Introduction

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Metals are emerging as principal agents in air-pollution particles that cause adverse respiratory effects. Vanadium compounds in particular have been identified as a source of occupational bronchial asthma and bronchitis in individuals working in the petrochemical industry (1, 2). Vanadium-containing residual oil fly ash particles from the emissions of petrochemical plants cause lung inflammation and airway hyperresponsiveness in rats (3, 4). Moreover, vanadium-bearing emission source particles or urban particulate matter containing trace levels of vanadium upregulate the expression of a variety of inflammatory cytokines or their receptors in vitro (5, 6). We have previously reported that vanadium instilled intratracheally in rats causes airway remodeling that includes airway smooth-muscle thickening, mucous-cell metaplasia, and peribronchiolar fibrosis (7). The cellular and molecular mechanisms that mediate vanadium-induced airway injury are not well understood. However, increasing evidence indicates that vanadium could cause cellular stress by activating a variety of signal transduction pathways, including those leading to the phosphorylation of mitogen-activated protein (MAP) kinases (8).

MAP kinases 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), Jun amino-terminal kinases (JNKs) (also known as stress-activated protein kinases), and p38 MAP kinases (reviewed in 11). JNKs and p38 MAP kinases 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 (PDGF) and epidermal growth factor (EGF) (11). Two major isoforms of ERK, p44 (ERK-1) and p42 (ERK-2), have been identified in mammalian systems (13). A major pathway involved in ERK-1 and ERK-2 stimulation in a variety of cell types requires the sequential activation of Ras, Raf, and MAP kinase kinase (MEK) (12). It is becoming increasingly clear that the ERK pathway, like p38 and JNK, is activated by environmental stress including reactive oxygen species (ROS) such as H₂O₂ and transition metals including vanadium (14).

Vanadium compounds have been reported to activate all three MAP kinase families (JNK, p38, and ERK), although the precise mechanism of activation of these MAP kinases remains unclear (8, 9). Pervanadate has been reported to activate ERK-1/2 via phosphorylation of the EGF receptor (EGF-R), and the presumed mechanism of EGF-R activation is via competitive inhibition of protein tyrosine phosphatase (PTP) activity (8). However, PTP inhibition may occur through oxidant-independent and oxidant-dependent mechanisms, depending on the vanadium compound. For example, vanadate is a competitive PTP inhibitor and inhibition is reversible with ethylenediaminetetraacetic acid (EDTA) (10). In contrast, pervanadate (a complex of vanadate and H₂O₂) inhibits by irreversibly oxidizing the catalytic cysteine of PTP (10). The possible contribution of vanadium-generated ROS in mediating MAP kinase activation, either through PTP inhibition or an alternative mechanism, has not been investigated.

In this study we have investigated the mechanism of MAP kinase activation in rat pulmonary myofibroblasts by vanadium pentoxide (V₂O₅), a largely insoluble form of vanadium present in ambient air pollution particles that has been associated with fibroproliferative lung disease (17). Titanium dioxide (TiO₂), a metal that does not cause pulmonary disease in vivo, was used as a negative control of comparable particle diameter (18). V₂O₅ was a potent activator of ERK-1/2, whereas TiO₂ did not activate ERK-1/2. The EGF-R-specific tyrosine kinase inhibitor AG1478 blocked V₂O₅-induced ERK-1/2 activation, but surprisingly EGF-R was only weakly phosphorylated by V₂O₅ as compared with activation of EGF-R by EGF. Instead, V₂O₅ primarily caused tyrosine phosphorylation of a 115-kD protein (p115) that was immunoprecipitated by an anti-Src homology 2 PTP (SH-PTP2) antibody. Moreover, activation of p115 was inhibited by AG1478. V₂O₅-induced ERK-1/2 activation and p115 phosphorylation were blocked by the antioxidant N-acetyl-L-cysteine (NAC). These data support the idea that V₂O₅ activates ERK-1/2 through an EGF-R signaling pathway that is initiated via the generation of ROS.

