Myocyte enhancer factor 2B is involved in the inducible expression of NOX1 ⁄ NADPH oxidase, a vascular superoxide-producing enzyme Masato Katsuyama*, Muhammer Ozgur Cevik*, Noriaki Arakawa, Tomoko Kakehi, Toru Nishinaka, Kazumi Iwata, Masakazu Ibi, Kuniharu Matsuno and Chihiro Yabe-Nishimura Department of Pharmacology, Kyoto Prefectural University of Medicine, Japan Reactive oxygen species including hydrogen peroxide, hydroxyl radical, singlet oxygen, peroxynitrite, and superoxide (O 2 – ) have been documented as intrinsic signaling molecules that modulate multiple cellular responses. The major source of O 2 – in vascular cells and cardiac myocytes is the NADPH oxidase family [1–3]. NADPH oxidase catalyzes the reduction of molecular oxygen to O 2 – using NADPH as an electron donor. A wealth of data has been collected on the phagocyte NADPH oxidase, an essential component of the host antimicrobial defense system [4]. The phago- cyte oxidase consists of the catalytic subunit gp91phox (NOX2), the regulatory subunits p22phox, p47phox, p40phox and p67phox, and the small GTPase Rac. Recent studies in nonphagocytic cells identified several homologs of the catalytic subunit NOX2. NOX1 is one of these homologs predominantly expressed in colon epithelial cells (CEC) and in vascular smooth muscle cells (VSMC). The expression of NOX1 in VSMC is induced by various vasoactive factors, such as angiotensin II, platelet-derived growth factor (PDGF), phorbol ester, and fetal bovine serum [5,6]. Among these factors, prostaglandin (PG) F 2a , one of the primary prostanoids Keywords activating transcription factor-1; myocyte enhancer factor 2; NADPH oxidase; NOX1; vascular smooth muscle cells Correspondence C. Yabe-Nishimura, Department of Pharmacology, Kyoto Prefectural University of Medicine, Kyoto 602–8566, Japan Fax: +81 75 251 5348 Tel: +81 75 251 5333 E-mail: nchihiro@koto.kpu-m.ac.jp *These authors contributed equally to this work (Received 2 June 2007, revised 26 July 2007, accepted 7 August 2007) doi:10.1111/j.1742-4658.2007.06034.x NADPH oxidase is a major source of the superoxide produced in cardio- vascular tissues. Expression of NOX1, a catalytic subunit of NADPH oxi- dase, is induced by various vasoactive factors, including angiotensin II, prostaglandin (PG) F 2a and platelet-derived growth factor (PDGF). To clarify the molecular basis of this transcriptional activation, we delineated the promoter region of the NOX1 gene. RT-PCR and 5¢-rapid amplifica- tion of cDNA ends-based analyses revealed a novel 5¢-terminal exon of the rat NOX1 gene located approximately 28 kb upstream of the exon contain- ing the start codon. Both PGF 2a and PDGF enhanced the transcriptional activity of the ) 3.6 kb 5¢-flanking region of the NOX1 gene in A7r5 cells, a rat vascular smooth muscle cell line. A PGF 2a -response element was located between )146 and )125 in the 5¢-flanking region containing a consensus binding site for myocyte enhancer factor 2 (MEF2), to which binding of MEF2 was augmented by PGF 2a . Gene silencing of MEF2B by RNA interference significantly suppressed the expression of NOX1, while silencing of activating transcription factor (ATF)-1, previously impli- cated in up-regulation of NOX1, abolished the PGF 2a - or PDGF-induced expression of MEF2B. These results indicate that superoxide production in vascular smooth muscle cells is regulated by the ATF-1–MEF2B cascade by induction of the expression of the NOX1 gene. Abbreviations ATF, activating transcription factor; CEC, colon epithelial cells; CRE, cAMP response element; CREB, cAMP response element-binding protein; dsRNA, double-stranded RNA; EMSA, electrophoresis mobility shift assay; MEF, myocyte enhancer factor; PDGF, platelet-derived growth factor; PG, prostaglandin; PI3, phosphoinositide 3; VSMC, vascular smooth muscle cells. 