Vascular Biology |
From the Departments of Geriatric Medicine (N.K., M. Minami, K.H., T.K.) and Neurosurgery (M. Morimoto, S.M., Y.U., H.K., N.H.), Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Correspondence to Noriaki Kume, MD, PhD, Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan. E-mail nkume{at}kuhp.kyoto-u.ac.jp
| Abstract |
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Key Words: early growth response factor lysophosphatidylcholine platelet-derived growth factor ERK signaling pathway
| Introduction |
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With regard to the signal transduction pathways, lyso-PC has
been shown to mobilize intracellular
calcium,15 stimulate the
tyrosine phosphorylation of platelet and
endothelial cell adhesion
molecule-1,16 disrupt a
receptorG protein
coupling,17 elevate
intracellular cAMP,18
activate mitogen-activated protein (MAP) kinases such
as extracellular signalregulated kinase
(ERK)19 and c-Jun N-terminal
kinase (JNK),19 20
protein kinase C,21
activator protein
(AP)-1,20 22 and
nuclear factor (NF)-
B23 in
cultured vascular endothelial cells. Furthermore,
reagents (such as forskolin and dibutyryl cAMP) that increase
intracellular cAMP suppress lyso-PCinduced expression of PDGF-B chain
and ICAM-124 ; however, it
remains unclear whether these signal transductions elicited by lyso-PC
are directly linked to gene transcription induced by lyso-PC. Cieslik
and colleagues25 26
have shown that lyso-PC can activate stimulatory protein
(Sp)-1dependent transcription of the ecNOS gene; however, it remains
unclear which factors are involved in the transcriptional regulation of
other genes, including PDGF-A chain.
Recent studies have revealed that the human PDGF-A chain gene contains a single TATA box 36 bp upstream from the transcription initiation site.27 28 29 The proximal promoter of the PDGF-A chain gene, which is located between -71 and -55 bp upstream from the transcription start site, contains overlapping recognition elements for the zinc-finger transcription factors, early growth response factor (Egr)-1 and Sp-1.30 31 32 This site is constitutively occupied by Sp-1; however, transcriptional activation of this gene involves the replacement of this binding site with Egr-1, whose expression is strongly elevated in atherosclerotic lesions33 and can rapidly be induced by biological stimuli such as fibroblast growth factor and fluid shear stress.34 35 36 37 38 In the present study, therefore, we have tested the hypothesis that lyso-PC may induce expression of Egr-1 and that lyso-PCinduced PDGF-A chain expression may depend on transcriptional activation of this site by Egr-1.
| Methods |
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Cell Culture
Bovine aortic endothelial cells
(BAECs) were harvested from bovine aortas by scraping with a sterile
glass coverslip and were cultured in DMEM supplemented with 10%
(vol/vol) FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin and
grown in an atmosphere of 95% air/5% CO2 at
37°C. Confluent BAECs with passage numbers between 5 and 20, which
were serum-starved for 24 hours, were used for
experiments.
Northern Blot Analysis
Total cellular RNA was isolated from BAECs by the
acid-guanidinium phenol-chloroform method. Equal amounts of RNA were
subjected to 1% agarose gel electrophoresis containing formaldehyde
and subsequently transferred onto nitrocellulose membranes (Schleicher
& Schuell, Inc). Northern blots were hybridized with cDNA probes for
Egr-1 and PDGF-A chain labeled with
[
-32P]dCTP with the use of random
hexanucleotide primers (DNA Labeling Kit, Pharmacia),
exposed to a phosphorimaging plate, and analyzed by Fujix
Bioimage Analyzer BAS 2000 (Fuji Photo Film Co
Ltd).
Nuclear Runoff Assay
A nuclear runoff assay was performed as previously
described,5 with minor
modification. Briefly, the cells were washed with ice-cold PBS and
lysed with 0.5% Nonidet P-40 solution (10 mmol/L Tris HCl,
10 mmol/L NaCl, 3 mmol/L MgCl2, and
0.5% NP-400 [vol/vol], pH 7.4). The nuclei were isolated by
centrifugation and resuspended in a 40% glycerol
buffer (50 mmol/L Tris HCl, 40% [vol/vol] glycerol, 5
mmol/L MgCl2, and 0.1 mmol/L EDTA, pH 8.3).
Nascent transcription in vitro was performed with
[32P]UTP and other unlabeled
nucleotides at 30°C for 30 minutes. Transcribed RNA was
isolated by a RNA isolation reagent (Isogen-LS, Wako Pure
Chemical), followed by denaturation with sodium hydroxide and ethanol
precipitation. Linearized target cDNAs (5 µg in plasmid form) were
alkali-denatured and immobilized onto Hybond-N+ membranes
by use of a slot-blot apparatus (Schleicher & Schuell Inc).
