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Am. J. Respir. Cell Mol. Biol., Volume 18, Number 4, April 1998 562-569

Hydrogen Peroxide Activates Extracellular Signal-regulated Kinase via Protein Kinase C, Raf-1, and MEK1

Mark K. Abe, Sreedharan Kartha, Alla Y. Karpova, Jing Li, Pai T. Liu, Wen-Liang Kuo, and Marc B. Hershenson

Department of Pediatrics, Department of Pharmacological and Physiological Sciences, University of Chicago, Chicago, Illinois


    Abstract
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have previously demonstrated that hydrogen peroxide (H2O2) treatment of bovine tracheal myocytes increases the activity of extracellular signal-regulated kinases (ERK), serine/threonine kinases of the mitogen-activated protein (MAP) kinase superfamily thought to play a key role in the transduction of mitogenic signals to the cell nucleus. Moreover, H2O2-induced ERK activation was partially reduced by pretreatment with phorbol 12,13-dibutyrate, which depletes protein kinase C (PKC). In this study, we further examined the signaling intermediates responsible for ERK activation by H2O2 in airway smooth muscle, focusing on MAP kinase/ERK kinase (MEK), a dual-function kinase which is required and sufficient for ERK activation in bovine tracheal myocytes; Raf-1, a serine/threonine kinase known to activate MEK; and PKC. Pretreatment of cells with inhibitors of MEK (PD98059), Raf-1 (forskolin), and PKC (chelerythrine) each reduced H2O2-induced ERK activity. In addition, H2O2 treatment significantly increased both MEK1 and Raf-1 activity. No activation of MEK2 was detected. Together these data suggest that H2O2 may stimulate ERK via successive activation of PKC, Raf-1, and MEK1.


    Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have previously demonstrated that treatment of bovine tracheal myocytes with hydrogen peroxide (H2O2) increases the activity of extracellular signal-regulated kinases (ERK), serine/threonine kinases of the mitogen-activated protein (MAP) kinase superfamily thought to play a key role in the transduction of mitogenic signals to the cell nucleus (1). Concentrations of H2O2 as low as 25 µM were effective in increasing ERK activity. Depletion of protein kinase C (PKC) by phorbol ester pretreatment significantly inhibited H2O2-induced ERK activation, suggesting that ERK activity was PKC-dependent.

In the present study, we examined H2O2-induced ERK activation in bovine tracheal myocytes following pretreatment with inhibitors of MAP kinase/ERK kinases (MEK), dual-function kinases thought to be the only known activators of ERK; Raf-1, a serine/threonine kinase known to activate MEK, and PKC, which has been demonstrated to activate Raf-1 by direct phosphorylation (2). In addition, we measured the effects of H2O2 treatment on MEK1, MEK2, and Raf-1 activities. Our data suggest that in airway smooth muscle, H2O2 may stimulate ERK via successive activation of PKC, Raf-1, and MEK1.

In related studies, we found that the hydroxyl radical scavenger N-(2-mercaptoproprionyl)glycine inhibits H2O2-induced ERK activation, and that H2O2 treatment increases the activity of another member of the MAP kinase superfamily, stress-activated protein kinase (SAPK), also known as Jun amino-terminal kinase or JNK (5). These studies suggest that H2O2 activates the ERK pathway via the formation of hydroxyl radical, and that H2O2 may stimulate alternative forms of MAP kinase in airway smooth muscle.