5128 FEBS Journal 274 (2007) 5128–5136 ª 2007 The Authors Journal compilation ª 2007 FEBS generated in the vascular tissue, was also shown to induce the expression of NOX1 mRNA and cause hypertrophy of VSMC through increased generation of O 2 – [7]. In PGF 2a -induced as well as PDGF-induced NOX1 expression, transactivation of the epidermal growth factor receptor, which depends on protein kinase C d, elicits activation of extracellular signal- regulated kinase 1 ⁄ 2 as well as of phosphoinositide 3 (PI3) kinase. Downstream of PI3 kinase, a transcrip- tion factor of the cAMP response element (CRE)- binding protein (CREB) ⁄ activating transcription factor (ATF) family, ATF-1, was suggested to take part in the induction of NOX1 expression [8–10]. Except for the involvement of ATF-1, the entire picture of the transcriptional regulation of the NOX1 gene has not been clarified yet. In the upstream region of the human NOX1 gene, an interferon-c- responsive element was recently identified, which reg- ulates the expression of NOX1 in CEC [11]. On the other hand, we recently depicted novel transcripts of the mouse NOX1 gene that were induced to express under phenotypic modulation of VSMC. Of particular interest is that these transcripts were governed by promoters different from the one utilized for expres- sion of the NOX1 transcript in CEC [12]. To clarify the molecular basis of the transcriptional activation of NOX1 in vascular tissue, we delineated the pro- moter region implicated in the up-regulation of NOX1 gene expression. We report here the predomi- nant role of the ATF-1-myocyte enhancer factor 2B (MEF2B) cascade in superoxide production in vascu- lar smooth muscle cells by inducing the expression of the NOX1 gene. Results Determination of the 5¢-end of the NOX1 mRNA expressed in VSMC We reported the expression of novel NOX1 transcripts (c- and f-types) in mouse VSMC, which encoded an extended N-terminal peptide sequence upstream of the form expressed in CEC (a-type) [12]. We searched the rat genomic database and found the counterpart of the mouse NOX1 exon 1c, the 5¢-terminal exon of the c-type mRNA. RT-PCR was then performed in primary VSMC or PGF 2a -stimulated A7r5 cells, a rat vascular smooth muscle cell line, using a forward primer for the rat counterpart (ex1c2F in Fig. 1A), and a reverse primer covering the start codon of the NOX1 mRNA (NR3 in Fig. 1A). Amplified products were sequenced and the 5¢-terminus of the NOX1 transcript was determined by 5¢-RACE. The 5¢-terminus encoded 433 bp of exon 1c, and it was placed upstream of exon 1a containing the start codon (Fig. 1B). Unlike the mouse c-type mRNA, however, the rat c-type-like NOX1 mRNA neither contained the counterpart of the mouse exon 1b nor encoded an additional N-terminal peptide. Although the counterpart of the mouse exon 1f was found in the genomic database, an f-type-like transcript was not detected by RT-PCR in A7r5 cells. PGF 2a - and PDGF-induced transcriptional activation of the NOX1 promoter To examine whether the promoter region of the rat NOX1 gene contains elements responsive to vasoactive NOX1 mRNA 1a 2 chrX 3 k28 k 1c ATG 209 96433 ATG Intron Exon A B Fig. 1. Structure of the rat NOX1 gene and mRNA. (A) 5¢-Nucleotide sequences of the rat NOX1 cDNA and deduced amino acid sequences at the beginning of the open reading frame. Primers used for RT-PCR and 5¢-RACE are indicated with arrows. (B) The exon ⁄ intron structure of the NOX1 gene and its splicing pathway. Open boxes indicate exons. Numbers in the box denote the size of each exon (bp). The size of the intron is indicated under the broken lines (bp). Closed boxes show the open reading frame of the transcript. M. Katsuyama et al. MEF2B regulates vascular NOX1 ⁄ NADPH oxidase FEBS Journal 274 (2007) 5128–5136 ª 2007 The Authors Journal compilation ª 2007 FEBS 5129 factors, approximately 3.6 kb of the 5¢-flanking region of exon 1c was isolated and subcloned into a luciferase vector. As demonstrated in Fig. 2A, the transcriptional activity of the NOX1 promoter, r3642Luc, in A7r5 cells was significantly enhanced when cells were treated with PGF 2a or PDGF. These findings suggest that the 3.6 kb of the 5¢-flanking region contains sequences responsible for PGF 2a -induced as well as PDGF- induced transcriptional activation. As the activation by PGF 2a was more prominent than that by PDGF, PGF 2a was used for the subsequent promoter analyses. MEF2-binding site was essential for transcriptional activation of the NOX1 promoter To identify the region responsible for the transcrip- tional activation, a series of deletion mutants of the NOX1 promoter-luciferase chimera plasmids were con- structed. As shown in Fig. 2B, deletion