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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 722-731, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.2002-0033OC

Oxidative Stress Induces Arachidonate Release from Human Lung Cells through the Epithelial Growth Factor Receptor Pathway

Rafal Pawliczak, Xiu-Li Huang, Uday B. Nanavaty, Marion Lawrence, Patricia Madara and James H. Shelhamer

Critical Care Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland; and Department of Clinical Immunology and Allergy, Medical University of Lodz, Lodz, Poland

Address correspondence to: James H. Shelhamer, M.D., Critical Care Medicine Department, Bldg. 10, Rm. D43, Warren Grant Magnuson Clinical Center, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892. E-mail: jshelhamer{at}nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Oxidative stress is thought to be a factor influencing many inflammatory responses, including arachidonic acid (AA) release. We have studied the effect of hydrogen peroxide on AA and prostaglandin E2 release, cytosolic phospholipase (cPLA2) steady-state mRNA, cPLA2 protein levels, cPLA2 enzyme activity, and cPLA2 phosphorylation in a human lung epithelial cell line: A549 cells. Hydrogen peroxide caused a dose-dependent increase of A23187-stimulated AA and prostaglandin E2 release, with a maximum effect at 1 h. This effect is associated with a maximum specific cPLA2 activity at 1 h, and with a significant increase in cPLA2 Serine 505 phosphorylation. All these effects were abolished, in a dose-related manner, by the epithelial growth factor receptor kinase inhibitor, AG 1478. To further investigate the pathway leading to the increase cPLA2 phosphorylation, we used cells transfected with a Ras dominant negative vector and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and p38 kinase inhibitors. Cells transfected with the Ras dominant negative vector exhibited diminished hydrogen peroxide–induced AA release and cPLA2 phosphorylation as compared with cells transfected with the Ras expression vector. Both MEK and p38 kinase inhibitors inhibited the hydrogen peroxide effect on AA release and specific cPLA2 activity. Finally, cells stably transfected with an antisense cPLA2 vector exhibited diminished A23187-stimulated AA release in response to hydrogen peroxide as compared with cells stably transfected with empty expression vector. Collectively, these data show that hydrogen peroxide increases cPLA2 activity through its phosphorylation utilizing an epithelial growth factor/Ras/extracellular signal-regulated kinase and p38 pathway.

Abbreviations: arachidonic acid, AA • cytosolic phospholipase A2, cPLA2 • epithelial cell growth factor, EGF • EGF receptor, EGFR • extracellular signal-regulated kinase, ERK • fetal calf serum, FCS • tritium labeled AA, 3H-AA • hydrogen peroxide, H2O2 • mitogen-activated protein, MAP • MAP kinase, MAPK • MAPK/ERK, MEK • platelet-activating factor, PAF • phosphate-buffered saline, PBS • RNase protection assay, RPA • tumor necrosis factor-{alpha}, TNF-{alpha}


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Phospholipase A2s are a group of enzymes that catalyze the hydrolysis of the sn-2-ester bond of phospholipids, resulting in the production of free fatty acids and lysophospholipids. The group IV 85 kD cytosolic phospholipase (cPLA2) exhibits a preference for arachidonic acid (AA) in the sn-2 position in substrate phospholipids (1, 2). This is a tightly regulated process because the availability of free AA may limit the level of eicosanoid production. cPLA2 expression and activity is regulated at the transcriptional level by a number of stimuli, including cytokines, growth factors, ATP, and glucocorticoids (3–6). cPLA2 activity is also regulated posttranslationally by phosphorylation and by the intracellular calcium level (2, 7, 8).

Hydrogen peroxide appears to be an important mediator of lung inflammation involved in pathogenesis of asthma (9), adult respiratory distress syndrome (10), chronic obstructive pulmonary disease (11), and airway function impairment in cigarette smokers. The main sources of hydrogen peroxide in the airways and exhaled air are inflammatory cells, neutrophils, eosinophils, and macrophages.

Knockout mice studies provide evidence that cytosolic phospholipase A2 might play a key role in facilitation of lung injury and ARDS (12). cPLA2 may also be involved in mediation of oxidative stress–mediated kidney epithelial cell (13) and neuronal cell damage (14). These data suggest that oxidative stress and cPLA2 may act synergistically and that AA metabolites might be important modulators of oxidative stress.

The influence of oxidative stress on AA metabolism is still far from clear. Data obtained so far suggest phosphorylation and/or increase in cPLA2 protein synthesis during exposure to oxidative stress (15). The role of the epithelial growth factor (EGF) receptor (EGFR) pathway in this process is not clear. The EGFR is known to be phosphorylated by oxidative stress in epithelial cells, which may lead to activation of Ras/mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK)1,2 and other pathways (16). The goal of this study was to investigate the influence of oxidative stress (using A549 cells and a hydrogen peroxide exposure model) on AA release, specific cPLA2 activity, steady-state cPLA2 mRNA levels, and cytosolic phospholipase A2 protein levels. We also wanted to elucidate the importance of the EGFR pathway in this phenomenon.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
Human lung A549 cells were obtained from the ATCC (Rockville, MD). Cells were grown in Ham's F-12K media with 10% FCS and 2 mM of L-glutamine in 175 cm2 flasks (Becton and Dickinson, Bedford, MA) for cPLA2 activity assay and RNase protection assay (RPA) or in six-well plates from the same manufacturer for immunoblotting and AA release. Before starting each experiment, medium was removed and cells were incubated in serum-free media (Ham's F-12K media with L-glutamine) for two hours.

