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(Hypertension. 2003;42:177.)
© 2003 American Heart Association, Inc.

Scientific Contributions

Cyclic AMP Response Element–Binding Protein Mediates Reactive Oxygen Species–Induced c-fos Expression

Toshihiro Ichiki; Tomotake Tokunou; Kae Fukuyama; Naoko Iino; Satoko Masuda; Akira Takeshita

From the Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan.

Correspondence to Toshihiro Ichiki, MD, Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, 812-8582 Fukuoka, Japan. E-mail ichiki{at}cardiol.med.kyushu-u.ac.jp

	Abstract

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Although the cyclic AMP response element–binding protein (CREB) plays an important role in the survival of neuronal cells and T lymphocytes, the role of CREB in vascular smooth muscle cells (VSMCs) is incompletely characterized. We examined the role of CREB in VSMCs stimulated with reactive oxygen species. Activation of CREB was examined by Western blot analysis with an antibody that specifically recognizes phosphorylation at serine 133 of CREB, which is a critical marker of activation. Hydrogen peroxide (H₂O₂) time-dependently induced phosphorylation of CREB, with a peak at 15 minutes. The H₂O₂-induced phosphorylation of CREB was partially blocked by inhibition of either extracellular signal–regulated protein kinase kinase by PD98059 or of p38 mitogen-activated protein kinase (MAPK) by SB203580. AG1478, an epidermal growth factor receptor (EGFR) inhibitor, suppressed the H₂O₂-induced phosphorylation of CREB and tyrosine phosphorylation of EGFR. Overexpression of the dominant-negative form of CREB by an adenovirus vector suppressed H₂O₂-induced c-fos expression. These findings suggest that H₂O₂ induces CREB phosphorylation through EGFR transactivation and mitogen-activated protein kinase pathways. CREB might be a novel redox-sensitive transcription factor involved in the regulation of VSMC gene expression.

Key Words: free radicals • cyclic AMP • kinase • epidermal growth factor receptor

	Introduction

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Agrowing body of evidence suggests that reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂), superoxide, and hydroxy radical play a critical role in the pathogenesis of atherosclerosis, hypertension, and heart failure.¹ ROS are also involved in signal transduction of receptors for growth factors and vasoactive peptides, such as platelet-derived growth factor² and Angiotensin (Ang) II.³ ROS added exogenously to cells also elicit intracellular signaling events similar to those activated by growth factors or cytokines. H₂O₂ activates extracellular signal–regulated protein kinase (ERK),⁴ p38 mitogen-activated protein kinase (MAPK),⁵ and c-Jun NH₂-terminal kinase.⁶ Recent studies have shown that H₂O₂ induces phosphorylation of epidermal growth factor receptor (EGFR)^4,6 as induced by Ang II⁷ and thrombin.⁸

The cyclic AMP response element–binding protein (CREB)⁹ is a 43-kDa nuclear transcription factor belonging to the CREB/activated transcription factor (ATF) family. CREB binds to the octanucleotide sequence TGACGTCA as a homodimer or a heterodimer in association with other members of the CREB/ATF family.^10,11 Previous studies have demonstrated that neurotransmitters, hormones, and growth factors in different cell types could activate CREB.^10,12 Phosphorylation of a serine residue at position 133 (Ser-133) is necessary for transcriptional activation by CREB. Phosphorylation of CREB at Ser-133 allows access for CREB-binding protein (CBP), which is a transcriptional coactivator and has histone acetylation activity.¹³ Phosphorylation of Ser-133 is mediated by a variety of protein kinases, such as p90^RSK2, in response to activation of a ras-dependent ERK pathway¹⁴and MAPK-activated protein (MAPKAP) kinase 2 in response to activation of p38 MAPK.¹⁵

Overexpression of a dominant-negative CREB transgene that has a mutation at Ser-133 and thus, is unable to bind to CBP induces apoptosis in T cells after growth factor stimulation.¹⁶ Transgenic mice that overexpress a dominant-negative CREB in cardiomyocytes developed dilated cardiomyopathy.¹⁷ These studies suggest that CREB might contribute to cell survival and development in these cell types.

