Introduction

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Inflammatory diseases of the respiratory tract, such as asthma, bronchiectasis, and adult respiratory distress syndrome (ARDS), are characterized by a large increase in inflammatory oxidants, such as hydrogen peroxide (H₂O₂) and superoxide (O₂). These reactive oxidants play a major role in lung injury, characterized by increased epithelial cell permeability and decreased lung function. The mechanisms and target molecules of these oxidants are mainly unknown but may involve modification of growth-factor receptors. It is crucial that we understand how signaling pathways relate to oxidant-generating systems, the nature and regulation of the enzymes involved, and the consequences of these enzymes' modification during episodes of stress.

Aerobic cells are constantly exposed to reactive oxygen intermediates (ROIs), which are generated under various physiologic and pathologic conditions, such as inflammation, reperfusion, sepsis, and irradiation (1). Increased intracellular levels of the ROIs O₂, hydroxyl radical (·OH), or H₂O₂ are referred to as oxidative stress. Free radicals and redox stress induced by an excess of ROIs have been shown to play important roles in carcinogenesis by directly damaging DNA and acting as tumor promoters (2). Because induced intracellular ROI levels are now thought to participate in cellular signaling (6), additional targets for ROIs may exist. Indeed, exposure of cells to H₂O₂ activates the transcription factors c-jun (11, 12) and nuclear factor-B (NF-B) (6, 13), stimulates mitogen-activated protein (MAP) kinase activity (14), and induces transcription of human immunodeficiency virus (HIV) (15). The observation that H₂O₂ can induce rapid tyrosine phosphorylation of multiple cellular proteins (16) suggests that ROIs can regulate protein tyrosine kinases (PTKs) and protein tyrosine phosphatases.

One group of proteins that may be importantly affected by inflammatory oxidants are membrane receptors for various growth factors or cytokines that are induced and activated during inflammation to promote wound-healing processes. Among these receptors is the epidermal growth factor (EGF) receptor, which is overexpressed and activated in response to epithelial injury, and which functions in epithelial repair (20). The EGF receptor is a member of the receptor tyrosine kinase superfamily and is involved in regulating the proliferation and differentiation primarily of epithelial cell types (23, 24). The EGF receptor is a 170-kD glycoprotein (25) that spans the membrane via one -helical segment of 23 amino acids, connecting a large, heavily glycosylated extracellular ligand-binding domain and an intracellular tyrosine kinase domain.

Activation of the EGF receptor occurs in the following stages: Binding of EGF or transforming growth factor- (TGF-) to individual EGF receptors stimulates formation of noncovalent dimeric (or oligomeric) structures involving two (or more) receptors. The enzymatic tyrosine kinase activity of one receptor transphosphorylates residues on the opposite member of the receptor pair or dimer. Once activated, the receptor initiates a series of signal-transduction events through the tyrosine phosphorylation of interacting proteins that belong to the SH2 family, resulting in a sequence of responses that are involved in the mitogenic signal-transduction pathways of cells, ultimately leading to stimulation of DNA replication and cell division (25, 27). Besides undergoing tyrosine phosphorylation, the EGF-activated receptors are phosphorylated in vivo on serine/threonine, a mechanism by which receptor functions are attenuated. Various cellular protein kinases, such as protein kinase C (PKC), MAP kinase, p34cdk2 kinase, and casein kinase II, are considered to be involved in this serine/threonine phosphorylation and receptor attenuation (33). As a final step, the activated EGF receptor/ligand complex is internalized and is eventually degraded in lysosomes or dissociated for EGF-receptor recycling.

Recently, the signaling mechanism of the EGF receptor has been shown to involve generation of H₂O₂ (37), and various reports have indicated that oxidative modification of a reduced cysteine residue in the EGF receptor may reversibly affect its activation (38). Thus, despite the lack of evidence for a specific receptor for O₂ or H₂O₂ (41), these molecules may nonetheless interact with the EGF receptor. We have previously shown that the EGF receptor mediates resistance to ionizing radiation in A431 cells by enhancing its tyrosine phosphorylation (42, 43). Other investigators have suggested that cellular effects of ionizing radiation and ultraviolet radiation may be mediated by ROIs (44, 13). Therefore, we designed studies to explore whether the EGF receptor is a more general target for reactive radicals and may therefore sense cellular redox status.

In experiments designed to investigate how the function of the EGF receptor is affected by H₂O₂, we measured receptor phosphorylation in growing cells, in isolated membranes, and in purified EGF receptor exposed to H₂O₂. Our findings show that H₂O₂ stimulates EGF- receptor tyrosine phosphorylation. In all situations, phosphorylation was restricted to tyrosine residues. Serine and threonine residues of the EGF receptor were not phosphorylated, and the half-life of the receptor was longer after H₂O₂ exposure. At the same time, phospholipase C- (PLC-), but not MAP kinase, was activated by H₂O₂, whereas PKC- was deactivated by its translocation to the cytoplasm.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Materials

EGF was obtained from Collaborative Research (Waltham, MA), [³⁵S]methionine, [³²P]orthophosphate, [methyl-³H]thymidine, and ¹²⁵I-labeled protein A were from DuPont-New England Nuclear. Antiphosphotyrosine antibody PY69 and genistein were purchased from ICN Biochemical Inc. (Cleveland, OH). Antiphosphotyrosine antibody PY20 and anti-MAP kinase antibody were purchased from UBI Inc. (Santa Cruz, CA). Antiphosphotyrosine Ab-1-agarose conjugate was from Oncogene Science (Manhasset, NY). Polyclonal antiphospholipase C- antibody was from Upstate Biotechnology Inc. (Lake Placid, NY). Monoclonal anti-EGF receptor antibodies 528 and LA22 were kindly provided by Dr. J. Mendelsohn (Memorial Sloan Kettering Cancer Center, New York, NY) and Dr. J. D. Sato (Alton Jones Center, Lake Placid, NY), respectively. Antiserum RK2 to the EGF receptor carboxyl-terminal peptide was kindly provided by Dr. J. Schlessinger (New York University Medical Center, New York, NY). All of the other chemicals were from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated.