    Materials and Methods
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials

H2O2, anti-human alpha -smooth muscle actin, N-(2-mercaptopropionyl)glycine, peroxidase-linked goat antirabbit IgG, o-phenanthroline, protein A sepharose beads, forskolin, phorbol 12,13-dibutyrate, and myelin basic protein (MBP) were purchased from Sigma Chemical (St. Louis, MO). Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and ERK1-GST agarose conjugate were obtained from Upstate Biotechnology (Lake Placid, NY). An enhanced chemiluminescence kit and [gamma -32P]-ATP were obtained from DuPont/NEN Research Products (Wilmington, DE). For in vitro phosphorylation assays, polyclonal antibodies against ERK2, Raf-1, MEK1, and MEK2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A monoclonal antibody against MEK2 was purchased from Transduction Laboratories (Lexington, KY). cDNAs encoding a kinase-inactive MEK1 and a glutathione S-transferase (GST)-JNK fusion protein were provided by Dr. Gary Johnson (6) and Dr. James Posada (7), respectively. An anti-MAP kinase antiserum (Ab283) was provided by Dr. Marsha Rosner (1), and an antibody against phosphorylated ERK was purchased from Promega (Madison, WI). PD98059 was purchased from New England Biolabs (Beverly, MA). Chelerythrine was obtained from LC Laboratories (Woburn, MA). Hela cells were purchased from American Tissue Type Collection (Rockville, MD).

Cell Culture

Bovine tracheal smooth muscle cells were isolated as described previously (1). Myocytes of passage number 5 or less were studied. Confluent cultures exhibited the typical "hill and valley" appearance and showed specific immunostaining for alpha -smooth muscle actin. At 24 h prior to each experiment, bovine tracheal myocytes and Hela cells were serum-starved in Dulbecco's modified Eagle's medium without serum.

General Methods for Immune Complex Assays

ERK2, MEK1, MEK2, and Raf-1 kinase activities were assessed by in vitro phosphorylation assay. Bovine tracheal myocytes were grown to confluence in 100-mm culture dishes and serum-starved for 24 h. After treatment (H2O2 for 10 min in the presence or absence of various inhibitors), cells were washed twice with phosphate-buffered saline (150 mM NaCl, 0.1 M phosphate, pH 7.5) and incubated in the appropriate lysis buffer (30 min at 4°C). Insoluble materials were removed by centrifugation (14,000 rpm for 10 min at 4°C). Unless otherwise noted, 200 µg of lysate protein were then incubated with 25 µl of protein-A sepharose beads precoupled with the relevant antibody. After successive washes in lysis buffer and kinase buffer, the immune complexes were incubated with kinase buffer supplemented with [gamma -32P]-ATP and the appropriate substrate (20 min at 30°C). Reactions were terminated by adding Laemmli buffer and boiling. Samples were resolved on a 10% sodium dodecyl sulfate (SDS) gel and the proteins transferred to a nitrocellulose membrane using a semi-dry transfer apparatus (Hoefer, South San Francisco, CA). After Ponceau staining, the membrane was exposed to film and substrate phosphorylation was measured by digital optical scanning and image analysis (Jandel Scientific, San Rafael, CA).

ERK2 Kinase Assay

ERK2 kinase activity was assessed by immunoprecipitation using a polyclonal anti-ERK2 antibody, followed by in vitro phosphorylation assay using MBP as a substrate. The lysis buffer contained 50 mM Tris-HCl, pH 7.5; 1% Triton X-100; 40 mM beta -glycerophosphate, 100 mM NaCl, 50 mM NaF; 2 mM ethylenediamenetetraacetic acid (EDTA); 200 µM Na3VO4; and 0.2 mM phenylmethylsulfonyl fluoride (PMSF). Lysates were incubated with antibody overnight at 4°C. The immune complexes were washed 4 times with lysis buffer and once with kinase buffer containing 20 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid (Hepes) (pH 7.4), 10 mM MgCl2, 1 mM dithiothreitol (DTT), 200 µM Na3VO4, and 10 mM p-nitrophenyl phosphate. Immune complexes were resuspended in a final volume of 40 µl kinase buffer with 0.25 mg/ml MBP and 5 µCi [gamma -32P]-ATP.

