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CELLULAR AND MOLECULAR

Hydrogen Peroxide-Induced Extracellular Signal-Regulated Kinase Activation in Cultured Feline Ileal Smooth Muscle Cells

Department of Pharmacology, College of Pharmacy, Chung Ang University, Seoul, Korea

Received July 15, 2004; accepted August 20, 2004.

	Abstract

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H₂O₂ has been shown to act as a signaling molecule involved in many cellular functions such as apoptosis and proliferation. In the present study, we characterized the effects of H₂O₂ on the activation of mitogen-activated protein (MAP) kinases and examined the factors involved in the process of extracellular signal-regulated kinase (ERK) activation by H₂O₂ in ileal smooth muscle cells (ISMC). ISMC were cultured and exposed to H₂O₂. Western blot analysis was performed with phosphospecific MAP kinase antibodies. Potent activation of ERK and moderate activation of stress-activated protein kinase/c-Jun NH₂-terminal kinase occurred within 30 min of 1 mM H₂O₂ treatment. However, p38 MAP kinase was not activated by H₂O₂. The activation of ERK by H₂O₂ was reduced by the mitogen-activated/ERK-activating kinase inhibitor PD98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one], Ras inhibitor S-farnesylthiosalicylic acid, removal of extracellular Ca²⁺, depletion of the intracellular Ca²⁺ pool by thapsigargin, or pretreatment of ISMC with the calmodulin antagonist W-7. Also, H₂O₂-induced ERK activation was attenuated by a receptor tyrosine kinase inhibitor, tyrphostin 51, but not by down-regulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate or by a PKC inhibitor, GF109203X [3-[1-(dimethylaminopropyl)indol-3-yl]-4-(indol-3-yl)maleimide hydrochloride]. Growth factor receptor antagonist suramin pretreatment inhibited H₂O₂-induced ERK activation, highlighting a role for growth factor receptors in this activation. Furthermore, the ERK activation by H₂O₂ was blocked by pretreatment with either N-acetyl-cysteine, o-phenanthroline, or mannitol indicating that metal-catalyzed free radical formation may mediate the initiation of signal transduction by H₂O₂. These data suggest that short-term stimulation with H₂O₂ activates the signaling pathways of cell mitogenic effects which are thought to be a protective response against intestinal oxidative stress.

In contrast with the wide belief that H₂O₂ has only harmful functions due to its relentless production with the damaging nature, recent data have shown that H₂O₂ stimulates cell growth/proliferation and DNA synthesis in different types of cells, indicating that H₂O₂ acts as an important signaling molecule (Rao and Berk, 1992; Fiorani et al., 1995). Exposure of cells to exogenous H₂O₂ has been used to investigate multiple signaling pathways related to smooth muscle contraction (Jin and Rhoades, 1997) and proliferation (Baas and Berk, 1995) as well as apoptosis (Li et al., 1997).

The activation and phosphorylation of extracellular signal-regulated protein kinases (ERKs), one subtype of MAP kinases, is a pivotal step in the signal transduction cascade regulating cell proliferation by extracellular stimuli such as epidermal growth factors (EGF) and platelet-derived growth factor. Two major isoforms of ERK, p44 (ERK₁) and p42 (ERK₂), have been identified. In contrast, p38 MAP kinase and p46 to p54 MAP kinases (SAPK/JNK), two other subtypes of MAP kinases, mediate signals in response to cytokines and stress. It has been suggested that a major pathway involved in ERK₁ and ERK₂ stimulation in various types of cells requires the sequential activation of Ras, Raf, and mitogen-activated/ERK-activating kinase (MEK) (Herlaar and Brown, 1999).

It has been reported that intracellularly generated H₂O₂ stimulates tyrosine kinase or MAP kinase activities in several types of cells (Sundaresan et al., 1995; Guyton et al., 1996). Also, three major MAP kinase signaling cascades have been evaluated in pancreatic acinar cells in response to H₂O₂, menadione, and other physiologic stimuli (Dabrowski et al., 2000).

In the present study, exposure to exogenous H₂O₂ at physiological levels was used to characterize the effects of H₂O₂ on activation of MAP kinases including ERK₁/ERK₂, SAPK/JNK, and p38 MAP kinase in cultured ileal smooth muscle cells (ISMC). The results clearly indicate that H₂O₂ is a very potent activator of the ERKs in ISMC. We also investigated the involvement of various factors in the process of ERK activation by H₂O₂ in ISMC, including the roles of protein kinase C (PKC), Ca²⁺/calmodulin, receptor tyrosine kinase, MEK, and Ras, as well as effects of several antioxidants and a growth factor receptor antagonist in H₂O₂-induced ERK activation. The findings suggest that short-term oxidative stress induced by H₂O₂ activates the signal transduction of cell mitogenic effects which are thought to be a protective response against oxidant injury in ISMC.

