Introduction

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Mitogen-activated protein (MAP) kinases are a group of 38- to 110-kD serine/threonine kinases. Activation of extracellular signal-regulated protein kinases (ERKs), one subtype of MAP kinases, is a key step in the cascade mediating cell proliferation in response to a variety of extracellular signals, including epidermal growth factors, platelet-derived growth factor (PDGF), and phorbol esters (1). In contrast, p38 MAP kinase and p46 to p54 MAP kinases (Jun-N-terminal kinases, JNK), two other subtypes of MAP kinases, mediate signals in response to cytokines and environmental stress (2). Two major isoforms of ERK, p44 (ERK₁) and p42 (ERK₂), have been identified. A major pathway involved in ERK₁ and ERK₂ stimulation in various cell lines requires the sequential activation of Ras, Raf, and MAP kinase kinase.

Chronic exposure to reactive oxygen species such as H₂O₂, O₂, and ·OH causes pulmonary arterial remodeling and pulmonary hypertension (3, 4). Vascular smooth muscle cell proliferation is a major pathophysiologic event that occurs in the development of pulmonary hypertension. Recent evidence has shown that H₂O₂ stimulates cell growth/proliferation and DNA synthesis in different types of cells (5). This proliferation is associated with expression of the early immediate genes, such as c-myc, c-fos and c-jun (7). The effects of H₂O₂ on MAP kinases in pulmonary arterial smooth muscle, however, are unknown. Although H₂O₂ has been reported to activate MAP kinases in aortic smooth muscle (10, 11), the mechanisms of MAP kinase activation in response to H₂O₂ are not clear. Therefore, the purpose of this study was to characterize the effects of H₂O₂ on activation of ERK₁ and ERK₂ (also referred to as MAP kinases in this study) in vascular smooth muscle cells cultured from rat pulmonary arteries. We also investigated the roles of protein kinase C (PKC), Ca²⁺/calmodulin, and tyrosine kinases in H₂O₂-induced MAP kinase activation. Our results show that H₂O₂ activates ERK₁, ERK₂, p46 JNK, and p38 MAP kinases, and induces the expression of c-jun or fra-1 in a concentration- and time-dependent manner. The activation of ERK₁ and ERK₂ by H₂O₂ seems to depend on PKC activation, and appears to be modulated in part by Ca²⁺/calmodulin and by activation of tyrosine kinases.

	Materials and Methods

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Materials

Calphostin C, calmidazolium chloride (R 24571), and protein G plus protein A agarose were purchased from Calbiochem (San Diego, CA). Polyclonal anti-phospho-specific MAP kinase, anti-phospho-JNK, and anti-phospho-p38 antibodies were purchased from New England BioLabs (Beverly, MA). Polyclonal anti-ERK₁, anti-JNK₁, and anti-p38 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Enhanced chemiluminescence reagents and [-³²P]deoxycytosine triphosphate ([-³²P]dCTP) were obtained from Amersham (Arlington Heights, IL). Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, amphotericin B, fetal bovine serum (FBS), catalase, H₂O₂, thapsigargin, genistein, myelin basic protein (MBP), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), phorbol-12-myristate-13-acetate (PMA), trypan blue solution, and trypsin were purchased from Sigma Chemical Co. (St. Louis, MO).

Culture of Pulmonary Arterial Smooth Muscle Cells

Cultures of pulmonary arterial smooth muscle cells (PASMC) were prepared through a modification of the explant method described by Ross (12). Adult male Sprague- Dawley rats (250 to 270 g) were anesthetized intraperitoneally with pentobarbital sodium (60 mg/kg), and were bled by cutting the abdominal aorta. The pulmonary arteries were excised and cleaned. The endothelium of pulmonary artery was removed by gently rotating the vessel on a lightly sanded surgical steel rod. The vessel was then placed into serum-free DMEM containing 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 µg/ml amphotericin B, and incubated at 4°C overnight. On the following day the vessel was cut into 2-mm pieces and incubated in DMEM supplemented with 20% FBS in a humidified atmosphere of 5% CO₂ and 95% air at 37°C. A week later, the tissue explants were removed and fresh DMEM containing 20% FBS was added. After reaching confluency, cells were subcultured once weekly after detachment with 0.05% trypsin in phosphate-buffered saline (PBS) (pH 7.4). Cells were maintained in DMEM containing 10% FBS, and the medium was changed every 48 h. Experiments were performed on cells from passages 2 through 5.