Cell Culture and Treatments

A549 cells (derived from lung adenocarcinoma) were obtained from the American Type Culture Collection (Rockville, MD) and were grown in monolayer culture in Ham's F-12 medium containing 10% fetal bovine serum (FBS) (both from GIBCO Laboratories, Grand Island, NY). For individual experiments, A549 cells were seeded into six-well plates in appropriate medium supplemented with 10% FBS, and were switched to 1% FBS on the following day. Freshly made 10 mM H₂O₂ solution was added to the medium to the final desired concentrations 12 h later. After incubation at 37°C for the indicated times, the medium was aspirated and the cells were washed with ice-cold phosphate-buffered saline (PBS), pH 7.4.

Cell Proliferation: Rate of DNA Synthesis as Measured by Thymidine Incorporation

Cells were cultured as described previously and were treated with EGF or H₂O₂ for 24 h. During the last 6 h of treatment, 1 mCi/ml of [methyl-³H]thymidine was present in the culture media. At the end of the labeling period, cells were washed in ice-cold PBS and then in 10% ice-cold trichloroacetic acid (TCA). The acid-insoluble material was washed twice in 10% TCA. The incorporated radioactivity was measured by scintillation counting (45).

Cell-Cycle Analysis

After treatments, cells were harvested by trypsinization, pelleted, and gently resuspended in PBS. These cells were held on ice until they could be lysed and the nuclei could be stained. The percentage of cells in different phases of the cell cycle was determined with flow cytometry (46). Briefly, cells were stained with propidium iodide (Sigma) and passed through the beam of an argon-ion laser tuned to 514 (FACScan; Becton Dickinson, Moutain View, CA). The resulting fluorescence signal was amplified, recorded in the instrument's memory, and analyzed in the form of a DNA histogram by using a computer program interfaced with the integrator.

Western Blot Analysis of Phosphotyrosines, EGF Receptors, PLC-, and MAP Kinase

After exposure to H₂O₂, cells were cultured for the indicated times in the presence or absence of EGF, followed by removal of the culture medium and by rinsing of the cells with cold PBS. Cells were lysed in a lysis buffer containing a mild detergent and protease and phosphatase inhibitors (50 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid [Hepes], pH 7.5; 1% Triton X-100; 10% glycerol; 1 mM phenylmethylsulfonyl fluoride (PMSF); 10 µg/ ml leupeptin; 10 µg/ml aprotinin; 2 mM sodium orthovanadate (Na₃VO₄); 1.5 mM magnesium chloride (MgCl₂); 1 mM ethyleneglycol-bis-(-aminoethyl ether)-N,N,N',N'-tetraacetic acid [EGTA]), and were incubated for 30 min at 4°C. The lysates were then mixed with concentrated sodium dodecyl sulfate (SDS) sample buffer to achieve final concentrations of 62.5 nM Tris-HCl, pH 6.8; 2% SDS; 0.5% 2-mercaptoethanol; and 10% glycerol, and were boiled for 5 min. Samples were analyzed with 7% SDS- polyacrylamide gel electrophoresis (PAGE) and were western blotted, and nitrocellulose membranes were incubated with antiphosphotyrosine monoclonal antibody (mAb) PY69 or PY20 (at concentrations of 4 µg/ml), with anti-EGF receptor peptide polyclonal antibody RK2, with anti-EGF receptor mAb LA22 (at a concentration of 1 µg/ml), with anti-PLC- polyclonal antibody, or with anti-MAP kinase mAb. Bound antibody was detected with ¹²⁵I-protein A, or with a peroxidase-conjugated second antibody and chemiluminescence (ECL kit from Amersham, Arlington Heights, IL) (45). Sample protein concentrations were equalized before loading onto the gels. Either whole lysates or the immunoprecipitated (with 528 mAb) EGF receptor (see the subsequent discussion) were transferred onto the membrane and incubated with antiphosphotyrosine antibody (either mAb PY69 or mAb PY20). For the determination of PLC- phosphorylation, either whole lysate or immunoprecipitated phosphotyrosine proteins (with antiphosphotyrosine Ab-1-agarose conjugate; see the subsequent discussion) were transferred onto the membrane and incubated with polyclonal anti-PLC- antibody. For the determination of tyrosine phosphorylation of MAP kinase, we also used immunoprecipitation with an antiphosphotyrosine antibody followed by Western blot analysis with an anti-MAP kinase mAb to show that the tyrosine-phosphorylated protein bands at 42 and 45 molecular weight correspond to activated MAP kinase (49). Unless indicated otherwise, data shown for all Western blots are representative of at least three experiments.

Cell Metabolic Labeling

Cells were plated in 10% FBS culture medium for 2 d to reach approximately 70% confluence. For [³⁵S]methionine labeling, cells were switched to the labeling medium (90% methionine-free culture medium, 10% regular medium, 100 µCi of [³⁵S]methionine/ml, and 1% dialyzed FBS) for 16 h. After removing the labeling medium, the cells were rinsed twice with fresh medium and cultures were chased in 1% dialyzed FBS medium for varying periods in the presence or absence of EGF or H₂O₂. For [³²P]orthophosphate labeling, cells were grown for 16 h in phosphate-free medium with the addition of 300 µCi of [³²P]orthophosphate/ml, 4 mM glutamine, 1 mM sodium pyruvate, and 1% dialyzed FBS. Cells were incubated with H₂O₂ or with EGF during the final 30 min of labeling (45). After incubation for the indicated times, cells were rinsed with cold PBS, incubated in Triton X-100 lysis buffer, and subjected to EGF receptor immunoprecipitation as described subsequently.

Immunoprecipitation

Cells in Triton X-100 lysis buffer were incubated for 30 min in an orbital shaker at 4°C. To remove insoluble material, cell lysates were centrifuged at 14,000 rpm for 5 min at 4°C, and supernatants were precleared by adding 50 µl of 20% pansorbin (Calbiochem) to each sample and incubating for 1 h at 4°C. Aliquots of supernatants containing equal amounts of protein were immunoprecipitated for 2 h at 4°C with either anti-EGF receptor mAb 528/rabbit antimouse IgG (Accurate Chemical & Scientific Corp.)/protein A-Sepharose (Pharmacia LKB Biotechnology, Inc.) conjugate or with antiphosphotyrosine Ab-1 agarose conjugate (45).

