JunB forms the majority of the AP-1 complex and is a target for redox regulation by receptor tyrosine kinase and G protein-coupled receptor agonists in smooth muscle cells.

To understand the role of redox-sensitive mechanisms in vascular smooth muscle cell (VSMC) growth, we have studied the effect of N-acetylcysteine (NAC), a thiol antioxidant, and diphenyleneiodonium (DPI), a potent NADH/NADPH oxidase inhibitor, on serum-, platelet-derived growth factor BB-, and thrombin-induced ERK2, JNK1, and p38 mitogen-activated protein (MAP) kinase activation; c-Fos, c-Jun, and JunB expression; and DNA synthesis. Both NAC and DPI completely inhibited agonist-induced AP-1 activity and DNA synthesis in VSMC. On the contrary, these compounds had differential effects on agonist-induced ERK2, JNK1, and p38 MAP kinase activation and c-Fos, c-Jun, and JunB expression. NAC inhibited agonist-induced ERK2, JNK1, and p38 MAP kinase activation and c-Fos, c-Jun, and JunB expression except for platelet-derived growth factor BB-induced ERK2 activation. In contrast, DPI only inhibited agonist-induced p38 MAP kinase activation and c-Fos and JunB expression. Antibody supershift assays indicated the presence of c-Fos and JunB in the AP-1 complex formed in response to all three agonists. In addition, cotransfection of VSMC with expression plasmids for c-Fos and members of the Jun family along with the AP-1-dependent reporter gene revealed that AP-1 with c-Fos and JunB composition exhibited a higher transactivating activity than AP-1 with other compositions tested. All three agonists significantly stimulated reactive oxygen species production, and this effect was inhibited by both NAC and DPI. Together, these results strongly suggest a role for redox-sensitive mechanisms in agonist-induced ERK2, JNK1, and p38 MAP kinase activation; c-Fos, c-Jun, and JunB expression; AP-1 activity; and DNA synthesis in VSMC. These results also suggest a role for NADH/NADPH oxidase activity in some subset of early signaling events such as p38 MAP kinase activation and c-Fos and JunB induction, which appear to be important in agonist-induced AP-1 activity and DNA synthesis in VSMC.

Redox control plays an important role in gene regulation (1)(2)(3)(4)(5)(6). Underlying the importance of redox mechanisms, oxidants have been implicated in the pathogenesis of cell prolif-erative diseases such as atherosclerosis and cancer and in aging (7)(8)(9). Although the exact mechanisms by which oxidant stress influences the pathogenesis of these diseases are not clear, several laboratories including ours have demonstrated that oxidants exhibit mitogenic activity in a variety of cell types including VSMC 1 at nontoxic doses (10 -15). In addition, oxidants mimic growth factors in several aspects: oxidants stimulate phosphorylation of growth factor receptor and nonreceptor tyrosine kinases (16 -18), they activate extracellular signalregulated kinases (ERKs) (19,20), and they induce expression of several protooncogenes such as c-Fos and c-Jun (21)(22)(23)(24)(25). More importantly, a number of recent studies have demonstrated that growth factors stimulate production of H 2 O 2 in a variety of cell types including VSMC (26 -31). In addition, a requirement for H 2 O 2 production in the mitogenic signaling events of growth factors has been reported (31).
NADH/NADPH oxidase is a flavin-containing plasma membrane-bound enzyme that generates superoxide anion upon activation (32). Superoxide anion can be dismutated enzymatically or nonenzymatically to H 2 O 2 , which is then scavenged by catalase or peroxidase (33). In an effort to identify the sources of oxidant generation, several investigators have reported that growth factors that induce either hyperplasia or hypertrophy stimulate NADH/NADPH oxidase activity in various cell types (34,35). It was also reported that NADH/NADPH oxidase activity can be regulated in a growth-dependent manner (35). A role for NADH/NADPH oxidase activity in tumor necrosis factor-␣-and fibroblast growth factor-induced c-Fos expression and tumor necrosis factor-␣-induced Jun-NH 2 -terminal kinase-1 (JNK1) activation in chondrocytes has been reported (36,37). In another study, inhibition of NADH/NADPH oxidase activity by antisense targeted depletion of its p22 phox component blunted the hypertrophic effect of angiotensin II in VSMC (38). Because of these findings, a role for this enzyme in growth factor-induced signaling events has been suggested. However, the molecular events underlying the growth-regulating effects of NADH/NADPH oxidase are not clear. The purpose of the present investigation was to determine the role of redox-sensitive mechanisms in the modulation of agonist-induced early response events in VSMC. Here we report the following observations. 1) NAC, a thiol antioxidant, blocks agonist-induced ERK2, JNK1, and p38 MAPK activation, c-Fos, c-Jun, and JunB expression, AP-1 activity, and DNA synthesis in VSMC.
