Peroxynitrite Targets the Epidermal Growth Factor Receptor, Raf-1, and MEK Independently to Activate MAPK*

Activation of ERK-1 and -2 by H2O2 in a variety of cell types requires epidermal growth factor receptor (EGFR) phosphorylation. In this study, we investigated the activation of ERK by ONOO− in cultured rat lung myofibroblasts. Western blot analysis using anti-phospho-ERK antibodies along with an ERK kinase assay using thephosphorylated heat- andacid-stable protein (PHAS-1) substrate demonstrated that ERK activation peaked within 15 min after ONOO− treatment and was maximally activated with 100 μm ONOO−. Activation of ERK by ONOO− and H2O2 was blocked by the antioxidant N-acetyl-l-cysteine. Catalase blocked ERK activation by H2O2, but not by ONOO−, demonstrating that the effect of ONOO−was not due to the generation of H2O2. Both H2O2 and ONOO− induced phosphorylation of EGFR in Western blot experiments using an anti-phospho-EGFR antibody. However, the EGFR tyrosine kinase inhibitor AG1478 abolished ERK activation by H2O2, but not by ONOO−. Both H2O2 and ONOO− activated Raf-1. However, the Raf inhibitor forskolin blocked ERK activation by H2O2, but not by ONOO−. The MEK inhibitor PD98059 inhibited ERK activation by both H2O2 and ONOO−. Moreover, ONOO− or H2O2 caused a cytotoxic response of myofibroblasts that was prevented by preincubation with PD98059. In a cell-free kinase assay, ONOO− (but not H2O2) induced autophosphorylation and nitration of a glutathioneS-transferase-MEK-1 fusion protein. Collectively, these data indicate that ONOO− activates EGFR and Raf-1, but these signaling intermediates are not required for ONOO−-induced ERK activation. However, MEK-1 activation is required for ONOO−-induced ERK activation in myofibroblasts. In contrast, H2O2-induced ERK activation is dependent on EGFR activation, which then leads to downstream Raf-1 and MEK-1 activation.

Inflammation following tissue injury is associated with increased generation of reactive oxygen species such as superoxide anion (O 2 . ) and hydrogen peroxide (H 2 O 2 ) (1). Moreover, nitric oxide (NO ⅐ ) is synthesized by inflammatory cells and has the potential to react with O 2 . via a nearly diffusion-limited reaction to form peroxynitrite (ONOO Ϫ ) (2)(3)(4)(5). These oxidants may serve several physiological or pathophysiological func-tions. For example, NO ⅐ is thought to play a major role in host defense, but is also presumed to contribute to tissue injury (2). H 2 O 2 and O 2 . released into the extracellular environment by mononuclear cells during an oxidative burst may also play a role in immune defense (1). ONOO Ϫ is a potent cytotoxic species that has been proposed to contribute to the pathophysiology of a wide variety of inflammatory diseases. Although ONOO Ϫ is extremely short-lived at physiological pH (1-s halflife) (6), the formation of 3-nitrotyrosine by ONOO Ϫ reaction with tyrosyl residues serves as a stable marker or "footprint" (7)(8)(9). ONOO Ϫ not only affects tyrosine residues on proteins, but also may induce oxidative reaction products through modifications of cysteine, methionine, and tryptophan (4). Increasing evidence supports the idea that oxidants serve as signaling intermediates required for receptor tyrosine kinase function and downstream activation of mitogen-activated protein kinases (MAPKs). 1 In particular, H 2 O 2 generated intracellularly following the binding of platelet-derived growth factor or epidermal growth factor (EGF) to their respective receptors appears to reversibly inhibit protein-tyrosine phosphatase activity, which is required for phosphorylation of receptor tyrosine kinases (10,11). In the absence of EGF, exogenous H 2 O 2 alone can cause phosphorylation of EGFR through reversible oxidative modification of cysteine residues, leading to downstream activation of a Raf/MEK/MAPK phosphorylation cascade (12)(13)(14). In contrast, ONOO Ϫ stimulates phosphorylation of EGFR tyrosine residues by causing irreversible dimerization of EGFR via dityrosine cross-links (15). More recently, several nitric oxide-related species, including ONOO Ϫ , have been reported to activate MAPKs (16 -18), yet the mechanism of MAPK activation by ONOO Ϫ remains unclear.