Stimulation of MAP Kinases

Confluent PASMC on 100-mm dishes were growth-arrested with DMEM supplemented with 0.1% FBS for 48 h. On the day of the experiment, the culture medium was removed and replaced with fresh DMEM containing 0.1% FBS in the presence or absence of test reagents. After incubation, the cells were washed twice with ice-cold PBS and harvested in a lysis buffer composed of 80 mM -glycerophosphate, 20.0 mM 4-(2-hydroxyethyl)-1-piperazine-N'-2-ethane sulfonic acid (Hepes) (pH 7.4), 10 mM ethylene glycol-bis- (-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 2.0 mM ethylenediamine tetraacetic acid (EDTA), 2.0 mM dithiothreitol (DTT), 0.2 mM Na₃VO₄, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF). Cell lysates were prepared by quickly freezing harvested cells in liquid nitrogen, thawing on ice, scraping, and sonicating. After centrifugation for 30 min at 14,000 rpm at 4°C, the protein concentration of supernatant was determined by the protein assay system from Bio-Rad (Hercules, CA), using bovine serum albumin (BSA) as the standard.

MAP Kinase Activity Assay

MAP kinase activity was assayed by determining the amount of incorporation of ³²P into MBP in immunoprecipitates, as described by Duff and colleagues (13). Cell lysates were incubated on ice with antiserum to ERK₁ for at least 2 h. Immunocomplexes were collected by precipitation with protein G plus protein A agarose, centrifuged at 14,000 rpm for 5 s, and washed three times with the lysis buffer, as described previously. The precipitates were washed one more time with lysis buffer and resuspended in a kinase buffer that contained 5 mM -glycerophosphate, 20 mM Hepes (pH 7.4), 10 mM MgCl₂, 2 mM DTT, 0.02% Triton X-100, and 100 µM Na₃VO₄. The immunoprecipitates were then incubated with 50 µM adenosine triphosphate (ATP), 2 µCi [-³²P]ATP, and 1 mg/ml MBP at 30°C for 20 min. The reaction was terminated by adding ice-cold 20% trichloroacetic acid (TCA) and 60 mM Na-pyrophosphate. The precipitates were collected on nitrocellulose filters and washed twice with 5% TCA. The filters were then dried and the radioactivity counted. Each sample was assayed in duplicate. MAP kinase activity was calculated from the quantity of MBP phosphorylated (pmol/mg protein/min), and was expressed as a ratio to control activity.

Western Blot Analysis of MAP Kinase Activation

Equal amounts of proteins (15 µg) from each sample prepared as described previously were separated on a 10% acrylamide gel by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were then transferred to a nitrocellulose membrane. The membrane was blocked for 2 h at room temperature with 2% BSA in TBS-T buffer (20 mM Tris, 500 mM NaCl, 0.05% Tween 20). The blot was then incubated with the anti-phospho-specific MAP kinase antibody (1:1,000 dilution) at 4°C overnight, followed by incubation for 1 to 2 h with the secondary antibody (1:2,000 dilution) of horseradish peroxidase-conjugated antirabbit IgG (Bio-Rad). The immunoblot was visualized through enhanced chemiluminescence. The same blot was subsequently stripped and reprobed with anti-ERK₁, anti-JNK₁, or anti-p38 antibodies as internal controls. The results were quantified by densitometry of autoradiograms, using a GS-670 imaging densitometer (Bio-Rad). Increases in MAP kinase activity were calculated as the ratios of phosphorylated MAP kinases to total MAP kinases.