	Materials and Methods

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Cultures of Ileal Smooth Muscle Cells. Adult cats of either sex weighing between 2.0 and 3.0 kg were anesthetized with ketamine (50 mg/ml/kg), and then the abdomen was opened with a midline incision. The ileum was excised and cleaned of fat tissue. Three-centimeter ileal segments were slipped over a glass rod, and the serosa and the longitudinal muscle layer were separated from the circular muscle layer by tangential stroking of the mesenteric attachment as described by Bitar and Makhlouf (1982). The ileal segments were then opened and cut into small pieces. The circular muscle layer was sliced 0.5-mm thick with a Stadie Riggs tissue slicer (Thomas Scientific Apparatus, Philadelphia, PA). The last slices containing mucosa and submucosa were discarded. The sliced tissue was then placed into DMEM supplemented with 50% FBS containing 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 µg/ml amphotericin B and incubated in a humidified atmosphere of 5% CO₂ and 95% air at 37°C. On the following day, fresh DMEM containing 10% FBS was added. Ten days later the tissue explants were removed, and the medium was changed with fresh DMEM containing 10% FBS. After reaching confluence, cells were detached with 1% trypsin in Hanks' balanced salt solution with bicarbonate. Cells were then counted, seeded at 1 x 10⁶ cells/ml on 100-mm culture dishes, and maintained in DMEM containing 10% FBS. The medium was changed every 48 h until the cells reached confluence. Experiments were performed on cells of passage 4.

Stimulation of MAP Kinases. When the cells reached confluence, they were serum starved by incubation in DMEM containing 0.1% FBS for 48 h to arrest cell growth and silence gene activity. The growth-arrested cells were then stimulated either with H₂O₂ for indicated time periods or at indicated concentrations. In addition, cells were stimulated with 1 mM H₂O₂ for 30 min in the presence or absence of different inhibitors. After incubation, the cells were washed twice with ice-cold PBS and harvested in 5 mM EDTA-PBS. The harvested cells were then maintained on ice for 10 min and centrifuged for 5 min at 1300 rpm. The pellets were washed with ice-cold PBS and recentrifuged. The obtained pellets were homogenized in a homogenizing buffer composed of 20 mM Tris(hydroxymethyl)aminomethane, 0.5 mM EDTA, 0.5 mM EGTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 10 mM -mercaptoethanol (pH 7.5). Sample homogenates were then sonicated (3 s, 5x) and centrifuged for 7 min at 12,500 rpm at 4°C to remove cellular debris, and the supernatants were collected.

Protein Determination. The protein concentration of the supernatant in each reaction vial was measured spectrophotometrically using the Bio-Rad assay (Bio-Rad, Hercules, CA). The absorption was monitored at a wavelength of 595 nm.

Western Blot Analysis of MAP Kinase Activation. Equal amounts of protein from each sample were resolved on a 10% SDS-polyacrylamide gel by electrophoresis. Rainbow prestained molecular mass markers (Amersham Biosciences Inc., Arlington Heights, IL) were also run in an adjacent lane to permit molecular mass determination. The separated proteins were transferred to a 0.45-µm nitrocellulose membrane in transfer buffer containing 25 mM Tris (pH 8.3), 192 mM glycine, and 20% (v/v) methanol using a power supply by Power Pac 1000 (Bio-Rad, Melville, NY). The membranes were incubated in a PBS buffer containing 5% nonfat dry milk for 1 h at room temperature to block nonspecific binding. After washing three times in PBS, the blots were incubated with 1:1000 phosphospecific p44/p42 MAP kinase (Tyr-202/Tyr-204) antibody, phospho-SAPK/JNK antibody, or p38 MAP kinase antibody (purchased from Cell Signaling Technology Inc., Beverly, MA) in a PBS solution containing 0.1% BSA at 4°C overnight. The membranes were washed using PBS containing 0.05% Tween 20 and then incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h. Immunoreactive bands were visualized by enhanced chemiluminescence (PerkinElmer Life and Analytical Sciences, Boston, MA) (Sohn et al., 1993). The same blot was subsequently stripped and reproved with p44/42 MAP kinase antibody, SAPK/JNK antibody, or p38 MAP kinase antibody. Developed films from enhanced chemiluminescence were scanned and analyzed densitometrically using Scion Image. Fold inductions in MAP kinases were calculated as the ratios of phosphorylated MAP kinases to total MAP kinases. Most of the other reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Cell Viability Assays. Trypan blue exclusion was used as a marker of cell viability. Under the same experimental conditions, the growth-arrested cells were treated with 1 mM H₂O₂ for the indicated time periods. At each time after incubation, cells were washed with Hanks' balanced salt solution and detached with 1% trypsin in PBS. Cells were stained with trypan blue solution and then counted using a phase contrast microscope (model ULWSD 0.30; Olympus, Tokyo, Japan), hemacytometer, and a counter. To prevent damage to cells, this experiment should be performed within 5 min after staining.