MAPKs are a family of serine/threonine kinases that regulate a diversity of cellular activities. Three major classes have been described: extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs; also known as stress-activated protein kinases), and p38 MAPK (reviewed in Ref. 19). JNKs and p38 MAPKs mediate signals in response to cytokines and environmental stress, whereas the ERK subtypes are classically recognized as key transducers in the signaling cascade mediating cell proliferation in response to growth factors such as platelet-derived growth factor and EGF. Two major isoforms of ERK, p44 (ERK-1) and p42 (ERK-2), have been identified in mammalian systems. A major pathway involved in ERK-1 and -2 phosphorylation in a variety of cell types requires the sequential activation of Raf and MEK (20,21). It is becoming increasingly clear that the ERK pathway, like those of p38 and JNK, is activated by environmental stress, including reactive oxygen species such as H 2 O 2 (12).
In this study, we have investigated the mechanism of ONOO Ϫ -induced ERK-1 and -2 activation. Similar to previous studies on the mechanism of H 2 O 2 -induced ERK activation, we found that ONOO Ϫ activates EGFR, Raf-1, and MEK-1. However, unlike H 2 O 2 , which requires EGFR phosphorylation to initiate a downstream signaling cascade for activation of ERK, we report that ONOO Ϫ induces autophosphorylation of EGFR, Raf-1, and MEK-1 independently.

EXPERIMENTAL PROCEDURES
Cell Culture-Primary passage rat pulmonary myofibroblasts were isolated and characterized as described previously (22). Immunohistochemical analysis demonstrated these cells to be positive for markers of smooth muscle cells (␣-smooth muscle actin and desmin) and fibroblasts (vimentin), indicating a myofibroblast phenotype (23). Following the isolation procedure, aliquots of cells (1 ϫ 10 6 /ml) were stored in liquid nitrogen. Cells (1 ϫ 10 6 ) were thawed from the liquid nitrogen and plated in a 175-cm 2 flask in 10% fetal bovine serum/Dulbecco's modified Eagle's medium supplemented with L-glutamine and Fungizone. After reaching confluence, the cells were liberated with trypsin, replated on 100-mm dishes, and grown to confluence in 10% fetal bovine serum/Dulbecco's modified Eagle's medium. At this point, the cells were designated as passage 2. Cells were then rendered quiescent for 24 h with serum-free defined medium (SFDM) consisting of Ham's F-12 medium supplemented with 0.25% bovine serum albumin and an insulin/transferrin/selenium mixture (Roche Molecular Biochemicals) for the experiments described below.
Oxidant Treatments-Peroxynitrite was synthesized from acidified nitrite and hydrogen peroxide (Upstate Biotechnology, Inc., Lake Placid, NY). Stocks of ONOO Ϫ were stored in 1.2 N NaOH at Ϫ80°C. Prior to experimentation, ONOO Ϫ was quantitated spectrophotometrically (extinction coefficient at 302 nm ϭ 1670 M Ϫ1 cm Ϫ1 ) (24). Cells in SFDM were washed with phosphate-buffered saline (PBS) and then equilibrated in PBS for 5 min. ONOO Ϫ was delivered as a single bolus (e.g. 10 l of 100 mM stock delivered in 1 ml to give a final concentration of 1 mM) against one side of the dish while rapidly swirling the medium to assure optimal exposure of the cells to ONOO Ϫ before decomposition (15,25). In control experiments, ONOO Ϫ was prediluted for 1 min in an equivalent volume of PBS prior to adding to the cells. This "delayed" PBS solution contained completely decomposed ONOO Ϫ as determined spectrophotometrically, but still contained contaminants such as nitrite and nitrate (24). In other control experiments, cells were treated with equal volumes of 1.2 N NaOH (vehicle control). PBS, rather than SFDM, was used as the medium for ONOO Ϫ treatments to avoid interfering reactions of ONOO Ϫ with media constituents.