In Vitro Membrane Phosphorylation

Membrane fractions were prepared and the in vitro phosphorylation assay was done as previously described (45). Briefly, cells were collected in SAT buffer (0.25 M sucrose, 10 mM acetic acid, 10 mM triethanolamine, pH 7.4) and lysed in SEAT buffer (1 mM ethylenediamine tetraacetic acid [EDTA] in SAT buffer), followed by centrifugation at 800 rpm for 5 min to pellet nuclei. The supernatant was centrifuged at 100,000 rpm for 15 min to pellet the membrane. The resulting pellet was resuspended in Hepes-Triton buffer (20 mM Hepes, pH 7.4; 1% Triton X-100; 0.2 mM EDTA; 10% glycerol) and centrifuged at 100,000 rpm for 15 min. The supernatant containing the solubilized membrane fraction was used. The reaction mixture for the in vitro phosphorylation assay (final volume: 30 µl) contained 20 mM Hepes, pH 7.4; 1 mM MnCl₂; 5 µg bovine serum albumin (BSA); 5 µM adenosine triphosphate (ATP); and 10 µg membrane protein. The reaction was initiated by the addition of [³²P]ATP (in a total of 5 µM ATP), incubated for 10 min, and terminated by the addition of Laemmli's sample buffer.

Analysis of Phosphoamino Acids by Two-Dimensional Electrophoresis

Two-dimensional phosphoamino acid analysis of EGF receptors labeled metabolically with [³²P]orthophosphate was performed as described previously (45). Briefly, the polyacrylamide gel band containing EGF receptors was excised and homogenized. Labeled EGF receptors were precipitated with 15% TCA and partly hydrolyzed in 100 µl of 6 N HCl at 110°C for 1 h, followed by three cycles of washing with deionized water and lyophilization. The recovery of total labeled material in individual samples was 65-70%. The hydrolysate was subjected to two-dimensional thin-layer electrophoresis (TD-TLE). The radioactivity associated with individual phosphoamino acids was measured by scraping spots from the thin-layer plate and counting in a beta-counter.

Immunokinase Assay for Measuring EGF-Receptor Tyrosine Protein Kinase Activity

Cells were lysed and immunoprecipitated as described previously. Immune complexes were washed three times with 0.1% Triton X-100 in PBS (pH 7.4) and resuspended in 25 µl of 20 mM Hepes (pH 7.4)-100 µM Na₃VO₄-0.1% Triton X-100. EGF-receptor kinase activity assay was initiated as described (31, 45) by the addition of 25 µl of 20 mM Hepes-0.1% Triton X-100-6 mM MnCl₂ buffer containing 10 µCi of [³²P]ATP (in a total of 5 µM ATP) to each sample. The reaction was incubated for 10 min at 4°C, and was terminated by the addition of sample buffer. Immunokinase complexes were subjected to 7% PAGE. Gels were dried and subjected to autoradiography, and EGF receptor-autophosphorylated bands were excised and quantified by Cerenkov counting, as previously described (45).

PKC Activity

A549 cells were harvested by scraping in cold buffer A (20 mM Tris-HCl, pH 7.5; 250 mM sucrose; 6 mM EDTA; 0.5 mM dithiothreitol [DTT], supplemented with the protease inhibitors PMSF [0.5 mM], leupeptin [50 µg/ml], and aprotinin [20 µg/ml]). The cells were sonicated for 1 min in a bath sonicator and centrifuged at 500 × g for 5 min at 4°C to remove nuclei and whole cells. The cytosolic fraction was separated from the membranes by centrifugation at 100,000 × g for 1 h. Membrane-bound PKC was solubilized by resuspending the pellet in buffer A containing 0.5% Triton X-100 for 20 min on ice, and centrifuging for 30 min at 100,000 × g to remove nonsoluble material. Both the cytoplasmic and the solubilized membrane fraction were applied to a 0.2 ml DEAE-cellulose (DE-52) anion-exchange chromatography column, washed with buffer B (20 mM Tris, pH 7.5; 2 mM EDTA; and 5 mM EGTA). Bound PKC was eluted batchwise with 500 µl buffer C (buffer B containing 0.15 M NaCl). PKC activity was detected with the PKC enzyme assay kit (Amersham) according to the manufacturer's instructions and as previously described (42). 12-O-tetradecanoyl phorbol-13-acetate (TPA) was used as a positive control.

Western Blotting of PKC

PKC-containing protein fractions that were eluted from the DE-52 columns (see the previous discussion) were separated on SDS-10% PAGE and Western blotted onto nitrocellulose membranes, and the membranes were blocked in 3% BSA in PBS. PKC was detected by incubating the membrane in specific antibodies directed against the various PKC isozymes (1:1,000 in PBS). Bound antibodies were detected with protein A-conjugated horseradish peroxidase (HRP). Blots were scanned with an LKB Ultrascan XL densitometer to quantify PKC immunoreactivity, as previously described (42).

Extraction and Analysis of 1,2-Diacylglycerol

Diacylglycerol (DAG) levels were determined with the DAG kinase assay according to Preiss and colleagues (50), and as previously described (42, 51). Briefly, cells were washed with cold PBS, scraped, and centrifuged, and lipids were extracted from cell pellet with 1 ml chloroform- methanol-HCl (100:100:1, vol/vol) and 0.3 ml of a buffered saline solution (135 mM NaCl; 4.5 mM KCl; 1.5 mM CaCl₂; 0.5 mM MgCl₂; 5.6 mM glucose; 10 mM Hepes, pH 7.2; and 10 mM EDTA). The organic phase containing DAG was evaporated. The DAG kinase assay was done as described (50). Briefly, the evaporated lipid fractions were resuspended in 20 µl of an octyl--D-glucoside/cardiolipin solution (7.5% octyl--D-glucoside, 5 mM cardiolipin in 1 mM diethylenetriamine pentaacetic acid by bath sonication, followed by incubation at room temperature for 15 min. The reaction buffer was prepared as a 2× solution, containing 100 mM imidazole HCl, pH 6.6; 100 mM NaCl; 25 mM MgCl₂; and 2 mM EGTA. To the 20 µl of solubilized lipid/octylglucoside solution, the following were added: 50 µl of the 2× reaction buffer, 2 µl of 100 mM DTT, and DAG kinase (0.022 U). The final volume of the reaction mixture was 100 µl. The reaction was started by the addition of 1 µl of 10 mCi/ml [-³²P]ATP, and was allowed to proceed for 30 min at 25°C. The reaction was stopped by the addition of chloroform-methanol-HCl (100:100:1, vol/ vol). The chloroform phase was then collected, evaporated, resuspended in 10 µl chloroform-methanol (1:1, vol/vol), and spotted on a Silica Gel 60 thin layer chromatography plate. The plate was developed with chloroform-methanol-acetic acid (65:15:5, vol/vol), air-dried, and autoradiographed. The spots corresponding to DAG were scanned in a densitometer and presented as density units.

