h). PAR accumulation was specific to PARP activity because treatment with the PARP inhibitor 3-AB abolished the signal. Accordingly, 3-AB decreased the extent of cell death in these cells, demonstrating that the accumulation of PAR correlated with the final outcome of necrosis. These results are in agreement with previous data showing that inhibition of PARP activity prevented oxidative stress (H2O2)-induced cell death in the same cell type (21). Therefore, we can conclude from these data that PARP activation is one of the mechanisms responsible for hyperoxia-induced lung epithelial cell death, as hypothesized in a recent study (45).

PAR accumulation was also detected in apoptotic fibroblasts during hyperoxia. It is known that during apoptosis, cleavage of PARP-1 results in the abrogation of its enzymatic activity (46). However, it has also been described that, in certain conditions, apoptotic cells can accumulate PAR, which might correspond to a transient activation of PARP in early apoptosis (47, 48). The treatment with 3-AB (or PARP-1 gene deletion) did not protect cells from hyperoxia-induced apoptosis. Because 3-AB, administered at the same concentration, efficiently inhibited H2O2-induced cell death in the same cells (not shown), we conclude that the absence of protection in hyperoxia was not due to the inefficacy of 3-AB but was due to PARP-independent apoptosis in these cells. Similarly, PARP-1 –/– primary embryonic fibroblasts exposed to TNF-{alpha} or anti-CD95 showed the same amount of apoptosis as wild-type fibroblasts (49). Our results suggest that hyperoxia-induced cell necrosis depends on PARP activity in A549 cells, whereas PAR accumulation detected in fibroblasts may be attributed to early apoptotic events and does not necessarily contribute to cell death.

Most of the pathophysiologic models where tissue injury is known to be dependent on PARP-1 activity are characterized by cell death and an intense inflammatory response (e.g., asthma, lipopolysaccharide-induced acute lung injury, ischemia-reperfusion brain and heart damage) (20–22). In mouse lungs, hyperoxia induced a strong activation of PARP, as shown by PAR accumulation in alveolar cells, without detectable PARP-1 cleavage. PARP activity was mainly due to PARP-1 because the genetic loss of PARP-1 mostly reduced PAR accumulation in lungs. The absence of PARP-1 or treatment with 3-AB did not prevent hyperoxia-induced lung injury. Several hypotheses can be formulated to explain these results:

  1. The absence of efficacy in preventing lung injury raises the unanswered question about the relative importance of the epithelial cell population in maintaining the tightness of the alveolo-capillary barrier. Indeed, inducible overexpression of keratinocyte growth factor in lung epithelial cells protected cells from hyperoxia-induced cell necrosis but did not prevent lung edema and animal death (50).
  2. In vivo, there is evidence of necrosis and apoptosis, as detected by the presence of DNA laddering and TUNEL-positive cells (2). Because it has been reported that under certain conditions the absence of PARP-1 may shift cells toward apoptosis (51), we might have expected that the absence of PARP-1 or the inhibition of its activity could have worsened alveolar apoptotic cell death during hyperoxia. DNA analysis showed a similar degree of internucleosomal DNA fragmentation in hyperoxia-exposed PARP-1 –/– and PARP-1 +/+ mouse lungs, suggesting that absence of PARP-1 did not affect apoptosis in vivo. Alternatively, the evidence that intermediate forms of cell death can be induced by hyperoxia in vitro and in vivo (4, 42) might explain why in vivo PARP inhibition is not sufficient for protection.
  3. It may be possible that PARP activation was induced in alveolar cells concomitantly to irreversible oxidative stress damage. The absence of a protective effect observed in vivo when PARP activity is abolished suggests that the energy failure due to the high level of oxidative stress is not solely dependent on PARP. Indeed, treatment with 3-AB was not sufficient to rescue ATP depletion and cell death in H2O2- and hyperoxia-exposed Chinese hamster ovary cells (52), suggesting that PARP inhibition might be inefficient in preventing hyperoxia-induced ATP depletion in vivo.
  4. Other members of the PARP family might be responsible for hyperoxia-induced PAR accumulation; 18 putative PARP homologs have been identified (53). Because mice treated with 3-AB, like PARP-1 –/– animals, were not protected from hyperoxia-induced lung injury and because PARP activity was reduced in PARP-1 –/– animals, it is unlikely that PARP activity observed in hyperoxia could be due to other PARP.

Taken together, our results show that, according to the cell type, PARP-1 is differentially regulated during hyperoxia and can influence the cell death response. Despite evidence that in vitro epithelial cell necrosis is dependent on PARP activation, absence of PARP-1 was not sufficient to prevent hyperoxic injury in vivo, supporting the concept that multiple mechanisms are involved in the death of the different alveolar cell types.


   Acknowledgments
 
The authors thank Zhao-Qi Wang and Dominique Gallendo for providing PARP-1 –/– mice; Ann Kato, Michel Aurrand-Lions, Walter Reith, and Alexandra Reverdin for manuscript revision; Lucas Liaudet, Marc Chanson, and Jean-Claude Martinou for their scientific advice; and Philippe Henchoz, Geneviève Leyvraz, and Lan Jornot for their technical assistance.


   Footnotes
 
This work was supported by the Swiss National Research Foundation #3200-067865.02, and by the Wölfermann-Naegele, Novartis, and Lancardis Foundations.

Originally Published in Press as DOI: 10.1165/rcmb.2004-0361OC on September 8, 2005

Conflict of Interest Statement: A.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. I.M.-R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Y.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.B.A. received $3,000 in the last 2 years as a member of the advisory board of Berna Biotech, which has nothing to do with the present study.

Received in original form November 17, 2004

Accepted in final form September 1, 2005


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 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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