Western Blot Analysis-Confluent cell monolayers on 100-mm dishes were growth-arrested in serum-free medium for 24 h prior to treatment with ONOO Ϫ , H 2 O 2 , or metabolic inhibitors. Cells were washed twice with PBS, scraped, resuspended in lysis buffer (50 mM HEPES, 150 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 20 g/ml aprotinin, 20 g/ml leupeptin, and 20 g/ml pepstatin), and clarified by centrifugation at 13,000 rpm for 10 min. Thirty g of protein/sample was separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and blocked for 2 h at 25°C with 0.5% nonfat milk in PBS buffer (20 mM Tris, 500 mM NaCl, and 0.01% Tween 20). The membrane was then incubated overnight at 4°C with an appropriate dilution of anti-phospho-ERK or anti-ERK (New England Biolabs Inc.), anti-phospho-MEK-1 or anti-MEK-1 (New England Biolabs Inc.), or anti-phospho-EGFR or anti-EGFR (Upstate Biotechnology, Inc.) polyclonal antibody at 4°C overnight, followed by incubation for 2 h with a 1:2000 dilution of the appropriate horseradish peroxidaseconjugated secondary antibody. The immunoblot signal was visualized through enhanced chemiluminescence.
PHAS-1 Kinase Assay-ERK activity in cell lysates was measured as described previously (26) by phosphorylation of PHAS-1, a substrate for ERK (27). Briefly, confluent cell monolayers on 100-mm dishes were growth-arrested in serum-free medium for 24 h, treated with ONOO Ϫ , chilled on ice, washed twice with PBS, and scraped with 800 l of lysis buffer. ERK was immunoprecipitated by incubating 200 l of lysate with 2 g of anti-ERK antibody (Santa Cruz Biotechnology, Inc.) for 2 h and then adding 20 l of protein A-agarose (Santa Cruz Biotechnology, Inc.). After an overnight incubation at 0 -4°C with end-over-end mixing, the immune complex was recovered by centrifugation and washed three times with lysis buffer and once with 250 mM HEPES (pH 7.4), 10 mM MgCl 2 , and 200 M Na 3 VO 4 . Immune complex kinase assays were performed using a MAPK assay kit (Stratagene). The ERK pellets were resuspended in Stratagene reaction buffer containing 120 g of PHAS-1 substrate along with 3-5 Ci of [␥-32 P]ATP in a final volume of 180 l. Kinase reactions took place for 30 min at room temperature and were stopped by adding 4ϫ SDS-polyacrylamide gel electrophoresis reducing sample buffer and boiling for 10 min. ERK-PHAS samples were resolved on SDS-polyacrylamide gels, dried, and autoradiographed.
Cell-free MEK-1 Phosphorylation Assay-This assay was a modification of a previously reported method (21). Recombinant non-activated GST-MEK-1 (0.5 g; Upstate Biotechnology, Inc.) was diluted in 20 l of assay dilution buffer (20 mM MOPS (pH 7.2), 25 mM ␤-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, and 1 mM dithiothreitol) supplemented with a magnesium/ATP mixture (75 mM magnesium chloride and 500 M unlabeled ATP). An increasing concentration of ONOO Ϫ (1-1000 M) was added to the solution and incubated for 15 min at 30°C. The reaction was stopped by the addition of polyacrylamide gel electrophoresis sample buffer containing 2-mercaptoethanol. Activated GST-MEK-1 (Upstate Biotechnology, Inc.) was loaded on gels as a positive control. As an additional control, activated GST-Raf-1 was incubated with non-activated GST-MEK-1. Phosphorylation of GST-MEK-1 was detected by Western blotting using an anti-phospho-MEK-1 antibody. Western blots were stripped and reblotted using an antinitrotyrosine antibody (Upstate Biotechnology, Inc.).