Data Analysis. The data are expressed as the means ± S.E.M. of n separate experiments, and the statistical differences between means were determined by Student's t test with p < 0.05 considered significant.

	Results

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Characterization of H₂O₂-Induced MAP Kinase Activation in ISMC. To characterize the response of the cultured cells to various doses of ROS donor H₂O₂, the growth-arrested ISMC were exposed for 30 min with 10^-6, 10^-5, and 10^-4 to 10^-3 M H₂O₂. In Fig. 1A, Western blot analysis showed a concentration-dependent increase in the activated forms of both ERK₁ and ERK₂. Those two distinct bands were identified with the phosphospecific MAP kinase antibody since the increase in density of protein at positions corresponding to 42 and 44 kDa indicated increased phosphorylation of ERKs at their tyrosine residues and therefore their activation. Under the same experimental conditions, 10% FBS-DMEM activated ERKs by 2.5-fold over basal levels. Exposure of cells to 10^-3 M H₂O₂ for 30 min significantly increased p44/42 MAP kinase phosphorylation by 2.8 ± 0.1 times over the level of control samples, which consisted of ISMC incubated without any stimulant (Fig. 1B).

View larger version (27K): [in this window] [in a new window]   Fig. 1. H2O2 activates ERKs in a concentration-dependent manner. A, Western blot analysis showing that phosphorylated 44- and 42-kDa ERKs were detected with anti-phospho-p44/42 MAP kinase antibody. Growth-arrested ISMC were stimulated with H2O2 at indicated concentrations or with 10% FBS-DMEM for 30 min. The same blot was stripped and probed with anti-p44/42 MAP kinase antibody to show equal loading of proteins. B, the integrated density of both bands (p44 and p42 MAP kinases), corresponding to active MAP kinase, was quantified and shown in the diagram. Each value is the mean ± S.E. of three individual experiments performed in duplicate. *, p < 0.05 compared with controls.

Figure 2A presents the time course of H₂O₂-induced ERK activation in ISMC. Incubation of ISMC with 1 mM H₂O₂ caused a time-dependent increase in the activation of ERKs. MAP kinase phosphorylation rapidly reached the maximal levels at 30 min, showing only slight activation at 15 min, and then slowly declined. Compared with control cells, cells exposed with 1 mM H₂O₂ for 30 min showed a 2.5 ± 0.1-fold increase in ERK₁ and ERK₂ activation (Fig. 2B).

View larger version (22K): [in this window] [in a new window]   Fig. 2. Time course of H2O2-induced ERK activation. A, Western blot analysis showing phosphorylated ERK1 and ERK2. Growth-arrested cells were incubated with 10-3 M H2O2 for the indicated time periods. Treatment of ISMC with H2O2 activated ERKs in a time-dependent manner. B, the integrated density of both bands (p44 and p42 MAP kinases), corresponding to active MAP kinases, was quantified and shown in the diagram. Each value is the mean ± S.E. of two individual experiments performed in duplicate. *, p < 0.05 compared with controls.

In most of the later experiments, therefore, ISMC were stimulated with exogenous H₂O₂ at a final concentration of 1 mM for 30 min according to the concentration-response and time course data. To investigate any harmful effect of this concentration and time treatment of H₂O₂ on cells, cell viability between H₂O₂-stimulated and -untreated cells was compared by using trypan blue methods. There was no difference between the percentages of control cells that excluded trypan blue (100 ± 0%) and cells treated with 1 mM H₂O₂ for 120 min (98.8 ± 1.2%, n = 4) that excluded it. In addition, under the treatment conditions, the total number of control and H₂O₂-treated cells were no different (25.2 ± 1.9 x 10⁵ versus 24.9 ± 0.4 x 10⁵ cells, n = 4, respectively).

The ability of H₂O₂ to activate SAPK/JNK was also evaluated in ISMC. Cells treated with 10^-6, 10^-5, and 10^-4 to 10^-3 M H₂O₂ for 30 min showed a concentration-dependent increase in the activated forms of SAPK and JNK, which were detected with phosphospecific SAPK/JNK antibody (data not shown). Incubation of ISMC with 10^-3 M H₂O₂ for 30 min caused a moderate increase by 1.6 ± 0.1 times over the level of control in the density of two bands of 54 and 46 kDa corresponding to SAPK and JNK.

Figure 3A shows a time course of H₂O₂-induced SAPK/JNK activation. Even though the time-dependent increase in the activated forms of SAPK/JNK was similar to the activation of p44/42 MAP kinases by H₂O₂ with maximal activation observed at 30 min, the activation of SAPK/JNK by H₂O₂ had a slight different characterization in time course. We found that incubation of ISMC with 1 mM H₂O₂ caused relatively rapid activation of JNK. An increase in the density of the band corresponding to active JNK was noticed at just 5 min. Thereafter, the JNK phosphorylation has been gradually increased, reaching a maximum at 30 min. Interestingly, the activation of JNK quickly declined, reaching close to the basal values at 60 min after an H₂O₂ challenge (Fig. 3B).

