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(Circulation Research. 1995;77:29-36.)
© 1995 American Heart Association, Inc.

Articles

Differential Activation of Mitogen-Activated Protein Kinases by H₂O₂ and O₂^- in Vascular Smooth Muscle Cells

Arnold S. Baas, Bradford C. Berk

From the Cardiology Division, Department of Internal Medicine, University of Washington, Seattle.

Correspondence to Bradford C. Berk, MD, PhD, Cardiology Division, RG-22, University of Washington, Seattle, WA 98195. E-mail bcberk@u.washington.edu.

	Abstract

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Abstract Increased generation of active oxygen species such as H₂O₂ and O₂^- may be important in vascular smooth muscle cell growth associated with atherosclerosis and restenosis. In previous work, we showed that H₂O₂ stimulated vascular smooth muscle cell growth and proto-oncogene expression. In the present study, we compared the effects of H₂O₂ and O₂^- on cultured rat aortic vascular smooth muscle cell growth and signal transduction. O₂^- was generated in a concentration-dependent manner by the naphthoquinolinedione LY83583. Vascular smooth muscle cell growth, as measured by [³H]thymidine incorporation, was stimulated by 200 µmol/L H₂O₂ (110% increase versus 0.1% serum) and 1 µmol/L LY83583 (175% increase) to levels comparable to 10 ng/mL platelet-derived growth factor (210% increase). Since activation of mitogen-activated protein kinase (MAP kinase) is one of the earliest growth factor signal events, the activity of MAP kinase was measured by changes in mobility on Western blot and by phosphorylation of myelin basic protein. There was a concentration-dependent increase in MAP kinase activity by LY83583 (maximum, 10 µmol/L) but not by H₂O₂. The time course for activation of MAP kinase by LY83583 showed a maximum at 5 to 10 minutes with return to baseline by 20 minutes. Activation of MAP kinase by LY83583 was protein kinase C dependent. Expression of MAP kinase phosphatase-1 (MKP-1), a transcriptionally regulated redox-sensitive protein tyrosine/threonine phosphatase, was also measured. Although H₂O₂ induced MKP-1 mRNA to a greater extent than did LY83583, the increased MKP-1 expression could not explain the inability of H₂O₂ to stimulate MAP kinase, because mRNA levels were not detected until 60 minutes. The findings that both O₂^- and H₂O₂ stimulate vascular smooth muscle cell growth but only O₂^- rapidly activates MAP kinase suggest that additional signal events are required for the mitogenic effects of H₂O₂.

Key Words: mitogen-activated protein kinase • signal transduction • H₂O₂ • O₂^- • mitogen-activated protein kinase phosphatase-1

	Introduction

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We have previously shown that active oxygen species (ie, H₂O₂, O₂^-, and · OH) stimulate vascular smooth muscle cell growth and DNA synthesis.¹ This proliferation was associated with the induction of several growth-related proto-oncogenes, including c-myc and c-fos. Using the combination of xanthine and xanthine oxidase, which generates both H₂O₂ and O₂^-, we demonstrated in vascular smooth muscle cells that active oxygen species induced proto-oncogene mRNA expression in a protein kinase C (PKC)–dependent manner.^{1 2 3} Other investigators have also demonstrated that H₂O₂ and O₂^- stimulate growth-related events such as cell alkalinization and proto-oncogene induction.^{4 5} Active oxygen species may act as growth factors by direct oxidation of critical protein sulfhydryl groups, leading to activation of growth-regulatory factors, or by formation of transition metal complexes, which may inhibit protein phosphatases. Examples of active oxygen species acting in this fashion include dimerization of Fos-Jun proteins,⁶ activation of NF-B,⁷ activation of endoplasmic reticulum tyrosine kinases,⁸ and stimulation of kinases involved in growth-related signal transduction.⁹ These previous studies indicate that active oxygen species share properties with growth factors.

Many growth factors cause vascular smooth muscle cells to proliferate.¹⁰ Most of these factors activate membrane receptors such as the tyrosine kinase–coupled receptors (eg, platelet-derived growth factor [PDGF], fibroblast growth factor, and epidermal growth factor) or heptahelical G protein–coupled receptors (eg, angiotensin II and thrombin). Receptor binding in turn activates a protein kinase cascade linking extracellular signal events present at the cell membrane with changes in gene expression in the nucleus. Critical enzymes in this cascade are the 42- and 44-kD mitogen-activated protein kinases (MAP kinases). MAP kinases are serine/threonine protein kinases that are activated by many stimuli involved in cell growth and differentiation.^{11 12} Activation of the 42- and 44-kD MAP kinases requires both tyrosine and threonine phosphorylation^{13 14} and is mediated by a dual-specificity protein tyrosine/threonine kinase, MEK.^{15 16} Activation of MAP kinase by mitogens such as fibroblast growth factor has been shown to be biphasic: a transient early activation of MAP kinase occurs within 5 to 10 minutes of agonist administration, whereas a sustained late activation appears after 1 to 2 hours and is required for cell cycle progression,¹⁷ suggesting that MAP kinase activity is regulated by multiple kinases and phosphatases. A protein threonine/tyrosine phosphatase, 3CH134 (recently named MAP kinase phosphatase, or MKP-1), has been shown to dephosphorylate and inactivate MAP kinase.^{18 19} Recent data demonstrate that MKP-1 plays a critical role in regulating the growth of ras-transformed cells²⁰ and is the primary protein phosphatase in vascular smooth muscle cells that dephosphorylates MAP kinase in response to angiotensin II.²¹ Because MKP-1 is the rat homologue of the redox-sensitive phosphatase, CL100,^{22 23} regulation of MKP-1 expression may be critical to the effects of H₂O₂ and O₂^- on MAP kinase activity.