MEK1 and MEK2 Kinase Assays

MEK1 and MEK2 kinase activities were assessed by immunoprecipitation with a polyclonal antibody against MEK1 and monoclonal antibody against MEK2, respectively, followed by in vitro phosphorylation assay using a recombinant, bacterially expressed, kinase-inactive ERK1 as substrate. The Triton X-100 lysis buffer (TLB) consisted of 20 mM Tris HCl (pH 7.9), 137 mM NaCl, 5 mM EDTA, 1 mM ethyleneglycol-bis-(beta -aminoethyl ether)-N-N'-tetraacetic acid (pH 8.0), 10% glycerol, 1% Triton X-100, 0.2 mM PMSF, 1 µg/ml aprotinin, 20 µM leupeptin, 1 mM Na3VO4, 10 mM NaF, 1 mM tetrasodium pyrophosphate, 1 mM beta -glycerophosphate (pH 7.4), and 0.01 g/ml p-nitrophenylphosphate. Lysates (200 µg for MEK1 assays, 600 µg for MEK2 assays) were incubated with protein-A precoupled with antibody for 1 h at 4°C. Immune complexes were washed once with TLB, twice with TLB with 0.5 M NaCl, and once with kinase buffer consisting of 40 mM Hepes (pH 7.8), 10 mM MgCl2, and 1 mM DTT. Next, immune complexes were incubated in kinase buffer supplemented with 20 µM ATP (final concentration), 10 µCi [gamma -32P]-ATP, and recombinant kinase-inactive ERK1 (approximately 2.5 µg/sample). The MEK2 activity of cultured Hela cells was also assessed.

Raf-1 Kinase Assay

Raf-1 kinase activity was assessed by immunoprecipitation with an anti-Raf-1 antibody, followed by in vitro phosphorylation assay using a recombinant, bacterially expressed, kinase-inactive MEK1 as substrate. The lysis buffer consisted of 10 mM Tris HCl, pH 7.4; 1% Triton X-100; 1 mM EDTA; 150 mM NaCl; 50 mM NaF; 0.1% bovine serum albumin; 20 µg/ml aprotinin; 200 µM Na3VO4; and 0.2 mM PMSF. Lysates were incubated with antibody for 90 min at 4°C; protein-A sepharose beads were then added for an additional 30 min. Each pellet was then washed twice with lysis buffer, twice with PAN (10 mM Pipes, pH 7.0, 20 µg/ml aprotinin, and 100 mM NaCl) plus 0.5% NP-40, and twice with PAN alone. Pellets were then resuspended in 20 µl of kinase buffer (20 mM Pipes, pH 7.0, 10 mM MnCl2, 20 µg/ml aprotinin, and 200 µM Na3VO4) containing approximately 1 ng of kinase-inactive MEK1 and 5 µCi [gamma -32P]-ATP.

Western Analysis of MEK2 Expression

As detailed above, MEK2 activity was assessed in bovine tracheal myocytes and Hela cells by immunoprecipitation with a monoclonal antibody against MEK2 followed by in vitro phosphorylation using a kinase-inactive ERK1 as substrate. To confirm the presence of MEK2 in bovine tracheal myocyte and Hela immunoprecipitates, nitrocellulose membranes were probed with a polyclonal anti-MEK2 antibody. Signals were amplified and visualized using peroxidase-linked goat antirabbit IgG and enhanced chemiluminescence.

ERK In-gel Kinase Renaturation Assays

In some experiments, the ERK activity of whole cell lysates was assessed by in-gel kinase renaturation assay, as described elsewhere (1). To confirm that the 44- and 42-kD MBP kinases identified by kinase renaturation assays were indeed ERK1 and ERK2, the lysates of cells treated with PDGF were partially purified by MonoQ ion exchange chromatography. The 44- and 42-kD MBP kinases were identified by an anti-MAP kinase antiserum to be ERK1 and ERK2, respectively (data not shown).

Measurement of Phosphorylated ERK by Western Analysis

In some experiments, the ERK activity of whole cell lysates was estimated by determining the level of phosphorylated ERK. These experiments utilized a phospho-specific antibody (8) which recognizes ERK only when phosphorylated at Thr183 and Tyr185, which are required for full enzymatic activity (9).