up to )489, but not to )930, reduced PGF 2a -induced transcriptional activation, suggesting the existence of an enhancer binding site between )930 and )489. Deletion up to )125, but not to )146, completely abolished the PGF 2a -induced transcriptional activation. Between )146 and )125, a consensus sequence of the MEF2- binding site, 5¢-CTA(A ⁄ T) 4 TAG ⁄ A-3¢, was located (Fig. 3A). The introduction of mutations at this site (5¢-CTATAAATAG-3¢ to 5¢-CTATAgccAG-3¢) abolished PGF 2a -induced transcriptional activation (Fig. 3B). These findings clearly indicate that the MEF2-binding site between )139 and )130 is essential for PGF 2a -induced activation of the NOX1 promoter. Binding of MEF2 to the consensus sequence in the NOX1 promoter To verify whether the transcription factor MEF2 actu- ally binds to the consensus sequence in the NOX1 pro- moter, an electrophoretic mobility shift assay (EMSA) was carried out using nuclear extracts obtained from A7r5 cells (Fig. 4). With the probe containing the con- sensus MEF2-binding site of the NOX1 promoter, several bands were observed (lane 1, Fig. 4A). Among these bands, those indicated by arrowheads were mark- edly diminished when the mutated probe was utilized (lane 5). Stimulation of A7r5 cells with PGF 2a increased the intensity of these bands (lane 2), whereas the bands were undetectable in the presence of an excess amount of the unlabeled wild-type probe (lane 3). By contrast, the bands persisted in the presence of an excess amount of the mutated probe (lane 4). As shown in Fig. 4B, pre-incubation of the nuclear extract with an anti- MEF2 IgG generated supershifted bands as indicated by arrowheads (lane 3). On the other hand, pre-incuba- tion with an anti-ATF-1 IgG did not affect the mobility of the specific bands (lane 4, Fig. 4B). These results suggest that PGF 2a increases the binding of MEF2 to the consensus-binding site located between )139 and )130 of the NOX1 promoter. Gene silencing of MEF2B attenuated PGF 2a -or PDGF-induced NOX1 expression There are four types of MEF2 – MEF2A, MEF2B, MEF2C, and MEF2D – and these subtypes are enco- ded by distinct genes [13]. In A7r5 cells, MEF2A, MEF2C and MEF2D were constitutively highly expressed, whereas the expression level of MEF2B was very low (see Fig. 5A, Cycles of PCR). Upon stimula- tion with PGF 2a or PDGF, however, the expression of 0123 0123 Relative Luciferase Activity –91 –125 –146 –235 –323 –489 –930 1c luc pGL3basic MEF2 –3642 AP-1 TATA-like Relative Luciferase Activity PGF 2 α A B PGDF * * * * * * * * Fig. 2. Analyses of the NOX1 promoter activity in A7r5 cells. (A) PGF 2a - or PDGF-induced transcriptional activation of the NOX1 promoter. The 3.6 kb of the 5¢-flanking region of exon 1c was cloned into pGL3-basic and the reporter construct was transfected into A7r5 cells. The b-galactosidase-expression vector was cotrans- fected as an internal control. Serum-starved cells were incubated with 100 n M PGF 2a or 20 ngÆmL )1 PDGF-BB for 24 h. The relative luciferase activity is denoted as the fold-increase induced by PGF 2a or PDGF. Bars represent means ±SE of three experiments. Open bar, pGL3-basic; closed bar, r3642Luc. *P < 0.01 versus pGL3-basic. (B) Deletion of the MEF2-binding site abolished PGF 2a -induced transcriptional activation. A schematic diagram of the promoter-luciferase fusion plasmids is shown on the left, where the 5¢⁄3¢ ends of the construct relative to the transcription initiation site are indicated. The relative luciferase activity is denoted as the fold-increase induced by PGF 2a . Bars represent means ±SE of three experiments. *P < 0.01 versus pGL3-basic. MEF2B regulates vascular NOX1 ⁄ NADPH oxidase M. Katsuyama et al. 5130 FEBS Journal 274 (2007) 5128–5136 ª 2007 The Authors Journal compilation ª 2007 FEBS MEF2B was markedly augmented (Fig. 6A, mock). We therefore focused on the role of MEF2B in the PGF 2a - or PDGF-induced up-regulation of NOX1 gene expression. An expression vector coding a dou- ble-stranded RNA (dsRNA) targeting nucleotides 470– 494 of the rat MEF2B mRNA sequence was intro- duced into A7r5 cells. Following single cell cloning of the transfectants, two clones stably expressing the dsRNA, MEF2B-RNAi-1 and MEF2B-RNAi-2, were isolated. In these clones, mRNA