On the basis of these findings, we hypothesized that active oxygen species should activate MAP kinase in vascular smooth muscle cells. In the present study, we demonstrate that (1) O₂^- but not H₂O₂ stimulates activation of MAP kinase, (2) the activation by O₂^- is PKC dependent, and (3) the differential activation of MAP kinase is not well explained by greater induction of MKP-1 by H₂O₂ than by O₂^-.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Culture
Vascular smooth muscle cells were isolated from aortas of 200- to 250-g male Sprague-Dawley rats and maintained in 10% calf serum supplemented with DMEM as described previously.¹⁰ Cells used in experiments were passages 5 to 15.

Measurement of O₂^- Production
Chemiluminescence of lucigenin (bis-N-methylacridinium nitrate) was measured to determine O₂^- production exactly as described by Griendling et al,²⁴ except that photon emission was detected with a scintillation counter (LS 7000, Beckman Instruments, Inc) in out-of-coincidence mode with a single active photomultiplier tube. A buffer blank was subtracted from each reading before conversion of the data.

DNA Synthesis and Cell Number
Vascular smooth muscle cells were growth-arrested for 48 hours in DMEM supplemented with 0.1% calf serum and labeled with 1 µCi/mL [methyl-³H]thymidine (specific activity, 20 Ci/mmol; Dupont NEN) in the presence or absence of agonist for 24 hours as previously described.¹⁰ After labeling, cells were washed with cold PBS, trypsinized, resuspended in 20% trichloroacetic acid (TCA), and vortexed vigorously to lyse the cells. The cell lysate was vacuum-filtered through a glass-fiber filter (GF/F, Whatman). After washing with cold 5% TCA followed by 70% ethanol, the filter was dried. Incorporated [³H]thymidine was measured in a liquid scintillation counter. Experiments were performed three times in duplicate 35-mm dishes. For cell number, cells were plated at a density of 2x10³ cells per square centimeter in DMEM with 10% calf serum. After 24 hours, the medium was changed to DMEM with 0.1% serum supplemented with agonists or the appropriate vehicle. The medium was changed every 48 hours, and after 7 days, cell counts were performed with a hemocytometer. Data are presented as mean±SEM.

MAP Kinase Activity Measured by Western Blot and In-Gel Kinase Assay
After treatment, the cells were washed with PBS, and 0.5 mL of lysis buffer (10 mmol/L HEPES, pH 7.4, 5 mmol/L EDTA, 5 mmol/L EGTA, 50 mmol/L sodium pyrophosphate, 50 mmol/L NaF, 50 mmol/L NaCl, 100 µmol/L Na₃VO₄, 0.01% Triton X-100, and fresh 0.5 mmol/L phenylmethylsulfonyl fluoride and 10 µg/mL leupeptin) was added. Cell lysates were prepared by freezing in liquid nitrogen, thawing on ice, scraping, and sonication. After centrifugation for 30 minutes at 14 000 rpm in a microfuge (4°C), protein concentration was determined, and the samples were stored at -80°C. For Western blot analysis, 20 µg protein was subjected to SDS-PAGE in a 10% gel under reducing conditions, and proteins were then transferred to nitrocellulose (Hybond-ECL, Amersham) as previously described.⁶ The membrane was blocked for 2 hours at room temperature with a commercial blocking buffer from GIBCO. The blots were incubated for 1 hour at room temperature with the primary antibodies (anti-ERK1 and anti-ERK2 from Santa Cruz Biotechnology, 1:2000), followed by incubation for 1 to 2 hours with secondary antibody (horseradish peroxidase conjugated). Immunoreactive bands were visualized by chemiluminescence (ECL, Amersham).