Measurement of JNK Activation

JNK activity was determined by in vitro phosphorylation assay using a recombinant GST-Jun (1-79) fusion protein as substrate (7). Cell lysates were prepared in buffer A consisting of 25 mM Hepes (pH 7.5), 0.3 M NaCl, 5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 1 mM PMSF, 1 mM benzamidine, 20 µg/ml aprotinin, and 200 µM Na3VO4. The cleared lysates were then diluted 1:2 in buffer B consisting of 20 mM Hepes (pH 7.5), 50 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.05% Triton X-100, 1 mM PMSF, 1 mM benzamidine, 20 µg/ml aprotinin, and 200 µM Na3VO4, and 20 µg of GST-Jun (1-79) was added to each sample. After 2 h of incubation on ice, the complexes were washed 3 times in buffer B and resuspended in 30 µl of phosphorylation buffer consisting of 20 mM Hepes (pH 7.5), 2 mM DTT, 5 mM MgCl2, and 5 µCi of [gamma -32P]-ATP for 20 min at 30°C. Reactions were terminated by adding Laemmli buffer and boiling. Samples were resolved on a 10% SDS gel. Gels were stained with Comassie blue, destained in 40% methanol and 5% acetic acid, and dried. Autoradiograms were developed by exposing film to the dried gel. Quantitation of Jun phosphorylation by JNK was measured by optical scanning.

Inhibitors

We employed PD98059 [2-(2'-amino-3'-methoxyphenol)- oxanaphthalen-4-one] to inhibit the activation of MEK-1. Previous experiments in bovine tracheal myocytes have demonstrated PD98059 to inhibit both MEK1 and ERK activation while failing to inhibit the kinase activities of either Src or Raf-1 in these cells (10). PD98059 has also been shown to have no inhibitory activity for cAMP-dependent kinase, PKC, EGF receptor kinase, insulin receptor kinase, PDGF receptor kinase, phosphatidylinositol 3-kinase, Jun kinase, and p38 high osmolarity glycerol kinase (11). To inhibit Raf-1, we used forskolin (50 µM for 15 min). Previous studies have shown that forskolin decreases growth factor-induced Raf-1 activity in bovine tracheal myocytes (12). Forskolin, an activator of adenyl cyclase, inhibits Raf-1 activation via cAMP-responsive protein kinase A (13). Finally, we depleted PKC by pretreatment with phorbol 12,13-dibutyrate (200 ng/ml for 24 h) and inhibited PKC activity with chelerythrine chloride (1 µM for 30 min). Chelerythrine has an inhibitory potency for PKC of 0.66 µM (14).

To examine the requirement of hydroxyl radical for H2O2-induced activation of the ERK signaling pathway, we pretreated cells with two agents thought to inhibit the accumulation of hydroxyl radical in the cell. N-(2-mercaptoproprionyl)glycine is a free radical scavenger with specificity for hydroxyl radical, whereas the heavy metal chelator o-phenanthroline inhibits the metal-catalyzed production of hydroxyl radical from H2O2.

Statistical Analysis

The effects of H2O2 on ERK, MEK, and Raf-1 activities were determined by one-way analysis of variance (ANOVA). Differences identified by ANOVA were pinpointed by Student-Newman-Keuls' multiple range test.

    Results
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

H2O2 Treatment Activates ERKs

We examined the effect of H2O2 treatment on ERK activation. ERK2 kinase activity was assessed by immunoprecipitation using the anti-ERK2 antibody, followed by in vitro phosphorylation assay using MBP as a substrate. As noted previously (1), H2O2 treatment increased ERK activation in a concentration-dependent manner (Figure 1).


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  		Figure 1.   ERK activation by H2O2 and suppression by inhibitors of Raf-1 or PKC. (a) H2O2-induced ERK2 activation was assessed by immunoprecipitation using an anti-ERK2 antibody, followed by in vitro phosphorylation assay using MBP as a substrate. (b) Pretreatment of cultures with either phorbol 12,13-dibutyrate (Pdbu), forskolin (Fsk), or chelerythrine (Ch) significantly decreased H2O2-induced ERK activation (mean ± SEM; n = 3, P < 0.05, ANOVA).