levels of MEF2B, but not those of other types of MEF2, were reduced com- pared to levels in the mock-transfected cells (Fig. 5A). As shown in Fig. 5B, induction of NOX1 mRNA expression by PGF 2a or PDGF was almost completely abolished in MEF2B knocked-down cells. In addition, a PGF 2a -induced increase in O 2 – production as well as the basal level of cellular O 2 – was reduced in these clones compared with the mock-transfected cells (Fig. 5C). These results highlight the pivotal role of MEF2B in the PGF 2a - or PDGF-induced up-regula- tion of NOX1 expression. Gene silencing of ATF-1 attenuated PGF 2a -or PDGF-induced MEF2B expression We previously reported the involvement of ATF-1, a transcription factor of the CREB ⁄ ATF family, in the up-regulation of NOX1 expression in VSMC [8]. ATF-1 elicits transcriptional activation by binding to the cAMP response element (CRE). As CRE was not found in the promoter region of the NOX1 gene, ATF-1 was assumed to indirectly regulate the expres- sion of the NOX1 mRNA. We therefore elucidated the role of ATF-1 in the MEF2B-dependent activation of NOX1 expression. As shown in Fig. 6, the expression of MEF2B mRNA induced by PGF 2a or PDGF was almost completely abolished in ATF-1 knocked-down clones that we previously isolated [8]. These findings provide a clear link between ATF-1 and MEF2B, in that expression of MEF2B in VSMC is governed by ATF-1 itself. Discussion The major lines of evidence provided by this study are as follows: (a) the 5¢-terminus of the rat NOX1 mRNA expressed in VSMC was a counterpart of the mouse c-type mRNA induced under phenotypic modulation of VSMC; (b) the promoter region of the NOX1 gene contained a consensus MEF2-binding site that confers the responsiveness to PGF 2a ; (c) stimulation with PGF 2a enhanced the binding of MEF2 to its consensus binding site in the NOX1 promoter; (d) RNA interfer- ence targeted at MEF2B abolished expression of the 0123 Relative Luciferase Activity –125 –146 luc pGL3basic –146mut 1c B A MEF2 AP-1 TATA-like * Fig. 3. PGF 2a -induced transcriptional activation of the NOX1 promoter was dependent on the MEF2-binding site. (A) A consensus sequence of the MEF2-binding site and the AP-1 site, and a TATA-like sequence located upstream of the transcription initiation site. (B) Introduction of mutations at the MEF2-binding site (5¢-CTATAAATAG-3¢ to 5¢-CTATAgccAG-3¢) abolished PGF 2a -induced transcriptional activation. A sche- matic diagram of the promoter-luciferase fusion plasmids is shown on the left, where the 5¢⁄3¢ ends of the construct relative to the tran- scription initiation site are indicated. The relative luciferase activity is denoted as the fold-increase induced by PGF 2a . Bars represent means ±SE of three experiments. *P<0.01 versus pGL3-basic. M. Katsuyama et al. MEF2B regulates vascular NOX1 ⁄ NADPH oxidase FEBS Journal 274 (2007) 5128–5136 ª 2007 The Authors Journal compilation ª 2007 FEBS 5131 NOX1 mRNA induced by PGF 2a or PDGF; (e) RNA interference targeted at ATF-1 attenuated the induc- tion of MEF2B expression by PGF 2a or PDGF. Based on these findings and those of our earlier studies [8–10], it is reasonable to conclude that the ATF-1- MEF2B cascade constitutes the major signaling path- way that leads to the up-regulation of NOX1 gene expression in VSMC. The 5¢-terminus of the NOX1 mRNA identified in rat VSMC was a counterpart of the mouse c-type mRNA which was expressed in dedifferentiated origin probe MEF2 PGF 2α α PGF 2 α –++++ wt wt wt wt mut x100 cold –– – wt mut supershift lane A B 12 345 anti MEF2 Ab ––+– anti ATF-1 Ab origin MEF2 –+++ –– lane 1234 –+ free probes Fig. 4. PGF 2a increased binding of MEF2 to the consensus sequence. (A) Specific bands detected by EMSA. Nuclear extracts were prepared from A7r5 cells incubated with 100 n M PGF 2a for 8 h. Binding specificity was evaluated with a 100-fold excess of unlabeled oligonucleotide (lane 3), and with a mutated oligonucleo- tide probe (lane 5). (B) Supershift bands demonstrated in the presence of an anti-MEF2 IgG. Nuclear extracts were pre-incubated in the presence or absence of an anti-MEF2 IgG or an anti-ATF-1 IgG. 0 2 4 6 NOX1 GAPDH NOX1/GAPDH mock MEF2B RNAi-1 MEF2B RNAi-2 Control PGF 2α α PGF 2 α PGF 2 α Control Control PDGF PDGF PDGF Cycles