Northern Blot Analysis

Total RNA was isolated with a phenol-chloroform method described by Ausserer (14), and was quantitated spectrophotometrically at 260 and 280 nm. RNA samples (10 µg) were separated by gel electrophoresis on a denaturing agarose-formaldehyde gel and transferred to nylon membranes. The membranes were then baked at 80°C for 2 h and hybridized with ³²P-labeled c-jun or fra-1 cDNA probes. To normalize the signals, membranes were stripped and reprobed with Chinese hamster ovary B (CHOB) cDNA. The results were quantified with a GS-670 imaging densitometer (Bio-Rad), and the target mRNA/CHOB ratios were calculated.

Data Analysis

Data are presented as means ± SE from three to six individual experiments. Statistical significance was determined with paired or unpaired one-tailed Student's t tests, with P < 0.05 considered significant.

	Results

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Characterization of H₂O₂-induced MAP Kinase Activation in PASMC

The ability of H₂O₂ to activate MAP kinases was investigated in PASMC. Growth-arrested PASMC were exposed to various concentrations (10 to 1,000 µM) of H₂O₂ for 15 min, and MAP kinase activation was determined by both Western blot analysis and assay of immune-complex kinase activity. In Figure 1A, cell lysates (15 µg protein) from H₂O₂-treated cells were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Two distinct bands of 44 kD and 42 kD, corresponding to ERK₁ and ERK₂, were identified with the anti-phospho-specific MAP kinase antiserum. The increase in density of protein at positions corresponding to 42 kD and 44 kD indicated increased phosphorylation of ERKs at their tyrosine residues, and therefore their activation. Under the same experimental conditions, FBS (10%) stimulated MAP kinase phosphorylation by an average of 7-fold over basal levels. Compared with controls, cells treated with 100, 200, 400, and 1,000 µM H₂O₂ showed a concentration-dependent increase in the activated forms of both ERK₁ and ERK₂ (Figure 1A). Exposure of cells to 400 µM H₂O₂ for 15 min increased MAP kinase phosphorylation 2.8-fold.

View larger version (20K): [in this window] [in a new window]   View larger version (11K): [in this window] [in a new window]   Figure 1.   H2O2 activates MAP kinases in a concentration-dependent manner. Growth-arrested PASMC were stimulated with H2O2 at indicated concentrations or with 10% FBS for 15 min. MAP kinase activity was determined as described in MATERIALS AND METHODS. (A) Phosphorylated 42-kD and 44-kD ERKs were detected with anti-phospho-MAP kinase (anti-P-ERKs) antiserum. The same blot was stripped and probed with anti-ERK1 antibody to show equal loading of proteins. The immunoblots are representative of three independent experiments. (B) MAP kinase activity was determined in ERK1-immunocomplexes. Data shown are means ± SE of three to six experiments, each performed in duplicate. *P < 0.05 compared with controls.

To confirm that the increased phosphorylation of ERK₁ and ERK₂ kinases represented activated MAP kinase activity, a kinase activity assay was performed, using MBP as a substrate with ERK₁ immune complexes. The immune complexes were prepared by using a polyclonal antibody against ERK₁, which cross-reacts with ERK₂. As with its effect on the phosphorylation of ERK₁ and ERK₂ kinases, H₂O₂ increased the phosphorylation of MBP by immune complexes containing ERK₁ in a concentration-dependent manner. As shown in Figure 1B, a significant increase in ERK activity in response to extracellular H₂O₂ was seen at an H₂O₂ concentration as low as 10 µM. Treatment of PASMC with 400 µM H₂O₂ for 15 min significantly increased MBP phosphorylation by 2.2 ± 0.2 times over the baseline activity.

Figure 2 shows that H₂O₂ (200 µM) caused a time- dependent increase in ERK₁ and ERK₂ activation (Figure 2A) and MBP phosphorylation (Figure 2B), with similar kinetics. Both MAP kinase phosphorylation and activation reached maximal levels at 15 min. Compared with untreated cells, cells stimulated with H₂O₂ (200 µM) for 15 min showed a 2.2-fold increase in ERK₁ and ERK₂ activation (Figure 2A) and a 2.5 ± 0.4-fold increase in MAP kinase activity (Figure 2B), respectively. By 90 min after H₂O₂ challenge, MAP kinase activity was not significantly different from the basal values. Again, these observations show that the levels of ERK₁ and ERK₂ activation correlated well with the MBP phosphorylation in response to H₂O₂, indicating that H₂O₂-induced ERK₁ and ERK₂ phosphorylation does indeed reflect enhanced MAP kinase activity.