It was reported that CREB was activated by hydrogen peroxide (H₂O₂) through the p38 MAPK pathway in a macrophage cell line.¹⁸ We previously reported that AngII¹⁹ and thrombin²⁰ activated CREB in an ERK- and a p38 MAPK–dependent manner and that CREB played an important role in the hypertrophy of vascular smooth muscle cells (VSMCs). Although ROS play an important role in the signaling of AngII and thrombin, the role of ROS in the activation of CREB in VSMCs has not been determined. In the present study, we examined the signaling pathway of H₂O₂-induced CREB activation and the role of ROS in AngII-induced CREB activation.

	Methods

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Reagents
Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were purchased from GIBCO BRL. PD98059, KN93, KN92, and wortmannin were obtained from Research Biochemicals International. SB203580 was a generous gift from GlaxoSmithKline Pharmaceuticals. H89 was obtained from Biomol Research Laboratories, Inc. AG1478, catalase, and N-acetylcysteine (NAC) were obtained from Sigma. Antibodies against CREB, ERK, p38 MAPK, and their phosphorylated forms were obtained from New England Biolabs, Inc. Horseradish peroxidase–conjugated secondary antibodies (anti-rabbit or anti-mouse immunoglobulin G) were from Vector Laboratories, Inc. [³²P]dCTP was obtained form Du Pont–New England Nuclear. Unless mentioned otherwise, other chemical reagents were purchased from Wako Pure Chemicals.

Cell Culture
VSMCs were isolated from the thoracic aortas of Sprague-Dawley rats and maintained as described previously.²¹ Passages between 5 and 15 were used. VSMCs were grown to confluence, growth-arrested in DMEM with 0.1% bovine serum albumin for 2 days, and then used for the experiments.

Transfection of c-fos/Luciferase Fusion DNA Construct to VSMCs
VSMCs (3x10⁵) were prepared in a 6-cm tissue-culture dish. After 48 hours, 5 µg c-fos(-436 to approximately +45 bp)/luciferase fusion DNA and 2 µg ß-galactosidase gene driven by the simian virus 40 promoter-enhancer sequence were introduced to VSMCs by the DEAE-dextran method (Promega Co), as described previously.²² After transfection, the cells were cultured in DMEM with 10% fetal bovine serum for 24 hours and stimulated with 10^-4 mol/L H₂O₂ for 3 hours in DMEM with 0.1% bovine serum albumin. The luciferase activity was measured and normalized to ß-galactosidase activity, as described previously.²² The promoter region of the rat c-fos gene promoter was cloned by polymerase chain reaction. The nucleotide sequence was confirmed by the dideoxy chain-termination method in both sense and antisense strands.

Western Blot Analysis
Cell lysis and Western blot analyses of CREB, ERK, p38MAPK, and their phosphorylated forms were performed by using enhanced chemiluminescence (Amersham Biosciences), as described previously.²⁰

Immunoprecipitation
VSMCs were lysed in NP-40 lysis buffer (0.5% NP-40; 10 mmol/L Tris-HCl, pH 7.5; 150 mmol/L NaCl; 2.5 mmol/L KCl; 20 mmol/L ß-glycerol phosphate; 50 mmol/L NaF; 1 mmol/L Na₃VO₄; 1% aprotinin; 0.5% leupeptin; and 1 mmol/L dithiothreitol). Equal amounts of cell lyaste were subjected to immunoprecipitation with an anti-EGFR antibody (Calbiochem) and protein A (Amersham Biosciences). The pellet was washed with NP-40 lysis buffer and resuspended in sample buffer. Western blot analysis was performed with anti-phosphotyrosine antibody (clone 4G10, Upstate Biotechnology Inc).