View larger version (45K): [in this window] [in a new window]   Fig. 3. A, time course of H2O2-induced SAPK/JNK activation. Western blot analysis shows that phosphorylated SAPK and JNK were detected with anti-phospho-SAPK/JNK antibody. Growth-arrested cells were incubated with 10-3 M H2O2 for the indicated time periods. Treatment of ISMC with H2O2 activated SAPK/JNK in a time-dependent manner. B, the density of active JNK band was quantified and shown in the diagram as a percentage of the average value at time 0. Each value is the mean ± S.E. of two individual experiments performed in duplicate. *, p < 0.05 compared with controls. C, Western blot analysis showing that phosphorylated p38 MAP kinase was not detected with anti-phospho p38 MAP kinase antibody. Growth-arrested cells were incubated with 10-3 M H2O2 for the indicated time periods. The immunoblots shown are representative of four independent experiments.

It has been recently reported that p38 MAP kinase was activated in pancreatic acini by H₂O₂ and menadione (Dabrowski et al., 2000). Therefore, the effect of H₂O₂ on p38 MAP kinase was also examined in cultured ISMC. In four independent experiments, there was, however, no significant change in p38 MAP kinase activation by 1 mM H₂O₂ during the time course studied, as detected with phosphospecific p38 MAP kinase antibody (Fig. 3C).

MEK Mediates the ERK Activation by H₂O₂ in ISMC. It has been known that in several different signaling cascades leading to ERK activation, MEK, a dual specific protein kinase, phosphorylates ERK₁ and ERK₂. To examine whether MEK is involved in H₂O₂-induced ERK activation, a specific MEK inhibitor was used in this study. Growth-arrested cells were treated with 10 µM 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) (Lee et al., 2002; Shin et al., 2002) for 40 min prior to 1 mM H₂O₂ stimulation for 30 min. Preincubation of PD98059 decreased the ERK activation by H₂O₂ in cultured ISMC (Fig. 4). The data suggest that MEK is an upstream regulator of MAP kinase, and the ability of H₂O₂ to stimulate ERK activation is mediated by MEK in ISMC.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Effects of MEK inhibitor PD98059 on H2O2-induced ERK activation. Growth-arrested cells were treated with 10 µM PD98059 for 40 min prior to 10-3 M H2O2 stimulation for 30 min. PD98059 pretreatment decreased the H2O2 effect on ERK activation.

PKC May Not Be Involved in H₂O₂-Induced ERK Activation. Several studies have suggested that MAP kinase activation is PKC-dependent (Seger and Krebs, 1995; Dabrowski et al., 1996; Zhang et al., 1998) and that H₂O₂ stimulates both cytosolic and membrane-associated PKC activities (Stauble et al., 1994). Therefore, we evaluated whether inhibition of PKC has any effect on the activation of MAP kinase by H₂O₂ in ISMC. Growth-arrested cells were pretreated with 100 nM PMA for 24 h to down-regulate PKC and were then stimulated with 1 mM H₂O₂ or 100 nM PMA for 30 min to activate PKC, since phorbol esters are known as potent activators of PKC as treated for the short time (Zhang et al., 1998). Although down-regulation of PKC caused about 90% inhibition of acute PMA-induced ERK activation, pretreatment of cells with PMA did not alter basal levels of ERK activation, as shown in Fig. 5A.

View larger version (31K): [in this window] [in a new window]   Fig. 5. PKC may not be involved in H2O2-induced ERK activation. A, effects of PKC down-regulation on H2O2-induced ERK activation. Cells were or were not pretreated with 100 nM PMA for 24 h and were then exposed to H2O2 (10-3 M) or PMA (100 nM) for 30 min. PKC down-regulation had no effect on ERK activation by H2O2. B, effects of PKC inhibitor GF109203X on H2O2-induced ERK activation. Growth-arrested ISMC were or were not pretreated with 20 µM GF109203X for 40 min and were then stimulated with PMA (100 nM) or H2O2 (10-3 M) for 30 min. Pretreatment of cells with GF109203X had no effect on ERK activation by H2O2.

To confirm that PKC is not involved in H₂O₂-induced ERK activation in ISMC, the effects of 3-[1-(dimethylaminopropyl)indol-3-yl]-4-(indol-3-yl)maleimide hydrochloride (GF109203X) (Dabrowski et al., 1997, 2000), a relatively specific PKC inhibitor, were also examined. Pretreatment of ISMC with 20 µM GF109203X for 40 min had no effect on ERK activation by H₂O₂ (Fig. 5B). These results imply that the signaling pathway of H₂O₂-induced ERK activation appears to be PKC-independent.