View larger version (22K): [in this window] [in a new window]   View larger version (19K): [in this window] [in a new window]   Figure 2.   Time course of H2O2-induced ERK activation. (A) Western blot analysis showing phosphorylated ERK1 and ERK2. Growth-arrested cells were incubated with 200 µM H2O2 for the indicated time periods. The immunoblots shown are representative of three independent experiments. (B) MAP kinase activity determined by MBP phosphorylation in ERK1-immunocomplexes. Each value is the mean ± SE of five individual experiments performed in duplicate. *P < 0.05 compared with untreated groups.

On the basis of the concentration-response and time-course data, PASMC were exposed to extracellular H₂O₂ at a final concentration of 400 µM for 15 min in most of the later experiments. To examine the effect of this concentration-time treatment on cell damage, trypan blue exclusion was used as a marker of cell viability. There was no difference between the percentage of control cells that excluded trypan blue (100 ± 0%) and cells treated with 400 µM H₂O₂ for 15 min (96.3 ± 3.7%, n = 3) that excluded it. In addition, under the treatment conditions, the total numbers of control and H₂O₂-treated cells were no different (13.5 ± 2.6 × 10⁴ versus 14.5 ± 1.7 × 10⁴ cells, n = 3).

The effects of H₂O₂ on the other two groups of MAP kinases, JNK and p38, were also examined in PASMC. Cells were treated with 200 µM H₂O₂ for the indicated time periods, and the activated forms of JNK and p38 were detected with anti-phospho-specific JNK and p38 MAP kinase antibodies, respectively. Whereas both p54 and p46 JNK were recognized by anti-phospho-specific JNK antibody, the anti-phospho-p38 antibody revealed a protein at a position of about 46 kD, as expected according to the manufacturer's protocol. As shown in Figures 3A and 3B, p46 JNK and p38 activity were increased in a time-dependent manner in response to H₂O₂ stimulation. The activation kinetics for p46 JNK and p38 were fast and were similar to that for activation of ERKs, with a maximal 3.5- and 2.5-fold increase for p46 JNK and p38, respectively, at 15 min. There was no significant change in p54 JNK activity during the time course studied.

View larger version (42K): [in this window] [in a new window]   Figure 3.   Time course of H2O2-induced JNK and p38 MAP kinase activation. Growth-arrested cells were incubated with 200 µM H2O2 for the indicated time periods. MAP kinase activity was determined as described in MATERIALS AND METHODS. In each panel, the same blot was probed separately with the antibodies indicated. P-JNK = phospho-JNK antibody; P-p38 = phospho-p38 antibody. Treatment of PASMC with H2O2 activated p46 JNK (A) and p38 (B) MAP kinases in a time-dependent manner. The immunoblots shown are representative of two independent experiments.

The specificity of H₂O₂ in activating MAP kinases was tested by treatment of PASMC with catalase (500 U/ml), a H₂O₂ scavenger. Cells treated with catalase alone did not show any change in MBP phosphorylation compared with controls. Coincubation of cells with catalase and 400 µM H₂O₂ completely (P < 0.05, n = 4) inhibited H₂O₂-mediated stimulation of MAP kinase activity (data not shown). These results suggest that the activation of MAP kinases is mediated by H₂O₂ rather than by nonspecific stimulation under the experimental conditions.