Treatments
Hydrogen peroxide (30%) was obtained from Fisher (Fair Lawn, NJ), PD 98059 (an MEK inhibitor), SB 203580 (a p38 kinase inhibitor), and AG 1478 (an EGFR kinase inhibitor) were obtained from Calbiochem (San Diego, CA). Cells were treated with 200 µM of hydrogen peroxide in serum-free media from 10 min to 24 h as specified below for time course studies. Quantities of 2, 20, and 200 µM of hydrogen peroxide and 1 h exposure time were used for dose–response experiments. Inhibitors were added to the serum-free media and incubated with cells for 30 min before the experiments. Cells were washed two times with warm PBS and exposed to hydrogen peroxide in serum-free media.

Cell Viability Assay
To confirm that hydrogen peroxide does not induce cell death, a cell viability assay was performed. A549 cells in 96-well plates were incubated with medium containing 200 µM of hydrogen peroxide in serum-free Ham's F12K with L-glutamine. Some wells were incubated with fresh medium and were used as controls. Cell viability was assessed after 30 min, 4 h, or 24 h. At the end of the indicated time point, cell viability was assayed using a MTS(3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium) and PMS (phenazine methosulfate)-based assay (Promega, Madison, WI). MTS and PMS solutions were mixed in 20:1 ratio, and that solution was added to the medium at 1:4 dilution. The assay medium containing MTS was added to cells after removing the conditioning medium. The cells were further incubated for 30 min at 37°C in the incubator. Dehydrogenase enzymes from viable cells convert MTS into an aqueous formazan product, the quantity of which was measured by the absorbance at 490 nm in a SpectraMax microplate spectrophotometer (Molecular Devices, Sunnyvale, CA). The medium containing MTS plated on wells without cells acted as a blank control. Because in all experiments the blank control value was negligible, the percentage of viability of cells was calculated as the ratio of experimental values compared with controls as follows: % viable cells = (A490 Experiment/A490 Control) x 100.

Arachidonic Acid Release from A549 Cells
Cells were grown on six-well dishes and were labeled for 16 h with 1 µCi/ml 5,6,8,9,11,12,14,15-3H AA (3H-AA) (214 Ci/mmol) (Amersham Pharmacia Biotech, Piscataway, NJ) in Ham's F-12K media with 10% FCS and 2 mM L-glutamine. Subsequently, some cultures were treated with hydrogen peroxide in serum free-media for specified times, whereas cells incubated with serum-free media served as controls. Following the incubation with hydrogen peroxide, and after three washes with warm 1x PBS, 1 ml of calcium ionophore (A23187 at concentration 10-6 M; Calbiochem), platelet-activating factor (PAF, 10-7 M; Cayman Chemicals, Ann Arbor, MI) or tumor necrosis factor (TNF)-{alpha} (20 ng/ml; R&D Systems, Minneapolis, MN) solution in Ham's F-12K media with 10% FCS and with 2 mM of L-glutamine was added to some wells on a six-well plate and incubated for 30 min. Cells incubated with DMSO or media served as a control for A23187, PAF, and TNF-{alpha}–stimulated cells, respectively. Media were collected, centrifuged at 1,000 x g for 5 min at 4°C, and 0.9 ml of medium was transferred to a scintillation vial containing 10 ml of Bio-Safe II scintillation fluid (Research International Products Inc., Mount Prospect, IL) and counted in a scintillation counter (Beckman, Columbia, MD). Data are expressed as mean DPM ± SEM.

Prostaglandin E2 Detection
After exposure of A459 cells to H2O2 for times and doses specified, media was centrifuged at 1,000 x g for 5 min, and supernatants were collected and frozen at -70°C. Prostaglandin E2 (PGE2) concentration was measured using an ELISA kit obtained from R&D Systems according to the manufacturer's instruction.

Antibodies
Rabbit anti-human cPLA2, phosphospecific anti-Serine 505 human cPLA2 and Rabbit anti-human EGFR and phosphospecific (anti-Tyrosine 1068) anti-human EGFR antibodies were obtained from Cell Signaling Technology, Inc. (Beverly, MA). Rabbit anti-human p38, anti-ERK1/2 antibodies were obtained from Calbiochem. Phosphospecific anti-p38 phosphorylated at Threonine 180 and Tyrosine 182, anti-ERK 1, 2, phosphorylated at Threonine 185 and Tyrosine 187 were obtained from Biosource (Camarillo, CA). Horseradish peroxidase–conjugated goat anti-rabbit IgG was obtained from Jackson Immunoresearch Laboratories (West Grove, PA).