[ 3 H]Thymidine Incorporation Assay-Confluent rat lung myofibroblasts on 24-well tissue culture plates were rendered quiescent in SFDM for 24 h; then the medium was switched to PBS, and ONOO Ϫ (1-1000 M) was added to the medium with constant swirling. After 15 min, the medium was removed, and fresh SFDM containing 5 Ci/ml [ 3 H]thymidine (Amersham Pharmacia Biotech) was added back. After 24 h at 37°C with 5% CO 2 and humidified air, the cells were rinsed three times with SFDM and chilled on ice for 30 min. The medium was removed, and 5% trichloroacetic acid (0.5 ml/well) was added for 10 min. After removing the trichloroacetic acid, the cells were washed three times with ice-cold water and solubilized with a solution of 1% Triton X-100 and 0.1% NaOH. Radioactivity was measured in a liquid scintillation counter.
Cytotoxicity Assay-Cytotoxicity was measured by the MTT cell viability assay (Roche Molecular Biochemicals) according to the manufacturer's instructions.

ONOO Ϫ Activation of ERK Requires Immediate Addition to
Cell Cultures-Since ONOO Ϫ has an extremely short half-life at neutral pH (ϳ1 s) (6), we assessed ERK activation in cultures of pulmonary myofibroblasts that received ONOO Ϫ immediately as compared with cell cultures that received ONOO Ϫ that had first been allowed to incubate (i.e. delayed) in PBS for 1 min at pH 7.4. The immediate addition strategy was performed by delivering a single bolus of the ONOO Ϫ stock against the side of the dish while swirling the PBS solution to allow for mixing. Direct addition resulted in strong ERK activation, whereas the delay of ONOO Ϫ in PBS for 1 min caused no activation of ERK (Fig. 1). This experiment was essential for demonstrating that ERK activation was due to ONOO Ϫ and not to trace amounts of H 2 O 2 , which is relatively stable at pH 7.4. Furthermore, this experiment ruled out the formation of stable nitrating agents that could potentially be formed by the reaction of ONOO Ϫ with components of the cell culture medium. The addition of the same volume of 1.2 N NaOH alone to PBS (10 l/1 ml), either immediately or delayed, did not cause ERK activation (data not shown).
ONOO Ϫ Activates ERK in a Time-and Concentration-dependent Manner-Treatment of cells with 1 mM ONOO Ϫ activated ERK maximally within 15 min as determined by Western blotting using an anti-phospho-ERK antibody or by an ERK kinase assay using the PHAS-1 substrate ( Fig. 2A). Phospho-ERK Western blotting and PHAS-1 experiments performed with increasing concentrations of ONOO Ϫ (1-1000 M) showed that maximal activation of ERK was reached at 100 M (Fig.  2B). In these experiments, Western blotting with an antibody that detects unphosphorylated ERK was used to show that ERK protein levels did not change appreciably as a consequence of ONOO Ϫ treatment. H 2 O 2 also maximally activated ERK at 100 M (data not shown). The concentrations of ONOO Ϫ or H 2 O 2 required to activate ERK in our study were within the micromolar range reported by other investigators for activation of cellular signaling pathways (12,15).
EGFR Phosphorylation Is Required for H 2 O 2 -induced ERK Activation, but Not for ONOO Ϫ -induced ERK Activation-We observed that EGF, ONOO Ϫ , and H 2 O 2 induced phosphorylation of EGFR as determined by Western blotting using an antibody specific for phospho-EGFR (Fig. 4A). Moreover, the EGFR tyrosine kinase inhibitor AG1478 (31) abolished phosphorylation of EGFR induced by all of these agents (Fig. 4A). However, although AG1478 significantly inhibited ERK activation induced by EGF and H 2 O 2 , AG1478 did not significantly inhibit ERK activation induced by ONOO Ϫ (Fig. 4, B and C).