Effects of H₂O₂ on Early Gene Expression

Because an increase in proto-oncogene expression is one of the earliest markers for cell proliferation and differentiation, we investigated the effects of H₂O₂ on the expression of the early immediate genes c-jun and fra-1. Growth-arrested PASMC were treated with FBS (10%) for 15 min or with H₂O₂ (200 µM) for 0 to 2 h. Total RNA was then isolated and hybridized with ³²P-labeled c-jun or fra-1 cDNA. The level of RNA encoding c-jun or fra-1 increased markedly after cells were treated with 10% FBS for 15 min. Exposure to H₂O₂ caused a time-dependent increase in c-jun and fra-1 mRNA levels, with optimal expression at 60 min for c-jun and 90 min for fra-1. There were maximal 4.5-fold and 3.5-fold increases in c-jun and fra-1 mRNA expression, respectively, in response to H₂O₂ treatment. The expression of the housekeeping gene CHOB was not affected by H₂O₂ (Figure 4).

View larger version (71K): [in this window] [in a new window]   Figure 4.   Time course of H2O2 effects on early gene expression. PASMC were incubated with 10% FBS for 15 min or with 200 µM H2O2 for 0 to 2 h. Total RNA was isolated and the Northern blot was hybridized separately with probes for c-jun, fra-1, or cDNA for the housekeeping gene CHOB. The results are representative of two separate experiments.

Role of Protein Kinase C in H₂O₂-induced MAP Kinase Activation

H₂O₂ has been reported to stimulate both cytosolic and membrane-associated PKC activities (15). Additional studies have also suggested that activation of PKC is associated with the activation of MAP kinases (18). Accordingly, we designed experiments to examine the possible involvement of PKC in H₂O₂ stimulation of MAP kinase activity. In the first series of experiments, cells were pretreated with 100 nM PMA for 24 h to downregulate PKC, and were then exposed to H₂O₂ (400 µM) for 15 min. Downregulation of PKC caused a 90% inhibition of acute PMA-induced ERK₁ and ERK₂ activation, as shown in Figure 5. Pretreatment of cells with PMA did not significantly alter basal levels of ERK₁ and ERK₂ activation. However, PKC downregulation resulted in a 66% decrease in the activated forms of ERK₁ and ERK₂ in response to H₂O₂.

View larger version (21K): [in this window] [in a new window]   Figure 5.   Effects of PKC downregulation on H2O2-induced MAP kinase activation. Cells were or were not pretreated with 100 nM PMA for 24 h, and were then exposed to H2O2 (400 µM) or PMA (100 nM) for 15 min. PKC downregulation significantly reduced ERK activation by H2O2. Immunoblots are representative of three independent experiments.

The effects of calphostin C, a relatively specific PKC inhibitor, on H₂O₂-induced MAP kinase activation are shown in Figure 6. Pretreatment of PASMC with 1 µM calphostin C for 1 h clearly inhibited the phosphorylation of ERK₁ and ERK₂ in response to PMA or H₂O₂ stimulation. In control cells, PMA (100 nM) increased MAP kinase phosphorylation 3.8-fold over basal levels. This effect of PMA was decreased to 1.5-fold in cells pretreated with calphostin C. Similar treatment of cells with calphostin C inhibited MAP kinase activation in response to 400 µM H₂O₂ by 74%. However, the MAP kinase activation induced by 10% FBS was essentially preserved despite calphostin C pretreatment, suggesting that inhibition of the H₂O₂ response by calphostin C was not due to nonspecific effects. These results show that inhibition or downregulation of PKC activity markedly reduced H₂O₂-mediated stimulation of MAP kinase activity, indicating that the H₂O₂ effect on MAP kinases appears to be mediated by a PKC-dependent pathway.

View larger version (22K): [in this window] [in a new window]   Figure 6.   Effects of PKC inhibitor calphostin C on H2O2-induced MAP kinase activation. Growth-arrested PASMC were or were not pretreated with 1 µM calphostin C for 1 h under bright light, and were then stimulated with PMA (100 nM), H2O2 (400 µM), or FBS (10%) for 15 min. Calphostin C pretreatment significantly inhibited H2O2- or PMA-increased ERK activity, but did not affect FBS-induced ERK activation. Results shown are typical of three separate experiments.