Northern Blot Analysis
Preparation of total RNA and Northern blot analyses of c-fos and rRNA were performed as described previously.²³

Expression of Dominant-Negative And Wild-Type CREB
A recombinant adenovirus vector overexpressing a mutant of CREB (AdCREBM1), in which the phosphorylation site at Ser-133 was changed to alanine, was a gift from Dr Anthony J. Zeleznik (University of Pittsburgh, Pittsburgh, Pa).²⁴ An adenovirus vector expressing wild-type CREB (AdWTCREB) was constructed according to the manufacturer’s instructions (Takara Biomedicals). VSMCs grown to confluence were washed with phosphate-buffered saline 3 times. Then the cells were incubated with AdCREBM1 or adenovirus vector expressing Lac Z (AdLacZ) under gentle agitation for 2 hours at room temperature. After infection, the cells were washed 3 times, cultured in DMEM with 0.1% bovine serum albumin for 2 days, and used for the experiments. Multiplicity of infection (MOI) indicates the number of viruses per cell added to the culture dish.

Statistical Analysis
Statistical analyses were performed by 1-way ANOVA and multiple-comparison (Fisher) tests when appropriate. A value of P<0.05 was considered significant. Data were expressed as mean±SE.

	Results

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Phosphorylation of CREB at Ser-133 by H₂O₂
To investigate whether CREB is phosphorylated in response to H₂O₂, we performed Western blot analysis with an antibody that recognizes CREB species phosphorylated at Ser-133. H₂O₂ strongly stimulated phosphorylation of CREB (Figure 1A, upper panel), with a peak at 15 minutes of stimulation. H₂O₂ dose-dependently induced phosphorylation of CREB at 15 minutes of stimulation (Figure 1B, upper panel). However, higher concentrations of H₂O₂ (10³ to 10⁴ µmol/L) reduced CREB phosphorylation (data not shown), suggesting that H₂O₂ at higher concentration might be toxic to our VSMCs. Incubation with xanthine and xanthine oxidase also induced phosphorylation of CREB (data not shown). Catalase (1000 U/mL) abolished the H₂O₂-induced CREB phosphorylation, confirming that H₂O₂ is responsible for the phosphorylation of CREB (Figure 1C). The basal phosphorylation level of CREB was slightly increased by preincubation with catalase; however, the mechanism is not known at this point. NAC, a potent antioxidant, also inhibited H₂O₂-induced CREB phosphorylation (Figure 1D) without affecting the basal CREB phosphorylation level. The total level of CREB protein expression as detected by Western blot analysis with an antibody against CREB was unchanged after H₂O₂ stimulation (Figure 1A through 1D, lower panels).

View larger version (52K): [in this window] [in a new window]   Figure 1. Phosphorylation of CREB by H2O2. A, VSMCs were stimulated with H2O2 (100 µmol/L) for the indicated periods. B, VSMCs were stimulated for 15 minutes with varying concentrations of H2O2 as indicated in the figure. C, VSMCs were preincubated with catalase (1000 U/mL) for 30 minutes and then stimulated with H2O2 (100 µmol/L) for 15 minutes. D, VSMCs were preincubated with NAC (10 mmol/L) for 30 minutes and then stimulated with H2O2 (100 µmol/L) for 15 minutes. Phosphorylation of CREB was detected by Western blot analysis with a phospho-specific CREB antibody (upper panel). The membrane was stripped and reprobed with an anti-CREB antibody (lower panel). A representative autoradiograph is shown. Right-hand graph shows densitometric analysis of Western blots (n=6). The ratio of phosphorylated CREB to total CREB (pCREB/CREB) is indicated as a percentage of unstimulated controls. *P<005 vs control.