	Materials and Methods

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Materials

V₂O₅ was purchased from Aldrich Chemical Co. (Milwaukee, WI). TiO₂ was obtained from Sigma Chemical Co. (St. Louis, MO). An anti-ERK-1/2 that recognizes total cellular ERK (i.e., both activated and inactivated forms) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). An anti-phospho-ERK-1/2 antibody that is specific for the activated forms of ERK-1/2 was purchased from Promega (Madison, WI). The phosphotyrosine monoclonal antibody (PY20) and antimouse immunoglobulin (Ig) G-horseradish peroxidase (HRP) were purchased from Transduction Laboratories (Lexington, KY). An anti-EGF-R polyclonal antibody, an anti-protein kinase (PK) C-1 antibody, and a polyclonal anti-SHPTP2 antibody-agarose conjugate were from Santa Cruz. NAC was from Sigma. Tyrphostin AG1478 was from Calbiochem (La Jolla, CA). The MEK-1 inhibitor PD98059 ([2-(2'-amino-3'-methoxyphenyl)-oxanapthalen-4-one]) was obtained from New England Biolabs, Inc. (Beverly, MA). The PKC inhibitors GF 109203X and Ro 31-8220 were from Calbiochem. Primary passage rat-lung myofibroblasts were isolated and characterized as described previously (18).

Western Blotting

Confluent rat lung myofibroblasts in 75-cm² dishes were growth-arrested in serum-free defined medium (SFDM) for 24 h, then treated with 10 µg/cm² of V₂O₅ or TiO₂ for 5 min to 2 h. The cells were washed twice with ice-cold phosphate-buffered saline (PBS) and scraped. The cell pellets were resuspended in a lysis buffer (50 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid [Hepes], 150 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride [PMSF], and 20 µg/ml aprotinin, leupeptin, and pepstatin) and clarified by centrifugation at 13,000 rpm for 10 min. The protein concentrations were determined by Bradford assay. A total of 20 µg of protein from each sample was loaded on a 10 to 20% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. Separated proteins were then transferred to a nitrocellulose membrane and the membrane was blocked for 2 h at room temperature with 5% nonfat milk in Tris-buffered saline- Tween buffer (20 mM Tris, 500 mM NaCl, and 0.01% Tween 20). The membrane was then incubated with the anti-phospho-ERK1/2 antibody (1:1,000 dilution) at 4°C overnight, followed by incubation for 2 h with a 1:2,000 dilution of HRP-conjugated antirabbit IgG. The immunoblot was visualized through enhanced chemiluminescence (Amersham, Arlington Heights, IL). The same blot was subsequently stripped at 50°C for 30 min in a buffer containing 62.5 mM Tris (pH 6.7), 2% SDS, and 100 mM -mercaptoethanol. The stripped blots were reprobed with anti-ERK1/2, anti- PKC-1, or anti-phosphotyrosine (PY20).

EGF-R and SH-PTP2 Immunoprecipitation

Whole cell lysates were prepared as described in WESTERN BLOTTING. For EGF-R immunoprecipitation, cell lysates containing equal amounts of protein were incubated with 10 µl (4 µg) of anti-EGF-R polyclonal antibody at 4°C overnight. A total of 20 µl of protein-A agarose was then added to the lysates and incubated for 1 h. For immunoprecipitation of SH-PTP2, 2 µl (4 µg) of an anti-SH-PTP2 polyclonal antibody-agarose conjugate was added to each cell lysate and incubated overnight. The immune complex bound to agarose beads was pelleted by centrifugation and washed three times with lysate buffer. A total of 15 µl of 5× SDS-PAGE loading buffer was added to the immunoprecipitates before electrophoresis and Western blotting for phosphotyrosine.