Calcium and Calmodulin Play a Role in H₂O₂-Induced ERK Activation. It has been reported that H₂O₂ induced increases in intracellular Ca²⁺ concentration and Ca²⁺ release from skeletal muscle sarcoplasmic reticulum (Favero et al., 1995). Also, Ca²⁺ influx and Ca²⁺/calmodulin have been shown to be involved in the activation of MAP kinases (Chao et al., 1992). Accordingly, we examined the possible involvement of Ca²⁺ in H₂O₂-induced ERK activation in ISMC. First, to investigate the effects of extracellular Ca²⁺ on H₂O₂-induced ERK activation, growth-arrested cells were treated with 1 mM H₂O₂ for 30 min in the Ca²⁺-free incubation buffer containing 2 mM EGTA (Zhang et al., 1998). As shown in Fig. 6A, the ability of H₂O₂ to stimulate the activation of ERKs was abolished. Second, we evaluated the effects of thapsigargin, a selective inhibitor of the sarcoplasmic reticulum Ca²⁺-ATPase, on the ERK activation by H₂O₂. It has been reported that treatment of cells with thapsigargin effectively depletes the inositol-1,4,5-triphosphate-releasable pool of intracellular Ca²⁺ (Sagara et al., 1992). In this study, pretreatment with 1 µM thapsigargin in the presence of extracellular Ca²⁺ for 2 h inhibited H₂O₂-induced ERK activation by 37%.

View larger version (24K): [in this window] [in a new window]   Fig. 6. A, effects of thapsigargin pretreatment on H2O2-induced ERK activation. Growth-arrested ISMC were or were not pretreated with 1 µM thapsigargin (TG) for 2 h prior to stimulation with 10-3 M H2O2 for 30 min in the presence or absence of extracellular Ca2+. Removal of extracellular Ca2+ or thapsigargin pretreatment resulted in a decrease on ERK activation by H2O2. B, effects of calmodulin antagonist on H2O2-induced ERK activation. Growth-arrested ISMC were pretreated with 50 µM W-7 for 1 h prior to incubation with 10-3 M H2O2 for 30 min. Treatment of cells with W-7 caused a decrease in ERK activation by H2O2.

We also investigated the effects of calmodulin on the phosphorylation of ERKs in response to H₂O₂ stimulation. Growth-arrested ISMC were pretreated with the calmodulin antagonists 50 µM N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) (Zhang et al., 1998) for 1 h prior to incubation with 1 mM H₂O₂ for 30 min. Pretreatment of ISMC with W-7 caused a significant decrease in ERK activation by H₂O₂ (Fig. 6B). The basal level of ERK activity was not affected by W-7 treatment in ISMC. Taken together, the data suggest that Ca²⁺/calmodulin as well as Ca²⁺ influx and the release of Ca²⁺ from intracellular stores seems to play a prominent role in the amplification of ERK stimulation by H₂O₂.

Growth Factor Receptor and Metal-Catalyzed Free Radical Formation Mediates the Initiation of Signal Transduction by H₂O₂. It has recently been reported that H₂O₂ stimulated tyrosine kinase activity and caused receptor tyrosine phosphorylation, suggesting that H₂O₂-induced mitogenic response is, in part, mediated through platelet-derived growth factor receptor in aortic smooth muscle cells (Jin et al., 2000). Among two classic tyrosine kinases, receptor and nonreceptor tyrosine kinases, receptor tyrosine kinases virtually are the growth factor receptors located in the inner side of the cytoplasmic membrane and subjected to dimerization and autophosphorylation upon activation. Therefore, we evaluated the effects of tyrphostin 51 on H₂O₂-induced ERK activation. Tyrphostin, a member of the genistein family of tyrosine kinase inhibitor, is specific to receptor tyrosine kinases (Majumdar et al., 1996). Pretreatment of ISMC with 90 µM tyrphostin 51 for 45 min prior to the addition of 1 mM H₂O₂ for 30 min significantly reduced ERK activation by H₂O₂ (Fig. 7A). The data imply that H₂O₂-induced ERK activation is partly mediated through growth factor receptor.

View larger version (25K): [in this window] [in a new window]   Fig. 7. A, effects of the receptor tyrosine kinase inhibitor on H2O2-induced ERK activation. Growth-arrested ISMC were pretreated with 90 µM tyrphostin 51 for 45 min prior to 10-3 M H2O2 stimulation for 30 min. The receptor tyrosine kinase inhibitor tyrphostin 51 pretreatment reduced ERK activation by H2O2. B, effects of suramin on H2O2-induced ERK activation. Growth-arrested ISMC were treated with suramin for 45 min prior to 10-3 M H2O2 stimulation for 30 min. Suramin inhibited ERK activation by H2O2.