ERK Activation by H 2 O 2 Is Dependent on Raf-1, yet ONOO Ϫinduced ERK Activation Is Not Raf-dependent-Using a Raf-1 kinase assay that utilized MEK-1 as the substrate, we observed that both H 2 O 2 and ONOO Ϫ stimulated Raf-1-dependent phosphorylation of MEK-1 (Fig. 5A). Furthermore, activation of Raf-1 by both of these oxidants was blocked by pretreatment with forskolin (Fig. 5A). Pretreatment of cells with forskolin prior to the addition of oxidant significantly blocked H 2 O 2induced ERK activation in phospho-ERK Western blot assays, yet forskolin did not affect ONOO Ϫ -induced ERK activation (Fig. 5, B and C (Fig. 6). Accordingly, the MEK inhibitor blocked the phosphorylation of MEK-1 by either ONOO Ϫ or H 2 O 2 in Western blot experiments using an anti-phospho-MEK-1 antibody.
ONOO Ϫ Causes Nitration and Autophosphorylation of Recombinant GST-MEK-1 Fusion Protein in a Cell-free System-To further investigate the mechanism through which ONOO Ϫ phosphorylates MEK-1, we treated recombinant nonactivated GST-MEK-1 with ONOO Ϫ in a buffer solution containing a magnesium/ATP mixture and performed Western blotting using an anti-phospho-MEK-1 antibody or an antinitrotyrosine antibody. As shown in Fig. 7, ONOO Ϫ induced phosphorylation of GST-MEK-1. Moreover, ONOO Ϫ caused a concentration-dependent increase in nitrotyrosine formation on GST-MEK-1 (Fig. 7). These experiments provide, for the first time, evidence of a direct biochemical modification of MEK-1 by ONOO Ϫ that results in autophosphorylation. In parallel experiments, H 2 O 2 did not phosphorylate GST-MEK-1 in this cell-free system (data not shown). Non-activated Raf-1 was not commercially available to perform a similar type of FIG. 1. Immediate addition of ONOO ؊ is required to activate ERK-1 and ERK-2. A, representative Western blot results for phospho-ERK and total ERK after the immediate addition of ONOO Ϫ versus addition of ONOO Ϫ to cultures after 1 min in PBS (pH 7.4) to allow for ONOO Ϫ degradation. Myofibroblasts were rendered quiescent for 24 h in SFDM and then switched to PBS (pH 7.4) for the addition of ONOO Ϫ to a final concentration of 1 mM. Immediate addition was performed by delivering a bolus of the ONOO Ϫ stock in 1.2 M NaOH directly against the side of the dish and immediately mixing the PBS solution overlaying the cells. Delayed addition was performed by adding the same amount of the ONOO Ϫ stock in 1.2 M NaOH to PBS for 1 min in the absence of cells and then adding the 1 mM ONOO Ϫ solution to the cells. After 15 min, the cell lysates were harvested and assayed for ERK activation by Western blot analysis using an anti-phospho-ERK antibody. The data shown are typical of four separate experiments. B, densitometric analysis of ERK activation from four separate experiments. The relative level of ERK activation was determined by densitometric scanning of the phospho-ERK bands and normalized to the ERK signal. **, p Ͻ 0.01 as compared with no addition. experiment. However, lysates from ONOO Ϫ -and H 2 O 2 -treated cells were immunoprecipitated with the anti-nitrotyrosine antibody, and Western blotting was performed using an antiphospho-Raf-1 antibody. Phosphorylated Raf-1 was co-immunoprecipitated with the anti-nitrotyrosine antibody from lysates of ONOO Ϫ -treated cells, but not from lysates of H 2 O 2treated cells (data not shown).
