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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 542-552, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.4775

Hydrogen Peroxide Induces Upregulation of Fas in Human Airway Epithelial Cells via the Activation of PARP-p53 Pathway

Tadashi Fujita, Muneharu Maruyama, Jun Araya, Kazuhiko Sassa, Yukio Kawagishi, Ryuji Hayashi, Shoko Matsui, Tatsuhiko Kashii, Naohiro Yamashita, Eiji Sugiyama and Masashi Kobayashi

The First Department of Internal Medicine, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan

Address correspondence to: Muneharu Maruyama, M.D., The First Department of Internal Medicine, Faculty of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan. E-mail: mmaruyam-tym{at}umin.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Fas mediates apoptosis following binding with Fas ligand. Fas is expressed in human airway epithelial cells and has a critical role in the pathophysiology of various pulmonary disorders. Hydrogen peroxide (H2O2) is an important mediator of airway epithelial injury. In this context, we hypothesized that H2O2 would increase the expression of cell surface Fas in human airway epithelial cells. To test this hypothesis, the modulation of Fas expression with H2O2 was assessed in normal human bronchial epithelial cells and A549 cells. The majority of Fas was cytoplasmic in both cell types without any stimulation. Hydrogen peroxide significantly increased Fas in the plasma membrane fraction, while decreasing Fas in the cytoplasmic fraction. Incubation with an agonistic antibody for Fas induced apoptosis in H2O2-treated cells in proportion to the level of surface Fas expression on those cells. Inhibitors of poly(ADP-ribose) polymerase abrogated the H2O2-induced Fas translocation to the plasma membrane and p53 activation. Expression of dominant–negative p53 also inhibited the Fas translocation induced by H2O2 in A549 cells. These results indicate that H2O2 induces Fas upregulation by promoting cytoplasmic transport of Fas to the cell surface in human airway epithelial cells, and that the activation of the poly(ADP-ribose) polymerase-p53 pathway may be involved in this mechanism.

Abbreviations: antibodies, Abs • 3-aminobenzamide, 3-AB • bronchoalveolar lavage fluid, BALF • 4',6-diamidino-2'-phenylindol dihydrochloride, DAPI • Fas ligand, FasL • green fluorescent protein, GFP • hydrogen peroxide, H2O2 • immunoglobulin, Ig • idiopathic pulmonary fibrosis, IPF • monoclonal Abs, mAbs • mean fluorescence intensity, MFI • normal human bronchial epithelial, NHBE • superoxide anion, O2- • poly(ADP-ribose) polymerase, PARP • phosphate-buffered saline, PBS • phycoerythrin, PE • propidium iodide, PI • reactive oxygen species, ROS • tumor necrosis factor, TNF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A large number of studies have demonstrated that reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), superoxide anion (O2-), and hydroxyl radical, play a critical role in the initiation and progression of a great diversity of diseases and in the regulation of many important biologic processes (1). The lung is a major target for oxidative stress because ROS are produced by inflammatory cells and by chemotherapeutic drugs that accumulate in the lung (1, 2). Moreover, a variety of lung diseases require oxygen therapy, which adds to the oxidant burden on the lung. In the lung, ROS are able to induce various forms of lesions in the airways as well as in the pulmonary vessels and parenchyma (3, 4).

Apoptosis is a highly conserved, and exquisitely efficient cellular process of cell death that is important for the development and homeostasis of multicellular organisms (5). It is induced by the ligation of cell surface receptors and a variety of environmental agents (5). In some instances, apoptosis is accompanied by an increase in the intracellular levels of ROS, and the addition of antioxidants prevents death of the cell (6, 7). Furthermore, the direct exposure of cells to H2O2- and O2--generating agents can induce apoptosis (7–10), indicating that ROS can act as mediators of apoptosis. There is also growing evidence that ROS are involved in apoptosis in a wide range of diseases such as pulmonary fibrosis, atherosclerosis, and acquired immunodeficiency syndrome (11–13). Therefore, an understanding of the mechanisms of ROS-induced apoptosis is needed to elucidate the apoptotic actions of stimuli that produce ROS and also to clarify the pathogenesis of numerous important diseases.

Fas is a Type I membrane protein belonging to the tumor necrosis factor (TNF)/nerve growth factor receptor family (14). Ligation and clustering of Fas receptors with either agonistic antibodies (Abs) to Fas or cells expressing Fas ligand (FasL) triggers a series of events inside the cells that lead to the rapid induction of apoptosis (15). Fas ligand is a Type II transmembrane protein that is homologous to TNF (16). The data of Hagimoto and colleagues suggest a role for the Fas pathway in bleomycin-induced lung injury and fibrosis in mice (17). The same group has also demonstrated that intratracheal administration of activating Abs to Fas was capable of inducing pulmonary fibrosis in mice (18). A recent study by Kuwano and coworkers has shown that the upregulation of Fas and FasL expression in bronchiolar and alveolar epithelial cells and in infiltrating lymphocytes or granulocytes, respectively, in lung tissues from patients with idiopathic pulmonary fibrosis (IPF) (19). These findings support the notion that the Fas-FasL pathway is involved in the pathogenesis of various fibrotic lung diseases. However, the detailed cellular mechanisms of Fas expression in human airway epithelial cells remains to be elucidated.

