Báo cáo khoa học: Myocyte enhancer factor 2B is involved in the inducible expression of NOX1⁄ NADPH oxidase, a vascular superoxide-producing enzyme

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

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

is induced by various vasoactive factors,

NADPH oxidase is a major source of the superoxide produced in cardio- vascular tissues. Expression of NOX1, a catalytic subunit of NADPH oxi- including angiotensin II, dase, prostaglandin (PG) F2a 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 PGF2a and PDGF enhanced the transcriptional activity of the ) 3.6 kb 5¢-flanking region of the NOX1 gene in A7r5 cells, line. A PGF2a-response element was a rat vascular smooth muscle cell 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 PGF2a. 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 PGF2a- 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.

(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).

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.

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Reactive oxygen species including hydrogen peroxide, hydroxyl radical, singlet oxygen, peroxynitrite, and –) have been documented as intrinsic superoxide (O2 signaling molecules that modulate multiple cellular – in vascular cells responses. The major source of O2 and cardiac myocytes is the NADPH oxidase family [1–3]. NADPH oxidase catalyzes the reduction of – using NADPH as an electron molecular oxygen to O2 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 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) F2a, one of the primary prostanoids

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MEF2B regulates vascular NOX1 ⁄ NADPH oxidase

Results

Determination of the 5¢-end of the NOX1 mRNA expressed in VSMC

generated in the vascular tissue, was also shown to induce the expression of NOX1 mRNA and cause hypertrophy of VSMC through increased generation of – [7]. In PGF2a-induced as well as PDGF-induced O2 the epidermal transactivation of NOX1 expression, 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].

is that

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 PGF2a-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.

PGF2a- 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 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 the human NOX1 gene, an interferon-c- region of 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 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.

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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.

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NOX1 promoter-luciferase chimera plasmids were con- structed. As shown in Fig. 2B, deletion up to )489, but not to )930, reduced PGF2a-induced transcriptional activation, suggesting the existence of an enhancer binding site between )930 and )489. Deletion up to to )146, completely abolished the )125, but not PGF2a-induced transcriptional activation. Between )146 and )125, a consensus sequence of the MEF2- located binding site, 5¢-CTA(A ⁄ T)4TAG ⁄ A-3¢, was (Fig. 3A). The introduction of mutations at this site to 5¢-CTATAgccAG-3¢) abolished PGF2a-induced transcriptional activation the (Fig. 3B). These findings clearly indicate that MEF2-binding site between )139 and )130 is essential for PGF2a-induced activation of the NOX1 promoter.

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Relative Luciferase Activity

(B) Deletion of

Fig. 2. Analyses of the NOX1 promoter activity in A7r5 cells. (A) PGF2a- 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 nM PGF2a or 20 ngÆmL)1 PDGF-BB for 24 h. The relative luciferase activity is denoted as the fold-increase induced by PGF2a or PDGF. Bars represent means ±SE of three experiments. r3642Luc. *P < 0.01 versus Open bar, pGL3-basic; closed bar, pGL3-basic. the MEF2-binding site abolished PGF2a-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 PGF2a. Bars represent means ±SE of three experiments. *P < 0.01 versus pGL3-basic.

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 PGF2a 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 PGF2a increases the binding of MEF2 to the consensus-binding site located between )139 and )130 of the NOX1 promoter.

Gene silencing of MEF2B attenuated PGF2a- or PDGF-induced NOX1 expression

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 PGF2a or PDGF. These findings suggest that the the 5¢-flanking region contains sequences 3.6 kb of responsible for PGF2a-induced as well as PDGF- induced transcriptional activation. As the activation by PGF2a was more prominent than that by PDGF, PGF2a was used for the subsequent promoter analyses.

MEF2-binding site was essential for transcriptional activation of the NOX1 promoter

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To identify the region responsible for the transcrip- tional activation, a series of deletion mutants of the 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 PGF2a or PDGF, however, the expression of

M. Katsuyama et al.

MEF2B regulates vascular NOX1 ⁄ NADPH oxidase

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Fig. 3. PGF2a-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 PGF2a-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 PGF2a. Bars represent means ±SE of three experiments. *P < 0.01 versus pGL3-basic.