An in-gel kinase assay²⁵ to measure MAP kinase phosphotransferase activity was performed on cell lysates, which were harvested as described above. Equal amounts of protein (5 to 10 µg) were separated by SDS-PAGE in a gel containing 0.4 mg/mL myelin basic protein (MBP). The gel was then incubated twice in buffer A (50 mmol/L HEPES, pH 7.4, and 5 mmol/L ß-mercaptoethanol) containing 20% isopropanol for 30 minutes, once in buffer A for 1 hour, twice in buffer A containing 6 mol/L guanidine-HCl for 30 minutes, twice in buffer A containing 0.04% Tween 20 at 4°C for 16 hours and 2 hours, once in buffer A containing 100 µmol/L Na₃VO₄ and 10 mmol/L MgCl₂ at 30°C for 30 minutes, and once in buffer A containing 100 µmol/L Na₃VO₄, 10 mmol/L MgCl₂, 50 µmol/L ATP, and 50 µCi [-³²P]ATP for 1 hour at 30°C. The reaction was terminated by washing the gel five to eight times in a fixative solution containing 10 mmol/L sodium pyrophosphate and 5% TCA for 15 minutes. The gel was dried and subjected to autoradiography. Autoradiographic signal intensity was quantified by densitometry in the linear range of film exposure by using a LaCie scanner and National Institutes of Health IMAGE 1.49 software. The statistics were computed with the program SYSTAT. A value of P<.05 was considered significant.

RNA Blot Analysis
Total RNA was prepared from vascular smooth muscle cells, and Northern blot analysis was performed exactly as described previously.²⁶ Radiolabeling of the MKP-1 cDNA probe (a full-length rat MKP-1 cDNA subcloned into pGEM)²⁷ was performed with a random primer labeling kit (Bethesda Research Laboratories) according to the manufacturer's protocol using [-³²P]dCTP (specific activity, 3000 Ci/mmol; Du Pont-New England Nuclear). Methylene blue staining of the 28S mRNA was used to normalize RNA loading. After hybridization, blots were washed once in 1x standard saline citrate (SSC; 20x SSC is composed of 3 mol/L NaCl and 0.3 mol/L sodium citrate at pH 7.0) and 1% SDS (30 minutes, room temperature) and once in 0.1x SSC and 0.1% SDS (30 minutes, 55°C).

Materials
LY83583 was kindly supplied by Lilly Pharmaceuticals or was purchased from Sigma Chemical Co. All other chemicals and reagents were purchased from Sigma, unless otherwise indicated.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Production of Oxygen Radicals
The naphthoquinolinedione LY83583 was used to generate O₂^- in vascular smooth muscle cells. This compound freely crosses cell membranes and generates O₂^- via metabolism by cytosolic and membrane-bound NAD(P)H oxidases.²⁴ The production of O₂^- by vascular smooth muscle cells in response to LY83583 was measured by lucigenin chemiluminescence as described previously.²⁴ Because lucigenin is in equilibrium across the cell plasma membrane, lucigenin chemiluminescence detects both extracellular and intracellular O₂^-.²⁴ As illustrated in Fig 1, LY83583 stimulated a concentration-dependent increase in O₂^-. In response to 1 µmol/L LY83583, O₂^- production increased 35-fold relative to control (3.5 chemiluminescence units versus 0.1 chemiluminescence unit). Similar results were obtained with the vitamin K derivative menadione, which also generates intracellular O₂^- via metabolism by NAD(P)H oxidase (data not shown). The O₂^- production by LY83583 was significantly inhibited (>60%) by the presence of 1000 U/mL superoxide dismutase (SOD), indicating specific scavenging of extracellular O₂^- by SOD. Tiron (4,5-dihydroxy-1,3-benzene disulfonic acid), a membrane-permeant nonenzymatic O₂^- scavenger, was also used to scavenge intracellular O₂^-.²⁴ Tiron (10 mmol/L) completely abolished LY83583-induced lucigenin chemiluminescence (data not shown), suggesting that it is able to scavenge both intracellular and extracellular O₂^-. In contrast, catalase (10 U/mL), a specific scavenger of H₂O₂, had no significant effect on LY83583-induced O₂^- generation (Fig 1). These results demonstrate the presence of both intracellular and extracellular enzymes in vascular smooth muscle cells capable of generating O₂^- from LY83583.

View larger version (28K): [in this window] [in a new window]   Figure 1. Graph showing that LY83583 (LY) generates O2- in vascular smooth muscle cells. Rat aortic smooth muscle cells were growth-arrested for 48 hours in 0.1% calf serum before stimulation with LY. Cells were harvested by using trypsin, and 106 cells per milliliter were used to measure O2- by chemiluminescence of lucigenin (bis-N-methylacridinium nitrate). Results are representative of three or four experiments. Catalase (cat.) was added at 10 U/mL; superoxide dismutase (SOD), at 1000 U/mL.