Oxidative stress leads to the damage of various biomolecules in the cells, such as formation of DNA single strand breaks and carbonyl proteins, and lipid peroxidation (20). The DNA strand breaks are known to induce the activation of DNA damage recognition proteins, p53 and poly(ADP-ribose) polymerase (PARP) (21, 22). Both proteins are functionally related, and are involved in the maintenance of genome integrity by means of the inhibition of cell cycle progression or the induction of apoptosis. There is growing evidence that poly(ADP-ribosyl)ation of p53 by PARP is required for the rapid accumulation and activation of p53 (23–25). As for the lung, Kuwano and colleagues have demonstrated that p53 and p21, a cyclin-dependent kinase inhibitor, were expressed in bronchiolar and alveolar epithelial cells in patients with IPF (26). Furthermore, they confirmed this observation in a murine model of bleomycin-induced pulmonary fibrosis (27). Guinee and colleagues have reported that apoptosis of Type II pneumocytes was identified in diffuse alveolar damage and was associated with p53 and p21 expression (28). Concerning PARP, it has been reported that PARP activity is increased in bleomycin-induced pulmonary fibrosis in hamsters (29), and that inhibitors of PARP such as nicotinamide and niacin attenuate the development of pulmonary fibrosis (30). All of these results imply that p53 and PARP might have relevance to the apoptosis of bronchial and alveolar epithelial cells in various interstitial lung diseases, including IPF.

In the present study, we investigated whether H2O2 was capable of inducing the expression of Fas on the cell surface of human airway epithelial cells. We found that H2O2 upregulated Fas expression by increasing cell surface trafficking of Fas in the cells, and that the activation of the PARP–p53 pathway was involved in this process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
Cryopreserved primary normal human bronchial epithelial (NHBE) cells were purchased from Clonetics (San Diego, CA) and grown in LHC-9 medium (Biofluids, Inc., Rockville, MD). The cells were seeded in 100-mm tissue culture dishes at a density of 1 x 104 cells/cm2 and incubated at 37°C in a humidified, 95% air/5% CO2 atmosphere. The cells were subcultured when they reached 60–80% confluency. The cells were used within the first five passages.

A549 cells, a tumor cell line from a human carcinoma with properties of Type II alveolar epithelial cells, were purchased from Human Science Research Resource Bank (Osaka, Japan). This cell line has wild-type p53 (31), and has been used as a model of human Type II alveolar epithelial cells in the literature (32). The culture medium was RPMI 1640 (Nissui Pharmaceutical, Tokyo, Japan), containing 10% heat-inactivated fetal calf serum (HIFCS; Gibco BRL, Rockville, MD), 2 mM L-glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin. The cells were grown in 100-mm tissue culture dishes or 6-well culture plates (Corning, New York, NY) in a humidified, 95% air/5% CO2 atmosphere.

Reagents
Monoclonal Abs (mAbs) against human Fas, clone 7C11 (immunoglobulin [Ig] M), and clone 13 (IgG) were purchased from Immunotech (Marseille, France) and Transduction Laboratories (Lexington, KY), respectively. Control IgM was also obtained from Immunotech. Phycoerythrin (PE)-labeled mouse antihuman Fas mAb, clone DX2, and PE-labeled mouse IgG1 were purchased from DAKO JAPAN Co., Ltd. (Kyoto, Japan). Catalase, paraformaldehyde, and 3-aminobenzamide (3-AB) were obtained from Sigma (Tokyo, Japan). Hydrogen peroxide and propidium iodide (PI) were from Wako Pure Chemical Industries (Osaka, Japan).

Flow Cytometric Detection of Fas
After treatment with various compounds, cells were harvested with 0.05% trypsin containing 0.53 mM ethylenediaminetetraacetic acid, and washed twice with staining solution containing 1.0% bovine serum albumin and 0.02% sodium azide in phosphate-buffered saline (PBS). The cell pellet was resuspended at 106 cells/ml. The cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, and then washed twice with staining solution. For measurement of total cellular Fas, cells were permeabilized by incubation in staining solution containing 10 µg/ml digitonin for 5 min, and washed twice with staining solution. For measurements of both cell surface and total cellular Fas, the cells were incubated with PE-labeled antihuman Fas mAb, DX2, or PE-labeled control mouse IgG1, in the dark for 30 min at 4°C. After incubation, cells were washed twice and resuspended in staining solution. Data acquisition and analysis were performed on a flow cytometer (FACScan using the Cell Quest software; Becton Dickinson, Mountain View, CA). Ten thousand cells were examined for each determination. Values for mean fluorescence intensity index (MFI index) were calculated using the formula: MFI index = [(MFI of anti-Fas Ab-stained cells–MFI of control IgG1-stained cells)/MFI of control IgG1-stained cells].

Induction of Fas-mediated Apoptosis
After treatment with 0–200 µM H2O2 for 12 h, cells were incubated with 1 µg/ml agonistic anti-Fas Ab, 7C11, in 6-well culture plates for 8 h at 37°C in a humidified 95% air/5% CO2 atmosphere. Then apoptotic cells were quantified as described below.

Determination of Apoptosis
Apoptotic cells were identified by one of the following two methods.