Effects of PKC Depletion and Inhibition on H2O2-induced ERK Activation

To assess the requirement of PKC for H2O2-induced ERK activation, cells were pretreated either with phorbol 12,13-dibutyrate (200 ng/nl for 24 h) or chelerythrine (10 µM for 30 min). Depletion of PKC by phorbol ester treatment and inhibition of PKC activity each caused a significant reduction in ERK2 activity, though the inhibitory effect was incomplete (Figure 1; P < 0.05, ANOVA). These data suggest that PKC activation is required for H2O2-induced ERK activation, although PKC-independent pathways may also exist.

Role of Raf-1 in H2O2-induced ERK Activation

We examined the effect of forskolin, an activator of adenylate cyclase, on H2O2-induced ERK activation. Previous experiments have demonstrated forskolin to be an inhibitor of Raf-1 in bovine tracheal myocytes (12). Forskolin pre-treatment (50 µM for 15 min) nearly abolished H2O2-induced ERK2 activity (Figure 1; P < 0.05, ANOVA), suggesting that H2O2-induced ERK activation requires the catalytic activation of Raf-1.

To further examine the requirement of Raf-1 for H2O2-induced ERK activation, we directly measured Raf-1 kinase activity by immunoprecipitation with an antibody against Raf-1, followed by in vitro phosphorylation assay using a recombinant, bacterially expressed, kinase-inactive MEK1 as substrate. H2O2, phorbol 12,13-dibutyrate (200 ng/ml for 10 min), and PDGF (30 ng/ml for 10 min) each significantly increased Raf-1 activity (Figure 2; P < 0.05, ANOVA). Together with the forskolin inhibitor experiments, these data suggest that Raf-1 activation is required for H2O2-induced ERK activation.


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  		Figure 2.   Activation of Raf-1 by H2O2. (a) Raf-1 kinase activity was assessed by immunoprecipitation with an antibody against Raf-1, followed by in vitro phosphorylation assay using a recombinant, bacterially expressed, kinase-inactive MEK1 as substrate. (b) H2O2, phorbol 12,13-dibutyrate (Pdbu), and PDGF treatment each significantly increased Raf-1 activity (mean ± SEM; n = 7, P < 0.05, ANOVA).

Role of MEKs in H2O2-induced ERK Activation

We have previously shown that the synthetic MEK inhibitor PD98059 inhibits PDGF-induced MEK1 and ERK activities in bovine tracheal myocytes (10). To examine the effect of PD98059 on H2O2-induced ERK activation, we measured ERK activation by in-gel kinase renaturation assay. PD98059 pretreatment of H2O2-exposed cells induced a concentration-dependent reduction in both ERK1 and ERK2 activity (Figure 3), suggesting that H2O2-induced ERK activation requires the catalytic activation of MEK.


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  		Figure 3.   The synthetic MEK inhibitor PD98059 inhibits H2O2-induced ERK activation. ERK activation was measured by in-gel kinase renaturation assay. Similar results were obtained in three separate experiments.

To further examine the requirement of MEK for H2O2-induced ERK activation, we directly measured MEK1 kinase activity by immunoprecipitation with an antibody against MEK1, followed by in vitro phosphorylation assay using a recombinant, bacterially expressed, kinase-inactive ERK1 as substrate. H2O2 treatment substantially increased MEK1 activity (Figure 4). Further, H2O2-induced MEK1 activation was significantly reduced by pretreatment with PD98059 (P < 0.05, ANOVA). These data confirm the requirement of MEK1 for H2O2-induced ERK activation.


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  		Figure 4.   Activation of MEK1 by H2O2 and inhibition by the synthetic MEK inhibitor PD98059 (30 µM). (a) MEK1 kinase activity was assessed by immunoprecipitation with an antibody against MEK1, followed by in vitro phosphorylation assay using a recombinant, bacterially expressed, kinase-inactive ERK1 as substrate. (b) H2O2-induced MEK1 activation was significantly reduced by pretreatment with PD98059 (mean ± SEM; n = 3, P < 0.05, ANOVA).