Vascular Smooth Muscle Cell DNA Synthesis and Cell Number Are Increased by LY83583
We previously found that H₂O₂ stimulated vascular smooth muscle cell growth.^{1 2 3} To determine whether LY83583 was also mitogenic for vascular smooth muscle cells, growth-arrested cells were exposed to varying concentrations of LY83583. LY83583 stimulated a concentration-dependent increase in [³H]thymidine incorporation, which was maximal at 0.3 to 1 µmol/L LY83583. At LY83583 concentrations of >10 µmol/L, [³H]thymidine incorporation was inhibited (data not shown) in a manner analogous to that observed with high concentrations of H₂O₂ and xanthine/xanthine oxidase.¹ As illustrated in Fig 2A, exposure of vascular smooth muscle cells to LY83583 increased DNA synthesis to nearly the same extent as did PDGF. Relative to 0.1% serum, 10 ng/mL PDGF caused a 210% increase in [³H]thymidine incorporation, whereas 1 µmol/L LY83583 and 200 µmol/L H₂O₂ caused 175% and 110% increases in DNA synthesis, respectively. As illustrated in Fig 2B, exposure of vascular smooth muscle cells to LY83583 for 7 days increased cell number in a fashion similar to that observed with PDGF. Relative to 0.1% serum, 1 µmol/L LY83583 increased cell number by 300%, whereas exposure to 5 ng/mL PDGF for 7 days increased cell number by 70%. The addition of 50 U/mL cell-permeant polyethylene-glycolated SOD (PEG-SOD) completely inhibited the increase in cell number stimulated by LY83583, suggesting that intracellular O₂^- was responsible for the mitogenic effect of LY83583. Addition of 1000 U/mL SOD failed to inhibit LY83583-stimulated vascular smooth muscle cell growth, probably because SOD is cell impermeant. The concentrations of LY83583 (0.3 to 1 µmol/L) that maximally increased DNA synthesis and cell number caused 5- to 35-fold increases in O₂^- production in vascular smooth muscle cells (Fig 1).

View larger version (17K): [in this window] [in a new window]   Figure 2. Bar graphs showing that LY83583 (LY) stimulates vascular smooth muscle cell growth and DNA synthesis. Rat aortic smooth muscle cells were growth-arrested for 48 hours in 0.1% calf serum before stimulation with LY, H2O2, xanthine (X) and xanthine oxidase (XO), or platelet-derived growth factor (PDGF). A, DNA synthesis. Cells were labeled with [3H]thymidine in the presence or absence of agonist for 24 hours. [3H]Thymidine incorporation was measured in a liquid scintillation counter. In response to 10% serum, [3H]thymidine incorporation was 66 220±3980 cpm per well. B, Cell number. Cells were plated at a density of 2x103 cells per square centimeter and grown in DMEM containing 10% calf serum. After 24 hours, the medium was changed to DMEM with 0.1% serum supplemented with agonists or appropriate vehicle. The medium was changed every 48 hours, and after 7 days, cell counts were performed with a hemocytometer. Polyethylene-glycolated superoxide dismutase (PEG-SOD) was added at 50 U/mL. In response to 10% serum, cell number increased to 121±20x103 cells per well. Results are mean±SEM of a single experiment and are representative of three experiments.

Active Oxygen Species Stimulate MAP Kinase
Since activation of MAP kinase is one of the earliest mitogenic signaling events in cells, we investigated the ability of active oxygen species to stimulate MAP kinase. Growth-arrested vascular smooth muscle cells were exposed for 10 minutes to LY83583 (0.03 to 1 µmol/L) or H₂O₂ (10 to 200 µmol/L). Cell lysates were size-fractionated by SDS-PAGE, and activation of MAP kinase was measured by "band shift" of the phosphorylated 42- and 44-kD MAP kinase species on Western blot. Phosphorylation of MAP kinase retards its mobility on SDS-PAGE, thereby enabling the activated MAP kinase to be detected by its increased apparent molecular weight. As shown in Fig 3, LY83583 (maximum, 1 µmol/L) caused a concentration-dependent activation of MAP kinase at 10 minutes, whereas H₂O₂ (range, 10 to 200 µmol/L) failed to activate MAP kinase. These findings demonstrate that activation of MAP kinase at 10 minutes is caused only by O₂^- and not by H₂O₂.

View larger version (34K): [in this window] [in a new window]   Figure 3. LY83583 (LY) activates 42- and 44-kD mitogen-activated protein kinases (ERK2 and ERK1, respectively). Rat aortic smooth muscle cells were growth-arrested in 0.1% calf serum for 48 hours, treated for 10 minutes with angiotensin II (Ang II), H2O2, or LY, and cell lysates were prepared (see "Materials and Methods"). Approximately 25 µg protein from each sample was size-fractionated by SDS-PAGE in a 10% gel, and proteins were then transferred to nitrocellulose. Blots were incubated for 1 hour with a mixture of anti-ERK1 and anti-ERK2 antibodies (rabbit polyclonal antibodies), followed by incubation for 1 to 2 hours with secondary antibody (horseradish peroxidase–conjugated goat anti-rabbit). Immunoreactive bands were visualized by chemiluminescence.