Therefore, we assumed the possible involvement of growth factor receptors in the initiation of ERK activation by H₂O_2. The effects of suramin on H₂O₂-induced ERK activation in ISMC were examined since suramin has been known to block the binding of ligands to several growth factor receptors and to inhibit ERK₂ activation by epidermal growth factor or by ultraviolet C irradiation (Sachsenmaier et al., 1994). As shown in Fig. 7B, pretreatment of ISMC with 0.4 mM suramin for 45 min before the direct addition of 1 mM H₂O₂ blocked ERK activation. The inhibition of H₂O₂-induced ERK activation by 0.4 mM suramin is greater than that by 0.3 mM suramin pretreatment.

H₂O₂ has been shown to produce its effect through the activation of MAP kinase since catalase or N-acetyl-cysteine block MAP kinase activation (Sundaresan et al., 1995; Guyton et al., 1996). We therefore explored the effects of several antioxidants on ERK activation by H₂O₂ in ISMC, as shown in Fig. 8. First, pretreatment of ISMC with the glutathione precursor N-acetyl-cysteine (20 mM) blocked H₂O₂-induced ERK activation. The data suggest that oxidative stress initiates ERK activation by H₂O₂. Second, mannitol (100 mM), a free radical scavenger with specificity for hydroxyl radical (Guyton et al., 1996; Chuang et al., 2000), also abolished the ability of H₂O₂ to activate ERKs. Third, the iron chelator o-phenanthroline (100 µM) (Guyton et al., 1996; Regan et al., 2001) also significantly reduced H₂O₂-induced ERK activation. This implies that metal-catalyzed reactions are required for ERK activation by H₂O₂. In fact, the major mechanism of the hydroxyl radical (OH^.) generation inside cells is from H₂O₂ via the Fenton reaction (H₂O₂ + Fe²⁺ or Cu⁺ OH^- + OH^. + Fe³⁺ or Cu²⁺). Besides, Fe²⁺ or Cu⁺ can be readily regenerated by cellular electron donors, such as reduced nicotinamide-adenine dinucleotide phosphate (NADPH), ascorbate, and various thiol compounds (Stadtman and Berlett, 1998). Accordingly, these data indicate that H₂O₂ can partly convert via metal-catalyzed reactions to other oxygen-derived free radical species including hydroxyl radical, and the oxidation by this free radical initiates signal transduction inducing to ERK activation. In addition, the findings provide that the ERK activation is mediated by H₂O₂ rather than by nonspecific stimulation under the experimental conditions since cells treated with an antioxidant alone did not show any change in ERK phosphorylation compared with controls.

View larger version (31K): [in this window] [in a new window]   Fig. 8. Role of free radicals in H2O2-induced ERK activation. A, growth-arrested ISMC were pretreated with N-acetyl-cysteine (20 mM), mannitol (100 mM), or o-phenanthroline (100 µM) for 2 h prior to 10-3 M H2O2 stimulation for 30 min. Treatment of ISMC with free radical inhibitors decreased ERK activation by H2O2. The inhibitors alone did not activate ERKs. B, the integrated density of both bands (p44 and p42 MAP kinases), corresponding to active MAP kinases, was quantified and shown in the diagram. Each value is the mean ± S.E. of three individual experiments performed in duplicate. * and #, p < 0.05 compared with controls and with H2O2-treated cells, respectively.

H₂O₂-Induced ERK Activation May Occur through Ras-Dependent Pathways. The membrane-associated small (21 kDa) G protein Ras is important in activation of MEK/ERK cascades by many types of stimuli. However, MAP kinase activation can also occur through Ras-independent pathways (Kolch et al., 1993). Therefore, we examined the inhibitory effects of S-farnesylthiosalicylic acid (FTS) (Gana-Weisz et al., 1997) on H₂O₂-induced ERK activation in ISMC to check whether Ras is a component of the signaling transduction leading to ERK activation. Growth-arrested ISMC were pretreated with 50 µM FTS for 24 h prior to incubation with 1 mM H₂O₂ for 30 min. Significant inhibition of ERK activation in ISMC by FTS was observed. The FTS did not, however, cause a decrease in the total amount of ERKs (Fig. 9). The data implies an involvement of Ras in mediating H₂O₂-induced ERK activation.

View larger version (16K): [in this window] [in a new window]   Fig. 9. Effects of Ras antagonist FTS on H2O2-induced ERK activation. Growth-arrested ISMC were pretreated with 50 µM S-farnesylthiosalicylic acid for 24 h prior to 10-3 M H2O2 stimulation for 30 min. Treatment of ISMC with Ras antagonist FTS decreased ERK activation by H2O2.