We also attempted to assess the role of MEK2 in H2O2-induced ERK activation by immunoprecipitating cell extracts with an antibody against MEK2, followed by in vitro phosphorylation assay using a recombinant, bacterially expressed, kinase-inactive ERK1 as substrate. However, we were unable to detect MEK2 activity in bovine tracheal myocytes even after stimulation with 10% fetal bovine serum (Figure 5, top panel ). The ERK kinase activity of bovine tracheal myocyte anti-MEK2 immunoprecipitates was no greater than that obtained using a control antibody (data not shown). The presence of MEK2 in immunoprecipitates of bovine tracheal myocyte cell extracts was confirmed by Western analysis (Figure 5, lower panel ). On the other hand, analysis of Hela cell extracts demonstrated basal activity in unstimulated cells and a modest increase in activity with EGF. These data demonstrate that our assay was indeed capable of detecting MEK2 activity in bovine tracheal myocytes, and that H2O2 activates MEK1 but not MEK2 in these cells.


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  		Figure 5.   H2O2 activates MEK1 but not MEK2 in bovine tracheal myocytes. (Upper panel) MEK2 activity was assessed by immunoprecipitating cell extracts with an antibody against MEK2, followed by in vitro phosphorylation assay using a recombinant, bacterially expressed, kinase-inactive ERK1 as substrate. There was no MEK2 activation in bovine tracheal myocytes (lanes 1-3), whereas Hela cell extracts demonstrated ample MEK2 activity (lanes 4 and 5). This experiment was repeated three times. (Lower panel) The presence of MEK2 in immunoprecipitates of bovine tracheal myocyte cell extracts was confirmed by Western analysis. (FBS, fetal bovine serum; EGF, epidermal growth factor.)

H2O2 Treatment Activates ERKs via the Production of Hydroxyl Radical

To examine the proximal mechanism by which H2O2 activates the ERK signaling pathway, we pretreated cells with either N-(2-mercaptoproprionyl)glycine, a scavenger of hydroxyl radicals, or o-phenanthroline, which inhibits the metal-catalyzed production of hydroxyl radical from H2O2. In these experiments, ERK activation was estimated by measuring the level of ERK phosphorylation in whole cell lysates using an antibody raised against a dually phosphorylated peptide that corresponds to the active form of ERKs. Pretreatment of bovine tracheal myocytes with the hydroxyl radical scavenger N-(2-mercaptoproprionyl)glycine (2 mM for 45 min) substantially reduced ERK phosphorylation (Figure 6), suggesting that hydroxyl radicals are required for H2O2-induced activation of the ERK signaling pathway. On the other hand, pretreatment with the heavy metal chelator o-phenanthroline (100 µM for 45 min) appeared to have no effect on H2O2-induced ERK phosphorylation.


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  		Figure 6.   H2O2 treatment activates ERK via the production of hydroxyl radical. Cells were pretreated with either N-(2-mercaptoproprionyl)glycine (MPG), a scavenger of hydroxyl radicals, or o-phenanthroline (PA), a metal chelator which inhibits the production of hydroxyl radical from H2O2. ERK activation was estimated by measuring the level of ERK phosphorylation in whole cell lysates using an antibody raised against a dually phosphorylated peptide that corresponds to the active form of ERK. This experiment was repeated twice.

H2O2 Treatment Activates JNK

We examined the effect of H2O2 treatment on the activity of another member of the MAP kinase superfamily, SAPK, also known as Jun amino-terminal kinase or JNK (5). Treatment of bovine tracheal myocytes with H2O2 substantially increased JNK activity (Figure 7). As has been noted previously (7), JNK activity appeared to peak at submaximal concentrations of stimulus.


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  		Figure 7.   H2O2 treatment activates JNK. (a) JNK activity was assessed by in vitro phosphorylation using a recombinant GST-Jun (1-79) fusion protein as a substrate. For comparison, the response to 50 ng/ml anisomycin (AN), a potent activator of JNK, is also shown. (b) Treatment of bovine tracheal myocytes with H2O2 increased JNK activity (mean ± SEM; n = 3).