Role of Calcium/Calmodulin in H₂O₂-induced MAP Kinase Activation

H₂O₂ has been shown to increase the intracellular Ca²⁺ concentration (21, 22) and to induce Ca²⁺ release from the sarcoplasmic reticulum (23). Increases in intracellular Ca²⁺ concentration, in turn, can induce Ca²⁺-dependent MAP kinase activation (24). Therefore, we investigated the possibility that increases in intracellular Ca²⁺ concentration are involved in MAP kinase activation by H₂O₂. We first examined the effects of extracellular Ca²⁺ on H₂O₂-mediated stimulation of MAP kinases. When PASMC were treated with H₂O₂ (400 µM, 15 min) in Ca²⁺-free incubation buffer containing 2 mM EGTA, the ability of H₂O₂ to stimulate the activation of ERK₁ and ERK₂ was decreased by approximately 56% (Figure 7A). Next, we investigated the effects of thapsigargin, a selective inhibitor of the sarcoplasmic reticulum Ca²⁺-ATPase, on the activation of MAP kinases by H₂O₂. Treatment of cells with thapsigargin has been shown to effectively deplete the inositol-1,4,5-triphosphate (IP₃)-releasable pool of intracellular Ca²⁺ (25). In the present study, when cells were pretreated with thapsigargin (1 µM) in calcium-free medium for 2 h, the ability of H₂O₂ to enhance MAP kinase activity was reduced by 45% compared with the activity in vehicle-treated controls (Figure 7A). However, the MAP kinase activation in response to FBS (10%, 15 min) was unaffected by the similar treatment of cells with thapsigargin (1 µM, 2 h) in the absence of extracellular Ca²⁺ (Figure 7B). Such results suggest that Ca²⁺ influx, as well as the release of Ca²⁺ from intracellular stores, may be important in the amplification of MAP kinase stimulation by H₂O₂.

View larger version (18K): [in this window] [in a new window]   View larger version (25K): [in this window] [in a new window]   Figure 7.   Effects of thapsigargin pretreatment on H2O2-induced activation of MAP kinases. (A) Growth-arrested PASMC were or were not pretreated with thapsigargin (TG; 1 µM) for 2 h prior to stimulation with 400 µM H2O2 for 15 min in the presence or absence of extracellular Ca2+. Removal of extracellular Ca2+ or thapsigargin pretreatment resulted in a respective 56% or 45% decrease on ERK activation by H2O2. (B) Similar treatment of cells with thapsigargin (1 µM, 2 h) in the absence of extracellular Ca2+ had no effect on MAP kinase activation by FBS (10%, 15 min). Data shown are representative of two independent experiments.

To determine the effects of calmodulin on H₂O₂-stimulated MAP kinase activity, PASMC were pretreated with the calmodulin antagonists W-7 or R 24571, which are chemically distinct from each other. Whereas pretreatment of cells with W-7 (50 µM) for 1 h inhibited H₂O₂- induced ERK₁ and ERK₂ activation by 41% (Figure 8A), R 24571 treatment (1 µM, 20 min) decreased the H₂O₂ effect by 44% (Figure 8B). Basal levels of MAP kinase activity were not significantly affected by W-7 or R 24571 treatment.

View larger version (26K): [in this window] [in a new window]   View larger version (35K): [in this window] [in a new window]   Figure 8.   Effects of calmodulin antagonists on H2O2-stimulated MAP kinase activation. Growth-arrested PASMC were pretreated with either (A) 50 µM W-7 for 1 h or (B) 1 µM R 24571 for 20 min prior to incubation with 400 µM H2O2 for 15 min. The activation of ERK1 and ERK2 was determined as described in MATERIALS AND METHODS. Treatment of cells with W-7 or R 24571 caused a respective 41% or 44% decrease in H2O2-stimulated ERK activity. Immunoblots are representative of four separate experiments.

Effects of Tyrosine Kinase Inhibition on H₂O₂-induced MAP Kinase Activation

Recent studies have shown that H₂O₂ causes activation of tyrosine kinases and regulates protein tyrosine phosphorylation (17, 21, 26). Therefore, it is possible that the observed increase in MAP kinase activity after stimulation with H₂O₂ is mediated by H₂O₂-induced activation of tyrosine kinases. To test this possibility, we performed experiments both in the presence and in the absence of the tyrosine-kinase inhibitor genistein. Western blot analysis (Figure 9) showed that pretreatment of PASMC with 92.5 µM genistein for 45 min resulted in a 44% reduction in the activation of MAP kinases by H₂O₂. This implies that tyrosine kinases may be involved in the H₂O₂-induced activation of ERK₁ and ERK₂ kinases.