PHAS-1 Kinase Assay

ERK activity in cell lysates was measured as described previously (19) by phosphorylation of PHAS-1, a substrate for ERK-1/2 (20). Briefly, confluent rat lung myofibroblasts in 75-cm² dishes were growth-arrested in SFDM for 24 h, then treated with 10 µg/cm² of V₂O₅ or TiO₂ for 5 min to 2 h. The cells were placed on ice, then washed twice with PBS and scraped off with 800 µl of lysate buffer consisting of 50 mM Hepes, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, and 20 µg/ml aprotinin, leupeptin, and pepstatin. Lysates were clarified by centrifugation at 13,000 rpm for 10 min and protein concentrations were determined by Bradford assay. Immunoprecipitation was performed by incubating 200 µl of lysate with 2 µg of anti-ERK antibody for 2 h, then adding 20 µl of protein-A/agarose (Santa Cruz). After an overnight incubation at 0 to 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 MgCl₂, and 200 µM Na₃VO₄. Immune-complex kinase assays were performed using a MAP Kinase Assay Kit (Stratagene, La Jolla, CA). The ERK pellets were resuspended in Stratagene reaction buffer containing 120 µg of PHAS-1 substrate along with 3-5 µCi [-³²P]adenosine triphosphate in a final volume of 180 µl. Kinase reactions took place for 30 min at room temperature and were stopped by adding 4× SDS-PAGE reducing sample buffer and boiling for 10 min. ERK-Phas samples were resolved on 4 to 20% PAGE gels, dried, and autoradiographed.

	Results

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Differential Activation of ERK-1/2 by V₂O₅ and TiO₂

V₂O₅ exposure stimulated activation of ERK-1/2 in rat pulmonary myofibroblasts as demonstrated by Western blotting using an antibody to the phosphorylated forms of ERK-1/2 (Figure 1A). In contrast, the inert metal TiO₂ did not activate ERK-1/2. The same blot was stripped and Western blotting was performed using an anti-ERK antibody that recognized total ERK-1/2 (activated and unactivated) to show equivalent amounts of MAP kinase in each lane (Figure 1A). To verify ERK-1/2 activity, immunoprecipitation was performed on cell lysates using the antibody raised against total ERK-1/2 and kinase assays were performed using PHAS-1 as a substrate (Figure 1B). These data clearly show that V₂O₅ was a strong activator of ERK- 1/2-induced phosphorylation of PHAS-1. Dose-response experiments using increasing concentrations of metal showed that 10 µg/cm² of V₂O₅ caused maximal activation of ERK-1/2, whereas 0.1 µg/cm² V₂O₅ did not activate ERK-1/2 and 1 µg/cm² induced an approximate half-maximal response (data not shown).

View larger version (50K): [in this window] [in a new window]   Figure 1.   Differential activation of ERK-1/2 by V2O5 and TiO2. Confluent, quiescent cultures of rat pulmonary myofibroblasts were treated with 10 µg/cm2 of V2O5 or TiO2 and cell lysates collected at the indicated time points as described in MATERIALS AND METHODS. (A) Western blot using an antibody to the phosphorylated ERK-1/2 (anti-P-ERK) or an antibody to both activated and unactivated ERK-1/2 (anti-ERK), showing that V2O5 activates both isozymes of ERK without altering the level of unactivated ERK-1/2. SFDM alone was used as a control. (B) Kinase assay showing phosphorylation of PHAS-1 substrate by activated ERK-1/2. Immunoprecipitation of ERK-1/2 from cell lysates was performed as described in MATERIALS AND METHODS.

V₂O₅-Induced Activation of ERK-1/2 Is Dependent on Raf and MEK, but not PKC

Activation of ERK-1/2 by V₂O₅ was inhibited by forskolin, indicating a Raf-dependent pathway (Figure 2A). Further, the MEK inhibitor PD98058 (21) abolished V₂O₅-induced activation of ERK-1/2 (Figure 2B). These data indicate that V₂O₅ was not directly causing ERK-1/2 phosphorylation but was mediating MAP kinase activation via a sequential Raf/MEK/ERK pathway. Because other investigators reported that ERK-1/2 activation by H₂O₂ was PKC-dependent, we investigated the possible involvement of PKC in V₂O₅-induced ERK-1/2 activation. Phorbol 12-myristate 13-acetate (PMA) treatment is known to cause acute activation of conventional and novel PKCs (within 5 min), while depleting the level of these PKCs after prolonged (24 h) treatment (22). Treatment of myofibroblasts with 1 µM PMA strongly activated ERK-1/2 within 5 min (Figure 3A), demonstrating that ERK-1/2 can be activated via a PKC-dependent pathway in these cells. However, ERK-1/2 activation by V₂O₅ was not affected by treatment with PMA for 24 h, which completely depleted levels of PKC-1 (Figure 3B). Further, pretreatment of myofibroblasts with specific inhibitors of conventional PKCs GF 109203X (100 µM) and Ro 31-8220 (100 µM) before V₂O₅ treatment did not affect ERK-1/2 activation (data not shown).