	Results

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A549 Cell Growth Is Modulated by H₂O₂

A549 cultures were examined for the effects of H₂O₂ and EGF on the rate of DNA synthesis and thymidine incorporation. In an epithelial tumor-cell line with increased numbers of EGF receptors, such as A549 cells, EGF can inhibit growth (43, 45, 46, 52). As expected, and as shown in Figure 1, addition of 5 nM EGF was antiproliferative for A549 cells, and reduced the rate of thymidine incorporation into DNA to 70% of control values. Exposure of cultures to H₂O₂ was also inhibitory to DNA synthesis and the rate of thymidine incorporation, as compared with these processes in untreated cells. The extent of H₂O₂- induced growth inhibition was dose dependent, and was maximized at 400 µM (50% inhibition). Moreover, when EGF-treated cultures were exposed to H₂O₂ (Figure 1A), the antiproliferative effects were additive, suggesting two independent effects. A simple additive response suggests a lack of interaction, in that real interactive effects would either produce synergy or antagonism.

View larger version (15K): [in this window] [in a new window]   Figure 1.   Growth inhibition of A549 cells by H2O2 and EGF analyzed by the rate of DNA synthesis (A) and by cell-cycle analysis (B-E). Cells were seeded at 5 × 106 cells/100-mm dish in F-12 medium supplemented with 1% FBS. At 12 h after seeding, cultures were untreated or treated with either H2O2 or EGF for 24 h as follows: (A) 0 to 100 µM H2O2 and 0 to 5 nM EGF, as indicated. (B) Untreated (control). (C) Treated with 100 µM H2O2. (D) Treated with 5 nM EGF. (E) Treated with 100 µM H2O2 + 5 nM EGF. For A, cells were labeled for the last 6 h of treatment with [3H]thymidine as described in MATERIALS AND METHODS. After the 24-h treatment, aliquots were precipitated with cold (4°C) 10% TCA, filtered, and counted in scintillation fluid. The results are expressed as means of triplicates with standard error bars. For B-E, nuclei were prepared after the 24-h treatment from the respective cultures, and flow cytometry was performed after nuclei were incubated with a propidium iodide solution as described in MATERIALS AND METHODS. The percentages of cells in the G1, S, and G2-M phases are summarized in Table 1. Representative DNA distribution of one of three studies is shown.

Flow-cytometric cell-cycle analysis performed on control, H₂O₂-treated, and EGF-treated A549 cells demonstrated that H₂O₂ exposure as well as EGF treatment increased the fraction of cells in the G₀-G₁ phase and reduced the number of cells in the S phase of the cell cycle (Figures 1B through 1E, and Table 1). However, the most prominent effect on the cell-cycle distribution was observed upon treatment with H₂O₂ in the presence of EGF (Figure 1E). After only 24 h of treatment in the presence of 200 µM H₂O₂ plus 5 nM EGF, the S-phase contribution was reduced from 37.3% to 13.4%. In comparison, treatment with either 5 nM EGF alone or with 200 µM H₂O₂ reduced the S-phase contribution to 28% and 25.4%, respectively (Table 1).

EGF-Receptor Tyrosine Phosphorylation Is Modulated by H₂O₂

The observation of added effects of H₂O₂ and EGF on cell proliferation led us to search for alterations in EGF receptor function following H₂O₂ exposure, and to compare these changes with the effects of EGF. A series of experiments were done to determine whether exposure of cells to H₂O₂ results in changes in phosphorylation of the EGF receptor. First, the effects of H₂O₂ were examined in intact cells either with whole lysates (Figure 2) or after immunoprecipitation of the EGF receptor with anti-EGF receptor mAb 528 (Figure 3). Then, to investigate the possibility that H₂O₂ may directly affect phosphorylation of the EGF receptor under cell-free conditions, we studied the effects of H₂O₂ either on membrane preparations or on purified, immunoprecipitated receptor (Figure 4).

View larger version (20K): [in this window] [in a new window]   Figure 2.   Dose and time responses of H2O2-induced EGF-receptor tyrosine phosphorylation. A549 cell cultures (2 × 105 cells/35-mm well) were exposed to varying treatments for the indicated times at 37°C. Cell lysates were prepared in Triton X-100 lysis buffer, subjected to 7% SDS-PAGE, and immunoblotted either with antiphosphotyrosine antibody PY20 (A, C, D, E) or with anti-EGF receptor antibody LA22 (B). (A) and (B) Fifteen-minute treatments of: lanes 1, no treatment (950 cpm); lanes 2, 20 nM EGF (5,200 cpm); lanes 3, 100 µM H2O2 (3,800 cpm); lanes 4, 100 µM H2O2 + 20 nM EGF (8,075 cpm). (C) EGF-receptor tyrosine phosphorylation responses to H2O2 and EGF demonstrated in A. Values (mean) represent data derived from duplicate points in three experiments. The mean range of values in C is 5%. (D) Time response of 100 µM H2O2 treatment. (E) Dose response of H2O2 treatment for 15 min. Representative autoradiograms of one of three experiments are shown.

View larger version (38K): [in this window] [in a new window]   Figure 3.   H2O2-induced EGF-receptor tyrosine phosphorylation analyzed after immunopreciptation with anti-EGF receptor 528 mAb (A), and the effect of genistein on tyrosine phosphorylation (B). (A) A549 cells were untreated (control), treated with 20 nM EGF, or exposed to 200 µM H2O2. After 20 min at 37°C, cell lysates were prepared in Triton X-100 lysis buffer, subjected to immunoprecipitation with the anti-EGF receptor mAb 528, analyzed with 7% SDS-PAGE, and immunoblotted with antiphosphotyrosine antibody PY20. Representative autoradiogram of one of three experiments. (B) A549 cells were untreated (control), treated with 20 nM EGF, or exposed to 200 µM H2O2 as in A. Increasing concentrations of genistein were added at the time of exposure. After 20 min at 37°C, cell lysates were prepared in Triton X-100 lysis buffer containing genistein at the same indicated concentrations. The lysates were separated with 7% SDS-PAGE and immunoblotted with antiphosphotyrosine antibody PY20. Representative autoradiogram of one of three experiments is shown.