2) NADH/NADPH oxidase activity plays a role in some subset of agonist-induced early response events such as induction of p38 MAPK activation, c-Fos and JunB expression, AP-1 activity, and DNA synthesis. 3) JunB appeared to be the predominant member of the AP-1 complex formed in response to both receptor tyrosine kinase and G protein-coupled receptor agonists. 4) AP-1 with c-Fos and JunB composition exhibited a higher transcriptional activity than AP-1 with other compositions tested. Together, these results strongly suggest that redox-sensitive mechanisms play an important role in the modulation of AP-1 activity and growth in VSMC.
DNA Synthesis-VSMC were plated onto 60-mm dishes, allowed to grow to 70 -80% confluence, and then growth arrested by incubation in Dulbecco's modified Eagle's medium containing 0.1% (v/v) calf serum for 72 h. Growth-arrested VSMC were exposed to various agonists in the presence and absence of the indicated inhibitors for 24 h. Cells were pulse labeled with 1 Ci/ml [ 3 H]thymidine for 2 h just before the end of the incubation period and harvested by trypsinization followed by centrifugation. The cell pellet was resuspended in cold 10% (w/v) trichloroacetic acid and vortexed vigorously to lyse the cells. The mixture was allowed to remain on ice for 20 min and was then passed through a GF/F glass microfiber filter. The filter was washed once with cold 5% trichloroacetic acid and once with cold 70% ethanol, dried, and placed in a liquid scintillation vial containing the mixture. The radioactivity was measured in a liquid scintillation counter (Beckman LS 3801).
Western Blot Analysis-Growth-arrested VSMC were treated with and without the agonists in the presence and absence of appropriate inhibitors for the indicated time periods at 37°C. Medium was aspirated, and cells were rinsed with cold phosphate-buffered saline (PBS) and frozen immediately in liquid nitrogen. 250 l of lysis buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 g/ml phenylmethylsulfonyl fluoride, 100 g/ml aprotinin, 1 g/ml leupeptin, 20 mM ␤-glycerophosphate, 2 mM sodium fluoride, 2 mM sodium pyrophosphate, and 1 mM sodium orthovanadate (Na 3 VO 4 )) was added to the frozen monolayers, thawed on ice for 15 min, and scraped into 1.5-ml Eppendorf tubes. The cell lysates were cleared by centrifugation at 12,000 rpm for 30 min at 4°C. The protein content of the supernatants was determined using Bradford reagent (Bio-Rad). Cell lysates containing equal amounts of protein were resolved by electrophoresis on a 0.1% SDS and 10% polyacrylamide gels. The protein was transferred electrophoretically to a nitrocellulose membrane (Hybond, Amersham Pharmacia Biotech). After blocking in 10 mM Tris-HCl buffer, pH 8.0, containing 150 mM sodium chloride, 0.1% Tween 20, and 5% (w/v) non-fat dry milk, the membrane was treated with appropriate primary antibodies followed by incubation with appropriate peroxidaseconjugated secondary antibodies. The antigen-antibody complexes were detected using chemiluminescence reagent kit (Amersham Pharmacia Biotech).