View larger version (28K): [in this window] [in a new window]   Figure 9.   Effects of the tyrosine kinase inhibitor genistein on MAP kinase activation by H2O2. Growth-arrested cells were treated with 92.5 µM genistein for 45 min prior to H2O2 (400 µM) stimulation for 15 min. Phosphorylated 42-kD and 44-kD ERKs were detected with anti-phospho-MAP kinase antibody, as described in MATERIALS AND METHODS. Genistein pretreatment decreased H2O2 effect on ERK activation by 44%. Results are representative of three independent experiments.

	Discussion

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MAP kinases play a key role in mediating smooth muscle growth and proliferation, which are important in the pathogenesis of pulmonary arterial remodeling and pulmonary hypertension induced by reactive oxygen species (1, 3). The present study provides evidence that ERK₁ and ERK₂, p46 JNK, and p38, as well as expression of the early immediate genes c-jun and fra-1, are activated in response to H₂O₂ in vascular smooth muscle cells cultured from rat pulmonary arteries. Exposure of PASMC to H₂O₂ resulted in a time- and concentration-dependent increase in ERK₁ and ERK₂ phosphorylation and MAP kinase activity, as determined by Western blot analysis and assay of immune-complex kinase activity, respectively. Catalase, an enzyme that neutralizes H₂O₂, blocked the effects of H₂O₂ on MAP kinases, demonstrating the specificity of H₂O₂-mediated activation of MAP kinases. These findings are consistent with those reported in rat aortic smooth muscle cells (10, 11), neutrophils (5), NIH-3T3 cells (10, 27), Jurkat T cells (17), and bovine tracheal myocytes (26), in all of which H₂O₂-stimulated MAP kinase activity has been demonstrated.

Our study and other investigations (5, 10, 11, 17, 26, 27) provide solid evidence that H₂O₂ activates ERKs. However, Baas and Berk (28) observed that H₂O₂ stimulation (1 µM to 2 mM, 1 to 60 min) did not increase MAP kinase activity, although it stimulated aortic smooth muscle cell growth. Several possibilities would explain the discrepancy of these observations. First, the superoxide-generating systems in PASMC may be different from that in aortic smooth muscles. Potential sources of superoxide anion include cyclooxygenase, xanthine oxidase, reduced nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, nitric oxide synthase (NOS), and ionizing radiation. Superoxide generated from different sources may have distinct signal-transduction cascades and mediate different cellular responses (29). Studies of superoxide-generating systems are needed and may provide more information. Another likely explanation is that H₂O₂-induced MAP kinase activation is a cell-specific response. Work by Guyton and colleagues (10) clearly demonstrated that the same treatment with H₂O₂ induced a 10- to 20-fold increase in ERK₂ activity in different types of cells. Our study showed that H₂O₂ was a moderate stimulus for ERKs in PASMC, with a magnitude of activation of ERK₁ and ERK₂ of approximately 3-fold. On the other hand, cells of the same type (i.e., rat aortic smooth muscle cells) have been shown not to respond, to respond weakly (1.5-fold increase), or to respond potently (10-fold increase) to similar H₂O₂ treatment (200 µM, 10 to 20 min), depending on the culture and experimental conditions, such as culture medium, FBS concentration, and treatment buffer (10, 11, 28). In addition, we observed that MAP kinase activation in response to H₂O₂ is weaker in pulmonary smooth muscle cells at higher passage numbers. Depending on the number of cell passages, few or none of the effects of H₂O₂ on ERK activation could be observed in PASMC. The mechanisms underlying this finding are not understood. Furthermore, the different response of MAP kinase to H₂O₂ suggests that H₂O₂ may be able to utilize multiple pathways to produce mitogenic effects in different cells. Possible pathways may involve activation of tyrosine kinase and phospholipase A₂ (PLA₂), inhibition of intracellular cyclic adenosine monophosphate (cAMP) formation and PKA activity, or inhibition of protein tyrosine phosphatase activity.

