Among these factors, prostaglandin PG F2a, one of the primary prostanoids Keywords activating transcription factor-1; myocyte enhancer factor 2; NADPH oxidase; NOX1; vascular smooth musc
Trang 1expression 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 (O2 ) have been documented as intrinsic
signaling molecules that modulate multiple cellular
responses The major source of O2 in vascular cells
and cardiac myocytes is the NADPH oxidase family
[1–3] NADPH oxidase catalyzes the reduction of
molecular oxygen to O2 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) F2a, 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) 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,
a rat vascular smooth muscle cell line A PGF2a-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 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
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.
Trang 2generated in the vascular tissue, was also shown to
induce the expression of NOX1 mRNA and cause
hypertrophy of VSMC through increased generation of
O2 [7] In PGF2a-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 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
NOX1 mRNA
1a 2
chrX
3 k
28 k
433
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.
Trang 3factors, 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
3.6 kb of the 5¢-flanking region contains sequences
responsible for PGF2a-induced as well as
PDGF-induced transcriptional activation As the activation by
PGF2a was more prominent than that by PDGF,
PGF2awas 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 PGF2a-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 PGF2a-induced transcriptional activation Between )146 and )125, a consensus sequence of the MEF2-binding site, 5¢-CTA(A ⁄ T)4TAG⁄ A-3¢, was located (Fig 3A) The introduction of mutations at this site (5¢-CTATAAATAG-3¢ to 5¢-CTATAgccAG-3¢) abolished PGF2a-induced transcriptional activation (Fig 3B) These findings clearly indicate that the MEF2-binding site between )139 and )130 is essential for PGF2a-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 PGF2aincreased 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
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
Relative Luciferase Activity
–91 –125 –146 –235 –323
–489
–930
pGL3basic
–3642
AP TA
Relative Luciferase Activity
PGF 2α
A
B
PGDF
*
*
*
*
*
*
*
*
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 n M PGF2aor 20 ngÆmL)1PDGF-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
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.
Trang 4MEF2B 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
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 PGF2aor PDGF was almost completely
abolished in MEF2B knocked-down cells In addition,
a PGF2a-induced increase in O2 production as well as
the basal level of cellular O2 was reduced in these
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
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 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 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 PGF2a; (c) stimulation with PGF2aenhanced the binding of MEF2 to its consensus binding site in the NOX1 promoter; (d) RNA interfer-ence targeted at MEF2B abolished expression of the
Relative Luciferase Activity
–125
–146
luc
pGL3basic –146mut
1c
B
A
A T
*
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.
Trang 5NOX1 mRNA induced by PGF2a or PDGF; (e) RNA
interference targeted at ATF-1 attenuated the
induc-tion of MEF2B expression by PGF2aor 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 2αα
PGF 2α
– + + + +
supershift
lane
A
B
1 2 3 4 5
anti ATF-1 Ab
origin
MEF2
– + + +
– –
– +
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 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.
0 2 4 6
NOX1 GAPDH
mock
MEF2B RNAi-1
MEF2B RNAi-2
Control PGF 2αα
PGF
2α
PGF
2α Control Control
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)
mock MEF2BRNAi-1 MEF2BRNAi-2
0 50 60 70 80
90 *
*
*
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 n M PGF2a(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 PGF2a.
Trang 6VSMC 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 PGF2a or PDGF In MEF2B knocked-down cells, the induction of NOX1 mRNA expression by PGF2a 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 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 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 PGF2a- 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
ControlPGF
2αα
Control PG
F 2α
Control PGF
2α
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.
Trang 7gene expression may lead to a better understanding
of regulatory mechanisms in vascular superoxide
production
Experimental procedures
Materials
PGF2awas 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-32P]-UTP, and [a-32P]-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 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]
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 25C 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 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 tRNAValpromoter 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 PGF2a 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 O2 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
Trang 8This 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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