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 the NOX1 gene, found in the promoter region of 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 PGF2a 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

MEF2B was markedly augmented (Fig. 6A, mock). We therefore focused on the role of MEF2B in the PGF2a- 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 two clones stably expressing the the transfectants, 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 PGF2a or PDGF was almost completely abolished in MEF2B knocked-down cells. In addition, – production as well as a PGF2a-induced increase in O2 – was reduced in these level of cellular O2 the basal clones compared with the mock-transfected cells (Fig. 5C). These results highlight the pivotal role of MEF2B in the PGF2a- or PDGF-induced up-regula- tion of NOX1 expression.

Gene silencing of ATF-1 attenuated PGF2a- or PDGF-induced MEF2B expression

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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 (c) stimulation with to PGF2a; the responsiveness PGF2a 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 We previously reported the involvement of ATF-1, a transcription factor of the CREB ⁄ ATF family, in the

M. Katsuyama et al.

MEF2B regulates vascular NOX1 ⁄ NADPH oxidase

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Fig. 4. PGF2a increased binding of MEF2 to the consensus sequence. (A) Specific bands detected by EMSA. Nuclear extracts were prepared from A7r5 cells incubated with 100 nM PGF2a 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.

Fig. 5. Gene silencing of MEF2B attenuated PGF2a- 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 PGF2a 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 nM PGF2a (closed bar) for 24 h. Mean values of ethidium fluorescence were calculated from four samples. *P < 0.05 versus control mock-transfected cells. (cid:2)P < 0.05 versus mock-transfected cells treated with PGF2a.

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NOX1 mRNA induced by PGF2a or PDGF; (e) RNA interference targeted at ATF-1 attenuated the induc- tion of MEF2B expression by PGF2a 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 expressed in dedifferentiated mRNA which was

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MEF2B regulates vascular NOX1 ⁄ NADPH oxidase

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Fig. 6. Gene silencing of ATF-1 attenuated PGF2a- 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- three experiments. *P < 0.05 versus resent mock control.

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 PGF2a or PDGF. In MEF2B knocked-down cells, the induction of NOX1 mRNA expression by PGF2a or PDGF was almost completely it was unaffected in MEF2A abolished, whereas knocked-down clones (data not shown). Accordingly, inducible expression of MEF2B appears to up-regulate the NOX1 gene expression in VSMC.

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 is expressed in human VSMC [14], oxidase, NOX5, but not in rodents.

strongly suggest

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 PGF2a [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 It should be transcription factors. that a consensus CRE sequence was pointed out 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 PGF2a- or PDGF-induced increase in MEF2B mRNA was almost completely abolished in ATF-1 knocked- down clones. These the results involvement of the ATF-1-MEF2B cascade in the reg- ulation of vascular NOX1 gene expression. We previously

expression of monocyte level of

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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 chemo- in inducing the 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- reported the pathophysiological significance of NOX1-derived reactive oxygen species in angiotensin II-induced chronic hypertension using the NOX1-deficient mice [19]. The basal 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 involved in the up-regulation of NOX1 cascade

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MEF2B regulates vascular NOX1 ⁄ NADPH oxidase

regulatory mechanisms in vascular gene expression may lead to a better understanding of superoxide production.

Experimental procedures

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 PGF2a or 20 ngÆmL)1 PDGF-BB. Luciferase activity in cell lysates was deter- mined and normalized with b-galactosidase activity as described previously [21].

Materials

PGF2a 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-32P]- ATP, [a-32P]-UTP, and [a-32P]-dCTP were from ICN Bio- medicals (Costa Mesa, CA).

EMSA

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].

Cell culture

as described The EMSA was performed essentially 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-32P]-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 (cid:2)C for 30 min, resolved in a 4% polyacrylamide gel, and analyzed using a Fujix BAS 2000 Bio-imaging Analyzer (Fuji Film, Tokyo, Japan).

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.

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 tRNAVal promoter. A7r5 clones stably expressing the anti- MEF2B dsRNA were obtained as described previously [7]. incubated for 48 h in DMEM containing 0.5% Cells fetal bovine serum were exposed to 100 nm PGF2a or 20 ngÆmL)1 PDGF-BB for 24 h, and used for the subse- quent isolation of total RNA. Northern blot analysis and – production using a flow measurement of intracellular O2 cytometer were performed as described previously [7,8]. The geometric mean of ethidium fluorescence intensity was used for analysis.

Reporter constructs and luciferase assay

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.

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

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Statistical analysis

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10 Fan C, Katsuyama M & Yabe-Nishimura C (2005)

Acknowledgements

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.

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.

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