of PCR mock RNAi-1 RNAi-2 anti-MEF2B A B C dsRNA GAPDH MEF2B MEF2A MEF2C MEF2D (40) (40) (25) (25) (25) (25) * * Mean Fluorescence Values mock MEF2BRNAi-1 MEF2BRNAi-2 0 50 60 70 80 90 * * * † † Fig. 5. Gene silencing of MEF2B attenuated PGF 2a - or PDGF- induced NOX1 expression. (A) Expression of anti-MEF2B dsRNA precursors and silencing of MEF2B expression in the MEF2B RNAi-1 and RNAi-2 clones. Total RNA was reverse-transcribed and the cDNA fragments were amplified by PCR. (B) Induction of the NOX1 mRNA expression by PGF 2a or PDGF was suppressed in RNAi-1 and RNAi-2. A representative northern blot is shown. Bars represent the mean ±SE of three experiments. *P<0.01 versus mock control. (C) Ethidium fluorescence in the cells untreated (control; open bar) or treated with 100 n M PGF 2a (closed bar) for 24 h. Mean values of ethidium fluorescence were calculated from four samples. *P<0.05 versus control mock-transfected cells. P<0.05 versus mock-transfected cells treated with PGF 2a . MEF2B regulates vascular NOX1 ⁄ NADPH oxidase M. Katsuyama et al. 5132 FEBS Journal 274 (2007) 5128–5136 ª 2007 The Authors Journal compilation ª 2007 FEBS VSMC. The rat NOX1 mRNA, however, did not con- tain the counterpart of the mouse exon 1b, which encoded an additional N-terminal peptide upstream of exon 1a. Unlike the NOX1 mRNA expressed in VSMC, the transcript identified in rat colon or intact aorta did not contain exon 1c at the 5¢-terminus (data not shown). The transcript expressed in VSMC con- tained an extended 5¢-untranslated region that differed from the colon-type NOX1 mRNA. Accordingly, the 5¢-flanking region of exon 1c appears to contain ele- ments essential for the specific expression of the NOX1 mRNA in dedifferentiated VSMC. In the aorta or in a vascular smooth muscle cell line (T ⁄ G HA-VSMC) of human origin, the c-type-like mRNA has not been identified so far (data not shown). Thus there seems to be considerable species–specific differences in the regu- lation of the NOX gene expression in VSMC. It should be noted that another catalytic subunit of NADPH oxidase, NOX5, is expressed in human VSMC [14], but not in rodents. The MEF2-binding site in the promoter region was shown to be elemental for induction of NOX1 expres- sion in VSMC. This site was also conserved in the mouse NOX1 gene. To date the functional role of MEF2 documented in VSMC has been somewhat con- troversial. MEF2A was reported to be involved in an angiotensin II-induced vascular hypertrophy [15], and in inducing the expression of monocyte chemo- attractant protein-1 [16], which plays a key role in the development of atherosclerosis and restenosis after angioplasty. Conversely, MEF2B was reported to bind to the promoter region of the smooth muscle myosin heavy chain gene to regulate the transcription of the smooth muscle-specific gene expression [17]. Intrigu- ingly, MEF2B was documented to be highly expressed in neointima of balloon-injured carotid artery [18], which suggests that the expression of MEF2B is up- regulated under phenotypic modulation of VSMC. Among four types of MEF2, MEF2A, MEF2C and MEF2D were constitutively expressed in A7r5 cells, and the levels of these transcripts were relatively high. By contrast, the basal level of the MEF2B mRNA was very low, but expression of MEF2B was increased upon stimulation with PGF 2a or PDGF. In MEF2B knocked-down cells, the induction of NOX1 mRNA expression by PGF 2a or PDGF was almost completely abolished, whereas it was unaffected in MEF2A knocked-down clones (data not shown). Accordingly, inducible expression of MEF2B appears to up-regulate the NOX1 gene expression in VSMC. Previously, the involvement of ATF-1, a transcrip- tion factor of the CREB ⁄ ATF family, was suggested in the up-regulation of NOX1 expression by various vasoactive factors [8]. ATF-1 is known to activate transcription of genes by binding to CRE. In the 3.6 kb promoter region of the rat NOX1 gene, how- ever, a canonical CRE was not found. This suggests an indirect involvement of ATF-1 in the NOX1 tran- scription, though there might be other ATF-1 binding sites elsewhere in the NOX1 gene. We also observed that phosphorylation of ATF-1 occurred within 5 min of stimulation with PGF 2a [8], whereas expression of the NOX1 