Flow cytometric DNA content analysis with PI. Following treatment with various reagents, cells (5 x 106) were harvested and immediately immobilized by ice cold 70% ethanol overnight. After washing with PBS, the pellet was resuspended in 40 mM citrate buffer for 30 min, incubated with 100 µg/ml RNase in PBS for 30 min at 37°C, and stained with 50 µg/ml PI in the dark. The quantity of cells with hypodiploid DNA was measured on a FACScan at the FL-2 channel. Ten thousand cells were examined for each determination.

Fluorescence microscopic analysis with 4',6-diamidino-2'-phenylindol dihydrochloride staining. We performed nuclear staining with 4',6-diamidino-2'-phenylindol dihydrochloride (DAPI) (Boehringer Mannheim Biochemicals, Indianapolis, IN). Harvested cells were washed with PBS, and stained with 10 µg/ml DAPI-methanol for 30 min in the dark, and then seeded on a glass slide and photographed with a fluorescence microscope (Nikon, Tokyo, Japan).

Subcellular Fractionation
Cells were fractionated into plasma membrane and cytosolic fractions with some modifications of the protocol described by Paul and coworkers (33). Briefly, cultured cells were scraped off the plates using a cell lifter in homogenization buffer (0.28 M sucrose, 10 mM MgCl2, 150 mM NaCl, 50 mM 2-[N-morphilino]-ethanesulfonic acid, 1 mM ethyleneglycol-bis-[ß-aminoethyl ether]-N,N'-tetraacetic acid, pH 6.0, 0.1 mM PMSF, and 0.1 mg/ml soybean trypsin inhibitor) and collected by centrifugation at 200 x g for 10 min at 4°C. The pellet was homogenized in a Teflon glass Potter-Elvehjem homogenizer (Curtin Matheson Scientific, Houston, TX) with a Wheaton Teflon pestle for 10 strokes and then centrifuged at 700 x g for 15 min at 4°C. The pellet was discarded and the resulting supernatant (S1) was centrifuged at 10,000 x g for 30 min at 4°C to sediment a crude membrane fraction (P1). The resulting supernatant (S2) was diluted with 50 mM HEPES, pH 7.5, and centrifuged at 100,000 x g for 1 h at 4°C in an ultracentrifuge (Beckman SW40-Ti; Beckman Instruments, Inc., Palo Alto, CA) to precipitate a second membrane fraction (P2). The resulting supernatant (S3) contained an enriched cytosolic fraction. Resolution of P1 fraction into purified membrane fraction was achieved by the following steps: the fraction was resuspended in 50 mM HEPES, pH 7.5, layered atop a sucrose cushion (48% wt/vol), and centrifuged at 100,000 x g for 1 h at 4°C in an ultracentrifuge (Beckman TLA-100.3; Beckman Instruments, Inc.); following this centrifugation, the region of a band floating above the sucrose cushion containing an enriched plasma membrane fraction was resuspended in 50 mM HEPES, pH 7.5, and centrifuged at 100,000 x g for 1 h at 4°C to remove sucrose. The pellet fraction was diluted with 50 mM HEPES, pH 7.5. Aliquots of both cytosolic and plasma membrane fractions were assayed for Western blot analysis as described below.

Western Blot Analysis
For detection of Fas, p53, p21, and PARP, we performed Western blot analyses. Following treatment, cells were lysed with NP-40–based lysis buffer. The cell lysates were centrifuged at 12,000 x g for 10 min at 4°C, and the supernatants were used for the subsequent experiments. Protein concentrations were measured by the Bradford method (Bio-Rad protein assay; Bio-Rad, Richmond, CA). Thirty µg samples of each of the protein preparations were fractionated on 7% or 10% sodium dodecyl sulfate-polyacrylamide gel and electrically transferred to a nitrocellulose membrane (Immobilon; Millipore, Bedford, MA). After blockage of nonspecific binding with 5% skim milk, blots were incubated for 16 h at 4°C with Abs to Fas (clone 13, 1:1,000 dilution; Transduction Laboratories), p53 (1:1,000 dilution; Transduction Laboratories), p21 (1:1,000 dilution; Transduction Laboratories), and poly(ADP-ribose) (1:500 dilution; BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA). Membranes were washed twice with PBS-TWEEN 80, and then incubated with antimouse IgG horseradish peroxidase-conjugated secondary Ab for 1 h. The filters were washed again with PBS-TWEEN 80 and developed with an ECL Western blotting detection system (Amersham Life Science, Tokyo, Japan) at room temperature before being exposed to Kodak X-Omat film (Eastman Kodak, Rochester, NY).

Transfection
The plasmid vector expressing a dominant negative mutant (pCMV-p53 mt135) was purchased from CLONTECH Laboratories, Inc. (Tokyo, Japan). Wild-type p53 gene and p53 mt135 gene differ by G to A conversion at nucleotide 1,017. A549 cells were plated in 6-well plates at 1 x 106 cells/ml in 2 ml of RPMI 1,640 containing 10% HIFCS. When the cells reached 70% confluency, each well was pretreated with 10 µg of the plasmid (pCMV-p53 mt135 or pUC19) and 20 µg of Lipofectin (Gibco BRL) in 2 ml of serum-free RPMI 1640 medium for 12 h. Then the medium was changed to fresh medium. Twenty-four hours later, the cells were treated with 200 µM H2O2 for 12 h. Then the cell surface expression of Fas was evaluated by flow cytometry and cell fractionation studies. When the control plasmid vector expressing green fluorescent protein (GFP) was transfected, over 40% of A549 cells expressed GFP, as determined by flow cytometric analysis (data not shown).