MAPK Pathways Mediate H₂O₂-Induced CREB Phosphorylation
Downstream kinases of ERK¹⁴ and p38 MAPK¹⁵ have been reported to phosphorylate CREB. H₂O₂-induced CREB phosphorylation was partially blocked by PD98059 (30 µmol/L), an ERK kinase inhibitor, or SB203580 (10 µmol/L), a p38 MAPK inhibitor (Figure 2A). PD98059 and SB203580 showed an additive effect. The same concentrations of PD98059 or SB203580 completely blocked H₂O₂-induced ERK or p38 MAPK activation, respectively, suggesting that partial inhibition might not be due to insufficient doses of the inhibitors (data not shown). Recent studies have shown that H₂O₂ activates MAPK through EGFR transactivation.^6,25 AG1478, an EGFR inhibitor, almost completely inhibited CREB phosphorylation by H₂O₂ (Figure 2B).

View larger version (50K): [in this window] [in a new window]   Figure 2. Effects of MAPK and EGFR inhibitors on H2O2-mediated CREB phosphorylation. A, VSMCs were preincubated with PD98059 (30 µmol/L) and/or SB203580 (10 µmol/L) for 30 minutes and then stimulated with H2O2 (100 µmol/L) for 15 minutes. B–E, VSMCs were preincubated with AG1478 (2.5 µmol/L) for 30 minutes and then stimulated with H2O2 (100 µmol/L) for 15 minutes. Western blot analyses of CREB (A and B) were performed as described in the legend to Figure 1. C, Cell lysates were subjected to immunoprecipitation with an anti-EGFR antibody, followed by Western blot analysis (Blot) with an anti-phosphotyrosine antibody (upper panel). The membrane was stripped and reprobed with an anti-EGFR antibody (lower panel). Western blot analyses of ERK (D) and p38MAPK (E) was performed with the same procedure as described in the legend to Figure 1. A representative autoradiograph is shown. Right-hand panels (A and B) show densitometric analysis of Western blots (n=6). *P<0.05 vs control; #P<0.05 vs H2O2 alone.

Immunoprecipitation with an anti-EGFR antibody, followed by Western blot analysis with an anti-phosphotyrosine antibody, revealed that H₂O₂ phosphorylated at the tyrosine residue of EGFR and AG1478 completely inhibited H₂O₂-induced tyrosine phosphorylation (Figure 2C). AG1478 also inhibited H₂O₂-induced ERK and p38 MAPK activation (Figure 2D and 2E), suggesting that EGFR might be upstream from MAPKs in H₂O₂ signaling and that the inhibitory effect of AG1478 on H₂O₂-induced CREB activation might be ascribed to inhibition of these MAPK pathways.

In addition to ERK and p38 MAPK, protein kinase A (PKA),⁹ phosphatidylinositol 3-kinase,²⁶ and calmodulin-dependent protein kinase II¹¹ are reported to mediate CREB phosphorylation. H89, a PKA inhibitor, did not affect H₂O₂-induced CREB phosphorylation (Figure 3A) but almost completely inhibited forskolin-induced CREB phosphorylation, suggesting that H89 sufficiently suppressed PKA activity. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, did not affect H₂O₂-induced CREB phosphorylation, whereas insulin-dependent CREB phosphorylation was inhibited by wortmannin at this concentration (Figure 3B). KN93, a calmodulin kinase II inhibitor, did not affect H₂O₂-induced CREB phosphorylation, whereas KN93 sufficiently inhibited A23187-induced CREB phosphorylation (Figure 3C). Finally, we examined the role of PKC in the phosphorylation of CREB by H₂O₂, because H₂O₂ is reported to activate certain PKC species.²⁷ Depletion of PKC by overnight exposure to 1 µmol/L phorbol myristate acetate (PMA) or a PKC inhibitor GF109203X did not affect H₂O₂-induced CREB phosphorylation, whereas these pretreatments sufficiently inhibited PMA-induced CREB phosphorylation. Because the PKC isoforms , ß, , , and are major PKC isoforms expressed in VSMCs²⁸ and are sensitive to GF109203X or PKC depletion by long-term exposure to PMA, PKC might not play a dominant role in CREB activation in response to H₂O₂.