mRNA was not observed until 3 h after the stimulation [7]. These findings support the view that ATF-1 indirectly regulates the NOX1 gene expression, possibly by up-regulating the expression of the genes encoding other transcription factors. It should be pointed out that a consensus CRE sequence was located approximately 2.6 kb upstream of the tran- scription start site of the rat MEF2B gene (rat MEF2B mRNA, GenBank BC079361; genomic sequence, rat chromosome 16). In accord with this observation, the PGF 2a - or PDGF-induced increase in MEF2B mRNA was almost completely abolished in ATF-1 knocked- down clones. These results strongly suggest the involvement of the ATF-1-MEF2B cascade in the reg- ulation of vascular NOX1 gene expression. We previously reported the pathophysiological significance of NOX1-derived reactive oxygen species in angiotensin II-induced chronic hypertension using NOX1-deficient mice [19]. The basal level of the NOX1 transcript is much lower than the levels of other NOX isoforms expressed in vascular tissue, whereas inducible expression of NOX1 has been docu- mented in association with various vascular disorders. In this context, identification of the ATF-1-MEF2B cascade involved in the up-regulation of NOX1 MEF2B GAPDH mock ATF-1 RNAi-5 ATF-1 RNAi-16 Control PGF 2 α α Control PGF 2 α Control PGF 2 α PDGF PDGF PDGF MEF2B/GAPDH 0 1 2 3 4 5 * Fig. 6. Gene silencing of ATF-1 attenuated PGF 2a - or PDGF-induced MEF2B expression. Expression of the MEF2B transcript was exam- ined by RT-PCR in the ATF-1 RNAi-5 and RNAi-16 clones. Bars rep- resent the mean ±SE of three experiments. *P<0.05 versus mock control. M. Katsuyama et al. MEF2B regulates vascular NOX1 ⁄ NADPH oxidase FEBS Journal 274 (2007) 5128–5136 ª 2007 The Authors Journal compilation ª 2007 FEBS 5133 gene expression may lead to a better understanding of regulatory mechanisms in vascular superoxide production. Experimental procedures Materials PGF 2a was purchased from Nacalai Tesque (Kyoto, Japan). Antibodies against MEF2 and ATF-1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). PDGF-BB was obtained from PEPROTECH (London, UK). [c- 32 P]- ATP, [a- 32 P]-UTP, and [a- 32 P]-dCTP were from ICN Bio- medicals (Costa Mesa, CA). Cell culture The A7r5 cell line, obtained from American Type Culture Collection (Rockville, MD), was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum. Primary VSMC were isolated from Sprague-Dawley rats by a migration method [20]. 5¢-RACE 5¢-RACE was carried out using a-3¢⁄5¢-RACE kit (Roche, Basel, Switzerland). Total RNA isolated from either A7r5 cells or primary VSMC was reverse transcribed with the primer NR1, which is complementary to nucleotides 207– 231 of the rat NOX1 mRNA (GenBank AF152963; Fig. 1A). The cDNAs were tagged with dATP using termi- nal deoxytransferase. The first round of amplification was carried out using the oligo dT-anchor primer and the sec- ond primer NR2, complementary to nucleotides 182–206 of the rat NOX1 mRNA. The resulting PCR products served as templates for the subsequent nested amplification of cDNAs specific for NOX1. For this amplification, the anchor primer and the primer cup1R or cup2R, comple- mentary to the sequence in exon 1c, were utilized (Fig. 1A). Based on the sequencing analyses, the 5¢-end of the longest cDNA clones was regarded as the transcriptional start site and denoted as +1. Reporter constructs and luciferase assay The rat genomic DNA was isolated from A7r5 cells with a PUREGENE DNA Isolation Kit (Gentra SYSTEMS, Min- neapolis, MN). The 5¢-flanking region of the rat NOX1 gene was amplified by PCR and cloned into the vector pGL3-basic (Promega, Madison, WI). The 3.6 kb 5¢-flank- ing region was cloned into the HindIII site of pGL3-basic. A series of 5¢-deletion constructs were made by cleavage with restriction enzymes or amplification by PCR. All con- structs were subjected to sequencing analyses to verify the orientation and fidelity of the insert. Luciferase plasmids (0.75 lgÆwell )1 ) and a pSV-b-galactosidase control vector (0.25 lgÆwell )1 ; Promega) were cotransfected into A7r5 cells with FuGENE 6 Transfection Reagent (Roche). The cells were then cultured for 24 h, and a further 24 h in serum- free DMEM. They were subsequently incubated for 24 h