Immunostaining and Confocal Laser Microscopy
A549 cells were treated with or without 100 µM H2O2 for 12 h at 37°C, and fixed for 30 min in PBS containing 3.7% formaldehyde. The fixed cells were rinsed twice with PBS and permeabilized with 0.5% Triton X-100 for 5 min at room temperature. After blockage with 3% skim milk for 30 min at room temperature, the cells were incubated with PE-labeled mouse anti-human Fas mAb, clone DX2, at 37°C for 30 min. The samples were visualized with immunofluorescence microscopy and examined with a Carl Zeiss confocal laser fluorescence inverted microscope (LSM 510, Carl Zeiss, Oberkochen, Germany).

Statistical Analysis
We repeated each type of experiment at least three times and confirmed that similar data were obtained. All values are presented as means ± SD. Comparisons were made with one-way ANOVA with Fisher's post hoc test. Differences between means were evaluated by Student's t test. A P value less than 0.05 was judged to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hydrogen Peroxide Promotes the Translocation of Cytoplasmic Fas to the Cell Surface
The initial experiment was performed to determine whether or not H2O2 was capable of enhancing the cell surface expression of Fas in NHBE cells and A549 cells. After incubation with H2O2 at concentrations from 0 to 200 µM for 12 h, we evaluated the Fas expression by flow cytometric analysis. H2O2 induced a dose-dependent increase in cell surface Fas in both kinds of cells (Figures 1A–1F). Catalase (1,000 U/ml), an enzyme that hydrolyzes H2O2 to O2 and H2O, inhibited the H2O2 (200 µM)-induced upregulation of cell surface Fas (Figure 1G). We also examined the time course of cell surface Fas expression on A549 cells following exposure to H2O2 (200 µM). The level of cell surface Fas gradually increased in 24 h culture with H2O2 (Figures 2A-E).



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  		Figure 1. Effect of H2O2 on the cell surface expression of Fas in NHBE cells and A549 cells. NHBE cells (A-C) and A549 cells (D-G) were incubated for 12 h with H2O2 at the concentrations of 0 µM (A, D), 50 µM (B), 100 µM (C, E), and 200 µM (F). G shows Fas expression in A549 cells treated with H2O2 (200 µM) and catalase (1,000 U/ml) for 12 h. Cell surface Fas expression was then analyzed by a FACScan flow cytometer. A bold solid line represents a histogram of cells stained with anti-Fas Ab (DX2). A solid line denotes a histogram of cells stained with control mouse IgG1. The number in each panel represents mean fluorescence intensity index (MFI index). Values for MFI index were calculated using the formula: MFI index = ([(MFI of anti-Fas Ab-stained cells) – (MFI of control IgG1-stained cells)]/MFI of control IgG1-stained cells).

 


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  		Figure 2. Time-dependent effect of H2O2 of the cell surface expression of Fas in A549 cells. A549 cells were incubated with 200 µM H2O2 for 0 h (A), 3 h (B), 6 h (C), 12 h (D), and 24 h (E). Cell surface Fas expression was then analyzed by a FACScan flow cytometer. A bold solid line represents a histogram of cells stained with anti-Fas Ab (DX2). A solid line denotes a histogram of cells stained with control mouse IgG1. The MFI index is indicated in each panel.

 
We next examined the effect of H2O2 on the cellular distribution of Fas in NHBE cells and A549 cells using cell fractionation studies. Cell fractionation studies demonstrated that Fas was predominantly located in the cytoplasmic fraction (Figure 3A), which is consistent with the study on A549 cells by Nambu and colleagues (34). Thus the majority of Fas is sequestered within intracellular compartments. We further determined whether H2O2 was capable of inducing the increase in plasma membrane Fas. Increase in plasma membrane Fas was determined after a 12 h incubation of NHBE cells and A549 cells in 100 µM and 200 µM H2O2, respectively (Figure 3A). Conversely, Fas in the cytoplasmic fraction was decreased following this treatment.



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  		Figure 3. Effect of H2O2 on the expression of plasma membrane Fas in NHBE cells and A549 cells. (A) NHBE cells and A549 cells were incubated in the presence or absence of H2O2 at the indicated concentrations for 12 h, and fractionated into cytoplasmic and plasma membrane fractions as described in the text. Immunoblot analysis of Fas in the cell fractions was then performed. (B) Visualization of H2O2-induced Fas receptor trafficking. A549 cells were treated with or without 100 µM H2O2 for 12 h at 37°C, and fixed for 30 min in PBS containing 3.7% formaldehyde. The fixed cells were rinsed twice with PBS, and permeabilized with 0.5% Triton X-100 for 5 min at room temperature. After blockage with 3% skim milk for 30 min at room temperature, the cells were incubated with PE-labeled mouse anti-human Fas mAb, clone DX2, at 37°C for 30 min. The samples were visualized with immunofluorescence microscopy and examined with a Carl Zeiss confocal laser fluorescence inverted microscope (LSM 510, Carl Zeiss, Oberkochen, Germany).