View larger version (39K): [in this window] [in a new window]   Figure 3. Effect of various protein kinase inhibitors on H2O2-induced CREB phosphorylation. A, VSMCs were incubated with H89 (10 µmol/L) for 30 minutes and then stimulated with H2O2 (100 µmol/L) for 15 minutes or forskolin (10 µmol/L) for 5 minutes. B, VSMCs were incubated with wortmannin (50 nmol/L) for 30 minutes and then stimulated with H2O2 (100 µmol/L) for 15 minutes or insulin (100 nmol/L) for 10 minutes. C, VSMCs were incubated with KN92 (10 µmol/L) or KN93 (10 µmol/L) for 30 minutes and then stimulated with H2O2 (100 µmol/L) for 15 minutes or A23187 (10 µmol/L) for 10 minutes. D, VSMCs were incubated with PMA (1 µmol/L) over night (O/N) or with GF109203X (10 µmol/L) for 30 minutes and then stimulated with H2O2 (100 µmol/L) for 15 minutes or with PMA (1 µmol/L) for 10 minutes. Western blot analyses were performed as described in the legend to Figure 1. The same results were obtained in other independent experiments (n=3), and a representative autoradiograph is shown.

Activation of CRE-Dependent Transcription by H₂O₂
CRE is 1 of the critical cis-DNA elements of the c-fos gene promoter. We tested whether H₂O₂ activated CRE-dependent gene transcription by using a c-fos promoter/luciferase reporter construct. As shown in Figure 4A, normalized luciferase activity after H₂O₂ stimulation (10^-4 mol/L) was increased by 2-fold compared with that of control (mean±SE, n=5, P<0.01). We confirmed the role of CREB in the c-fos gene promoter by overexpression of a dominant-negative form of CREB (ie, AdCREBM1) with an adenovirus vector. Infection of AdCREBM1 inhibited H₂O₂-induced c-fos promoter activity. Infection of AdCREBM1 inhibited H₂O₂-induced c-fos mRNA expression at 30 minutes of stimulation. Overexpression of wild-type CREB increased the basal and H₂O₂-induced CREB phosphorylation levels and increased c-fos mRNA expression. Infection of AdLacZ did not affect H₂O₂-induced c-fos mRNA expression (Figure 4B). Western blot analysis with an antibody against -tubulin suggests that equal amounts of protein were loaded on each lane.

View larger version (53K): [in this window] [in a new window]   Figure 4. Suppression of H2O2-induced c-fos expression by AdCREBM1. A, A luciferase vector driven by the c-fos gene promoter (5 µg) was transfected to VSMCs with an expression vector of LacZ (2 µg). The next day, AdCREBM1 (30 MOI) or an empty adenovirus (Ad Empty) was infected. The next day, the VSMCs were stimulated with H2O2 for 3 hours, and then luciferase and ß-galactosidase (ß-gal) assays were performed. Data are expressed as mean±SEM (n=5). *P<0.05 vs control. B, VSMCs were infected with 30 MOI of AdCREBM1 (CREBM1), 30 MOI of Ad WTCREB (WTCREB), or 30 MOI of AdLacZ (LacZ) and stimulated with H2O2 (100 µmol/L) for 15 or 30 minutes. Northern blot analysis of c-fos mRNA (30-minute stimulation) and Western blot analyses (15-minute stimulation) of phospho-CREB, CREB, and -tubulin were performed. Right-hand panel shows ratio of c-fos mRNA to total rRNA (black bars) and of pCREB to CREB (hatched bars). *P<0.05 vs without H2O2 (n=6).

Recent results suggest that ROS play a critical role in AngII signaling.^3,5 We recently reported that AngII induced CREB phosphorylation.¹⁹ We examined whether ROS are involved in AngII-induced CREB phosphorylation. NAC inhibited AngII-induced activation of ERK and p38 MAPK (Figure 5A and 5B). NAC also inhibited AngII-induced CREB phosphorylation (Figure 5C). However, NAC did not affect AngII-induced c-fos mRNA expression (Figure 5D). These results suggest that ROS mediate AngII-induced CREB phosphorylation, but a different signaling pathway might be involved in AngII-induced c-fos mRNA induction.