View larger version (27K): [in this window] [in a new window]   Figure 4.   Effect of H2O2 on EGF-receptor tyrosine phosphorylation in isolated membranes (A and B), and on immunoprecipitated EGF receptor (C). Isolated membranes were treated with 20 nM EGF (lanes 2) or exposed to 200 µM H2O2 (lanes 4). Lanes 1 represent the control for EGF treatment, and lanes 3 represent the control for H2O2 exposure. After 20 min at 37°C, membranes were solubilized in Triton X-100 lysis buffer, analyzed with 7% SDS-PAGE, and immunoblotted either with PY20 (A) or with anti-EGF receptor antibody LA22 (B). Representative autoradiogram of one of three experiments is shown. The effect of H2O2 on isolated EGF-receptor protein kinase activity (C). Immunoprecipitated EGF receptor was exposed to 200 µM H2O2 for the indicated times at 27°C. The immunocomplexes were then heated in sample buffer and subjected to 7% SDS-PAGE. The gel was dried and subjected to autoradiography, and EGF-receptor-autophosphorylated bands were excised and quantified by Cerenkov counting. Representative autoradiogram of one of three experiments is shown. Cerenkov counts (cpm) of the bands were as follows: 0 min = 1,900; 3 min = 2,600; 6 min = 3,500; 12 min = 4,700; 25 min = 5,400; 45 min = 5,700; 60 min = 6,060. Values (mean) are derived from triplicate determinations from one experiment representative of three similar studies.

As shown in Figure 2, A549 cell cultures were exposed to 100 µM H₂O₂, 20 nM EGF, or both. Cells were lysed and equal amounts of total protein lysates were subjected to SDS-PAGE analysis. Paired Western blots were immunodetected either with PY20 antiphosphotyrosine antibody (Figure 2A) or with LA22 anti-EGF receptor antibody (Figure 2B). Only antiphosphotyrosine immunoblots showed changes in the intensity of the phosphorylated tyrosines of the EGF receptor (PY20 immunoblots, Figure 2A); no changes were observed in the intensity of the receptor itself (LA22 immunoblots, Figure 2B). This indicates that the amount of EGF receptor remained unchanged, whereas its tyrosine phosphorylation increased with either H₂O₂ or EGF treatment. In addition, Figures 2A and 2C show that 100 µM H₂O₂ enhanced EGF-receptor tyrosine phosphorylation 4-fold (from 950 cpm to 3,800 cpm; Figures 2A and 2C, lane 3 versus lane 1), whereas EGF augmented tyrosine phosphorylation 5.5-fold (from 950 cpm to 5,200 cpm; Figures 2A and 2C, lane 2 versus lane 1). Moreover, H₂O₂ further enhanced the EGF-induced EGF-receptor tyrosine phosphorylation: as shown, EGF plus 100 µM of H₂O₂ raised the degree of receptor tyrosine phosphorylation 8.5-fold (from 950 cpm to 8,075 cpm; Figures 2A and 2C, lane 4 versus lane 1). This clearly demonstrates that at a saturating EGF concentration, additional tyrosine phosphorylation could be caused by H₂O₂.

The kinetics of H₂O₂-induced EGF-receptor tyrosine phosphorylation is demonstrated in Figure 2D. As shown, EGF-receptor tyrosine phosphorylation was stimulated to a level almost 4-fold that of the control after 15 min of treatment with 100 µM H₂O₂, and remained elevated for several hours (see Figure 5, illustrating the kinetics of tyrosine phosphorylation after exposure to 100 µM H₂O₂, for longer time points). This increase in tyrosine phosphorylation was also shown to be H₂O₂-dose dependent (Figure 2E).

View larger version (42K): [in this window] [in a new window]   Figure 5.   Compared kinetics of EGF receptor (A and B) and PLC- (C and D) tyrosine phosphorylation by H2O2 or EGF. A549 cells were cultured with 10 nM EGF or with 100 µM H2O2 at 37°C for the indicated periods. After incubation, cell lysates were prepared in Triton X-100 lysis buffer at 4°C, separated with 7% SDS-PAGE, and immunoblotted either with antiphosphotyrosine antibody PY20 (A) or with anti-EGF receptor antibody RK2 (B). After incubation, cell lysates were immunoprecipitated with antiphosphotyrosine antibody Ab-1 on agarose beads. The phosphoryl proteins were eluted with phenyl phosphate, subjected to 7% SDS-PAGE, and then immunoblotted with anti-PLC- antibody (C). After incubation, the entire cell lysates were subjected to 7% SDS-PAGE and immunoblotted with anti-PLC- antibody (D). Representative autoradiograms of one of three experiments are shown.

The experiments represented by Figure 2 were repeated with similar results when Western blotting was performed with antiphosphotyrosine antibody (PY20) after the immunoprecipitation of EGF receptors with mAb 528 (Figure 3). To investigate further the mechanism of EGF-receptor tyrosine phosphorylation after H₂O₂ exposure, we compared the responses to EGF and to H₂O₂ in the presence of genistein, a known specific inhibitor of protein tyrosine kinases (55). Figure 3B shows that when A549 cells were cultured in the presence of different concentrations of genistein, dose-dependent genistein inhibition of EGF-receptor tyrosine phosphorylation was observed. Both EGF- and H₂O₂-induced tyrosine phosphorylation of the receptor were partially attenuated in the presence of genistein. These results provide additional evidence that H₂O₂-induced tyrosine phosphorylation of EGF receptor is due to PTK activation.

We also performed a cell-free, in vitro phosphorylation assay by using either membrane fractions prepared from A549 cells (Figures 4A and 4B) or immunoprecipitated receptors in an immunokinase assay (Figure 4C). An increase in EGF-receptor tyrosine phosphorylation was observed when membrane fractions were exposed to H₂O₂ (Figures 4A and 4B), indicating a direct effect of H₂O₂ on membrane components to enhance EGF-receptor tyrosine phosphorylation. Moreover, when immunoprecipitated receptors in an immunokinase assay (Figure 4C) were directly exposed to H₂O₂, this observation was extended. An increase in receptor phosphorylation was observed again, suggesting that H₂O₂ may directly affect the phosphorylation of EGF receptor tyrosine residues and thus the tyrosine kinase activity of the receptor.