 
We confirmed these observations by immunocytochemistry using confocal microscopy in A549 cells. In control cells, Fas had intracellular punctate fluorescence consistent with organelles in the cytoplasm (Figure 3B). In contrast, treatment with H2O2 increased fluorescence on the cell surface, indicating increased Fas on the plasma membrane (Figure 3B).

Percent cell death measured by the exclusion of trypan blue dye was 17.6 ± 2.9% in H2O2 (100 µM, 12 h)-exposed NHBE cells and 7.5 ± 5.1% in H2O2 (200 µM, 12 h)-exposed A549 cells. Higher concentrations of H2O2 were not tested because they may cause cell damage manifested as morphologic changes of the monolayer or cell detachment (data not shown).

We determined the effect of H2O2 on the total amount of Fas protein by flow cytometric analysis. H2O2 did not significantly modulate the level of total Fas antigen in digitonin-treated NHBE cells (Figure 4).



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  		Figure 4. Effect of H2O2 on the expression of cell surface and total Fas in NHBE cells. NHBE cells were incubated in the presence or absence of H2O2 (50 µM, and 100 µM) for 12 h. Some of the H2O2-treated cells were permeabilized by treatment with 10 µg/ml digitonin, and then Fas expression of cells was analyzed by a FACScan flow cytometer. A bold solid line represents a histogram of permeabilized cells stained with anti-Fas Ab (DX2) (total Fas). A solid line denotes a histogram of cells stained with DX2 (cell surface Fas). A dashed line represents a histogram of permeabilized cells stained with control mouse IgG1. The MFI index is indicated in each panel.

 
These findings indicate that H2O2 promotes the trafficking of Fas receptors to the plasma membrane.

H2O2-induced Fas Is Able to Mediate Apoptosis
To examine whether H2O2-induced Fas was capable of mediating apoptosis, we evaluated the effect of an agonistic anti-Fas Ab, 7C11, on the apoptosis of NHBE cells by measuring the percentage of cells having hypodiploid DNA using PI staining followed by flow cytometric analysis (Figure 5). The cells in control culture showed a small apoptotic population (6.9 ± 1.7%). Incubation with 100 µM H2O2 slightly increased the apoptotic population (19.5 ± 3.3%) compared with that of control cells. On the other hand, incubation with anti-Fas Ab, 7C11, significantly increased the apoptotic population (51.2 ± 3.9%) in H2O2 (100 µM)-treated cells. However, anti-Fas Ab did not significantly increase the rate of apoptosis in cells without H2O2 pretreatment (10.6 ± 2.5%). Similar data were obtained in A549 cells (data not shown).



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  		Figure 5. Flow cytometric DNA analysis of the effects of H2O2 and anti-Fas IgM on NHBE cells. After incubation with H2O2, NHBE cells were stained with 10 µg/ml of PI, and DNA content in each cell was analyzed by FACScan flow cytometer. The number in each panel represents the percentage of cells with hypodiploid DNA. (A) NHBE cells were incubated in medium for 20 h. (B) NHBE cells were treated with 100 µM H2O2 for 12 h, and then incubated with 1 µg/ml control IgM for a further 8 h. (C) NHBE cells were treated with 100 µM H2O2 for 12 h, and then incubated with 1 µg/ml agonistic anti-Fas IgM (7C11) for a further 8 h. (D) NHBE cells were incubated in medium for 12 h, and then exposed to 1 µg/ml agonistic anti-Fas IgM (7C11) for a further 8 h. A significant increase (P < 0.001) in apoptosis was noted in the cells treated with H2O2 plus anti-Fas IgM compared with those with any other treatment. A representative of three experiments is shown in each panel.

 
To confirm that H2O2-induced Fas has the ability to mediate apoptosis in NHBE cells, we morphologically analyzed the agonistic anti-Fas Ab-triggered apoptosis by counting cells with nuclear condensation and fragmentation using DAPI staining followed by fluorescence microscopy. Incubation with an anti-Fas Ab, 7C11, greatly increased the rate of cells with apoptotic features in H2O2-treated cells (Figure 6). However, in the cells without H2O2 pretreatment, the apoptotic population did not significantly increase following incubation with an anti-Fas Ab. This phenomenon was also observed in A549 cells (data not shown).



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  		Figure 6. Nuclear morphology of NHBE cells after treatment with H2O2 and anti-Fas IgM. NHBE cells were preincubated in the presence or absence of 100 µM H2O2 for 12 h and then exposed to 1 µg/ml agonistic anti-Fas IgM or control IgM for a further 8 h. Cells were stained with 10 µg/ml DAPI, and observed with a fluorescence microscope for morphologic changes of apoptosis. Original magnification, X400. Arrows indicate nuclear condensation and arrow heads point to nuclear fragmentation as morphologic signs of apoptosis. (A) NHBE cells were incubated in medium for 20 h. (B) NHBE cells were treated with 100 µM H2O2 for 12 h, and then incubated with 1 µg/ml control IgM for a further 8 h. (C) NHBE cells were treated with 100 µM H2O2 for 12 h, and then incubated with 1 µg/ml agonistic anti-Fas IgM (7C11) for a further 8 h. (D) NHBE cells were incubated in medium for 12 h, and then exposed to 1 µg/ml agonistic anti-Fas IgM (7C11) for a further 8 h. A representative of three experiments is shown in each panel.