Immunoblotting of cPLA2, p38, ERK1/2, and EGFR
A549 cells were grown on six-well culture dishes until 90% confluent and then exposed to hydrogen peroxide in serum-free media. Cells were washed with cold PBS. Cell lysate was collected by scraping and transferred to 1.5-ml tubes. After 30 s sonication (repeated three times), 20 µl of cell lysate was boiled for 5 min before electrophoresis on 8% polyacrylamide gels (Invitrogen, Carlsbad, CA) (for cPLA2), 10% (for p38), 4–20% (for ERK1/2), or 4% for (EGFR) using 1x Tris-Glycine/SDS buffer. The separated proteins were electrophoretically transferred onto a nitrocellulose membrane, blocked with 5% nonfat milk for 2 h, and then probed with a 1:1,000 dilution of first antibody as specified above. Blots were washed three times with 0.1% Tween 20 in PBS for 5 min, followed by three washes with 0.3% Tween 20 in PBS for 5 min. The blots were then probed with a 1:1,000 dilution of horseradish peroxidase–labeled goat anti-Rabbit IgG and developed using the ECL Western blotting detection system (Amersham) and exposed to Kodak (Rochester, NY) MR radiographic film.

Specific cPLA2 Activity Assay
A549 cells grown on 175-cm2 culture flasks were washed with cold PBS, and cells were harvested and resuspended in 0.5 ml of homogenization buffer (50 mM HEPES, pH 7.4, 1 mM EGTA, 50 mg/ml leupeptin, 1 mM dithiothreitol, 0.5 mM phenyl-methylsulfonyl fluoride, 10 mM phosphoramidon, 10 mg/ml soybean trypsin inhibitor, 100 mg/ml aprotinin). Cells were disrupted by repeated sonication for 30 s on ice. The lysate was centrifuged at 1,000 x g for 5 min to remove nuclei, unbroken cells, and debris.

As the physiologic cellular Ca2+ level is normally at the submicromolar level (0.1 µM in normal resting cells and 0.3–1.0 µM in activated cells) and the cPLA2 activity is measurable under micromolar concentrations of Ca2+, a physiologically relevant Ca2+ concentration (0.5 µM free Ca2+) was used for all the assays (1 mM EGTA and 0.96 mM CaCl2). Two microliters of 14C Phosphatidylcholine were added to 95 µl of crude cell lysate. The reaction was started by addition of 2.5 µl of 116 mM CaCl2. The assays were incubated at 37°C for 1 h and terminated by the addition of 300 µl of 2:1 chloroform/methanol containing 1% acetic acid and 1 mg/ml free AA. Release of free fatty acid was analyzed using silica gel H thin layer chromatography plates. Data are expressed as percentage activity of control cells incubated in the presence of serum-free media only ± SEM.

RNase Protection Assay
A549 cells were exposed to hydrogen peroxide (200 µM) as described. The medium was removed and cells were washed three times with cold PBS, carefully scraped, and collected by spinning for 5 min at 300 x g at 4°C. Total RNA was isolated using the RNAqueous kit from Ambion (Austin, TX). RNA was quantified using 260 nm optical density. To construct the probe for cPLA2 mRNA, a 307-bp product of cPLA2 cDNA was amplified by PCR using the following sets of sense and antisense primers: 5'primer: 5'-ATTCTGGATTGTGCTACCTACG-3'(corresponding to bases 787–808 of the human cPLA2 cDNA sequence, GenBank accession number: M68874); 3'primer: 5-CTTTTTCCTTCAAACTGCTCAG-3' (which correspond to bases 1093–1072 of the cPLA2 cDNA sequence). The product was cloned into the pGEM-T Easy vector (Promega). Orientation of the insert was determined by DNA sequencing. The cPLA2 RNA probe and a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe (Ambion) were radiolabeled using an in vitro transcription kit (Ambion) with T7 polymerase and ({alpha}-32P) UTP (800 µCi [29.6TBq]/mmol; NEN Life Science, Boston, MA). An RPA assay kit (RPA III; Ambion) was used to quantitate target mRNA. Two micrograms of total RNA were mixed with 5,000 cpm (for GAPDH) or 20,000 cpm (for cPLA2) of cRNA. Then, the mixture was hybridized at 42°C overnight and digested by the addition of 1:100 dilution RNase A/T1 at 37°C for 30 min. Digestion was terminated by the addition of RNase inactivation and precipitation mixture. The protected fragments were separated on 6% polyacrylamide 8 M urea gels (Invitrogen) and visualized by autoradiography using Kodak MR-X film at -70°C for 24–48 h.

Ras Dominant Negative and Ras Expression Experiments
Ras N17 dominant negative vector and Ras expression vector were obtained from Clontech (Palo Alto, CA). A594 cells were grown in six-well dishes and were transiently transfected with 1.8 µg of the aforementioned vectors in serum-free media using Lipofectamine Plus (Invitrogen) for 6 h. The media was replaced with media containing 10% FCS (with 3H-AA for AA release experiments), and cells were grown for 24 h. AA release experiments were performed as described above.

Stable Expression of Antisense cPLA2 RNA in A549 Cells
The cPLA2 antisense expression vector was made using a PCR-based technique as described previously (17). Briefly, a 310-bp PCR product was obtained using the following pair of primers: 5'- CTCGAGGAATTCTTCGGAGCTGAA-3' and 5'-GCTAGCAGGGGTTGTAGAGATAAA-3' using coding cDNA for cPLA2 as a template. The product was cloned in pCR 2.1 vector (Invitrogen). The insert was cut out using XhoI and NheI restriction enzymes and subsequently cloned into pCDNA 3.1 (+) vector (Invitrogen). The identity and orientation of the insert was confirmed by DNA sequencing. A549 cells grown in T-175 flasks were transfected with 20 µg cPLA2 antisense expression vector (AS) or with empty pCDNA 3.1 (+) plasmid using Lipofectamine Plus reagent in Ham's F-12 serum-free media as described above. After 6 h, medium was removed and cells were maintained in Ham's F-12 media with Glutamine and 10% FBS containing 1.2 mg/ml of geneticin (Calbiochem). Passages 2–4 were used for experiments.