Several observations in our study suggest that activation of ERK₁ and ERK₂ by H₂O₂ appears to be mediated through both PKC-dependent and -independent mechanisms. PKC downregulation significantly decreased H₂O₂ activation of MAP kinases by 66%. Calphostin C pretreatment resulted in a 74% inhibition of the H₂O₂ response without affecting FBS-induced MAP kinase activation. These data suggest that H₂O₂ may activate ERK₁ and ERK₂ primarily through the simulation of PKC. On the other hand, calphostin C and PKC downregulation were insufficient to abolish the effects of H₂O₂ on MAP kinase activation. This implies that activation of MAP kinases by H₂O₂ may also occur via a PKC-independent pathway. These observations are consistent with the reports of Abe and collegues (26) and Whisler and coworkers (17) that H₂O₂-induced activation of MAP kinases was both PKC-dependent and PKC-independent. These findings are also consistent with the reports of Rao and associates that early immediate gene (c-fos and c-jun) induction by H₂O₂ is mediated by both PKC-dependent and -independent mechanisms (7). Alternatively, H₂O₂ may cause MAP kinase activiation by stimulating PKC isoforms that are insensitive to either calphostin C or PMA treatment. Further studies are needed to identify the PKC isoforms involved in H₂O₂-mediated activation of MAP kinases.

Our results also suggest that H₂O₂-induced ERK₁ and ERK₂ activation may be mediated by Ca²⁺ influx, Ca²⁺- release, and Ca²⁺/calmodulin. We found that: (1) removal of extracellular Ca²⁺ caused a 56% inhibition of H₂O₂ effects on MAP kinase activation; (2) depletion of the intracellular IP₃-sensitive Ca²⁺ pool by thapsigargin reduced H₂O₂-mediated MAP kinase activation by 45% but had no effect on FBS response; and (3) treatment of cells with the calmodulin antagonist W-7 or calmidazolium chloride decreased H₂O₂ activation of MAP kinases by 41% and 44%, respectively. Taken together, these results indicate that Ca²⁺ influx, Ca²⁺ release from intracellular stores, and Ca²⁺/calmodulin-dependent kinases are probably involved in H₂O₂-mediated ERK₁ and ERK₂ activation in PASMC.

Additionally, our data show that treating cells with genistein, a tyrosine kinase inhibitor, decreased H₂O₂ stimulation of MAP kinase activity by approximately 44%. These results, in agreement with previous observations that H₂O₂ increases tyrosine phosphorylation of proteins in different cell types (17, 26, 30), suggest that the mechanisms by which H₂O₂ activates ERK₁ and ERK₂ may include activation of tyrosine kinases.

Beyond this, our results demonstrate that p46 JNK and p38 MAP kinases are also activated in response to H₂O₂. Compared with its activation of ERKs, H₂O₂ activates p46 JNK to a greater degree (3.5-fold) and p38 to an equal degree (2.5-fold). This observation is not unexpected, since JNKs and p38 MAP kinases are involved in transducing the signals involved in cellular responses to a variety of stresses (2). Similar effects of H₂O₂ on JNK and p38 MAP kinases have been previously reported in NIH 3T3 cells (10). However, the functional importance and the mechanisms of activation of JNKs and p38 MAP kinases by H₂O₂ remain to be determined.

In summary, the present study has shown that H₂O₂ activates ERK₁, ERK₂, p46 JNK, and p38 MAP kinases, and increases the expression of the early immediate genes c-jun and fra-1 in smooth muscle cells cultured from pulmonary arteries. This activation of MAP kinases by H₂O₂ is concentration- and time-dependent. It appears that the H₂O₂-induced ERK₁ and ERK₂ stimulation occurs primarily through a PKC-dependent pathway and is partly mediated by Ca²⁺/calmodulin and by activation of tyrosine kinases.