ONOO Ϫ -induced Cytotoxicity Is Blocked by Pretreatment with MEK Inhibitor-To address the biological relevance of ONOO Ϫ -induced MAPK activation, we sought to determine first whether ONOO Ϫ caused mitogenesis of rat lung myofibroblasts using a [ 3 H]thymidine incorporation assay. As shown in Fig. 8A, recombinant EGF (50 ng/ml) caused a severalfold increase in [ 3 H]thymidine uptake. However, ONOO Ϫ caused a concentration-dependent decrease in [ 3 H]thymidine incorporation (Fig. 8A). Interestingly, the decrease in [ 3 H]thymidine incorporation caused by ONOO Ϫ treatment was prevented by pretreatment of cells with 50 M PD98059 for 1 h prior to the addition of ONOO Ϫ (Fig. 8B). Since the decrease in mitogenesis by ONOO Ϫ suggested a cytotoxic effect, we utilized the MTT assay to measure ONOO Ϫ -induced cytotoxicity. Treatment with ONOO Ϫ (1 mM) caused a rapid change in cell morphology (within 30 min) that was characterized by cell shrinkage and loss of focal adhesions (Fig. 9A). Pretreatment of the cells with 50 M PD98059 for 1 h protected the cells from the ONOO Ϫinduced change in cell morphology. ONOO Ϫ also caused a concentration-dependent decrease in cell survival as measured by the MTT cytotoxicity assay (Fig. 9B). Pretreatment with 50 M PD98059 significantly protected the cells against ONOO Ϫ -induced cytotoxicity. Using the MTT assay, we also observed that a 30-min treatment with 100 M H 2 O 2 induced 80 -90% cytotoxicity in cells that were pretreated for 1 h with Me 2 SO vehicle alone, whereas H 2 O 2 treatment induced only 10 -15% cytotoxicity in cells pretreated with 50 M PD98059 for 1 h (data not shown). DISCUSSION In this study, we have investigated possible differences in the mechanism of ERK activation by H 2 O 2 and ONOO Ϫ in vitro using primary passage rat pulmonary myofibroblasts. ONOO Ϫ was found to be a potent activator of ERK and also caused phosphorylation of EGFR, Raf-1, and MEK-1. Although these results initially suggested a mechanism of MAPK activation similar to that stimulated by H 2 O 2 , we observed that inhibitors of EGFR (AG1478) and Raf-1 (forskolin) did not block ONOO Ϫinduced MAPK activation. Since H 2 O 2 requires EGFR to activate MAPK, these data suggested a different mechanism of MAPK activation between ONOO Ϫ and H 2 O 2 . We further explored the mechanism of activation and found that ONOO Ϫ , but not H 2 O 2 , caused nitration and autophosphorylation of recombinant GST-MEK-1 fusion protein in a cell-free system. Additionally, phosphorylated Raf-1 was co-immunoprecipitated with an anti-nitrotyrosine antibody from lysates of ONOO Ϫ -treated cells. Collectively, these observations indicate that nitration of Raf-1 and MEK-1 causes autophosphorylation.
In our experiments, it was essential to test the possibility that H 2 O 2 was contributing to the activation of ERK since our preparations of ONOO Ϫ were synthesized from acidified nitrite and H 2 O 2 . Thus, these stocks of ONOO Ϫ could contain trace amounts of H 2 O 2 . Moreover, a recent study by Kirsch and de Groot (33) demonstrated that H 2 O 2 can be formed by the reaction of ONOO Ϫ with nicotinamide nucleotides. We excluded the possible contribution of H 2 O 2 by demonstrating that ONOO Ϫinduced phosphorylation of ERK required the immediate addition of ONOO Ϫ to cell cultures due to the rapid degradation of FIG. 2. ONOO ؊ activates ERK-1 and ERK-2 in a time-and concentration-dependent manner. A, myofibroblasts were rendered quiescent for 24 h in SFDM and then switched to PBS and exposed to 1 mM ONOO Ϫ for the indicated time points. Cell lysates were collected for Western blotting using an antibody specific for phosphorylated ERK (upper panel) or an antibody against total ERK (middle panel). ERK activity was measured by kinase assay following immunoprecipitation of ERK and using PHAS-1 as a substrate (lower panel). B, cells were exposed to an increasing concentration of ONOO Ϫ for 15 min prior to collecting cell lysates for phospho-ERK and ERK Western blotting (upper and middle panels, respectively) or immunoprecipitation of ERK followed by PHAS-1 kinase assay (lower panel).