Statistical Analysis
Comparisons were made using two-tailed unpaired Student's t tests. Dose-related effects were evaluated by one-way ANOVA. Differences were considered to be significant at P < 0.05. Microsoft Excel 2001 software (Microsoft Corp., Redmond, WA) running on an iMac computer (Apple Computer Inc., Cupertino, CA) was used to perform statistical analysis.

Quantification of Autoradiographs
Molecular Dynamics 301 computing densitometer (Molecular Dynamics, Sunnyvale, CA) was used to digitalize images. The optical density of the bands was analyzed with background subtraction using Image Quant software (Molecular Dynamics).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Influence of Hydrogen Peroxide on Calcium Ionophore–Stimulated AA and PGE2 Release from A549 Cells
Incubation of A549 cells in the presence of hydrogen peroxide resulted in a dose-dependent increase in AA release, with a maximum effect at 30 min and 1 h (Figure 1A) and at 200 µM (Figure 1B). The effect of hydrogen peroxide on AA release appeared to be more prominent with exposure to the calcium ionophore (A23187, 1 µM), suggesting that this effect is associated with an increase in activity of a calcium-dependent phospholipase. Hydrogen peroxide (200 µM) did not influence cell viability as measured by the MTS viability assay (percentage of viable cells after 24 h incubation with 200 µM of hydrogen peroxide was 103 ± 4.52% as compared with control). Moreover, this effect was also present in a similar time and dose fashion when PGE2 was measured as shown in Figures 2A and 2B.



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  		Figure 1. Hydrogen peroxide induces arachidonic acid release from A549 cells. (A) The effect of hydrogen peroxide on arachidonic acid release is maximal at 30 min and 1 h. 3H-AA–labeled cells were exposed to hydrogen peroxide. Media were changed and cells were exposed to A23187 (1 µM; closed bars) or DMSO (vehicle; open bars) for 30 min (n = 5–6, *P < 0.05 as compared with control). (B) The effect of hydrogen peroxide on arachidonic acid release is dose-dependent. 3H-AA–labeled cells were exposed to hydrogen peroxide for 1 h. Media were changed and cells were exposed to A23187 (1 µM; closed bars) or DMSO (open bars) for 30 min. See MATERIALS AND METHODS for details. n = 5–6, P < 0.05 by ANOVA.

 


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  		Figure 2. Hydrogen peroxide induces PGE2 release from A549 cells. (A) The effect of hydrogen peroxide on PGE2 release is maximal at 30 min and 1 h. Cells were exposed to hydrogen peroxide (200 µM) for 30 min, 1 h, or 4 h. Media were changed and cells were exposed to A23187 (1 µM) for 30 min. n = 5–6, *P < 0.05 as compared with control. (B) The effect of hydrogen peroxide on PGE2 release is dose-dependent. Cells were exposed to 2, 20, or 200 µM of hydrogen peroxide for 1 h. Media were changed and cells were exposed to A23187 (1 µM; n = 5–6, P < 0.05 by ANOVA).

 
Influence of Hydrogen Peroxide on PAF- and TNF-{alpha}– Stimulated Arachidonic Acid Release from A549 Cells
To confirm the aforementioned effect of hydrogen peroxide, we investigated its influence on arachidonate release in cells stimulated with other stimuli such as PAF and TNF-{alpha}. In this experimental design, hydrogen peroxide treatment was associated with a maximum arachidonate release at 30 and 60 min for cells treated with PAF and at 30 min for cells treated with TNF-{alpha} (Figures 3A and 3B).



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  		Figure 3. Hydrogen peroxide induces an increase in arachidonic acid release from A549 cells stimulated with PAF and TNF-{alpha}. The effect of hydrogen peroxide on arachidonic acid release is maximal at 30 min and 1 h. Cells were exposed to hydrogen peroxide (200 µM) for 30 min, 1 h, or 4 h. Media were changed and cells were stimulated by PAF (10-7 M; closed bars) or DMSO (vehicle; open bars) for 30 min (A) or by TNF-{alpha} (20 ng/ml; closed bars) or media (open bars) for 30 min (B). n = 5–6, *P < 0.05 as compared with control.

 
Influence of an EGFR Kinase Inhibitor (AG 1478), an MEK Inhibitor (PD 98059) and p38 Kinase Inhibitor (SB 203580) on Arachidonic Acid Release
We studied the effect of the EGFR kinase inhibitor, AG 1478, on AA release. Preincubation of cells with AG 1478 inhibited the effect of hydrogen peroxide (200 µM) on ionophore-stimulated AA release (Figure 4A). To further explore the role of downstream kinases on AA release, we used an MEK inhibitor, PD 98059, and a p38 kinase inhibitor, SB 203580, to study the effect of inhibition of the aforementioned kinases on hydrogen peroxide–induced AA release. Preincubation with PD 98059 (10 µM) resulted in a decrease of hydrogen peroxide–induced AA release from A549 cells (Figure 4B). Similarly, preincubation with SB 203580 (10 µM) significantly reduced the effect of hydrogen peroxide on ionophore-stimulated AA release (Figure 4C). Coincubation of cells with both inhibitors present in the media abolished the hydrogen peroxide effect on AA release, as shown in Figure 4D.