in the presence or absence of 100 nm PGF 2a or 20 ngÆmL )1 PDGF-BB. Luciferase activity in cell lysates was deter- mined and normalized with b-galactosidase activity as described previously [21]. EMSA The EMSA was performed essentially as described previously [22]. A double-stranded probe containing an MEF2-binding site was prepared by annealing complemen- tary synthetic oligonucleotides. The sense sequence was 5¢-GATTCTTCTATAAATAGGTACTTTCCCTCA-3¢. The sequence of the mutated probe was 5¢-GATTCTTCT ATAgccAGGTACTTTCCCTCA-3¢. Probes were labeled at the 5¢-end with [c- 32 P]-ATP and T4 polynucleotide kinase. Nuclear extracts of A7r5 cells were prepared as described previously [8]. The nuclear extracts and the labeled probe were incubated at 25 °C for 30 min, resolved in a 4% polyacrylamide gel, and analyzed using a Fujix BAS 2000 Bio-imaging Analyzer (Fuji Film, Tokyo, Japan). Gene silencing of MEF2B The anti-MEF2B dsRNA was designed against nucleotides 470–494 of the rat MEF2B mRNA sequence (GenBank BC079361). Sense and antisense oligonucleotides containing the hairpin sequence, the terminator sequence, and over- hanging sequences were synthesized. By annealing over- hanging sequences of the synthetic oligonucleotides, PCR was performed to amplify the sequence encoding the dsRNA, which was inserted into pPUR-KE harboring a tRNA Val promoter. A7r5 clones stably expressing the anti- MEF2B dsRNA were obtained as described previously [7]. Cells incubated for 48 h in DMEM containing 0.5% fetal bovine serum were exposed to 100 nm PGF 2a or 20 ngÆmL )1 PDGF-BB for 24 h, and used for the subse- quent isolation of total RNA. Northern blot analysis and measurement of intracellular O 2 – production using a flow cytometer were performed as described previously [7,8]. The geometric mean of ethidium fluorescence intensity was used for analysis. Statistical analysis Values were expressed as the mean ± SE. The statistical analysis was performed with Student’s t-test. For multiple treatment groups, a one-way anova followed by Bonferroni’s t-test was applied. MEF2B regulates vascular NOX1 ⁄ NADPH oxidase M. Katsuyama et al. 5134 FEBS Journal 274 (2007) 5128–5136 ª 2007 The Authors Journal compilation ª 2007 FEBS Acknowledgements This work was supported in part by Grant-in-Aid for Young Scientists (B) 17790173 from The Ministry of Education, Culture, Sports, Science and Technology of Japan (MK). The nucleotide sequences reported in this paper have been submitted to DDBJ ⁄ EMBL ⁄ GenBank with accession number AB258525. We thank Dr S. Tsuchiya, Graduate School of Pharmaceutical Sciences, Kyoto University, for valuable discussions and advice. References 1 Griendling KK, Sorescu D & Ushio-Fukai M (2000) NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res 86, 494–501. 2 Irani K (2000) Oxidant signaling in vascular cell growth, death, and survival: a review of the roles of reactive oxygen species in smooth muscle and endothelial cell mitogenic and apoptotic signaling. Circ Res 87, 179– 183. 3 Guzik TJ, West NE, Black E, McDonald D, Ratna- tunga C, Pillai R & Channon KM (2000) Vascular superoxide production by NAD(P)H oxidase: associa- tion with endothelial dysfunction and clinical risk factors. Circ Res 86, E85–E90. 4 Gallin JI (1993) Delineation of the phagocyte NADPH oxidase through studies of chronic granulomatous dis- eases of childhood. Int J Tissue React 15, 99–103. 5 Suh YA, Arnold RS, Lassegue B, Shi J, Xu X, Sorescu D, Chung AB, Griendling KK & Lambeth JD (1999) Cell transformation by the superoxide-generating oxidase Mox1. Nature 401, 79–82. 6 Lassegue B, Sorescu D, Szocs K, Yin Q, Akers M, Zhang Y, Grant SL, Lambeth JD & Griendling KK (2001) Novel gp91 (phox) homologues in vascular smooth muscle cells: nox1 mediates angiotensin II- induced superoxide formation and redox-sensitive sig- naling pathways. Circ Res 88, 888–894. 7 Katsuyama M, Fan C & Yabe-Nishimura C (2002) NADPH oxidase is involved in prostaglandin F2alpha- induced hypertrophy of vascular smooth muscle cells: induction of NOX1 by PGF2alpha. J Biol Chem 277, 13438–13442. 8 Katsuyama M, Fan C, Arakawa N, Nishinaka T, Miy- agishi M, Taira K & Yabe-Nishimura C (2005) Essen- tial role of ATF-1 in induction of NOX1, a catalytic subunit of NADPH oxidase: involvement of mitochon- drial respiratory chain. Biochem J 386, 255–261. 