Two-Dimensional Phosphoamino Acid Analysis of the H₂O₂-Induced Phosphorylation of EGF Receptor

As shown in Figure 6 and Table 2, exposure of A549 cells to EGF induced tyrosine phosphorylation of the EGF receptor, accompanied by an increase in phosphorylation of serine and threonine residues. This observation has previously been well documented in other cell lines (43, 45, 51, 58). Therefore, it was important to investigate the effect of H₂O₂ on the distribution of phosphorylation of these three phosphoamino acids in the EGF receptor. The next experiments were designed to measure H₂O₂-induced changes in the phosphorylation of serine, threonine, and tyrosine in EGF receptors (Figure 6). A549 cells were equilibrium-labeled with [³²P]orthophosphate and then treated with 20 nM EGF for 20 min at 37°C, or exposed to 200 µM H₂O₂ for 20 min before being lysed. The EGF receptors were immunoprecipitated from the lysates with mAb 528 and subjected to SDS-PAGE. EGF receptor bands were cut and hydrolyzed in HCl. The amino acids were separated by TD-TLE, and ³²P incorporation into tyrosine, threonine, and serine was measured (Figure 6 and Table 2). The results show that in contrast to EGF, which stimulated a marked increase in phosphorylation of tyrosine, threonine, and serine, H₂O₂ selectively stimulated tyrosine phosphorylation, with little effect on threonine and serine phosphorylation. The total observed increase in receptor phosphorylation was about 3-fold with EGF treatment and only about 1.6-fold with H₂O₂ exposure (Table 2). Thus, H₂O₂-induced phosphorylation of EGF receptor differs fundamentally from that induced by the ligand, EGF. Compared with phosphorylation induced by EGF, the molecular distribution among the sites of phosphorylation is shifted by H₂O₂ predominantly toward tyrosine phosphorylation.

View larger version (35K): [in this window] [in a new window]   Figure 6.   Two-dimensional phosphoamino acid analysis of the EGF receptor. (A) A549 cells were labeled in phosphate-free medium with [32P]orthophosphate at 37°C for 16 h. At 20 min prior to the end of the labeling the following treatments were applied: (A) No treatment; (B) 20 mM EGF; (C) 200 µM H2O2. Subsequently, cells were lysed and the EGF receptor was isolated by immunoprecipitation and 7% SDS-PAGE, as shown in Figure 3A. The isolated receptors were eluted from the gel and subjected to partial acid hydrolysis. The [32P]phosphoamino acids were resolved by two-dimensional thin-layer electrophoresis, localized by autoradiography, and identified by ninhydrin staining of carrier molecules of phosphoserine (S), phosphothreonine (T), and phosphotyrosine (Y). Representative autoradiogram of one of three experiments is shown. The phosphoamino acids were scraped from the electrophoresis plate and radioactivity was assayed by scintillation counting. The quantitative data are presented in Table 2. Values (mean) are derived from triplicate determinations from one experiment representative of three similar studies.

EGF-Receptor Turnover Is Modulated by H₂O₂

Pulse/chase experiments were done to determine the effects of H₂O₂ and of the natural ligand, EGF, on the EGF receptor rate of turnover (Figure 7). A549 cells were prelabeled with [³⁵S]methionine and were then cultured in fresh medium for varying intervals after the medium was supplemented with either 10 nM EGF or 100 µM H₂O₂. SDS-PAGE patterns of immunoprecipitated EGF receptors showed that the half-life of receptors in control cells was approximately 12 h. This fell markedly to a half-life of less than 8 h in the presence of 10 nM EGF. On the other hand, for cells cultured after being exposed to 100 µM H₂O₂, the half-lives were increased to 18 h (Figure 7).

View larger version (13K): [in this window] [in a new window]   Figure 7.   Kinetics of EGF receptor turnover by H2O2 and EGF. (A) A549 cells were labeled with [35S]methionine for 16 h. The labeling medium was removed and cultures were chased in fresh medium containing 1% FBS for the indicated periods in the presence of 10 nM EGF or 100 µM H2O2. EGF receptors were immunoprecipitated with mAb 528 and subjected to 7% SDS-PAGE and autoradiography. Representative autoradiogram of one of three experiments is shown. (B) The corresponding bands were excised and counted, and the plotted data were used to estimate the half-lives of labeled EGF receptors under the various conditions. Values (mean) represent data derived from duplicate points in three experiments. The SEM of the values was 10%.

Kinetics of EGF-Receptor and PLC- Tyrosine Phosphorylation by H₂O₂

Figure 5 shows the kinetics of EGF-receptor tyrosine phosphorylation (Figures 5A and 5B), as well as the kinetics of PLC- tyrosine phosphorylation (Figures 5C and 5D) in A549 lung epithelial cells cultured with 10 nM EGF or with 100 µM H₂O₂ for varying intervals of up to 4 h. The cells were lysed in Triton X-100 lysis buffer at 4°C at each time point. The lysates were then subjected to SDS- PAGE and immunoblotted with antiphosphotyrosine antibody PY20 (Figure 5A), with anti-EGF receptor antibody RK2 (Figure 5B), or with anti-PLC- antibody (Figure 5C and 5D) after (Figure 5C) or before (Figure 5D) immunoprecipitation with antiphosphotyrosine Ab-1.

EGF-induced EGF-receptor tyrosine phosphorylation began to decline by 1 h after initiating culture with the ligand, and fell to low levels by 3 h of incubation (Figure 5A). This was expected, because of the rapid downregulation of EGF receptors caused by EGF (Figure 7), which resulted in decreased receptor levels in these cultures (Figure 5B). In contrast, induction of EGF-receptor tyrosine phosphorylation by H₂O₂ gradually increased with the duration of culture with H₂O₂, reaching peak levels at 0.25 to 0.5 h and remaining elevated for at least 4 h. The observed changes in tyrosine phosphorylation were associated with EGF receptors, since immunoprecipitation of lysates with anti-EGF receptor mAb, followed by SDS-PAGE and immunoblotting with antiphosphotyrosine antibody PY20 (as demonstrated in Figure 3), gave identical results (data not shown).