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  		Figure 4. The influence of an EGF receptor kinase inhibitor and MEK and p38 kinase inhibitors on arachidonic acid release. (A) An EGF receptor kinase inhibitor, AG 1478, abolished the effect of hydrogen peroxide on arachidonic acid release. Cells were preincubated with AG 1478 and exposed to hydrogen peroxide (200 µM) for 1 h. Then, cells were exposed to A23187 (closed bars) or media (open bars). Data are expressed as DPM ± SEM. n = 4–6, *P < 0.05 as compared with cells treated with hydrogen peroxide alone; P < 0.05 by one-way ANOVA for AG 1478 dose-related effects. (B) A MEK inhibitor, PD 98059 inhibited the effect of hydrogen peroxide on arachidonic acid release from A549 cells. Cells were preincubated with PD 98059 and exposed to hydrogen peroxide (200 µM) for 1 h. Then, cells were exposed to A23187 (1 µM; closed bars) or media with DMSO (vehicle; open bars). Data are expressed as DPM ± SEM. n = 4–6, *P < 0.05 as compared with cells exposed to hydrogen peroxide. (C) An MEK inhibitor, SB 203580, inhibited the effect of hydrogen peroxide on arachidonic acid release from A549 cells. Cells were preincubated with SB 203580 and exposed to hydrogen peroxide (200 µM) for 1 h. Cells were exposed to A23187 (1 µM; closed bars) or media with DMSO (vehicle; open bars). Data are expressed as DPM ± SEM. n = 4–6, *P < 0.05 as compared with cells exposed to hydrogen peroxide alone. (D) The effect of PD 98059 and SB 203580 on hydrogen peroxide–stimulated arachidonic acid release. Cells were preincubated with PD 98059 (10 µM) and SB 203580 (10 µM) and exposed to hydrogen peroxide (200 µM) for 1 h. Cells were exposed to A23187 (1 µM; closed bars) or media (open bars). Data are expressed as DPM ± SEM. n = 4–6, *P < 0.05 as compared with cells incubated with hydrogen peroxide alone.

 
Influence of Hydrogen Peroxide on Cytosolic Phospholipase A2 Activity
Because AA release may involve multiple phospholipases, a specific cPLA2 assay was employed to study the influence of hydrogen peroxide on cytosolic phospholipase A2–specific activity in A549 cells. The exposure of A549 cells to hydrogen peroxide resulted in a time-dependent (Figure 5A) and dose-dependent (Figure 5B) increase of phospholipase A2 activity, with maximum effect at the 1-h time point, suggesting cPLA2 involvement in this process.



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  		Figure 5. The influence of hydrogen peroxide on cPLA2 activity. (A) The influence of hydrogen peroxide on cPLA2 activity over time. A549 cells were exposed to hydrogen peroxide (200 µM) in for 30 min, 1 h, and 4 h. Cells exposed to media served as controls. Cells were harvested and cPLA2 activity was assayed as described in MATERIALS AND METHODS section. Data are shown as percentage of control activity. n = 3–4, *P < 0.05 as compared with control. (B) The influence of hydrogen peroxide on cPLA2 activity is dose-dependent. A549 cells were exposed to 2, 20, or 200 µM of hydrogen peroxide for 1 h. Cells exposed to media served as control. Data are shown as percentage of control activity ± SEM. n = 3–4, P < 0.05 by one-way ANOVA.

 
Influence of an EGFR Kinase Inhibitor, AG 1478, an MEK Inhibitor, PD 98059, and a p38 Kinase Inhibitor, SB 203580, on Specific cPLA2 Activity
An EGFR kinase inhibitor (AG 1478, 10 µM) abolished the hydrogen peroxide–induced increase in cPLA2 activity, suggesting that oxidative stress–induced increase of cPLA2 activity at least in part is mediated through an EGF receptor pathway (Figure 6). To further explore the role of downstream kinases on AA release, we used an MEK inhibitor, PD 98059, and a p38 kinase inhibitor, SB 203580, to study the effect of inhibition of the aforementioned kinases on hydrogen peroxide–induced cPLA2 activity. Preincubation with PD 98059 (10 µM) resulted in a decrease of hydrogen peroxide–induced cPLA2 activity in A549 cells (Figure 6). Similarly, SB 203580 (10 µM) preincubation significantly reduced the effect of hydrogen peroxide on cPLA2 activity, as shown in Figure 6. Coincubation of cells with both inhibitors abolished the hydrogen peroxide effect on cytosolic phospholipase A2 activity, as shown in Figure 6.