View larger version (38K): [in this window] [in a new window]   Figure 2.   V2O5 activates ERK-1/2 via Raf-1 and MEK. Confluent cultures of rat lung myofibroblasts were pretreated with 50 µM forskolin (Raf-1 inhibitor) or 100 µM PD98059 (MEK inhibitor) for 1 h, then exposed to 10 µg/cm2 V2O5 or TiO2 for 30 min before collecting cell lysates. (A) Western blot using activated ERK-1/2 antibody showing that V2O5-induced phosphorylation of ERK was inhibited by the Raf-1 inhibitor forskolin. (B) V2O5-induced activation of ERK-1/2 was also abolished by the MEK inhibitor PD98059.

View larger version (38K): [in this window] [in a new window]   Figure 3.   Conventional PKC activity is not required for activation of ERK-1/2 by V2O5. Confluent, quiescent cultures of rat lung myofibroblasts were pretreated with 1 µM PMA in dimethyl sulfoxide (DMSO) or DMSO vehicle alone for 24 h before activation of ERK-1/2 with 10 µg/cm2 V2O5 or TiO2. Western blot analysis of cell lysates was performed using antibodies against PKC-1 or phosphorylated ERK-1/2. (A) PMA treatment for 5 min activated ERK, demonstrating that PKC activation can cause ERK phosphorylation. (B) PKC-1 protein was completely depleted in cell lysates from myofibroblasts treated for 24 h with PMA, yet V2O5-induced activation of ERK-1/2 was not affected by PKC depletion.

The EGF-R-Specific Tyrosine Kinase Inhibitor AG1478 Inhibits V₂O₅-Stimulated ERK-1/2 Activation and V₂O₅-Induced Tyrosine Phosphorylation of a 115-kD SH-PTP2-Binding Protein

A previous report by Zhao and coworkers showed that pervanadate could induce tyrosine phosphorylation of EGF-R, and these investigators suggested that activation of MAP kinase by pervanadate was due at least in part to EGF-R activation (8). Moreover, EGF stimulates the ERK pathway via Raf and MEK. To investigate the possible role of EGF-R signaling in V₂O₅-induced ERK-1/2 activation, we used a tyrosine kinase inhibitor (AG1478) that is specific for EGF-R (23). AG1478 blocked V₂O₅- induced ERK-1/2 activation > 80% as determined by Western blotting with a phospho-ERK-1/2 antibody (Figure 4A). Western blotting with the PY20 phosphotyrosine antibody revealed that V₂O₅ caused tyrosine phosphorylation of a protein with a molecular mass of 115 kD and to a lesser extent induced phosphorylation of a ~ 66-kD protein (p66) (Figure 4B). The phosphorylation of both p115 and p66 were inhibited 60 to 70% by 100 µM AG1478 (Figure 4B). Surprisingly, no detectable phosphorylation was observed at the molecular mass corresponding to the EGF-R (170 kD) (Figure 4B). However, immunoprecipitation with an EGF-R antibody followed by phosphotyrosine Western blotting revealed that EGF-R tyrosine phosphorylation did occur as a result of V₂O₅ treatment, although the relative amount of phosphorylated EGF-R was < 5% of the amount of tyrosine phosphorylation observed in EGF-treated cells (Figure 5A). EGF also stimulated phosphorylation of 115- and 66-kD proteins (Figure 5B). EGF-R immunoprecipitation did not result in coimmunoprecipitation of either 115- or 66-kD phosphorylated proteins in either V₂O₅- or EGF-treated cells (Figure 5A). The 115-kD protein that was tyrosine phosphorylated after V₂O₅ treatment or EGF stimulation was identified as an SH-PTP2-binding protein, inasmuch as this phospho-protein was immunoprecipitated with a polyclonal anti- SH-PTP2 antibody (Figure 5C). Anti-SH-PTP2 did not coimmunoprecipitate the EGF-R.