FIG. 3. Effect of antioxidants N-acetyl-L-cysteine and catalase on ONOO ؊ -and H 2 O 2 -induced ERK-1 and ERK-2 activation.
Confluent quiescent cultures of rat pulmonary myofibroblasts were incubated with N-acetyl-L-cysteine (NAC; 50 mM) or catalase (3000 units/ml) and then treated with 1 mM ONOO Ϫ (upper panel) or H 2 O 2 (lower panel) for 15 or 30 min prior to collecting cell lysates for Western blotting using an anti-phospho-ERK antibody. this oxidant at neutral pH (Fig. 1). Premixing the ONOO Ϫ in PBS (pH 7.4) for 1 min resulted in a complete loss of ERKphosphorylating activity. In contrast, this premixing delay did not diminish the activity of H 2 O 2 in activating ERK. However, to definitively exclude the possibility that trace levels of H 2 O 2 present in the preparations of ONOO Ϫ were mediating activation of ERK, catalase was used to eliminate H 2 O 2 as has been reported previously (10,11). Pretreatment of cells with catalase abolished activation of ERK by H 2 O 2 , but did not inhibit ERK activation by ONOO Ϫ (Fig. 3). These data confirmed that acti-vation of ERK following treatment with ONOO Ϫ was not due to the presence of H 2 O 2 .
H 2 O 2 -induced ERK activation has been reported to require phosphorylation of EGFR (12,13). For this reason, we investigated the role of EGFR in ONOO Ϫ -induced ERK activation. Using a Western blot technique with an antibody specific for the phosphorylated form of EGFR, we showed that ONOO Ϫ , as well as EGF and H 2 O 2 , strongly induced phosphorylation of EGFR within 5 min (Fig. 4). We then employed the EGFRspecific tyrphostin analog AG1478, which has been reported to   (Fig. 6). This observation suggests that ERK is not activated directly by ONOO Ϫ , but instead requires MEK.
Our findings demonstrate important differences in the mechanisms of H 2 O 2 -and ONOO Ϫ -induced signal transduction involving MAPKs. The effect of H 2 O 2 on MAPK activation is dependent on the phosphorylation of EGFR, whereas ONOO Ϫ is capable of bypassing EGFR and Raf-1 blockades to activate MAPK. Although H 2 O 2 can be a source of cellular stress generated from exogenous environmental insult, it is becoming increasingly clear that intracellular H 2 O 2 functions as a signaling molecule in normal physiological processes to regulate the phosphorylation of receptor tyrosine kinases such as EGFR and the platelet-derived growth factor receptor (10,11). The effect of H 2 O 2 on EGFR activation to trigger a MAPK signaling cascade appears to be due to the quenching of protein-tyrosine phosphatase activity (11,34). In contrast, ONOO Ϫ appears to activate EGFR via nitration of tyrosine residues, which results in receptor dimerization and autophosphorylation (15). We have also observed that ONOO Ϫ treatment of rat pulmonary myofibroblasts causes nitration and autophosphorylation of EGFR and the platelet-derived growth factor receptor. 2 We provide evidence for the first time that ONOO Ϫ directly activates MEK (i.e. autophosphorylation) in a cell-free system that excluded other proteins (Fig. 7). H 2 O 2 did not activate MEK-1 in the cell-free system. As has been shown for ONOO Ϫinduced autophosphorylation of EGFR (15), it is likely that a nitration reaction is responsible for autophosphorylation of MEK-1 by ONOO Ϫ , and we showed that GST-MEK-1 was nitrated in a concentration-dependent manner that correlated with the amount of ONOO Ϫ that was required to induce autophosphorylation (Fig. 7). We excluded the possible contribution of phosphatase inhibition as a mechanism of ONOO Ϫ -induced 2 P. Zhang   MEK-1 activation since the cell-free phosphorylation assay excluded phosphatases.