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  		Figure 6. The influence of an EGF receptor kinase inhibitor and MEK and p38 kinase inhibitors on specific cPLA2 activity. A549 cells were preincubated with AG 1478 (10 µM) or with the MEK inhibitor, PD 98059 (10 µM) or with the p38 kinase inhibitor, SB 203580 (10 µM) for 30 min and exposed to hydrogen peroxide (200 µM) for 1 h. n = 3–4, *P < 0.05 as compared with cells incubated without inhibitors and exposed to hydrogen peroxide.

 
cPLA2 Steady-State mRNA and Protein Levels are Unchanged after Incubation with Hydrogen Peroxide
To investigate whether hydrogen peroxide increased the cPLA2 activity through de novo cPLA2 mRNA or protein production, an RPA and immunoblotting were performed. The steady-state cPLA2 mRNA levels were unchanged during the incubation period, with hydrogen peroxide up to 24 h (control cPLA2/GAPDH ratio was 0.96 ± 0.16, and after 24 h incubation with hydrogen peroxide was 0.82 ± 0.19, NS), suggesting no change in de novo synthesis of cPLA2 mRNA in response to hydrogen peroxide exposure (Figure 7). cPLA2 protein levels as assessed by immunoblotting remained unchanged up to 4 h of incubation (Figure 8B) (99 ± 5% at 4 h as compared with control, NS).



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  		Figure 7. Steady-state cPLA2 mRNA levels are unchanged during the incubation with hydrogen peroxide. Cells were exposed to hydrogen peroxide (200 µM) for 1, 4, or 24 h. There was no change in steady-state levels of cPLA2 mRNA. Steady-state levels of GAPDH mRNA are presented as a control.

 


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  		Figure 8. Exposure of A549 cells to hydrogen peroxide causes cPLA2 phosphorylation. Cells were exposed to hydrogen peroxide (200 µM) for 30, 60, or 240 min. (A) Immunoblot with phosphospecific anti-Serine 505 human cPLA2 antibody. The immunoblot shown is representative of four separate experiments. (B) Immunoblot with anti cPLA2 antibody demonstrating equal amounts of cPLA2 protein. The immunoblot shown is representative of four separate experiments.

 
cPLA2 Is Phosphorylated as a Result of Hydrogen Peroxide Exposure
To determine the phosphorylation status of cPLA2 protein, immunoblotting using a phosphospecific antibody detecting the phosphorylation of Serine 505 of cPLA2 was used. As shown in Figure 8, cellular cPLA2 protein is phosphorylated at Serine 505 at 30 min (optical density increase +29 ± 2% above control) and 1 h after (+59 ± 20% as compared with control) hydrogen peroxide exposure. This effect corresponds with an increase in cellular AA release and specific cPLA2 activity.

Hydrogen Peroxide Induces Phosphorylation of p38, ERK1, and ERK2
To further investigate the enzymes directly involved in cPLA2 phosphorylation, immunoblotting for common type and phosporylated forms of p38 kinase, ERK1,2 kinases, were performed. As shown in Figure 9A, hydrogen peroxide (200 µM) induces a time-dependent increase in phosphorylation in p38 (maximum at 10 min, +95 ± 5% above control, P < 0.05), and ERK1,2 kinase phosphorylation with a maximum at 10 and 30 min (+65 ± 19% above control, P < 0.05) (Figure 9B). The protein levels of common type p38 and ERK1,2 were blotted to ensure even loading and were unchanged throughout the incubation as shown in Figures 9A and 9B.



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  		Figure 9. Hydrogen peroxide causes phosphorylation of p38 kinase and ERK 1 and 2. Cells were exposed to hydrogen peroxide (200 µM) for 10, 30, or 60 min. (A) An immunoblot using phosphospecific anti-p38 phosphorylated at Threonine 180 and Tyrosine 182 (P-p38) or anti p38 antibody (p38). The immunoblots shown are representative for three separate experiments. (B) Immunoblots using phosphospecific antibody anti-ERK1,2, phosphorylated at Threonine 185 and Tyrosine 187 (P-ERK1,2) or anti ERK1,2 (ERK1,2). The immunoblots shown are representative of three separate experiments.

 
Hydrogen Peroxide Induces Phosphorylation of Tyrosine 1068 of EGFR
EGFR immunobloting, using antibodies recognizing the common form of EGFR and the EGFR phosphorylated at Tyr 1068, was performed. Figure 10 presents immonoblotting showing that EGFR phosphorylation is increased at 10 min of incubation with hydrogen peroxide (200 µM) and lasts at least up to 30 min of incubation (increase +93 ± 6.5% above control, P < 0.05 at 10 min and +46 ± 4%, P < 0.05 at 30 min). Total EGFR protein levels remained unchanged during the incubation period, as shown on Figure 10.



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  		Figure 10. Hydrogen peroxide causes phosphorylation of the EGF receptor. Cells were exposed to hydrogen peroxide (200 µM) in serum-free media for 10, 30, or 60 min. Cells exposed to serum-free media served as control. (A) Immunoblotting using phospho-specific (anti-Tyrosine 1068) anti-human EGFR antibody (P-Y1068-EGFR). The immunoblot shown is representative for three separate experiments. (B) Immunoblotting using anti-EGFR antibody (EGFR) demonstrating equal loading. The immunoblot shown is representative of three separate experiments.