    Discussion
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have shown in cultured bovine tracheal myocytes that (1) H2O2 treatment induces a concentration-dependent increase in ERK activation; (2) H2O2-induced ERK activation is significantly reduced by inhibitors of MEK (PD98059), Raf-1 (forskolin), and PKC (chelerythrine); and (3) H2O2-induced ERK activation is accompanied by activation of MEK1 and Raf-1. Together these data suggest that H2O2 treatment may activate ERK via successive activation of PKC, Raf-1, and MEK1.

These data confirm and extend previous work in bovine tracheal myocytes demonstrating that H2O2 induces phosphorylation of ERK1 and ERK2 (1). Furthermore, bovine tracheal myocyte ERK2 activity was reduced both by PKC depletion (by phorbol 12,13-dibutyrate) and inhibition (chelerythrine), confirming the role of PKC in ERK activation. PKC has been previously demonstrated to play a role in bovine tracheal smooth muscle ERK activation by serotonin (12), bradykinin, and endothelin-1 (15). Whereas we did not examine PKC expression in these cells, analyses of canine tracheal myocyte PKC expression suggest that a wide variety of both calcium-dependent and calcium-independent isoforms may be expressed in airway smooth muscle (16). PKC-alpha , -beta , -gamma , and -epsilon have each been demonstrated to activate the serine/threonine kinase Raf-1 by direct phosphorylation (2); and activated Raf-1 may phosphorylate and activate MEK, the dual-function kinase required and sufficient for ERK activation (10, 17). We therefore examined the roles of Raf-1 and MEK in H2O2-induced ERK activation.

We found that H2O2 treatment of bovine tracheal myocytes induced Raf-1 activation, and that pretreatment of cells with forskolin, an inhibitor of Raf-1 in this system (12), nearly abolished H2O2-induced ERK activity. Although we have not ruled out the possibility that H2O2 activates ERK via an alternative forskolin-sensitive pathway, our data strongly suggest that H2O2-induced ERK activation in bovine tracheal myocytes is dependent on catalytic activation of Raf-1. Raf-1 has been previously demonstrated to play a role in tracheal smooth muscle ERK activation by serotonin (12) and endothelin (7). The sensitivity of H2O2-induced ERK activation to forskolin contrasts with the insensitivity of PDGF-induced ERK activation to this reagent (12), the latter of which implies the existence of Raf-1-independent ERK activation pathways.

We also found that pre-treatment of H2O2-stimulated cells with PD98059 inhibited both MEK1 and ERK activation, strongly suggesting that MEK1 is required for H2O2-induced ERK activation. However, we were unable to demonstrate MEK2 activity in bovine tracheal myocytes, implying that MEK2 is not involved. This conclusion is consistent with previous data demonstrating that MEK1 is sufficient for ERK activation in bovine tracheal myocytes (10). Although the notion that MEK1 and MEK2 play different roles in cell signaling may be surprising in light of their sequence homology, other examples of selective activation of MEK1 but not MEK2 exist. For example, it has been demonstrated that tumor necrosis factor-alpha preferentially activates MEK1 in mouse macrophages (21).

Pretreatment of cells with the hydroxyl radical scavenger N-(2-mercaptoproprionyl)glycine inhibited H2O2-induced ERK activation, suggesting that H2O2 activates the ERK pathway via the formation of this reactive oxygen intermediate. These data are consistent with previous work demonstrating that mannitol, another free radical scavenger with specificity for hydroxyl radical, inhibits H2O2-induced ERK activation in NIH 3T3 cells (22). On the other hand, pretreatment with o-phenanthroline, a metal chelator thought to inhibit the generation of hydroxyl radical from H2O2, had no apparent effect on ERK activation. The explanation for this discrepancy is unclear but may relate to differences in cellular permeability or subcellular compartmentalization (23).