 
These findings indicate that H2O2-induced cell surface Fas functions to mediate apoptosis in human airway epithelial cells.

PARP and p53 Are Involved in H2O2-induced Fas Expression
We next investigated what kind of molecules would be involved in the H2O2-induced upregulation of cell surface Fas in human airway epithelial cells. It is well known that H2O2 induces DNA damage, and that this DNA cleavage activates PARP and p53 (21, 22). Hence, we examined whether PARP and p53 would be implicated in the H2O2-induced cell surface Fas expression in NHBE cells and A549 cells.

We determined whether H2O2 increased the activity of PARP in human airway epithelial cells. Using anti-poly-(ADP-ribose) Ab, we performed Western blot analyses of whole cell lysates obtained from NHBE cells prepared 30 min and 1.5 h after exposure to H2O2, with or without pretreatment with an inhibitor of PARP, 3-AB. Exposure to H2O2 obviously increased poly(ADP-ribosyl)ation of the proteins in NHBE cells, and preincubation with 3-AB markedly inhibited H2O2-induced poly(ADP-ribosyl)ation (Figure 7). Similar data were obtained in A549 cells (data not shown). These results indicate that H2O2 stimulates the activity of PARP in human bronchial epithelial cells, and that 3-AB is potent in inhibiting its activity.



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  		Figure 7. H2O2-induced poly(ADP-ribosyl)ation of the proteins in NHBE cells, and its inhibition by PARP inhibitor, 3-AB. NHBE cells were preincubated in the presence or absence of 10 mM 3-AB for 1 h, and then treated with or without H2O2 at the concentration of 100 µM for 0 h, 0.5 h, and 1.5 h. Thirty µg of each of the protein preparations was subjected to 7% sodium dodecyl sulfate-polyacrylamide gel and blotted. Immunoblotting was performed with specific Ab to poly(ADP-ribose).

 
To assess whether the activation of PARP would be necessary for H2O2-induced Fas trafficking in human airway epithelial cells, we stimulated A549 cells with H2O2 (100 µM and 200 µM) in the presence or absence of 3-AB (10 mM), a potent inhibitor of PARP, and evaluated the cell surface expression of Fas using a flow cytometer. Preincubation of A549 cells with 3-AB prevented the H2O2-induced increase in cell surface Fas (Figure 8A). We further confirmed the involvement of PARP activity in the H2O2-induced increase in plasma membrane Fas by cell fractionation studies (Figure 8B). Similar findings were observed in NHBE cells (data not shown). These findings indicate that PARP activity is implicated in the H2O2-induced increase in cell surface trafficking of Fas.



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  		Figure 8. Effect of PARP inhibitor, 3-AB, on the expression of plasma membrane Fas in A549 cells. (A) A549 cells were preincubated in the presence or absence of 10 mM 3-AB for 1 h, and then treated with or without H2O2 at the concentrations of 100 µM and 200 µM for 12 h. Cell surface Fas expression was then analyzed by a FACScan flow cytometer. A bold solid line represents a histogram of cells stained with anti-Fas Ab (DX2). A solid line denotes a histogram of cells stained with control mouse IgG1. (B) Cells were fractionated into cytoplasmic and plasma membrane fractions as described in the text. Immunoblot analysis of Fas in the cell fractions was then performed.

 
We verified that the extent of apoptosis of these H2O2 and/or 3-AB–treated cells after ligation with an agonistic Ab to Fas was proportional to the amount of cell surface Fas receptors (Figure 9).



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  		Figure 9. Effect of PARP inhibitor, 3-AB, on anti-Fas IgM-induced apoptosis in H2O2-treated A549 cells. A549 cells were preincubated for 12 h in the presence or absence of 10 mM 3-AB and exposed to 200 µM H2O2 for 12 h. A549 cells were then incubated for a further 8 h with 1 µg/ml anti-Fas IgM (7C11) (closed bars) or control IgM (open bars). DNA analysis of apoptosis by flow cytometry was performed as described in the text. Values are mean ± SD of three individual experiments. *P < 0.05, significantly different from H2O2 (200 µM)-exposed cells with anti-Fas IgM treatment.

 
Consistent with the knowledge that H2O2 activates p53 and p21 (35), H2O2 caused A549 cells to increase the levels of p53 and p21 in a dose- and time-dependent fashion (Figure 10). The kinetic study demonstrated that the increase in p53 began at 3 h after H2O2 treatment, and occurred 3 h earlier than the increase in p21, indicating that the p53 activated by H2O2 transcriptionally upregulates p21. We then examined whether the increase in PARP activity would be necessary for the activation of p53 in human airway epithelial cells. An inhibitor of PARP, 3-AB, was a strong inhibitor to the H2O2-induced accumulation of p53 and p21 in A549 cells (Figure 11). These results obviously indicate that H2O2 increases and activates p53 via PARP activation.



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  		Figure 10. Dose- and time-dependent effects of H2O2 on the accumulation of p53 and p21 in A549 cells. (A) A549 cells were incubated for 6 h with H2O2 at the indicated concentrations. (B) A549 cells were treated with 200 µM H2O2 for the indicated times before harvest. Thirty µg of each of the protein preparations were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel and blotted. Immunoblotting was performed with specific mAbs to p53 and p21.