9 Fan C, Katsuyama M, Nishinaka T & Yabe-Nishimura C (2005) Transactivation of the EGF receptor and a PI3 kinase-ATF-1 pathway is involved in the upregulation of NOX1, a catalytic subunit of NADPH oxidase. FEBS Lett 579, 1301–1305. 10 Fan C, Katsuyama M & Yabe-Nishimura C (2005) PKCdelta mediates up-regulation of NOX1, a catalytic subunit of NADPH oxidase, via transactivation of the EGF receptor: possible involvement of PKCdelta in vascular hypertrophy. Biochem J 390, 761–767. 11 Kuwano Y, Kawahara T, Yamamoto H, Teshima- Kondo S, Tominaga K, Masuda K, Kishi K, Morita K & Rokutan K (2006) Interferon-gamma activates tran- scription of NADPH oxidase 1 gene and upregulates production of superoxide anion by human large intesti- nal epithelial cells. Am J Physiol Cell Physiol 290, C433–C443. 12 Arakawa N, Katsuyama M, Matsuno K, Urao N, Tab- uchi Y, Okigaki M, Matsubara H & Yabe-Nishimura C (2006) Novel transcripts of NOX1 are regulated by alternative promoters and expressed under phenotypic modulation of vascular smooth muscle cells. Biochem J 398, 303–310. 13 Black BL & Olson EN (1998) Transcriptional control of muscle development by myocyte enhancer factor-2 (MEF2) proteins. Annu Rev Cell Dev Biol 14, 167–196. 14 Banfi B, Molnar G, Maturana A, Steger K, Hegedus B, Demaurex N & Krause KH (2001) A Ca(2+)-activated NADPH oxidase in testis, spleen, and lymph nodes. J Biol Chem 276, 37594–37601. 15 Suzuki E, Nishimatsu H, Satonaka H, Walsh K, Goto A, Omata M, Fujita T, Nagai R & Hirata Y (2002) Angiotensin II induces myocyte enhancer factor 2- and calcineurin ⁄ nuclear factor of activated T cell-dependent transcriptional activation in vascular myocytes. Circ Res 90, 1004–1011. 16 Suzuki E, Satonaka H, Nishimatsu H, Oba S, Takeda R, Omata M, Fujita T, Nagai R & Hirata Y (2004) Myocyte enhancer factor 2 mediates vascular inflammation via the p38-dependent pathway. Circ Res 95, 42–49. 17 Katoh Y, Molkentin JD, Dave V, Olson EN & Perias- amy M (1998) MEF2B is a component of a smooth muscle-specific complex that binds an A ⁄ T-rich element important for smooth muscle myosin heavy chain gene expression. J Biol Chem 273, 1511–1518. 18 Firulli AB, Miano JM, Bi W, Johnson AD, Casscells W, Olson EN & Schwarz JJ (1996) Myocyte enhancer binding factor-2 expression and activity in vascular smooth muscle cells. Association with the activated phenotype. Circ Res 78, 196–204. 19 Matsuno K, Yamada H, Iwata K, Jin D, Katsuyama M, Matsuki M, Takai S, Yamanishi K, Miyazaki M, Matsubara H et al. (2005) Nox1 is involved in angiotensin II-mediated hypertension: a study in Nox1-deficient mice. Circulation 112, 2677–2685. 20 Qin H, Ishiwata T, Wang R, Kudo M, Yokoyama M, Naito Z & Asano G (2000) Effects of extracellular matrix on phenotype modulation and MAPK transduc- tion of rat aortic smooth muscle cells in vitro. Exp Mol Pathol 69, 79–90. M. Katsuyama et al. MEF2B regulates vascular NOX1 ⁄ NADPH oxidase FEBS Journal 274 (2007) 5128–5136 ª 2007 The Authors Journal compilation ª 2007 FEBS 5135 21 Nishinaka T & Yabe-Nishimura C (2005) Transcription factor Nrf2 regulates promoter activity of mouse aldose reductase (AKR1B3) gene. J Pharmacol Sci 97, 43–51. 22 Nishinaka T, Fu YH, Chen LI, Yokoyama K & Chiu R (1997) A unique cathepsin-like protease isolated from CV-1 cells is involved in rapid degradation of retino- blastoma susceptibility gene product, RB, and transcrip- tion factor SP1. Biochim Biophys Acta 1351, 274–286. MEF2B regulates vascular NOX1 ⁄ NADPH oxidase M. Katsuyama et al. 5136 FEBS Journal 274 (2007) 5128–5136 ª 2007 The Authors Journal compilation ª 2007 FEBS . Myocyte enhancer factor 2B is involved in the inducible expression of NOX1 ⁄ NADPH oxidase, a vascular superoxide-producing enzyme Masato Katsuyama*, Muhammer Ozgur Cevik*, Noriaki Arakawa,. Katsuyama M, Fan C, Arakawa N, Nishinaka T, Miy- agishi M, Taira K & Yabe-Nishimura C (2005) Essen- tial role of ATF-1 in induction of NOX1, a catalytic subunit of NADPH oxidase: involvement of mitochon- drial. mitochon- drial respiratory chain. Biochem J 386, 255–261. 9 Fan C, Katsuyama M, Nishinaka T & Yabe-Nishimura C (2005) Transactivation of the EGF receptor and a PI3 kinase-ATF-1 pathway is involved in