	Discussion

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In this study, we demonstrate that short-term treatment of ISMC with H₂O₂ causes a significant increase in the density of protein at positions corresponding to 42 and 44 kDa, indicating the increased phosphorylation of ERKs. Cell viability in the ISMC treated with 1 mM H₂O₂ showed no difference from that of controls at up to a 2-h time point, whereas treatment of ISMC with 1 mM H₂O₂ for 48 h finally induces the significant decrease in the cell viability. Our data indicate that short-term stimulation with H₂O₂ activates the signaling pathways of cell mitogenic responses. Convincing evidence has been presented to show the opposing effects of H₂O₂ on cell growth by stimulating proliferation (Guyton et al., 1996; Goldkorn et al., 1998) and triggering apoptosis (Li et al., 1997). These dual effects might depend on the time of H₂O₂ treatment, the concentration of H₂O₂, and the types of cells. We also showed that moderate activation of SAPK/JNK occurred within 30 min of 1 mM H₂O₂ treatment in ISMC in a time-dependent manner. Our findings are consistent with previous observations that ERK and SAPK/JNK were activated by H₂O₂ in different cell types including rat aortic smooth muscle cells (Guyton et al., 1996), pulmonary arterial smooth muscle cells (Zhang et al., 1998), and bovine tracheal myocytes (Abe et al., 1994). On the contrary, there has been a report in which H₂O₂ stimulation (1 µM to 2 mM, 1-60 min) did not increase MAP kinase activity, although it stimulated aortic smooth muscle cell growth (Baas and Berk, 1995).

Our results, interestingly, provided that p38 MAP kinase was not activated at any time period when ISMC were stimulated with 1 mM H₂O₂ for 5, 15, 30, 60, and 120 min. These observations are different from those findings of the previous studies since p38 MAP kinase activity was increased by H₂O₂ stimulation in aortic smooth muscle cells (Guyton et al., 1996) and pulmonary arterial smooth cells (Zhang et al., 1998). The first possible explanation for such discrepancy is that H₂O₂-induced MAP kinase activation is a cell-specific response. Guyton et al. reported that H₂O₂ stimulation under the same experimental conditions induced ERK activation with various extents in different types of cells. Experimental conditions, such as culture medium, FBS concentration, and treatment buffer could affect H₂O₂-induced MAP kinase activation (Baas and Berk, 1995; Guyton et al., 1996). Another explanation is that superoxide-generating systems in ISMC may differ from those in other types of cells. A recent report has suggested that superoxide, generated from different sources such as cyclooxygenase, xanthine oxidase, NADPH oxidase, etc., may have distinct signal transduction pathways and mediate different cellular responses (Lander, 1997). We also observed that ERK activation in response to H₂O₂ is weaker in ISMC at higher passage numbers. Although, the mechanisms underlying this observation are not fully understood, it seems to be deterioration of the cell protective mechanism against oxidant injury with aging as a broad range of view since ERK has been shown to play a critical role in cell survival.

PKC is present in the cell cytoplasm and upon agonist stimulation rapidly translocates to the particulate or membrane fraction observed by Western blot analysis (Sohn et al., 1997a,b). Our findings suggest that H₂O₂-induced ERK activation may not be mediated through PKC-dependent mechanisms. These are consistent with recent reports that, in pancreatic acinar cells, ROS activates MAP kinases through a PKC-independent mechanism (Dabrowski et al., 2000). On the other hand, in some studies, activation of the MAP signaling cascade was PKC-dependent (Seger and Krebs, 1995; Dabrowski et al., 1996). Interestingly, there have been several reports suggesting that H₂O₂-induced activation of MAP kinases was mediated by both PKC-dependent and -independent mechanisms (Abe et al., 1994; Zhang et al., 1998). Possible interpretation for the discrepancy of these observations is that H₂O₂ may be able to utilize multiple pathways to produce mitogenic effects depending on cell types. Also, H₂O₂ may cause MAP kinase activation by stimulating PKC isoforms that are insensitive to either GF109203X or PMA treatment.

Our investigation provides evidence that H₂O₂-induced ERK activation appears to be mediated by Ca²⁺ influx, Ca²⁺ release from intracellular stores, and Ca²⁺/calmodulin. The data imply that the Ca²⁺ influx triggers the intracellular Ca²⁺ channel opening responsible for releasing Ca²⁺ from the intracellular stores because the removal of extracellular Ca²⁺ abolished H₂O₂ effects on ERK activation close to the basal level. The process of Ca²⁺-induced Ca²⁺ release is important in regulating the way in which cells mobilize Ca²⁺ from their internal stores. The most important diffusible messenger for activating the intracellular Ca²⁺ stores is Ca²⁺ itself. The data suggest that the release of Ca²⁺ from intracellular stores is also critical to the amplification of ERK stimulation by H₂O₂, since depletion of the intracellular Ca²⁺ pool by thapsigargin in the presence of extracellular Ca²⁺ reduced ERK activation by 37%. These findings are consistent with the previous study that Ca²⁺ influx and Ca²⁺/calmodulin were involved in H₂O₂-induced activation of MAP kinases in pulmonary artery smooth muscle cells (Zhang et al., 1998).