We then explored whether EGF-receptor tyrosine kinase that was activated by exposure to H₂O₂ could phosphorylate a substrate other than the receptor itself. PLC- was chosen as a well-described substrate of the EGF receptor that is known to be activated by EGF (30). When cells were cultured with EGF or H₂O₂ for intervals comparable with those in the previous experiment, tyrosine phosphorylation of PLC- was also observed (Figure 5C). Following addition of EGF, the time course of phosphorylation of PLC- on tyrosine residues (Figure 5C) corresponded with the kinetics of EGF-receptor tyrosine phosphorylation (Figure 5A) and degradation (Figure 5B and Figure 7), decaying after 3 h. On the other hand, the level of tyrosine-phosphorylated PLC- increased when the duration of culture with H₂O₂ was extended to 4 h (whereas the amount of total PLC- protein was unchanged for both H₂O₂ and EGF treatments [Figure 5D]). The kinetics pattern of PLC- tyrosine phosphorylation (Figure 5C) paralleled the time course of EGF-receptor phosphorylation on tyrosine residues in response to either EGF or H₂O₂ (Figure 5A). The observation that the kinetics of tyrosine phosphorylation on EGF receptors and on PLC- were parallel when cells were cultured with EGF (rapid elevation, followed by decay in both cases) and when cells were cultured with H₂O₂ (less rapid elevation followed by much slower decay in both cases) leads to two conclusions: (1) there is a close association between the levels of EGF-receptor phosphorylation and PLC- tyrosine phosphorylation not only in the presence of EGF but also in the presence of H₂O₂; and (2) even though PLC- is tyrosine-phosphorylated by H₂O₂, the kinetics of its tyrosine phosphorylation are completely different than those induced by EGF, which may affect activation of PLC- and its ability to catalyze phosphatidylinositol-2-phosphate (PIP2) hydrolysis.

PKC and MAP Kinase Modulation by H₂O₂

In a regular pattern of signal transduction (Figure 8), PLC- is phosphorylated on its tyrosine residues to enhance the catalysis of PIP2 to inositol-1,4,5-trisphosphate (IP3) and DAG, thereby enhancing the activities of PKC and MAP kinase (59). However, although exposure to 100 to 300 µM H₂O₂ increased PLC- tyrosine phosphorylation in parallel with raising EGF-receptor tyrosine phosphorylation (Figure 5), no increase in MAP kinase phosphorylation and activation could be observed (data not shown). At the same time, within minutes after H₂O₂ exposure, PKC activity decreased 2-fold in the membrane and simultaneously increased 2-fold in the cytosol (Figure 9A). The opposite was observed when cells were treated with 200 nM TPA (Figure 9B). Because PKC- is the major isozyme in A549 cells (42), Western blot analysis demonstrated that blotted PKC- decreased in the membrane and increased in the cytosol with kinetics paralleling those of the changes in PKC activity after H₂O₂ exposure (data not shown). The levels of DAG, the physiologic activator of PKC- (64), were also decreased upon exposure of A549 cells to H₂O₂ (Figure 9C). Both the decrease in DAG levels (Figure 9C) and the translocation of PKC- from the membrane to the cytosol (Figure 9A) occurred with the same kinetics, within minutes after H₂O₂ exposure. Even though it could be expected (Figure 8) that a tyrosine-phosphorylated and thus an activated PLC- would increase the cellular levels of DAG, Figure 9 shows that DAG levels decreased and PKC activity decreased as well. As shown in Figure 6, the EGF receptor is aberrantly phosphorylated by H₂O₂, and transmits its downstream signal to phosphorylate PLC- with slower kinetics than occur with its ligand, EGF. This may be reflected in the observed decreased levels of DAG, accompanied by translocation of PKC from the membrane to the cytosol and no MAP kinase tyrosine phosphorylation.

View larger version (17K): [in this window] [in a new window]   Figure 8.   Scheme of H2O2 cellular redox effects on EGF-receptor (EGFR) phosphorylation and downstream signals.

View larger version (15K): [in this window] [in a new window]   Figure 9.   Effects of H2O2 on PKC activity and changes in DAG levels. A549 cells were treated with 200 µM H2O2 (A, C) or with 200 nM TPA (B). At the indicated times, cells were collected, cytoplasmic and membranal PKC purified, and PKC activity measured (A, B). (C) Alternatively, at the indicated times, cells were extracted with chloroform-methanol-1 N HCl (100:100:1). Lipid extracts were assayed for DAG levels with the DAG kinase reaction. Each value represents mean ± SEM of triplicate determinations from three experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The present study provides mechanistic insights into the molecular effects of H₂O₂ exposure on the EGF receptor and its downstream signaling in lung epithelial cells. Cell-cycle analysis (Figure 1) suggests that both EGF and H₂O₂ cause G₁ arrest in A549 cells. This growth arrest may be the basis of the growth inhibition observed at nanomolar to micromolar concentrations of EGF (43, 45, 46, 52, 65) and H₂O₂. Moreover, we have shown that H₂O₂ potentiates EGF-induced G₁ arrest, which is again consistent with the observed additive growth-inhibitory effects of H₂O₂ and EGF (Figure 1). Furthermore, we suggest that these additive responses indicate a lack of interaction between the EGF- and H₂O₂-induced pathways, because any such interaction would have produced either synergy or contrariety.

We have shown that when lung epithelial cells are exposed to H₂O₂, the EGF receptor is preferentially phosphorylated on tyrosine residues (Figures 2-4, Figure 6). This is supported by similar findings in some other cells. Gamou and Shimizu showed, in the human squamous carcinoma cell line NA, that exposure to H₂O₂ (0.25 to 1 mM) enhanced EGF-receptor tyrosine phosphorylation. Tryptic phosphopeptide mapping of these receptors revealed that H₂O₂ enhanced the phosphorylation of tyrosine 1,173 and three other residues but not of serine and threonine residues (66).

The mechanism by which H₂O₂ activates tyrosine phosphorylation of the EGF receptor is not yet clear. EGF- receptor autophosphorylation is thought to be a trans event, involving dimerization or oligomerization followed by tyrosine phosphorylation on the opposite receptor molecule (67, 31, 32). Therefore, H₂O₂ could act by releasing EGF receptors from constraints that prevent their dimerization under normal physiologic conditions, such as intracellular inhibitors or structural elements attached to intact plasma membranes (68). Another possibility is that H₂O₂ leads to separation of critically important tyrosine phosphatases from their association with the receptor kinase. Consequently, it is possible that tyrosine phosphorylation induced by H₂O₂ treatment is due to activation of a kinase, inhibition of a phosphatase, or both. Treatment of A549 cells with a phosphatase inhibitor, pervanadate, induced EGF-receptor phosphorylation (data not shown), perhaps through a mechanism similar to that of oxidative stress. However, treatment of these cells with H₂O₂ in the presence of pervanadate enhanced the increase in tyrosine phosphorylation, suggesting that H₂O₂-induced tyrosine phosphorylation of the EGF receptor involves more than phosphatase inhibition, and that H₂O₂ treatment induces an increase in phosphorylation not only by reducing the rate of dephosphorylation.