ERK, JNK, and p38 MAPK Assays-After appropriate treatments, cells were washed with cold PBS and solubilized on ice for 15 min in lysis buffer containing 20 mM Hepes, pH 7.4, 2 mM EGTA, 1 mM dithiothreitol, 50 mM ␤-glycerophosphate, 1% Triton X-100, 10 units/ml aprotinin, 2 M leupeptin, 1 mM Na 3 VO 4 , and 400 M phenylmethylsulfonyl fluoride. The cell lysates were cleared by centrifugation at 13,000 rpm for 10 min at 4°C. Cell lysates normalized for protein were immunoprecipitated by incubating with anti-ERK2 and anti-JNK1 rabbit IgG for ERK2 and JNK1 assays, respectively, for 2 h followed by the addition of 40 l of 50% (w/v) protein A-Sepharose beads for an additional hour. The beads were washed three times with lysis buffer, three times with wash buffer (100 mM Tris-HCl, pH 7.6, 500 mM lithium chloride, 0.1% Triton X-100, and 1 mM dithiothreitol), and three times with kinase buffer (12.5 mM Mops, pH 7.5, 12.5 mM ␤-glycerophosphate, 7.5 mM MgCl 2 , 2 mM EGTA, 0.5 mM sodium fluoride, and 0.5 mM Na 3 VO 4 ). The ERK2 activity present in the immunoprecipitates was determined by resuspending in 30 l of kinase buffer containing 5 g of myelin basic protein, 20 M ATP, and 1 Ci of [␥-32 P]ATP per reaction and incubating at 30°C for 20 min. For the JNK1 assay, incubation with the kinase buffer was the same as that for the ERK2 assay except that 1 g of GST-c-Jun was used instead of myelin basic protein. The reactions were stopped by adding 20 l of 4 ϫ Laemmli sample buffer. The samples were heated at 95°C for 5 min and analyzed by SDS-gel electrophoresis on 12% acrylamide gels. The dried gel was exposed to X-Omat AR x-ray film and developed. Activation of p38 MAPK was measured by Western blotting using phospho-p38 MAPK antibodies.
Electrophoretic Mobility Shift Assay-Growth-arrested VSMC were treated with and without agonists for the indicated time periods, and nuclear extracts were prepared as described earlier (39). Protein-DNA complexes were formed by incubating 5 g of nuclear protein in a total volume of 20 l consisting of 15 mM Hepes, pH 7.9, 3 mM Tris-HCl, pH 7.9, 60 mM KCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 4.5 g of bovine serum albumin, 2 g of poly(dI-dC), 15% glycerol, and 100,000 cpm of 32 P-labeled oligonucleotide probe for 20 min at 30°C. For supershift analysis, antibodies (4 g) were added to the reaction mix after the initial 20-min incubation, and incubation continued for an additional 2 h. Protein-DNA complexes were resolved on a 4% polyacrylamide gel using 0.25 ϫ TBE buffer (1 ϫ TBE ϭ 50 mM Tris borate, pH 8.3, and 1 mM EDTA). Double-stranded oligonucleotides (AP-1, 5Ј-CGCTTGATGAGTCAGCCGGAA-3Ј) were labeled with [␥-32 P]ATP using a T4 polynucleotide kinase kit per the supplier's protocol (Promega). Unincorporated nucleotides were removed by chromatography in a G-25 spin column (Bio-Rad).
Transient Transfection and CAT Assays-VSMC culture was split evenly into 100-mm dishes the day before transfection and grown in Dulbecco's modified Eagle's medium containing 10% calf serum to 80 -90% confluence. Cells were transfected with collagenase (coll)-CAT plasmid DNA (20 g/100-mm dish) using a calcium phosphate precipitation method as described by Angel et al. (40). Cells were washed with PBS 16 h after transfection, incubated in Dulbecco's modified Eagle's medium containing 0.1% calf serum for 36 h at 37°C, and then stimulated with and without agonists in the presence and absence of appropriate inhibitors for 6 h. In the other set of experiments, VSMC were cotransfected with expression plasmids (8 g each) for c-Fos or Jun family members alone or in various combinations along with AP-1 reporter plasmid, coll-CAT, and 48 h after transfection cells were processed for CAT activity. Vector DNA was used to normalize the amount of DNA transfected with the cells. Cells were washed with PBS, scraped in 1 ml of TEN buffer (40 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 8.0, 150 mM NaCl) into an Eppendorf tube and pelleted by centrifugation at 12,000 g for 1 min at 4°C. The cell pellet was suspended in 100 l of cold 250 mM Tris-HCl buffer, pH 7.5, and the cells were lysed by three repeated freeze-thaw cycles. Cell debris was removed by centrifugation at 12,000 ϫ g for 5 min at 4°C. The protein concentration of the supernatant was determined as described above. CAT activity was measured by the method of Gorman et al. (41). In brief, 50 g of protein from each condition was incubated with 20 l of 4 mM acetyl-CoA, 32.5 l of 1 M Tris-HCl buffer, pH 7.5, 4 l of 50 Ci/ml [ 14 C]chloramphenicol in a total volume of 150 l at 37°C for 2-4 h. Controls without cell extract and/or with nontransfected cell extracts were incubated simultaneously. Acetylated and nonacetylated chloramphenicol was extracted with ethyl acetate and separated by thin layer chromatography on Silica Gel 1B plates using a chloroform:methanol mixture (19:1) as solvent, and the air-dried silica plates were subjected to autoradiography.