 


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  		Figure 11. Effect of PARP inhibitor, 3-AB, on the H2O2-induced accumulation of p53 and p21 in A549 cells. A549 cells were preincubated for 2 h in the presence or absence of 10 mM 3-AB, and then treated with or without 200 µM H2O2 for a further 6 h. Thirty µg of each of the protein preparations were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel and blotted. Immunoblotting was performed with specific mAbs to p53 and p21.

 
To examine whether the enhanced cell surface Fas expression by H2O2 was mediated through p53 activation, we transfected A549 cells with a vector expressing a dominant negative p53 mutant (pCMV-p53 mt135) or a control plasmid (pUC19). After 36 h, the cells were exposed to H2O2 for 12 h, after which the surface expression of Fas was assessed by flow cytometry and cell fractionation studies. The transfection with a dominant negative p53 mutant partially inhibited the H2O2-induced increase in cell surface Fas (Figure 12A). The average inhibiting ratio of MFI index was 39.7 ± 5.3%. Cell fractionation studies demonstrated that the transfection with pCMV-p53 mt135 suppressed the H2O2-induced increase in plasma membrane Fas (Figure 12B). As expected, the inhibition of p53 function by a dominant negative mutant decreased the susceptibility of H2O2-exposed A549 cells to the induction of Fas-mediated apoptosis (Figure 12C). These observations indicate that H2O2 increases the cell surface Fas expression in human airway epithelial cells via PARP-p53 activation pathway.




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  		Figure 12. Transfection of dominant-negative p53 inhibits H2O2-induced upregulation of cell surface Fas. A549 cells were transfected with control pUC19 or a plasmid expressing dominant–negative p53 (pCMV-p53 mt135) for 36 h. Then the medium was changed to fresh medium, and the cells were treated with 200 µM H2O2 for a further 12 h. (A) The cells were analyzed for the cell surface expression of Fas by flow cytometry. A solid line represents a histogram of cells stained with anti-Fas Ab (DX2). A dashed line denotes a histogram of cells stained with control mouse IgG1. The MFI index is indicated in each panel. The ratio of MFI index (dominant–negative p53 transfected cells/control vector transfected cells) after H2O2-treatment was 60.3 ± 5.3% (mean ± SD). A representative of three experiments is shown in each panel. (B) The cells were analyzed for cell fractionation studies. Immunoblot analysis of Fas in the cell fractions was then performed. A representative gel from three experiments is shown. (C) The cells were examined for flow cytometric DNA analysis following treatment with anti-Fas IgM. The cells were further incubated with 1 µg/ml anti-Fas IgM (7C11) (closed bars) or control IgM (open bars) for 8 h, and stained with 10 µg/ml of PI. Then, DNA content in each cell was analyzed by FACScan flow cytometer as described in the text. Values are means ± SD of three individual experiments. *P < 0.05, significantly different from the cells transfected with a control plasmid (pUC19) and subsequently exposed to H2O2 plus anti-Fas IgM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Fas is known to be a transmembrane receptor that mediates apoptosis, and has been reported to express other diverse functions, including the induction of angiogenesis and the activation of various tyrosine kinases (36, 37). Other studies have revealed that Fas is expressed by a variety of cell types in the lung, including bronchiolar and alveolar epithelial cells, fibroblasts, alveolar macrophages, and lymphocytes under appropriate stimulation (19, 38, 39). The increased level of Fas expression has been detected in epithelial cells and alveolar macrophages in various pathologic conditions, such as hypersensitivity pneumonitis and IPF (19, 40). In the present study, we investigated the effect of H2O2 on the cell surface expression of Fas in human bronchial epithelial cells. H2O2 induced the upregulation of cell surface Fas that served to mediate apoptosis. This H2O2-induced Fas upregulation appears to be mediated via the PARP-p53 activation pathway.

Clinically, increased levels of exhaled H2O2 have been reported in patients with various inflammatory lung diseases, such as acute respiratory distress syndrome, chronic obstructive pulmonary disease, and bronchiectasis (41–43). Baughman and colleagues demonstrated spontaneous H2O2 release from alveolar macrophages in cigarette smokers and patients with active sarcoidosis (44, 45). Moreover, therapeutic application of oxygen radical scavengers has demonstrated some effectiveness in a variety of pulmonary diseases (46). These findings indicate that ROS, including H2O2, have an important role in the pathogenesis of a number of lung diseases.

The exposure time to and concentration of H2O2 are critical for this kind of study. High concentrations of H2O2 exert direct toxic effects on susceptible cells, culminating in cell death, whereas low concentrations of this diffusible ROS alter cellular functions by modulating intracellular signal transduction in cells. We carefully chose the concentrations of H2O2 that did not significantly induce cell death following 24 h exposure (data not shown). The conditions of our study were in keeping with those of previous studies in which the effects of H2O2 on various cellular functions, other than direct toxicity, in A549 cells were investigated (47, 48).