Activation of tyrosine kinase is one of the most early events in response to a variety of growth stimulators. Inhibition of tyrosine kinase not only blocks mitogen-stimulated growth response but also leads to cell apoptosis, suggesting tyrosine kinase plays a key role in the balance between cell growth and death (Knebel et al., 1996). H₂O₂ has also been shown to activate receptor tyrosine kinases, such as fibroblast growth factor receptors and EGF receptors (Rao, 1997; Goldkorn et al., 1998), as well as several nonreceptor tyrosine kinases, such as p56^lck and p72^syk (Schieven et al., 1993; Hardwick and Sefton, 1995). Our data imply that the activation of growth factor receptor may play a role in H₂O₂-induced ERK activation, since H₂O₂-induced ERK activation was inhibited by receptor tyrosine kinase inhibitor, tyrphostin 51. In the future study, the data need to be validated by dominant-negative approaches such as dominant-negative EGF receptor. It has been known that mitogen-stimulated signal cascade leading to ERK activation is initiated through the interaction of peptide growth factors with their receptors (Guyton et al., 1996). Many growth factor receptors have cysteine-rich motifs, the oxidation of which can simulate ligand binding (Heldin, 1995). Through receptor activation mediated by sulfhydryl oxidation, H₂O₂ has been postulated to mimic the actions of insulin and other receptor-binding proteins (Schieven et al., 1994). Our results show that suramin blocked H₂O₂-induced ERK activation, suggesting that oxidation of such cell surface receptors may mediate signal initiation by H₂O_2. We confirmed the sulfhydryl reactivity of the oxidant signal generated from H₂O₂ since pretreatment with the glutathione precursor, N-acetyl-cysteine, abolished the ability of H₂O₂ to activate ERK. Free radical species generated from H₂O₂ may directly oxidize and thereby activate cell surface receptors, although the oxidative modification of other molecules may also function in the regulation of ERKs by H₂O₂. Our findings suggest that free radicals or other redox mechanisms may constitute a pivotal component of the signaling cascade to ERK activation normally utilized by growth factors and other stimuli.

Ras proteins function as intermediates in signaling pathways activated by a variety of cell-surface receptors involved in cell growth and differentiation (Ullrich and Schlessinger, 1990; Fields et al., 1996). Ras has been known to activate several signal transduction pathways including the Raf-1/MEK/ERK cascade and the MEK kinase/JNK cascade (Herlaar and Brown, 1999). To transduce growth signals, Ras must be anchored to the inner surface of the plasma membrane. FTS might affect Ras membrane association and interfere specifically with Ras functions, thereby interfering with Ras-dependent signals associated with cell growth and proliferation. It has been demonstrated that inhibition of the Ras-dependent Raf-1 MAP kinase cascade is achieved by FTS (Gana-Weisz et al., 1997). They suggest that FTS affect various types of Ras proteins, unlike specific farnesyltransferase inhibitors. Our study also showed that pretreatment of ISMC with FTS significantly inhibited H₂O₂-induced ERK activation in ISMC. The data implies that Ras appears to be involved in mediating H₂O₂-induced ERK activation. Such Ras involvement has been proposed by Guyton and colleagues since H₂O₂-induced ERK activation was abolished in PC12 cells by inducible or constitutive expression of the dominant-negative Ras-N-17 allele (Guyton et al., 1996).

In summary, we focused on the cellular consequences of modulating the ERK signaling cascade induced by H₂O₂ in ISMC. Our data imply that H₂O₂ undergoes metal-catalyzed conversion to a hydroxyl-radical-like species and that oxidation by this free radical initiates signal transduction leading to ERK activation. In these signaling pathways to ERK activation by H₂O₂, receptor tyrosine kinase, Ca²⁺- and calmodulin-dependent kinase, Ras, and MEK are probably involved. The present study provides insight into mechanisms of MAP kinase regulation by H₂O₂ and suggests that ERK signaling pathways play a critical role in controlling cellular protection in the early stage in response to oxidative stress.

	Footnotes

 
doi:10.1124/jpet.104.074401.

ABBREVIATIONS: ROS, reactive oxygen species; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; EGF, epidermal growth factor; SAPK, stress-activated protein kinase; JNK, c-Jun NH₂-terminal kinase; MEK, mitogen-activated/ERK-activating kinase; ISMC, ileal smooth muscle cells; PKC, protein kinase C; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; PD98059, 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; PMA, phorbol 12-myristate 13-acetate; GF109203X, 3-[1-(dimethylaminopropyl)indol-3-yl]-4-(indol-3-yl)maleimide hydrochloride; W-7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; FTS, S-farnesylthiosalicylic acid.

Address correspondence to: Professor Uy Dong Sohn, 221 Heuksukdong, Dongjakgu, Department of Pharmacology, College of Pharmacy, Chung Ang University, Seoul 156-756 Korea, Republic of Korea. E-mail: udsohn{at}cau.ac.kr

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