Superoxide Anion Production Assay-Superoxide anion production in growth-arrested VSMC in response to agonists was measured according to the method described by Griendling et al. (34). Cells were washed with PBS and pelleted by centifugation at 1,000 rpm for 5 min at 4°C. Cells were then resuspended in assay buffer consisting of 130 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 1.5 mM CaCl 2 , 35 mM phosphoric acid, and 20 mM Hepes, pH 7.4. Superoxide anion production in VSMC in response to agonists was measured in the darkroom with a AutoLumat LB 953 luminometer. To start the assay, lucigenin was added to a final concentration of 250 M to 1 ϫ 10 6 cells followed by agonist in a total volume of 1 ml of assay buffer. Wherever appropriate, 20 mM NAC was added to cells 2 h before the assay, and DPI (12 M) was added 30 min before the assay. Photoemission in terms of relative light units was measured every minute for 5 min.
All of the experiments were repeated at least three to four times with similar results. All samples were normalized for protein content before Western blot analysis, immunoprecipitation, electrophoretic mobility shift assay and CAT assay. Statistical analysis on [ 3 H]thymidine uptake data was performed using Student's t test.

RESULTS
To study the role of redox-sensitive mechanisms in agonistinduced cell growth, growth-arrested VSMC were treated with and without 10% serum, 10 ng/ml PDGF-BB, or 0.1 unit/ml thrombin in the presence and absence of 20 mM NAC or 12 M DPI for 24 h, and DNA synthesis was measured by [ 3 H]thymidine uptake. As shown in Fig. 1, A and B, all three agonists caused a significant increase in DNA synthesis in VSMC, and this effect was blocked by NAC and DPI.
To investigate the underlying mechanisms of NAC-and DPIinhibited VSMC growth, we first determined the time course of agonist-induced expression of AP-1 components, c-Fos, c-Jun, JunB, and JunD. As shown in Fig. 2, A and B To test whether agonist-induced increases in the levels of c-Fos and members of the Jun family proteins are mediated via the generation of oxidants, we treated growth-arrested VSMC with serum, PDGF-BB, or thrombin in the presence and absence of NAC, a thiol antioxidant, and the protooncogene levels were determined. NAC significantly decreased c-Fos (70%), c-Jun (50%), and JunB (70%) expression in response to serum, PDGF-BB, and thrombin (Fig. 3). In contrast, NAC had no significant effect on JunD levels in either control or agonisttreated VSMC. NADH/NADPH oxidase has been implicated in cytokine-and growth factor-induced c-Fos expression and cytokine-induced JNK1 activation and c-Jun expression in chondrocytes (36,37). To test the role of NADH/NADPH oxidase activity in agonist-induced expression of c-Fos and members of the Jun family proteins, growth-arrested VSMC were treated with and without serum, PDGF-BB, or thrombin in the presence and absence of DPI, and the protooncogene levels were determined. Expression of c-Fos in response to these agonists was almost completely (85%) blocked by DPI (Fig. 4). DPI also significantly (70%) inhibited JunB expression induced by these agonists. DPI alone induced c-Jun expression by 3-fold, and it exhibited an additive effect with agonist-induced expression of this protooncogene (Fig. 4). DPI had no significant effect on JunD levels in either control or agonist-treated cells.