View larger version (41K): [in this window] [in a new window]   Figure 5. Effect of NAC on AngII-induced ERK, p38 MAPK, and CREB activation and on c-fos mRNA induction. VSMCs were preincubated with or without NAC (10 mmol/L) for 1 hour and then stimulated with AngII (100 nmol/L) for 5 minutes. Western blot analyses of (A) phosphorylated ERK and ERK, (B) phosphorylated p38 MAPK and p38 MAPK, and (C) phosphorylated CREB and CREB were performed as described in the legend to Figure 1. D, VSMCs were preincubated with or without NAC (10 mmol/L) for 1 hour and then stimulated with AngII (100 nmol/L) for 30 minutes. Northern blot analysis of c-fos mRNA was performed as described in the legend to Figure 4. Right-hand panel shows densitometric analyses of Northern and Western blots, (n=4). *P<0.05 vs control; #P<0.05 vs H2O2 alone.

	Discussion

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A growing body of evidence suggests that oxidative stress plays an important role in the progression of cardiovascular diseases, such as unstable angina, myocardial infarction, and heart failure. Therefore, it is important to understand the intracellular signals that are elicited by ROS to obtain insights into the pathogenesis of cardiovascular diseases. It is also generally accepted that ROS mimic the signaling of cytokines or growth factors, indicating that studying ROS signaling might contribute to understanding the signaling pathway of cytokines and growth factors. H₂O₂ activates CREB in macrophage cells line¹⁸ but inhibits it in neuronal cells,²⁹ suggesting that ROS differentially regulate CREB function in different cells types. We therefore studied the role of ROS in the activation of CREB in VSMCs, and this study is, to our knowledge, the first report showing that H₂O₂-induced CREB phosphorylation and CRE-dependent gene transcription involve transactivation of EGFR.

Recently, nerve growth factor (NGF)¹⁴ and EGF³⁰ were reported to phosphorylate and activate CREB through an ERK-p90^rsk2-dependent pathway. In contrast to EGF or NGF, fibroblast growth factor (FGF)–induced CREB phosphorylation is mediated by MAPKAP kinase-2,¹⁵ which lies immediately downstream from p38 MAPK. PD98059 or SB203580 suppressed H₂O₂-induced CREB phosphorylation. Therefore, p90^rsk2 and MAPKAP kinase-2 might be responsible for phosphorylation of CREB downstream from ERK and p38 MAPK, respectively. PD98059 and SB203580 showed additive effects, suggesting that the ERK pathway and the p38 MAPK pathway might be independent in terms of CREB activation. We showed that other inhibitors for protein kinase that might phosphorylate CREB failed to inhibit H₂O₂-induced CREB phosphorylation, suggesting that MAPK pathways are dominant for phosphorylation of CREB in response to H₂O₂.

Transactivation of EGFR is a critical signaling step for certain G protein–coupled receptors, such as endothelin, thrombin,⁸ and AngII⁷ receptor. A recent report showed that AngII, an ROS-inducing agonist, activated ERK and p38 MAPK through EGFR in VSMCs.³¹ We have shown that H₂O₂ activated ERK and p38 MAPK in an EGFR-dependent manner, which is consistent with results reported by Frank et al.²⁵ These data suggest that inhibition of H₂O₂-induced CREB phosphorylation by AG1478 might be ascribed to the suppression of these MAPK pathways. Although AngII is reported to transactivate EGFR through a src- and Pyk2-dependent manner in endothelial cells,⁶ a recent result showed that H₂O₂ activated EGFR through matrix metalloproteinase but not through Pyk2 in VSMCs.³² We have not examined the role of Pyk2 or matrix metalloproteinase in CREB activation. However, our results suggest that H₂O₂ might activate matrix metalloproteinase and induce EGFR transactivation, which are followed by CREB phosphorylation.