To confirm that the 42- and 44-kD MAP kinase species, as identified by band shift, represent activated kinases, an in-gel kinase assay was performed by using MBP as a substrate.²⁵ Cell lysates from vascular smooth muscle cells were size-fractionated by SDS-PAGE in a 10% gel containing 0.4 mg/mL MBP, and renatured protein kinase activity was determined by phosphorylation of MBP. As demonstrated in Fig 4A, lysates from vascular smooth muscle cells exposed to LY83583 (maximum, 10 µmol/L) showed increased activity of the 42- and 44-kD MAP kinases in a concentration-dependent fashion. The time course for MAP kinase activation by 10 µmol/L LY83583 was rapid and transient, with maximal activation at 5 minutes and return to baseline by 20 minutes (Fig 4B). This time course is similar to that shown previously for angiotensin II in vascular smooth muscle cells.¹² As demonstrated in Fig 5A, H₂O₂ (concentration range, 1 µmol/L to 2 mmol/L) failed to stimulate MAP kinase activity at 10 minutes as determined by in-gel kinase assay. At 200 µmol/L H₂O₂, a concentration of H₂O₂ that caused maximal increases in DNA synthesis and cell growth (see Fig 2),¹ the time course for H₂O₂ stimulation (2 to 60 minutes) showed no significant increase in 42- or 44-kD MAP kinase phosphotransferase activity (Fig 5B). When normalized to control conditions, the LY83583-mediated activation of MAP kinase was significantly increased compared with the effect of H₂O₂ (P<.005, n=4). We determined that the 42- and 44-kD kinases activated by LY83583 were ERK2 (42-kD MAP kinase) and ERK1 (44-kD MAP kinase),²⁷ respectively, by immunoprecipitation with a polyclonal anti-ERK antibody followed by in-gel kinase assay of the immunoprecipitated proteins (data not shown). In summary, LY83583 but not H₂O₂ specifically activated 42- and 44-kD MAP kinases as measured by both Western blot band shift and in-gel kinase assays.

View larger version (36K): [in this window] [in a new window]   Figure 4. LY83583 (LY) activates 42- and 44-kD mitogen-activated protein (MAP) kinases. A, Dose response. Rat aortic smooth muscle cells were growth-arrested in 0.1% calf serum for 48 hours and treated for 10 minutes with angiotensin II (Ang II), phorbol 12-myristate 13-acetate (PMA), or LY. Ethanol (ETOH) was added at the concentration required to prepare 30 µmol/L LY. Cell lysates from vascular smooth muscle cells were size-fractionated by SDS-PAGE in a 10% gel containing 0.4 mg/mL myelin basic protein (MBP), and renatured protein kinase activity was determined by phosphorylation of MBP. MAP kinase activity was measured by in-gel kinase assay of 42- and 44-kD bands detected by autoradiography. Autoradiographic density was quantified by using a visible light scanner and National Institutes of Health IMAGE 1.49 software. Results were calculated as percent control based on the activation of MAP kinase by control at 10 minutes for each experiment (autoradiographic intensity, 1.0). B, Time course. LY (10 µmol/L) was added for the indicated times, and MAP kinase activity was detected by in-gel kinase assay.

View larger version (37K): [in this window] [in a new window]   Figure 5. H2O2 does not activate mitogen-activated protein (MAP) kinase. A, Dose response. Rat aortic smooth muscle cells were growth-arrested in 0.1% calf serum for 48 hours and treated for 10 minutes with angiotensin II (Ang II), phorbol 12-myristate 13-acetate (PMA), or H2O2. Cell lysates from vascular smooth muscle cells were size-fractionated by SDS-PAGE in a 10% gel containing 0.4 mg/mL myelin basic protein (MBP), and renatured protein kinase activity was determined by phosphorylation of MBP. MAP kinase activity was measured by in-gel kinase assay of 42- and 44-kD bands detected by autoradiography. Autoradiographic density was quantified by using a visible light scanner and National Institutes of Health IMAGE 1.49 software. Results were calculated as percent control based on the activation of MAP kinase by control at 10 minutes for each experiment (autoradiographic intensity, 1.0). B, Time course. H2O2 (200 µmol/L) was added for the indicated times, and MAP kinase activity was detected by in-gel kinase assay.

Tiron Inhibits O₂^--Induced Early MAP Kinase Activation
To demonstrate that O₂^- generated by LY83583 was responsible for activation of MAP kinase, vascular smooth muscle cells were treated with Tiron (10 mmol/L) to scavenge O₂^-. Simultaneous addition of 10 mmol/L Tiron with 10 µmol/L LY83583 significantly inhibited LY83583-mediated activation of MAP kinases detected by in-gel kinase assay (Fig 6). Addition of Tiron did not significantly inhibit MAP kinase activation by angiotensin II and minimally inhibited activation by phorbol 12-myristate 13-acetate (PMA), demonstrating that Tiron did not cause a nonspecific inhibition of MAP kinase activity. These results suggest that O₂^- is responsible for MAP kinase activation by LY83583 in vascular smooth muscle cells.