The Fos and Jun family proteins constitute the transcription factor AP-1 (42). To investigate the relationship between the induction of expression of c-Fos and members of the Jun family proteins and AP-1-dependent transcription, we studied the expression of AP-1-responsive reporter gene in growth-arrested VSMC stimulated with serum, PDGF-BB, or thrombin in the presence and absence of NAC or DPI. VSMC were transiently transfected with a collagenase-CAT (coll-CAT) reporter plasmid in which CAT expression was driven by collagenase gene promoter containing a single AP-1 site, growth arrested, and then treated with and without the agonists in the presence and absence of NAC or DPI. All three agonists caused induction of the AP-1-dependent reporter gene expression, and this response was significantly blocked by both NAC and DPI (Fig. 5,  A and B). Because NAC and DPI inhibited the AP-1 activity despite the differential effects of these compounds on serum-, PDGF-BB-, and thrombin-induced c-Jun expression, we next determined the composition of AP-1 formed in response to these agonists. Gel supershift analysis showed the presence of c-Fos and JunB in the AP-1 complex formed in response to serum and thrombin (Fig. 6). Similar results were obtained with PDGF-BB (data not shown). To determine the functional activity of AP-1 with c-Fos and JunB composition, VSMC were cotransfected with expression plasmids for c-Fos and members of the Jun family in various combinations along with the AP-1 reporter plasmid coll-CAT, and CAT activity was measured 48 h after transfection. Consistent with the gel supershift analysis, AP-1 with c-Fos and JunB composition exhibited a higher transcriptional activity than AP-1 with other combinations tested (Fig. 7).
ERKs are a group of serine/threonine kinases that respond preferentially to various growth stimulants. These kinases are implicated in the expression of c-Fos in response to various agonists (43)(44)(45)(46). To understand the possible mechanisms by which NAC and DPI inhibit c-Fos expression induced by these agonists, we investigated the effects of these compounds on serum-, PDGF-BB-, and thrombin-induced activation of ERK2 in VSMC. All three agonists caused significant increases (8 -15-fold) in ERK2 activity as measured by an immunocomplex kinase assay using myelin basic protein as a substrate (Figs. 8  and 9). Although NAC completely inhibited the ERK2 activity induced by serum and thrombin, it had no significant effect on PDGF-BB-induced ERK2 activation (Fig. 8). Serum-, PDGF-BB-, and thrombin-induced ERK2 activity, however, was not affected by DPI (Fig. 9). NAC and DPI by themselves had no significant effect on ERK2 activity in growth-arrested VSMC.
JNKs are a group of MAPKs that are related to ERKs but respond preferentially to cellular stressors such as UV irradiation and agents that do not primarily cause growth (47)(48)(49). A role for JNKs has been suggested in the induction of expression of both c-Fos and c-Jun (43,44,50). To understand the possible mechanisms by which antioxidants affect the agonist-induced expression of c-Fos and members of the Jun family proteins, we studied the effects of NAC and DPI on serum-, PDGF-BB-, and thrombin-induced JNK1 activity. All three agonists activated JNK1 (10 -25-fold) as determined by an immunocomplex kinase assay with GST-c-Jun (1-79) as a substrate (Figs. 8 and  9). NAC significantly inhibited (50 -80%) the agonist-induced JNK1 activity (Fig. 8). On the contrary, DPI alone caused an increase (35% over control) in JNK1 activity, and it exhibited an additive effect with agonists on the activation of this enzyme (Fig. 9). It was reported that p38 MAPK cooperates with ERKs in the induction of expression of c-Fos in response to UV irradiation (51). To find the possible role of p38 MAPK in agonistinduced c-Fos expression, we tested the effect of serum, PDGF-BB, and thrombin on p38 MAPK activation. As determined by the phosphorylation of the enzyme, all three agonists activated p38 MAPK, and this effect was sensitive to inhibition by both NAC and DPI (Fig. 10).
To gain further evidence for a role of oxidants in receptor tyrosine kinase and G protein-coupled receptor agonist-induced growth signaling events, we tested the effect of serum, PDGF-BB, and thrombin on superoxide anion production in growtharrested VSMC. As shown in Fig. 11, all three agonists signif-

FIG. 2. Time course of serum, PDGF-BB, and thrombin induction of c-Fos, c-Jun, JunB, and JunD expression in VSMC.
Growth-arrested VSMC were treated with and without 10% serum (CS, panel A), 10 ng/ml PDGF-BB (panel A), or 0.1 unit/ml thrombin (panel B) for the indicated times, and cell lysates were prepared. Cell lysates containing equal amounts of protein from each condition were analyzed by Western blotting for c-Fos, c-Jun, JunB, and JunD levels using their respective antibodies.

FIG. 3. Effect of NAC on serum-, PDGF-BB-, and thrombininduced expression of c-Fos, c-Jun, JunB, and JunD in VSMC.