Although H₂O₂ stimulated CREB phosphorylation by several-fold, H₂O₂ increased CRE luciferase activity only 2-fold. The reason for this discrepancy is unclear. One explanation is that the basal luciferase activity might be relatively high in our VSMCs. Another possibility is that competition of activated CREB between endogenous CRE sites and CRE luciferase might occur and that most of the activated CREB might participate in the activation of endogenous genes, resulting in the weak activation of CRE luciferase by H₂O₂. Previously, Brindle et al³³and Ginty et al³⁴ reported that the ability to activate CRE-dependent gene transcription is different among signaling pathways despite the similar level of CREB phosphorylation. A recent report by Mayr et al³⁵ might explain this differential effect on CREB phosphorylation and CRE-dependent gene transcription. They showed that the CREB-CBP complex induced by mitogenic signals such as NGF or EGF is less stable than that induced by cAMP in the nucleus. Therefore, the relative instability of the H₂O₂-induced CREB/CBP complex might account for the weak activation of CRE-dependent gene transcription by H₂O₂.

Overexpression of wild-type CREB increased c-fos mRNA induction, and overexpression of dominant-negative CREB decreased c-fos mRNA expression in response to H₂O₂, suggesting the critical and essential role of CREB for c-fos gene expression. Overexpression of wild-type CREB strongly enhanced basal and H₂O₂-induced phosphorylation of CREB. However, basal c-fos mRNA expression is not so prominent in wild-type, CREB-overexpressing cells. The mechanism is presently unknown. An explanation could be that activation of CREB alone is not sufficient for c-fos gene expression, and simultaneous activation of an additional transcription factor(s) is necessary for induction.

The mechanism by which CREBM1 inhibits CREB function is believed to be the replacement of endogenous CREB with the mutated CREB, rather than inhibition of phosphorylation of endogenous CREB.³⁶ Because CREB can dimerize with ATF-1, it is possible that the effect of CREBM1 might be ascribed to sequestration of ATF-1. This possibility cannot be excluded at this point.

Although H₂O₂ and AngII use similar signaling mechanisms in terms of CREB activation, AngII but not H₂O₂ requires PKA activity for the activation of CREB.²⁰ Recently, Impey et al³⁷ reported that PKA activity is necessary for the translocation of ERK activated by NGF and that basal PKA activity might be required for AngII-induced ERK and CREB activation but not for H₂O₂-induced ERK activation. Inhibition of CREB function suppressed H₂O₂-induced c-fos mRNA expression. Although NAC inhibited AngII-induced MAPK activation and CREB phosphorylation, AngII-induced c-fos mRNA induction was not inhibited by NAC. These data suggest that AngII-induced c-fos mRNA induction might involve a ROS-independent pathway, although ROS play an important role in AngII-induced MAPK activation. Because NAC did not completely inhibit AngII-induced activation of MAPKs and CREB, the remaining activity of these pathways might be sufficient to induce c-fos mRNA. ROS seem to play a critical role in AngII signaling; however, signaling mechanisms are not identical between AngII and H₂O₂.

Perspective
We have shown that H₂O₂ activates ERK and p38 MAPK through EGFR transactivation. These MAPKs mediate phosphorylation of CREB, and activated CREB plays an important role in the induction of c-fos mRNA expression. Our results suggest that G protein–coupled receptor–induced CREB activation might involve ROS/EGFR transactivation and play an important role in the regulation of gene expression in VSMCs. However, the signaling mechanisms of gene activation are not identical between H₂O₂ and AngII, and further study is necessary.

	Acknowledgments

 
This study was supported in part by a grant from the Yamanouchi Foundation for Research on Metabolic Disorders and the Takeda Medical Research Foundation and by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to T.I.).

Received January 29, 2003; first decision February 24, 2003; accepted May 19, 2003.

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