View larger version (34K): [in this window] [in a new window]   Figure 6. Tiron, a scavenger of O2-, inhibits O2- activation of mitogen-activated protein (MAP) kinase. Rat aortic smooth muscle cells were growth-arrested in 0.1% calf serum for 48 hours and treated for 10 minutes with angiotensin II (Ang II), phorbol 12-myristate 13-acetate (PMA), or LY83583 (LY). Tiron (10 mmol/L) was added simultaneously to the control and agonist-treated cells as shown in lanes 5 to 8. Cell lysates from vascular smooth muscle cells were size-fractionated by SDS-PAGE in a 10% gel containing 0.4 mg/mL myelin basic protein (MBP), and renatured protein kinase activity was determined by phosphorylation of MBP. MAP kinase activity was measured by in-gel kinase assay of 42- and 44-kD bands detected by autoradiography.

Early MAP Kinase Activation by O₂^- is PKC Dependent
Since we previously found that active oxygen species stimulate proto-oncogene expression primarily via a PKC-dependent mechanism,^{1 2 3} we investigated the role of PKC in O₂^--mediated MAP kinase activation. Vascular smooth muscle cells were pretreated with 1 µmol/L phorbol 12,13-dibutyrate (PDBU) for 24 hours to downregulate PKC.²⁸ As shown in Fig 7, after PDBU treatment, LY83583 (10 µmol/L) failed to activate the 42- and 44-kD MAP kinases, indicating that LY83583 activation of MAP kinase is PKC dependent. Downregulation of PKC was demonstrated by the finding that PMA (200 nmol/L) was also no longer able to activate MAP kinase. In contrast, angiotensin II (100 nmol/L) activated MAP kinase to the same extent in PDBU-treated cells as in control cells, demonstrating that PDBU treatment had not altered the ability of MAP kinase to be activated. In summary, the results of these experiments indicate that O₂^- activates MAP kinase in a PKC-dependent fashion.

View larger version (20K): [in this window] [in a new window]   Figure 7. LY83583 (LY) activation of mitogen-activated protein (MAP) kinase is protein kinase C dependent. Rat aortic smooth muscle cells were growth-arrested in 0.1% calf serum for 48 hours and treated for 10 minutes with the angiotensin II (Ang II), phorbol 12-myristate 13-acetate (PMA), LY, or H2O2 (left). Protein kinase C was downregulated by 24-hour pretreatment with 1 µmol/L phorbol 12,13-dibutyrate (PDBU) (right). Cell lysates from vascular smooth muscle cells were size-fractionated by SDS-PAGE in a 10% gel containing 0.4 mg/mL myelin basic protein (MBP), and renatured protein kinase activity was determined by phosphorylation of MBP. MAP kinase activity was measured by in-gel kinase assay of 42- and 44-kD bands detected by autoradiography.

Role of MKP-1 in O₂^--Induced MAP Kinase Activation
Recently, a transcriptionally regulated dual-specificity protein phosphatase, MKP-1, was shown to dephosphorylate and inactivate MAP kinase.^{18 19 22} In several cell types, MKP-1 has been shown to be induced by oxidative stress,^{22 23} suggesting that it may play a role in signal transduction events stimulated by active oxygen species.^{29 30} In vascular smooth muscle cells stimulated by angiotensin II, we have shown that MKP-1 is the dominant mechanism by which MAP kinase is dephosphorylated.²¹ We hypothesized that MKP-1 mRNA would be induced to a greater extent by H₂O₂ than by LY83583, thereby explaining the failure of H₂O₂ to activate MAP kinase. To investigate the role of MKP-1 in regulation of MAP kinase by active oxygen species, MKP-1 mRNA levels were measured in vascular smooth muscle cells by using the 3CH134 cDNA.³¹ As illustrated in Fig 8, angiotensin II and PMA increased MKP-1 mRNA levels with a peak at 30 minutes. H₂O₂ also increased MKP-1 mRNA levels, but the peak was at 60 minutes, and mRNA levels were much less than observed with angiotensin II or PMA. LY83583 failed to induce MKP-1 expression significantly. No significant differences were observed for 28S mRNA levels between growth-arrested and treated cells.

View larger version (49K): [in this window] [in a new window]   Figure 8. H2O2 induces MAP kinase phosphatase-1 (MKP-1), a transcriptionally regulated phosphatase. Rat aortic smooth muscle cells were growth-arrested in 0.1% calf serum for 48 hours and treated for 15 to 60 minutes with the angiotensin II (Ang II), phorbol 12-myristate 13-acetate (PMA), LY83583 (LY), or H2O2. Total RNA was purified as described in "Materials and Methods." Northern blot analysis was performed by using a [-32P]dCTP–labeled cDNA probe for MKP-1. Methylene blue staining of the 28S mRNA was used to normalize RNA loading. Results are representative of four experiments.