View larger version (32K): [in this window] [in a new window]   Figure 4.   V2O5-induced activation of ERK-1/2 and p115 is inhibited by the EGF receptor tyrosine kinase inhibitor AG1478. Confluent, quiescent cultures of rat lung myofibroblasts were pretreated with 100 µM AG1478 for 1 h before activation of ERK-1/2 by 10 µg/cm2 V2O5 for 30 min and then collection of cell lysates. TiO2 (10 µg/cm2) was used a negative control metal. (A) Western blotting using an antibody to the phosphorylated forms of ERK-1 and -2 showed that AG1478 reduced ERK-1 and -2 activation by V2O5 > 80%. (B) Western blotting using antiphosphotyrosine (PY20), demonstrating > 70% inhibition of V2O5-induced p115 tyrosine phosphorylation by AG1478.

View larger version (29K): [in this window] [in a new window]   Figure 5.   V2O5-induced tyrosine phosphorylation of EGF-R and a SH-PTP2-binding protein. Confluent, quiescent cultures of rat lung myofibroblasts were treated with 10 µg/cm2 V2O5 (30 min) or 50 ng/ ml EGF (5 min). (A) Immunoprecipitation (IP) of cell lysates was performed with an EGF-R antibody before Western blotting (WB) using a phosphotyrosine (PY20) antibody as described in MATERIALS AND METHODS. This method allowed detection of EGF-R tyrosine phosphorylation after V2O5 treatment, albeit the phosphotyrosine was < 5% of that obtained after EGF stimulation. The anti-EGF-R IP did not coimmunoprecipitate p115 in either V2O5- or EGF-treated cells. (B) Tyrosine phosphorylation of EGF-R (170 kD) and p115 was observed in EGF-stimulated cell lysates that were not subjected to IP. (C) IP of cell lysates was performed with a SH-PTP2 antibody before Western blotting (WB) using a phosphotyrosine (PY20) antibody as described in MATERIALS AND METHODS. The anti-SH-PTP2 antibody immunoprecipitated a 115-kD phospho-protein (p115) in either V2O5- or EGF-treated cells, but did not coimmunoprecipitate the EGF-R (p170).

Activation of ERK-1/2 and Tyrosine Phosphorylation of the p115 SH-PTP2-Binding Protein by V₂O₅ Is Oxidant-Dependent

Pretreatment of cells with 50 mM NAC before V₂O₅ exposure inhibited activation of ERK-1/2 and ERK-mediated phosphorylation of PHAS-1 > 95% (Figure 6). NAC alone did not cause activation of ERK-1/2. Further, V₂O₅-induced tyrosine phosphorylation of p115 was inhibited > 90% by pretreatment with 50 mM NAC (Figure 7).

View larger version (33K): [in this window] [in a new window]   Figure 6.   Activation of ERK-1/2 by V2O5 is inhibited by the antioxidant NAC. Confluent, quiescent cultures of rat pulmonary myofibroblasts were treated with 50 mM NAC or SFDM for 1 h, then exposed to 10 µg/cm2 of V2O5 or TiO2 for 30 min before collecting cell lysates. (A) Western blot using an antibody to phosphorylated ERK-1/2 shows that V2O5-induced ERK activation is blocked by NAC (50 mM). (B) Kinase assay showing that V2O5 activated ERK-1/2 to phosphorylate PHAS-1 substrate and NAC pretreatment blocks activation of ERK-1/2 by V2O5.

View larger version (42K): [in this window] [in a new window]   Figure 7.   V2O5-induced tyrosine phosphorylation of p115 is oxidant-dependent. Confluent, quiescent cultures of rat lung myofibroblasts were treated with 50 mM NAC or SFDM for 1 h, then exposed to 10 µg/cm2 of V2O5 or TiO2 for 30 min before collecting cell lysates. Western blotting was performed using a phosphotyrosine (PY20) antibody or EGF-R antibody. As shown in Figure 4B, V2O5 primarily phosphorylated a protein of 115 kD (p115) with no detectable phosphorylation at the molecular mass of the EGF-R (170 kD). However, EGF-R phosphorylation could be detected after immunoprecipitation with an EGF-R antibody and phosphotyrosine Western blotting (Figure 5A). Therefore, in this experiment the same blot was stripped and Western blotting was performed with an anti-EGF-R antibody, which verified the presence of EGF-R in all samples.