Our results have shown that H2O2 induces the increase in cell surface expression of Fas in human airway epithelial cells. The concentrations of H2O2 used in the present study were well within the range found in the proximity of stimulated granulocytes, and are enough to cause DNA strand breaks but not cell death in a variety of target cells (49, 50). This DNA cleavage induces the accumulation and activation of both PARP and p53 (21–22), which are implicated in the maintenance of cellular genomic integrity. The activity of PARP has been reported to be involved in the regulation of p53 and its downstream pathways (23–25). The reduced p53 induction in cells deficient in PARP synthesis supports the potential role of poly(ADP-ribosyl)ation in the activation of p53 (24). We have demonstrated that the activation of p53 is necessary for the H2O2-induced cell surface expression of Fas in human airway epithelial cells. We also confirmed that poly(ADP-ribosyl)ation by PARP is a prerequisite to the activation of p53 because the inhibition of PARP activity abolished H2O2-induced accumulation in p53, resulting in the suppression of cell surface Fas expression.

There has been mounting evidence in the literature that Fas is shuttled to the cell surface by a protein secretory pathway involving the Golgi apparatus, and that several signaling molecules are implicated in this process (51, 52). Bennett and coworkers have revealed that the activation of p53 is responsible for the redistribution of death receptors, such as Fas and TNF receptor-1, from the cell interior to the cell surface (51). Other investigators have reported that protein kinase C is involved in the vesicular trafficking of several secretory molecules to the plasma membrane (52, 53). Based on the evidence that, in human lung epithelial cells, the majority of Fas was cytoplasmic, as assessed by cell fractionation (Figure 3A), H2O2 appears to increase the preexisting cytoplasmic Fas receptors to the plasma membrane. Because brefeldin A is known as an inhibitor of Golgi-dependent protein secretion (54), we examined the effect of brefeldin A on the cell surface expression of Fas in H2O2-treated cells. However, because brefeldin A was cytotoxic to the cells in the presence of H2O2, we were not able to determine the effect of this specific membrane transport inhibitor on the H2O2-induced trafficking of Fas in the cells. Considering the fact that the expression of dominant negative p53 markedly inhibited H2O2-induced Fas expression on the cell surface of A549 cells, it is likely that H2O2 induces the activation of p53 that, in turn, contributes to the shuttling of intracellular Fas to the plasma membrane in airway epithelial cells.

Aside from DNA damage, H2O2 has been reported to have a variety of other effects on cells. H2O2 has been demonstrated to be involved in cellular signaling pathways via plasma membrane-anchored receptors (55, 56). For instance, H2O2 can induce the phosphorylation of the epidermal growth factor receptor, and can activate its downstream signaling (55, 56). Conversely, there is growing evidence that H2O2 acts as an intracellular messenger in receptor signaling pathways (57). It has been reported that stimulation of rat vascular smooth muscle cells with platelet-derived growth factor induces a transient increase of the intracellular concentration of H2O2 that is required for platelet-derived growth factor–induced protein tyrosine phosphorylation (57). Using a phosphatase inhibitor, vanadate, we investigated whether the activation of protein tyrosine kinase is involved in the mechanism of H2O2-induced Fas expression. We found that vanadate enhanced H2O2-induced expression of cell surface Fas (data not shown). These results suggest that H2O2 may upregulate Fas expression by increasing tyrosine kinase activity in airway epithelial cells. Interestingly, the study by Huang and colleagues provides evidence that vanadate induces p53 activation, mainly through H2O2 generation (58). Therefore, the principal mechanism of vanadate-induced Fas upregulation in this study might be not the activation of tyrosine kinase, but the generation of H2O2.

Recent studies have provided evidence that the Fas–FasL pathway is closely implicated in the pathogenesis of a variety of pulmonary diseases (17–19, 59, 60). Using clinical specimens and animal models, the group of Kuwano and coworkers have repeatedly emphasized in a number of reports that Fas-mediated apoptosis of alveolar epithelial cells plays a crucial role in the development of fibrotic lung diseases (7, 17–19). Moreover, there are several reports that the levels of soluble FasL in bronchoalveolar lavage fluids (BALF) obtained from patients with acute respiratory distress syndrome correlates with their clinical courses, and that the BALF is capable of inducing apoptosis of human lung epithelial cells (60). We have shown that an agonistic anti-Fas IgM is capable of inducing apoptosis of the airway epithelial cells with H2O2-enhanced Fas expression. Taken together, it is possible that soluble FasL in BALF and FasL-expressing inflammatory cells, such as cytotoxic T lymphocytes in the alveolar space, may actively participate in the epithelial cell damage associated with acute and chronic lung injury.

In conclusion, this study indicates that H2O2 is capable of increasing the cell surface expression of Fas in airway epithelial cells, and that the activation of the PARP-p53 pathway and subsequent increase in Fas trafficking to the plasma membrane may be involved in this process. These observations are likely to be pathophysiologically important because increased amounts of H2O2 are produced in the local milieu in a variety of pulmonary diseases (41–43). This study therefore contributes to understanding of one mechanism for apoptosis that is probably a key process of repair and remodeling following lung injury, although future investigation will be necessary to determine the in vivo relevance of our findings.


   Acknowledgments
 
The authors wish to acknowledge the thoughtful suggestions and scientific review provided by Drs. Takashi Kondo and Toshio Miyawaki.

Received in original form November 16, 2001

Received in final form June 18, 2002


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 Introduction
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 Results
 Discussion
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