 
Influence of a Ras Dominant Negative Vector on cPLA2 Phosphorylation
As Ras may mediate EGFR-induced MAP kinase activation, we used a Ras dominant negative vector to study the role of Ras in this hydrogen peroxide–induced effect. A549 cells were transfected with a Ras dominant negative vector, and 24 h after transfection were incubated with hydrogen peroxide (200 µM). Immunoblotting using phosphospecific anti-cPLA2 antibodies revealed that the Ras dominant negative vector markedly reduced hydrogen peroxide–induced cPLA2 phosphorylation as compared with cells transfected with control vector (increase +65 ± 8.5% above control cells for cells incubated with hydrogen peroxide, and an increase +60 ± 10% above control cells for cells transfected with Ras expression vector and treated with hydrogen peroxide [Figure 11A]). Immunoblotting for cPLA2 demonstrated equal protein loading (Figure 11B).



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  		Figure 11. A Ras-dominant negative vector inhibits cPLA2 phosphorylation induced by hydrogen peroxide. Cells are transfected with a Ras expression vector or with Ras dominant negative expression vector (N17). WT cells were not transfected. Cells were exposed to hydrogen peroxide (200 µM) in serum-free media for 60 min. (A) Immunobloting using anti–phospho-serine 505-cPLA2 antibody. The immunoblot shown is representative of three separate experiments. (B) Immunobloting using anti-cPLA2 antibody. The immunoblot shown is representative for three separate experiments.

 
Influence of a Ras Dominant Negative Vector on Arachidonic Acid Release from A549 Cells
A549 cells were transfected with a Ras dominant negative vector, and 4 h after transfection, cells were labeled with 3H-AA. Twenty-four hours after transfection, cells were incubated with hydrogen peroxide (200 µM) for 1 h, followed by ionophore stimulation. After supernatant processing as described in MATERIALS AND METHODS, released labeled AA was assayed. Cells transfected with the Ras dominant negative vector and exposed to hydrogen peroxide revealed significantly decreased calcium ionophore–induced release of AA as compared with cells transfected with empty vector (Figure 12). These data demonstrate a role for Ras and the EGFR pathway in hydrogen peroxide–stimulated AA release.



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  		Figure 12. Ras-dominant negative vector inhibits cellular arachidonic acid release induced by hydrogen peroxide. Cells are transfected with a Ras expression vector or with Ras dominant negative expression vector (N17). WT cells were not transfected. 3H-AA–labeled cells were exposed to A23187 (1 µM; closed bars) or media with DMSO (vehicle; open bars). Data are expressed as DPM ± SEM. n = 4–6, *P < 0.05 as compared with cells transfected with Ras dominant negative vector (N17).

 
Stable Expression of cPLA2 Antisense RNA Decreases Hydrogen Peroxide–Induced Arachidonic Acid Release from A459 Cells
To confirm that cPLA2 is an enzyme that is responsible for hydrogen peroxide–induced AA release, we obtained A549 cells stably transfected with a vector expressing cPLA2 antisense RNA. These cells exhibit reduced cPLA2 protein production by immunoblotting (Figure 13). Cells transfected with the antisense vector exhibited diminished calcium ionophore–stimulated AA release in response to hydrogen peroxide as compared with cells transfected with the empty control vector (Figure 13B). These data suggest that cPLA2 might be the predominant phospholipase involved in oxidative stress–induced increase of AA release.



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  		Figure 13. Cells stably expressing cPLA2 antisense mRNA have diminished response to oxidative stress. A549 cells were transfected stably a with vector expressing cPLA2 antisense mRNA (AS) or with an empty vector (VC) serving as a control. (A) Immunoblotting using anti-cPLA2 antibody. (B) The effect of hydrogen peroxide on arachidonic acid release from A549 cells transfected stably with a vector expressing cPLA2 antisense mRNA (AS) or with an empty vector (VC). Cells were labeled with 3H-AA. Media were changed and cells were exposed to A23187 (1 µM; closed bars) or DMSO (vehicle; open bars) for 30 min. Data expressed as DPM ± SEM. n = 6, *P < 0.05 for VC cells exposed to hydrogen peroxide and ionophore compared with AS cells exposed to hydrogen peroxide and ionophore.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Oxidative stress is one of the important mechanisms causing tissue injury through generation of lipid hydroperoxides (18). Several models have been established to investigate the influence of oxidative stress on cells cultured in vitro: UV light B exposure, incubation with PMA, intracellular generation of oxygen radicals, hyperbaric oxygen exposure, and hydrogen peroxide administration. The latter model seems to be an ideal way to study the effect of oxidative stress on epithelial cells, due to the fact that most of the oxidants in the airways have a nonepithelial cell origin. This model also allows the study of extracellular oxidative stress delivery, which may alter cell functions via cell surface receptors.

Hydrogen peroxide is produced by migratory inflammatory cells such as neutrophils and eosinophils, as well as by tissue macrophages (19). At the physiologic level, it causes epithelial cell damage and mucous secretion, and may influence eicosanoid production by either epithelial cells or inflammatory cells. Hydrogen peroxide levels in the exhaled air are important markers of airways inflammation and correlate with impairment of lung function (9). Hydrogen peroxide modifies expression of various genes, including those involved in AA metabolism such as cycloxygenase-2 (20, 21).

cPLA2 is one of several phospholipases involved in AA metabolism during airway inflammation. It is expressed not only by inflammatory cells such as neutrophils, macrophages, eosinophils, and basophils, but also by airway epithelial cells, which might be an important source of eicosanoids in the setting of airway inflammation. cPLA2 activity is tightly regulated by intracellular calcium concentration, cytokines (e.g., IFN-{gamma}, TNF-{alpha}, IL-1ß) (3–6, 22) and growth factors (e.g., EGF) (23). cPLA2 activity is modified at the transcriptional level, mRNA half-life, and posttranslationally by phosphorylation (2). Although multiple phosphorylation sites are described (24, 25), the increase of cPLA2 activity is most commonly related to phosphorylation at Serine 505 and 727. This effect may be caused by TNF-{alpha}, IFN-{gamma}, phorbol esters, and other stimuli, including oxidative stress.