	Discussion

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In this report we extend what is known regarding the activation of MAP kinases by vanadium compounds and demonstrate that V₂O₅-induced ERK-1/2 phosphorylation in rat pulmonary myofibroblasts is oxidant-dependent. Moreover, we report for the first time that a vanadium compound induces tyrosine phosphorylation of an SH-PTP2- binding protein that appears to be a substrate for EGF-R tyrosine kinase and leads to downstream ERK-1/2 activation. Zhao and coworkers previously reported that pervanadate activation of ERK-1/2 was mediated at least in part through an EGF receptor tyrosine kinase-coupled and Ras-coupled pathway that led to downstream activation of Raf and MEK (8). However, these investigators attributed pervanadate-induced ERK-1/2 activation to the well-known ability of certain vanadium compounds (e.g., vanadate) to act as competitive inhibitors of PTP, and they did not consider the role of oxidants. Also, the mechanism of EGF-R activation was not explored in this previous study.

A recent study by Huyer and colleagues showed that pervanadate and vanadate inhibit PTP activity through completely different mechanisms (10). These investigators demonstrated that vanadate is a competitive inhibitor for PTP1B and that EDTA (a vanadate chelator) caused an immediate and complete reversal of PTP1B inhibition due to vanadate. In contrast, pervanadate (a general term for complexes formed between vanadate and H₂O₂) inhibited PTP1B by irreversibly oxidizing the catalytic cysteine of PTP1B as determined by mass spectrometry (10). Because V₂O₅-induced ERK-1/2 activation and tyrosine phosphorylation of the 115-kD SH-PTP2-binding protein was blocked by pretreatment with NAC, our data support the hypothesis that V₂O₅ inhibition of PTP requires ROS. Other investigators have reported that H₂O₂ is sufficient to inhibit PTP activity (24). Thus, the ROS-dependent mechanism of PTP inhibition by V₂O₅ could be similar to PTP inactivation by pervanadate.

It is becoming increasingly clear that oxidation plays an important role in the phosphorylation of receptor tyrosine kinases. For example, the binding of EGF to the EGF-R extracellular domain transiently increases intracellular H₂O₂, which serves to inhibit PTP activity and thereby favors tyrosine phosphorylation of the EGF-R (25). Lee and coworkers reported that H₂O₂ inactivated PTP1B in a reversible manner by oxidizing its catalytic site cysteine, and also suggested that inhibition of PTP by H₂O₂ might be required for receptor tyrosine kinase activation by binding of the growth factor (23). Further, interleukin-1 (26) and lysophosphatidic acid (27, 28) cause ERK activation via the generation of ROS. Cunnick and coworkers have suggested that ROS could act by inhibiting PTP activity rather than directly increasing the EGF receptor kinase activity in the lysophosphatidic acid-stimulated MAP kinase pathway (27). PDGF signal transduction requires ROS, and ligand-induced autophosphorylation of the PDGF receptor is blocked by NAC (29, 30). These studies provide evidence that ROS are likely essential for normal signaling events associated with receptor tyrosine kinases including EGF-R and PDGF-R. However, there is growing evidence that environmental factors can disrupt normal cellular signaling via the generation of ROS. For example, asbestos causes ERK activation in rat pleural mesothelial cells via phosphorylation of the EGF-R, and this event appears to be mediated by oxidants (31). Moreover, the addition of H₂O₂ to cultured NIH 3T3 cells strongly activates ERK-1/2 and this is blocked by an EGF-R tyrosine kinase inhibitor (14), and H₂O₂ has been reported to stimulate phosphorylation of the EGF-R in A549 lung epithelial cells (32). Thus, the EGF-R appears to be a central target for activation of MAP kinase by variety of environmental factors that generate oxidative stress.

Our data indicate that the EGF-R is involved in V₂O₅-induced ERK-1/2 activation. Tyrosine phosphorylation of EGF-R was detected after treatment with V₂O₅, and AG1478 (an inhibitor of EGF-R tyrosine kinase activity) blocked ERK-1/2 activation by V₂O₅. However, the major phospho-protein detected by phosphotyrosine Western blotting of V₂O₅-treated cell lysates was a 115-kD protein rather than the 170-kD EGF-R. We identified p115 as SH-PTP2-binding because this protein was immunoprecipitated by an anti-SH-PTP2 antibody. We further showed that p115 was an EGF-R substrate, inasmuch as AG1478 blocked ~ 70% of V₂O₅-induced p115 tyrosine phosphorylation. Moreover, we showed that EGF treatment induced tyrosine phosphorylation of a 115-kD protein in addition to the 170-kD EGF-R.