The events linking hydroxyl radical accumulation and signaling though the ERK pathway are uncertain. H2O2-induced signaling through the ERK pathway has been previously noted in several cell types, including NIH 3T3 fibroblasts (22, 24), pheochromocytoma PC12 cells (25), neutrophils (26), and vascular smooth muscle (22, 27). In several reports, H2O2-induced ERK signaling appeared to occur via activation of the 21-kD membrane-bound GTPase, Ras (22, 25, 29). Ras activation was proceeded by phosphorylation of the EGF receptor in one report (29) and blocked by suramin, an inhibitor of ligand-receptor interactions, in another (22). Together these data suggest that H2O2 may induce signaling through the ERK pathway via growth factor receptor activation. However, it is unlikely that activation of an EGF receptor pathway is the basis of H2O2-induced ERK activation in bovine tracheal myocytes because, as mentioned earlier, EGF activation of ERK is forskolin-insensitive in these cells (12). Oxidative conditions have also been demonstrated to selectively modify the phorbol ester-binding, regulatory domain of PKC, resulting in a constitutively active kinase (30). H2O2 could also stimulate PKC activation by inhibiting a protein tyrosine phosphatase (31). Finally, H2O2 has been shown to activate Src-family kinases in hematopoietic cells (32), and Src in turn has been shown to phosphorylate PKC-delta (33).

The precise role of ERK activation following H2O2 treatment of airway smooth muscle cells is unclear. We have not observed an increase in bovine airway smooth muscle cell DNA synthesis after H2O2 treatment, as measured either by [3H]-thymidine incorporation or bromodeoxyuridine labeling (unpublished data). Indeed, hyperoxic exposure of tracheal myocyte cultures has been noted to reduce growth and induce apoptosis (34, 35). The absence of cell growth upon H2O2 treatment may relate to the transient nature of ERK activation (1); in several cell types, including bovine tracheal myocytes, signaling outcomes such as growth or differentiation have been associated with sustained ERK activation (36). Alternatively, the absence of DNA synthesis following H2O2 treatment may relate to the activation of inhibitory, antimitogenic pathways. We found that H2O2 treatment increases the activity of another member of the MAP kinase superfamily, SAPK, also known as Jun amino-terminal kinase or JNK (5). Based on the types of signals which activate JNK, it is likely that this pathway is involved in growth inhibition rather than mitogenesis (39). The possibility that ERK and JNK have opposing effects is supported by studies in PC12 pheochromocytoma cells demonstrating that activation of JNK and concurrent inhibition of ERK are critical for apoptosis, whereas activation of ERK inhibits apoptosis (40). Indeed, it has recently been shown that activation of the Ras/ERK pathway protects against H2O2-induced cell death in NIH 3T3 cells (22). Thus, while ERK activation does not appear to mediate tracheal myocyte DNA synthesis following H2O2 treatment, it may serve to promote cell survival in this instance. The precise roles of these MAP kinase signaling pathways in tracheal myocyte growth, differentiation, and programmed cell death remain to be determined.

    Footnotes

Address correspondence to: Marc B. Hershenson, M.D., University of Chicago Children's Hospital, 5841 S. Maryland Ave., MC 4064, Chicago, IL 60637-1470. E-mail: mhershen{at}midway.uchicago.edu

(Received in original form March 12, 1997 and in revised form August 13, 1997).

Acknowledgments: This work was supported by National Heart, Lung and Blood Institute Grants HL54685, HL56399, HL02731 (M.B.H.) and HL09074 (M.K.A.). One author (A.Y.K.) is the recipient of a Lillian Gertrude Selz Scholarship.

Abbreviations EDTA, ethylenediamenetetraacetic acid; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinases; GST, glutathione S-transferase; Hepes, N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid; JNK, Jun amino-terminal kinase; MAP, mitogen-activated protein; MBP, myelin basic protein; MEK, MAP kinase/ERK kinases; PDGF, platelet-derived growth factor; PKC, protein kinase C; PMSF, phenylmethylsulfonyl fluoride; Raf-1, a serine/threonine kinase known to activate MEK; SAPK, stress-activated protein kinase.

    References
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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