If increased MKP-1 expression in response to H₂O₂ compared with O₂^- explains the failure of H₂O₂ to stimulate MAP kinase, then inhibiting MKP-1 expression should cause MAP kinase activation by H₂O₂. Because MKP-1 regulation is controlled at the level of transcription,³¹ we used actinomycin D to block transcription and, hence, MKP-1 mRNA induction. Previously, we found that actinomycin D pretreatment potentiated and prolonged MAP kinase activity stimulated by angiotensin II.²¹ Treatment of vascular smooth muscle cells for 30 minutes with actinomycin D (3 µg/mL) failed to enhance MAP kinase activity in response to H₂O₂ or LY83583 at 10 minutes compared with cells not treated with actinomycin D (Fig 9). In contrast, actinomycin D potentiated angiotensin II–stimulated activity at 60 minutes as previously reported.²¹ These data indicate that a redox-regulated transcriptional event (eg, MKP-1 induction) is unlikely to explain MAP kinase activation by O₂^- and not by H₂O₂.

View larger version (32K): [in this window] [in a new window]   Figure 9. Actinomycin D fails to enhance activation of mitogen-activated protein (MAP) kinase by LY83583 (LY) and H2O2 . Rat aortic smooth muscle cells were growth-arrested in 0.1% calf serum for 48 hours. mRNA transcription was inhibited by addition of actinomycin D (3 µg/mL) for 30 minutes where indicated. Cells were then treated for 10 or 60 minutes with the angiotensin II (Ang II), LY, or H2O2. Cell lysates from vascular smooth muscle cells were size-fractionated by SDS-PAGE in a 10% gel containing 0.4 mg/mL myelin basic protein (MBP), and renatured protein kinase activity was determined by phosphorylation of MBP. MAP kinase activity was measured by in-gel kinase assay of 42- and 44-kD bands detected by autoradiography.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The major finding of the present study is that both O₂^- and H₂O₂ stimulate vascular smooth muscle growth, yet only O₂^- causes rapid activation of MAP kinase. Both O₂^- and H₂O₂ cause vascular smooth muscle cell growth as measured by increases in [³H]thymidine incorporation and cell number. However, only O₂^- causes activation of MAP kinase at 5 to 10 minutes. These results indicate that early activation of MAP kinase is not required for vascular smooth muscle cell growth and proto-oncogene expression, since H₂O₂ stimulates these events yet does not activate MAP kinase at 5 to 10 minutes. Furthermore, these results indicate significant differences in the immediate effects of differential active oxygen species on vascular smooth muscle cell signal transduction.

Recent data suggest that active oxygen species may play a role in the progression of atherosclerosis and development of restenosis following balloon angioplasty. Epidemiological studies show a correlation between antioxidant therapy and a decreased incidence of coronary events in both men and women.^{32 33} The beneficial effects of antioxidants may be related to altered vessel redox, as demonstrated by findings that aortas from hyperlipidemic rabbits³⁴ and coronary arteries from balloon-injured pigs³⁵ generate increased levels of O₂^- compared with control vessels. We have previously demonstrated that antioxidants such as probucol³⁶ or the combination of vitamins C and E³⁷ produced a beneficial effect on vessel remodeling in a porcine coronary balloon injury model, suggesting a role for oxidative stress in the vessel response to injury. Active oxygen species have also been demonstrated to act as potent vasoconstrictors in vessel preparations.^{38 39} Together, these studies point to an important role for active oxygen species in many of the vascular responses to injury and hyperlipidemia.

The O₂^- generated in vascular smooth muscle cells by LY83583 appears to come from extracellular and intracellular sources, since SOD (which is cell impermeant) inhibits 60% to 70% of the O₂^- production, whereas Tiron (which is cell permeant) completely inhibits O₂^- production. Vascular smooth muscle cell growth was inhibited only by the addition of the cell-permeant O₂^- scavenger PEG-SOD, suggesting an important role for intracellular O₂^- in stimulating vascular smooth muscle cell growth. Within the cell, O₂^- may be scavenged by SOD to produce H₂O₂. However, the concentration of LY83583 (1 µmol/L) that caused maximal smooth muscle cell growth was much less than the concentration of H₂O₂ (200 µmol/L) that caused maximal cell growth, suggesting that the dismutation of O₂^- to H₂O₂ by intracellular SOD was not responsible for the growth response stimulated by LY83583. Since both O₂^- and H₂O₂ cause vascular smooth muscle cell growth yet only O₂^- activates MAP kinase, it is intriguing to speculate regarding the site(s) of action of these active oxygen species. Intracellular activation of critical growth enzymes by active oxygen species appears likely, because many growth-regulating enzymes are located within the cell. In addition, active oxygen species may also act on extracellular sites such as receptors or matrix to initiate a cellular growth response. The recent demonstration that 42- and 44-kD MAP kinases are present in vessels and regulated⁴⁰ suggests that O₂^- may activate these kinases in vivo.