Several groups have shown that expression of catalytically inactive SH-PTP2 results in the hyperphosphorylation of 115- to 120-kD proteins (33). Yamauchi and Pessin observed a 115-kD protein in NIH 3T3 cells expressing high levels of the human EGF-R (3T3/ER) that was tyrosine-phosphorylated after EGF stimulation, and they identified the p115 in 3T3/ER cells as an SH-PTP2- binding protein (33). These investigators found that immunoprecipitation of p115 by anti-SH-PTP2 did not coimmunoprecipitate the EGF-R, suggesting that only a small fraction of SH-PTP2 associates with the EGF receptor and that p115 is the major SH-PTP2-binding protein in EGF-stimulated cells. A recent study by Frearson and Alexander showed that inactive SHP-2 (i.e., SH-PTP2) was targeted to the cell membrane in Jurkat T cells, resulting in a selective increase in tyrosine phosphorylation of three membrane-associated candidate SHP-2 substrates, one of which was a 110-kD protein, and this association was critical to linking SHP-2 to the Ras/MAP kinase pathway (36). In the present study, our data support the hypothesis that p115 is a SH-PTP2-binding protein and EGF-R substrate in V₂O₅-stimulated myofibroblasts that plays a major role in MAP kinase activation.

In contrast to some other studies of H₂O₂-induced ERK activation that reported a dependence on PKC (15), we clearly showed that depletion of conventional PKC activity by 24 h pretreatment with PMA did not inhibit V₂O₅- induced ERK-1/2 activation. In our experiments, ERK was activated by PMA within 5 min, and because PMA has been demonstrated to rapidly activate PKC (22), this showed that PKC could mediate phosphorylation of ERK-1/2 in our system. However, chronic 24 h treatment by PMA depletes conventional and novel isozymes of PKC (22), and in our hands this treatment had no effect on V₂O₅-induced ERK activation. In addition, we demonstrated that inhibitors of conventional PKCs GF 109203X and Ro 31-8220 did not block V₂O₅ activation of ERK. Although PMA depletes conventional and novel isozymes of PKC, it does not deplete the atypical isozymes PKC- and PKC-. In fact, Liao and coworkers recently reported that angiotensin II activation of ERK-1/2 in vascular smooth-muscle cells is mediated by PKC- (37). Therefore, we cannot rule out these PMA-insensitive, atypical PKC isozymes as mediators of V₂O₅-induced ERK-1/2 activation.

The role of V₂O₅-induced MAP kinase activation in the progression of fibroproliferative lung disease in vivo is unknown. The finding that an EGF-R signaling pathway is activated by V₂O₅ in vitro is intriguing, inasmuch as we recently reported that administration of a tyrosine kinase inhibitor specific for the EGF-R (AG1478) prevents V₂O₅-induced pulmonary fibrosis in rats in vivo (38). It is unclear whether the in vivo delivery of AG1478 inhibits the progression of fibrosis by simply blocking autophosphorylation of the EGF-R and subsequent mitogenesis of connective tissue cells stimulated by endogenous EGF-R ligands (i.e., EGF, transforming growth factor-, heparin-binding EGF), or whether V₂O₅ exposure directly induces EGF-R phosphorylation and MAP kinase activation that results in increased connective tissue cell proliferation in the absence of endogenous EGF-R ligands.

In summary, we have shown that V₂O₅-induced MAP kinase activation is oxidant-dependent and requires components of an EGF-R signaling cascade. Importantly, we show for the first time that the major tyrosine-phosphorylated EGF-R substrate in vanadium-treated cells is a 115-kD SH-PTP2-binding protein that appears to play a role in ERK-1/2 activation. Further study is required to address the consequence of EGF-R signaling and subsequent MAP kinase activation to cell survival or cell death after exposure to metals that generate oxidative stress.