Growth-arrested VSMC were treated with and without 10% serum (CS), 10 ng/ml PDGF-BB, or 0.1 unit/ml thrombin in the presence and absence of 20 mM NAC for 2 h, and cell lysates were prepared and analyzed for c-Fos, c-Jun, JunB, and JunD levels as described in the legend of Fig. 2. icantly stimulated superoxide anion production by 5 min, and this effect was blocked by both NAC and DPI. DISCUSSION The important finding of the present study is that JunB is the major constituent of the AP-1 complex formed in response to, and is a target for redox regulation by both receptor tyrosine kinase and G protein-coupled receptor agonists in VSMC. The present findings also show that antioxidant NAC inhibits serum-, PDGF-BB-, and thrombin-induced JNK1 and p38 MAPK activation, c-Fos, c-Jun, and JunB expression, and DNA synthesis in VSMC. Although NAC completely blocked the serumand thrombin-induced ERK2 activity, it had no significant effect on PDGF-BB-induced activation of ERK2. These findings suggest a role for both redox-sensitive and redox-insensitive mechanisms in agonist-induced ERK2 activation in VSMC. ERKs are implicated in the induction of expression of c-Fos via phosphorylation and activation of the transcription factor Elk1 in response to growth factors (44,52). Because NAC had no effect on PDGF-BB-induced ERK2 activity, and it inhibited completely the c-Fos expression and DNA synthesis induced by PDGF-BB, it is likely that other mechanisms that are independent of ERKs modulate the induction of expression of c-Fos by PDGF-BB. This also implies that activation of ERK2 alone may not be sufficient for growth induction by PDGF-BB. In fact, others have also reported that activation of ERKs alone is not sufficient for VSMC mitogenesis induced by PDGF-BB and angiotensin II (53,54). It was reported that induction of c-Fos expression by UV requires a cross-talk between p38 MAPK and ERKs (51). Because all three agonists activated p38 MAPK, and this effect was inhibited by NAC, one likely mechanism by which NAC inhibits the PDGF-BB-induced c-Fos expression could be via down-regulating the p38 MAPK activity. A similar mechanism can be extrapolated to the inhibitory effect of DPI on agonist-induced c-Fos expression as DPI, although having no effect on agonist-induced ERKs activation, blocked p38 MAPK activation.
Reactive oxygen species production by NADH/NADPH oxidase has been implicated in cytokine-and growth factor-induced c-Fos expression and cytokine-induced JNK1 activation and c-Jun expression (36,37). Because DPI, a potent inhibitor of NADH/NADPH oxidase, blocked only the c-Fos and JunB expression and p38 MAPK activation but not the c-Jun expression or ERK2 and JNK1 activation induced by serum, PDGF-BB, and thrombin, it is likely that NADH/NADPH oxidase activity is required only for some subset of agonist-stimulated early response events. Despite the differential effects of NAC and DPI on agonist-induced ERK2 and JNK1 activation and c-Jun expression, both caused a decrease in agonist-stimulated AP-1 activity and growth. Because c-Fos and JunB were present in the AP-1 complex formed in response to serum, PDGF-BB, and thrombin, and the expression of these protooncogenes was inhibited by both NAC and DPI, it is possible that these two protooncogenes account for the agonist-induced AP-1 activity and growth in VSMC. Indeed, cotransfection of VSMC with expression plasmids for c-Fos and members of the Jun family revealed that AP-1 with c-Fos and JunB composition possess more gene transactivating activity than AP-1 with the other compositions tested.
Superoxide anion and H 2 O 2 are the frequently invoked reactive oxygen species implicated in growth factor-induced cellular signaling (10, 28, 31, 55, 56). In addition, growth factors have FIG. 8. Effect of NAC on serum, PDGF-BB, and thrombin activation of ERK2 and JNK1. After treatment of growth-arrested VSMC for 5 min (for ERK assay) or 10 min (for JNK assay) with and without 10% serum (CS), 10 ng/ml PDGF-BB, or 0.1 unit/ml thrombin in the presence and absence of 20 mM NAC, cell lysates were prepared. Cell lysates containing equal amounts of protein from each condition were immunoprecipitated with anti-ERK2 or JNK1 antibodies, and the ERK2 and JNK1 activities were measured by immunocomplex kinase assays using myelin basic protein or GST-c-Jun as substrates, respectively, as described under "Experimental Procedures." FIG. 9. Serum-, PDGF-BB-, and thrombin-induced ERK2 and JNK1 activities are insensitive to inhibition by DPI. Growtharrested VSMC were treated with and without 10% serum (CS), 10 ng/ml PDGF-BB, or 0.1 unit/ml thrombin in the presence and absence of 12 M DPI for 5 min (for the ERK2 assay) or 10 min (for the JNK1 assay), and cell lysates were prepared and assayed for ERK2 and JNK1 activities as described in the legend of Fig. 8.