We have previously shown that H₂O₂ and the combination of xanthine and xanthine oxidase (which produces H₂O₂, O₂^-, and uric acid) induce c-myc and c-fos expression in a PKC-dependent fashion.^{1 2 3} By activating PKC, active oxygen species would be expected to activate MAP kinase, because PMA activates MAP kinase in vascular smooth muscle cells and MAP kinase translocation is dependent on PKC activity in vascular smooth muscle cells.⁴¹ In fact, early activation of MAP kinase by O₂^- is PKC dependent, because downregulation of PKC blocks O₂^- activation of MAP kinase. However, the MAP kinase activation by LY83583 is small compared with the MAP kinase activation by PMA, yet the induction of c-fos and c-myc mRNA by active oxygen species and that by PMA are similar, suggesting a MAP kinase–independent pathway.

Three mechanisms may explain the apparent paradox of greater activation of MAP kinase by O₂^- than by H₂O₂, with similar activation of PKC. First, there may be activation of different upstream kinases. For example, direct oxidation of critical protein sulfhydryl groups by O₂^- may activate upstream tyrosine kinases.⁸ Second, O₂^- may reduce vanadate to form vanadyl and vanadyl hydroperoxides, which are potent inhibitors of protein tyrosine phosphatases^{29 30} as well as activators of tyrosine kinases.⁴² Preliminary data show that LY83583 (1 µmol/L) but not H₂O₂ (200 µmol/L) causes a 25% inhibition of protein tyrosine phosphatase activity in vascular smooth muscle cells (measured by hydrolysis of p-nitrophenyl phosphate) and resultant larger increases in phosphotyrosine-containing proteins (M. Marrero, J. Duff, B. Berk, unpublished data, 1994). Third, H₂O₂ induces MKP-1 but O₂^- does not (Fig 8), suggesting that there may be unopposed activation of MAP kinase by O₂^-. It is unlikely that the failure to induce MKP-1 completely explains the difference between H₂O₂ and O₂^-, because both angiotensin II and PMA induce MKP-1 to a greater extent than O₂^-, yet they also activate MAP kinase to a greater extent than O₂^-. In addition, since induction of MKP-1 mRNA by H₂O₂ is detected at 60 minutes and activation of MAP kinase is present as early as 5 minutes, the preferential induction of MKP-1 mRNA by H₂O₂ appears insufficient to explain the differences in MAP kinase activation. This discussion suggests that the differences in activation of MAP kinase by O₂^- and not by H₂O₂ are due to effects on upstream signaling pathways rather than downstream regulatory mechanisms.

The facts that both H₂O₂ and O₂^- increase vascular smooth cell growth, DNA synthesis, and proto-oncogene expression, yet H₂O₂ does not stimulate early MAP kinase activity, suggest that an alternative mechanism, independent of early MAP kinase activation, is required for these events. Since proto-oncogene expression is detected as early as 30 minutes after stimulation by active oxygen species,^{1 2 3} we propose that a PKC-dependent MAP kinase–independent pathway is responsible for early events, such as induction of c-fos and c-myc mRNA. Among MAP kinase–independent pathways, two kinase cascades appear likely as potential mediators: (1) There are several PKC isozymes stimulated by cis-unsaturated fatty acids, such as arachidonic acid and linoleic acid.⁴³ We have previously demonstrated that active oxygen species activate phospholipase A₂ and generate arachidonic acid.² Recent data suggest that products of these fatty acids may be involved in mitogenesis in response to growth factors, such as epidermal growth factor.⁴⁴ (2) A MAP kinase–independent pathway recently shown to be activated by cellular stress, tumor necrosis factor, and UV light (a form of oxidative stress) is represented by the stress-activated protein kinases, a newly described family of serine/threonine kinases.^{45 46 47} Finally, preliminary data from our laboratory suggest that both LY83583 and H₂O₂ stimulate a delayed activation of MAP kinase, first detectable 120 minutes after agonist stimulation (A. Baas, B. Berk, unpublished data, 1995). This late activation of MAP kinase is similar to that stimulated by fibroblast growth factor¹⁷ and appears to be required for cell-cycle progression.¹⁶ Because both H₂O₂ and O₂^- are mitogenic for vascular smooth muscle cells, the late activation of MAP kinase by active oxygen species may represent a MAP kinase–dependent pathway necessary for vascular smooth muscle cell growth and DNA synthesis. Future studies will be required to elucidate the roles of late MAP kinase activation, PKC, and stress-activated protein kinases in vascular smooth muscle cell growth stimulated by active oxygen species.

	Acknowledgments

 
This study was supported by grants from the National Institutes of Health to Dr Berk (RO1 HL49192) and Dr Baas (F32 HL09027). Dr Berk is an Established Investigator of the American Heart Association.

Received December 12, 1994; accepted March 27, 1995.

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