Oxidative stress was reported as one of factors (including calcium influx, cytokines, and growth factors) inducing AA release through increased cPLA2 synthesis and/or cPLA2 phosphorylation at least at Serine 505 (26, 27). UVB and free oxygen radicals were reported to cause not only cPLA2 phosphorylation, but also to increase cPLA2 mRNA levels in skin (27). In this study, we did not observe a change in cPLA2 steady-state mRNA level in human lung cells.

It has not been clear whether arachidonate release in response to oxidative stress depends on cPLA2 phosphorylation only or also involves cPLA2 protein synthesis, or involves other phospholipases including sPLA2 and iPLA2 (28, 29). The pathway leading to this effect has not been clearly defined.

Several studies have implicated the involvement of various kinases in cPLA2 phosphorylation. These include ERK1, ERK2 (30), JNK (15), and p38 (31, 32). Some studies postulated that an additional unidentified MAP kinase related kinase may exist, and that these enzymes might be predominantly involved in cPLA2 phosphorylation (33).

The EGFR belongs to the CGRP receptor family and contains at least three tyrosine residues which may undergo phosphorylation, causing several downstream events, including Ras activation and increasing activity of ERK1, ERK 2, p38 kinase, and SAPK/JNK kinase. Hydrogen peroxide is one of the stimuli which may cause EGFR phosphorylation and be the source of EGFR-dependent downstream signal transduction events (16).

In this article, using a type II pneumocyte model, we report that exposure of human airway epithelial cells to oxidative stress caused by hydrogen peroxide leads to an increase in EGFR phosphorylation, which caused activation of the Ras, MAP, and p38 kinase pathway, resulting in cPLA2 Serine 505 phosphorylation and an increase cPLA2-specific activity and cPLA2-dependent AA release. The effect of hydrogen peroxide seems to be significant on A23187-induced arachidonate release, suggesting involvement of calcium-dependent phospholipase. Although calcium ionophore allows for the study calcium-dependent phospholipase activity, it is a potent and nonspecific stimulus. Therefore, we repeated these experiments with other stimuli—PAF and TNF-{alpha}. Both inflammatory mediators may be more physiologic stimuli of arachidonate release. Data obtained from these experiments confirmed that hydrogen peroxide induced AA release in A549 cells. An increase in arachidonate release (as a result of H2O2 exposure) is accompanied by an increase in PGE2 synthesis. We have shown that hydrogen peroxide caused early (at 10 min) phosphorylation of Tyrosine 1068 of EGFR. The EGFR kinase inhibitor (AG 1478) decreases AA release, Serine 505 of cPLA2 phosphorylation, and cPLA2 activity in a dose-dependent manner. In a similar fashion, MAP p38 kinase and ERK1,2 phosphorylation were observed after cells were exposed to hydrogen peroxide. Preincubation of A549 cells with an MEK inhibitor (PD 98059) or a p38 kinase inhibitor (SB 203580), used separately or together, significantly decreased both A23187-stimulated AA release and specific cPLA2 activity. We assume that EGFR activation results in the parallel activation of the p38 and ERK MAP kinase pathways. This raises the possibility of cross-talk between pathways in the MAP kinase cascade. Although the cross-talk between various types of kinases has been postulated (34), there is not sufficient data available to support this hypothesis. It seems possible that proteins which bind to the docking groove of MAPKs might mediate such cross-talk (35). To confirm that this effect is mediated through an EGFR/Ras/p38 or ERK pathway, a Ras dominant negative vector and a Ras overexpression vector were used. Cells transiently transfected with the Ras dominant negative vector exhibited little change in calcium ionophore–induced AA release following exposure to hydrogen peroxide. Similarly, cPLA2 phosphorylation was decreased (no difference as compared with control cells) when Ras dominant negative cells were exposed to hydrogen peroxide.

Finally, we confirmed that exposure to oxidative stress increases A23187-induced AA release mainly through cPLA2 by demonstrating that cells stably expressing cPLA2 antisense RNA have a diminished hydrogen peroxide –stimulated AA release.

Taken together, these data suggest that hydrogen peroxide increases cPLA2 activity through its phosphorylation at least at Serine 505 by p38 kinase and/or ERK 1, 2. This effect is mediated through EGFR activation by direct activation of EGFR kinase and phosphorylation of at least Tyrosine 1068. This suggests that the EGFR might play an important role in modulating calcium-dependent AA release by oxidative stress.

An increase in cPLA2 activity during oxidative stress might be an important factor in the inflammation leading to increased synthesis of various AA metabolites. It may also influence gene expression through lipid nuclear signaling, and this may ultimately affect the development of lung epithelial cell injury.

Received in original form March 21, 2002

Received in final form July 8, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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