FIG. 10. NAC and DPI inhibit the serum-, PDGF-BB-, and thrombin-induced activation of p38 MAPK in VSMC.
Growtharrested VSMC were treated with and without 10% serum (CS), 10 ng/ml PDGF-BB, or 0.1 unit/ml thrombin in the presence and absence of 20 mM NAC or 12 M DPI for 10 min, and cell lysates were prepared. Cell lysates containing an equal amount of protein from each condition were analyzed by Western blotting for phospho-p38 MAPK using specific antibodies.
FIG. 11. Effect of serum, PDGF-BB, and thrombin on the production of superoxide anion in VSMC. Growth-arrested VSMC were treated with and without 10% serum, 10 ng/ml PDGF-BB, or 0.1 unit/ml thrombin in the presence and absence of 20 mM NAC or 12 M DPI, and superoxide anion production was measured by photoemission as described under "Experimental Procedures." Superoxide anion production was expressed as relative light units/1 ϫ 10 6 cells for 5 min. been reported to activate NADH/NADPH oxidase, a major reactive oxygen species-generating system in a variety of cell types including VSMC (28,34,35). Our results show that both receptor tyrosine kinase and G protein-coupled receptor agonists stimulate the production of superoxide anion in VSMC via a mechanism involving NADH/NADPH oxidase activity. However, although serum-, PDGF-BB-, and thrombin-induced superoxide anion production was completely blocked by NAC and DPI, the early growth response events of these agonists exhibited differential sensitivity of inhibition to these compounds. Specifically, DPI inhibited agonist-induced p38 MAPK activation and c-Fos and JunB expression. On the other hand, NAC inhibited ERKs, JNKs, and p38 MAPK group of MAPK activation and c-Fos, c-Jun, and JunB expression. Considering these findings, it is clear that besides NADH/NADPH oxidase, other mechanisms are involved in the redox regulation of these early response signaling events of receptor tyrosine kinase and G protein-coupled receptor agonists. It has been reported that agents such as hemin and HgCl 2 , which oxidize thiol groups, activate Ras and cause mitogenesis in lymphocytes (57,58). NAC facilitates synthesis of glutathione, a major intracellular thiol donor, by providing cysteine (59). Glutathione, besides scavenging reactive oxygen species, modulates the levels of intracellular thiols (59). Therefore, the broader inhibitory effects of NAC on agonist-induced early growth response events indicate a role for thiols. In fact, oxidation/reduction of thiols has been regarded as a critical regulatory event in the modulation of activities of several proteins including growth factor receptors, ion channels, and transcriptional factors (1-6, 60 -63).
Another possible molecule that is involved in agonist-induced NAC-sensitive early response events could be arachidonic acid. Both receptor tyrosine kinase and G protein-coupled receptor agonists stimulate the release of arachidonic acid in a variety of cell types including VSMC (64,65). In addition, a requirement for arachidonic acid release and its subsequent metabolism via the lipoxygenase/cytochrome P-450 monoxygenase pathways has been reported to be required for both receptor tyrosine kinase and G protein-coupled receptor agonistinduced growth in many cell types including VSMC (64,66). Furthermore, activation of secretory type phospholipase A 2 s (also known as group II phospholipase A 2 s), a group of ratelimiting enzymes for arachidonic acid release, requires disulfide bond formation (67). It was also reported that expression of group II phospholipase A 2 in transgenic mice results in epidermal hyperplasia (68). Although a role for arachidonic acid in agonist-induced thiol-sensitive mechanisms of oxidant generation can be extrapolated from these findings, future studies are required to test this hypothesis. In any case, the present study provides evidence for a role of several redox-sensitive mechanisms including NADH/NADPH oxidase activity in the early signaling events of both receptor tyrosine kinase and G proteincoupled receptor agonists in VSMC. In addition, the present study identifies JunB as the major component of the AP-1 complex formed in response to both receptor tyrosine kinase and G protein-coupled receptor agonists in VSMC and its possible role in redox-sensitive AP-1-mediated gene regulation.