We addressed the biological relevance of ONOO Ϫ -induced MAPK activation. Since mitogenesis is the paradigm of EGFR activation and ONOO Ϫ activated EGFR, we first investigated the effect of ONOO Ϫ on DNA synthesis using a [ 3 H]thymidine incorporation assay. Although EGF was a potent stimulator of mitogenesis in rat lung myofibroblasts, ONOO Ϫ caused a concentration-dependent decrease in mitogenesis (Fig. 8), and this was due to a reduction in cell survival as determined by the MTT cytotoxicity assay (Fig. 9). Our finding that the MEK inhibitor PD98059 prevented ONOO Ϫ -and H 2 O 2 -induced cytotoxicity was surprising since activation of the ERK pathway is necessary for proliferative and cell survival responses (35). Indeed, both EGF-induced DNA synthesis and ONOO Ϫ -induced cytotoxicity in myofibroblasts were blocked by PD98059. Thus, in the same cell type, MEK is critical for both proliferative and cytotoxic responses, depending on the stimulus. Other investigators have made observations similar to ours in other systems. Bhat and Zhang (36) reported that PD98059 protected oligodendrocytes from the cytotoxic effects of H 2 O 2 . Jimenez et al. (37) showed that asbestos-and H 2 O 2 -induced pleural mesothelial cell death was abrogated by the MEK inhibitor PD98059. The mechanism through which MEK or ERK mediates cytotoxicity in response to ONOO Ϫ or other oxidants is unclear. One possibility is that activation of ERK along with simultaneous activation of other kinases (e.g. p38 or JNK) could lead to a cell death response, whereas EGF does not activate these other kinases in myofibroblasts. It is known that oxidants, including H 2 O 2 and ONOO Ϫ , activate all three families of MAPKs (12,18). Thus, we suggest that activation of the ERK pathway by ONOO Ϫ or H 2 O 2 may be required (but not sufficient) for a cytotoxic response.
In summary, we report that ONOO Ϫ independently activates components of the MAPK signaling cascade that involves EGFR, Raf-1, and MEK-1. Although H 2 O 2 -induced ERK activation was completely blocked at the level of EGFR phosphorylation, ONOO Ϫ could bypass blockade of either EGFR tyrosine kinase or Raf-1 kinase and cause ERK activation. ONOO Ϫ caused nitration and autophosphorylation of GST-MEK-1 fusion protein in a cell-free system, suggesting that formation of nitrotyrosine activates MEK-1. These findings indicate that the mechanism of MAPK activation by ONOO Ϫ is fundamentally different from that of H 2 O 2 -induced MAPK activation.

FIG. 8. Effect of ONOO ؊ on [ 3 H]thymidine uptake by rat lung myofibroblasts.
A, cells were rendered quiescent in SFDM for 24 h and then switched to PBS and treated with ONOO Ϫ . After 15 min, the medium was aspirated, and fresh SFDM was added back along with 5 Ci/ml [ 3 H]thymidine for 24 h. B, quiescent cells were pretreated for 1 h with 50 M PD98059 (PD) in SFDM, and then the medium aspirated and PBS was added back. The medium was aspirated again 15 min after adding 1 mM ONOO Ϫ , and fresh SFDM with 5 Ci/ml [ 3 H]thymidine were added back for 24 h.