EGF-receptor tyrosine phosphorylation induced by EGF was increased markedly when the receptor was exposed to H₂O₂ (Figure 2), thereby indicating that H₂O₂- induced tyrosine phosphorylation enhances receptor activation by the natural ligand. This clearly demonstrates that at saturating EGF concentrations, additional tyrosine phosphorylation could be caused by H₂O₂. Moreover, our data provide strong evidence that H₂O₂-induced EGF-receptor phosphorylation results from PTK activation, because the presence of genistein, a competitor of ATP binding to tyrosine kinases, can attenuate H₂O₂-induced tyrosine phosphorylation (Figure 3). H₂O₂-induced EGF-receptor tyrosine phosphorylation was reproduced in isolated membrane fractions as well as in isolated receptors in an immunokinase assay (Figure 4), suggesting that H₂O₂ may activate the intrinsic tyrosine kinase of the EGF receptor and phosphorylate additional tyrosine residues that are only slightly phosphorylated under physiologic conditions resulting from EGF stimulation.

Because there was no stimulation by H₂O₂ of receptor serine/threonine phosphorylation, the total change in receptor phosphorylation was lower than the change observed with the ligand, EGF (Figure 6 and Table 2). The low level of serine/threonine phosphorylation may result in an insufficient attenuation of receptor function, such as lack of receptor internalization. Indeed, additional observations (Figure 7) suggest that receptor turnover and downregulation are far slower following cell exposure to H₂O₂ than to EGF. This is also reflected in the kinetics of EGF-receptor phosphorylation caused by H₂O₂ and the consequent tyrosine phosphorylation of PLC- (Figure 5). When cells are cultured with EGF, the kinetics of PLC- phosphorylation has a pattern of rapid elevation followed by a decay, whereas exposure to H₂O₂ results in a less rapid elevation of PLC- tyrosine phosphorylation followed by a much slower decay in the phosphorylation (Figure 5). This difference in the kinetics pattern of phosphorylation may explain the observed interruption of the normal flow in the downstream signaling produced by H₂O₂. Normally, in the ligand-induced pattern of signal transduction, a tyrosine-phosphorylated and thus activated PLC- catalyzes the break of PIP2 into IP3 and DAG, and the latter activates PKC to stimulate MAP kinase activation (Figure 8). In contrast, the atypical kinetics of EGF-receptor phosphorylation induced by H₂O₂ may have resulted in a failure of the signal to propagate. Even though H₂O₂ activates EGF-receptor tyrosine phosphorylation and its downstream PLC- substrate, DAG levels are not increased (Figure 9), but are rather decreased, and the downstream PKC and MAP kinase are not activated.

It is interesting, however, that the membrane-associated molecular events induced by H₂O₂ cannot be generalized. Although it is known that growth factors such as EGF and TPA activate MAP kinase signaling (59), different cells respond differently to redox exposure. For instance, in smooth-muscle cells (60) and in human umbilical-vein endothelial cells (61), H₂O₂ did not activate the MAP kinase cascade, whereas exposure of T cells to H₂O₂ did stimulate the MAP kinase route via PKC-dependent pathways (62). In yet another model (rat hepatoma cells), H₂O₂ caused persistent activation of MAP kinase independently of PKC activation (63). In our model, the effect of H₂O₂ on MAP kinase activity does seem to depend on intact PKC signaling, because the depletion of PKC in A549 cells significantly inhibited the ability of EGF to activate MAP kinase (data not shown). Therefore, the fact that no stimulatory effect on MAP kinase tyrosine phosphorylation is observed with A549 cells exposed to H₂O₂ could be simply a consequential outcome of the immediate translocation of PKC- from the membrane to the cytoplasm. This interpretation is supported by Gopalakrishna and Anderson's original findings that oxidant tumor promoters can induce the oxidative modification of PKC, resulting in either activation or inactivation of the kinase (69). They showed that treatment of MCF7 cells with phorbol ester to induce membrane association of PKC, followed by exposure to H₂O₂, resulted in increased inactivation of PKC, suggesting that membrane association of PKC increases its susceptibility to oxidative inactivation (70, 71).

As discussed previously (42), PKC downmodulates EGF-receptor tyrosine kinase activity via phosphorylation of EGF-receptor Thr⁶⁵⁴ (58) (see also scheme in Figure 8). Thus, an H₂O₂-induced decrease in membrane PKC may further augment EGF-receptor tyrosine kinase activity. The same may apply to MAP kinase. It has been shown that MAP kinase downmodulates EGF-receptor tyrosine kinase activity via phosphorylaton of the EGF-receptor Thr⁶⁶⁹ (72). Thus, the inability of H₂O₂ to induce MAP kinase tyrosine phosphorylation and activation may further promote EGF-receptor tyrosine kinase activity.

There is ample evidence in the literature to support the notion that in epithelial cells with increased levels of EGF receptor, there may be a quantitative relationship between EGF-receptor kinase activity and growth response. When an optimal kinase activation is exceeded, growth inhibition may result (43, 45, 46, 52, 65). Hence, the capacity of H₂O₂ to modify lung epithelial cell proliferation may partly reside in its ability to act as a direct biomodulator of the phosphorylation and function of the EGF receptor, followed by modification of the receptor's downstream signaling to affect PLC-, DAG, PKC, and MAP kinase.

In summary, the results in this study demonstrate that exposure of A549 lung epithelial cells to H₂O₂-mediated oxidative stress causes tyrosine phosphorylation of the EGF receptor without the accompanying effects of threonine/serine phosphorylation. Such exposure therefore creates an aberrantly activated receptor that fails to be turned over in a regular manner and to propagate a predictable, regular downstream signal. Although it is too early to speculate on the potential implications of aberrant EGF-receptor phosphorylation by inflammatory oxidants such as H₂O₂, such a modification may be important under conditions of increased EGF-receptor expression and activation (i.e., after epithelial injury or in malignant tumors) (73, 74)situations that are also commonly associated with increased production of inflammatory oxidants. Therefore, such oxidant-induced EGF-receptor alterations may affect epithelial-cell growth and repair processes.