The membranes were hybridized with transcribed RNAs containing an equal
amount of radioactivity in a solution containing 50% (vol/vol)
formamide, 5x SSPE (1x SSPE consists of 0.15 mol/L NaCl, 10
mmol/L NaH2PO3, and
1 mmol/L EDTA, pH 7.4), 0.1% (wt/vol) SDS, 10% (vol/vol)
Denhardts solution, and denatured salmon sperm DNA at 42°C for 36
hours. Filters were washed in 1x SSC with 0.1% (wt/vol) SDS for 15
minutes at room temperature, washed with 0.2x SSC supplemented with
0.1% (wt/vol) SDS for 10 minutes at 42°C, and then autoradiographed
with Fujix Bioimage Analyzer BAS2000 (Fuji Photo
Film).
Plasmid Constructs
Oligonucleotides were
synthesized by CyberSyn and purified by using reverse-phase C18
cartridges. The nucleotide sequence of oligo A, which
consists of the nucleotide sequence corresponding to the
PDGF-A chain core promoter located between -76 and -47 bp upstream
from the transcription start site, is
5'-GGGGGGGGCGGGGGCGGGG-GCGGGGGAGGG-3' (sense strand), and
the complementary strand was annealed. The nucleotide
sequence of mutant oligo A is
5'-GGGGGGGGCGGGGGGCGGGGGGCGGGGGGAGGG-3'
(G insertion sites are underlined). Oligo A and the mutant oligo A were
subcloned into the
Xho/HindIII
site of the pGL2 vector (Promega) and were designated A.pGL2-Luc and
Am.PGL2-Luc, respectively.
DNA Transfection and Luciferase Assay
Transfections were performed with 2 µg of each
construct in combination with 50 ng of pRL-SV40 vector (Promega), which
contains the simian virus 40 (SV40) early enhancer/promoter region and
thereby provides strong and constitutive expression of Renilla
luciferase as an internal control. BAECs cultured in a 12-well plate
were transfected with each plasmid construct by the lipofection method
with the use of LipofectAMINE (Life Technologies, Inc; GIBCO-BRL).
Eight hours after transfection, the cells were washed with PBS,
replaced with DMEM with 1% FCS, and grown to confluence and near
quiescence (3 or 4 days after transfection). After reaching confluence,
BAECs were incubated for 1 hour with lyso-PC in serum-free DMEM and
subsequently incubated with DMEM in the absence of lyso-PC for an
additional 6 hours. Firefly and Renilla luciferase activities in the
BAEC lysates were measured by using the Dual-Luciferase Reporter Assay
System (Promega). Luciferase activities were normalized for the protein
concentrations of cell lysates.
Nuclear Protein Extraction
BAECs were lysed in buffer A (10 mmol/L HEPES,
pH 7.9, 1.5 mmol/L MgCl2, 20 mmol/L
KCl, 0.5 mmol/L dithiothreitol, 0.5 mmol/L
phenylmethylsulfonyl fluoride, 1 µg/mL leupeptin, and 0.5%
Nonidet P-40) by incubation for 10 minutes at 4°C. The cell lysate
was recentrifuged at 16 000 rpm, and the pelleted nuclei were
lysed in buffer C (20 mmol/L HEPES, pH 7.9, 25% glycerol,
420 mmol/L NaCl, 0.2 mmol/L EDTA, 1.5 mmol/L
MgCl2, 0.5 mmol/L dithiothreitol, and
0.2 mmol/L phenylmethylsulfonyl fluoride) by gentle
shaking for 20 minutes at 4°C. The nuclear extract was clarified by
centrifugation, and the supernatant was stored at
-80°C until use.
Western Blot Analysis
Nuclear protein extracts were prepared as described
above. Protein concentrations were determined by Bradford protein assay
reagent (Bio-Rad Laboratories). Nuclear fractions of proteins were
resuspended in 20 µL of SDS sample buffer, boiled with 15%
2-mercaptoethanol, and then subjected to
SDSpolyacrylamide (8%) gel electrophoresis. Electrophoresed
proteins were electroblotted at 100 V for 1 hour onto nitrocellulose
membranes (ECL, Amersham) in a buffer containing 25 mmol/L
Tris-Cl, 192 mmol/L glycine, and 5% methanol. The membranes were
incubated with a rabbit polyclonal antibody directed to Egr-1 (Santa
Cruz) for 1 hour as a primary antibody (1:10 000 dilution), washed
vigorously, and then incubated with peroxidase-conjugated anti-rabbit
IgG (Amersham) for 1 hour. After a wash with PBS containing 0.05%
Tween 20, the bands were visualized by ECL reagents (Amersham). Blots
were exposed to x-ray films for 1 to 10 minutes.
Oligonucleotide Synthesis and
Radiolabeling
Oligo A (5'-GGGGGGGGCGGGGGCGGGGGCGGGGGAGGG-3') was
annealed with the antisense strand, and the double-stranded
oligonucleotides were end-labeled with
[
-32P]dATP (Amersham) by use of T4
polynucleotide kinase (New England Biolabs, Inc). The
unbound nucleotides were removed by Chromaspin-10 columns
(Clontech Laboratories).
Electrophoretic Mobility Shift Assay
Binding reactions were carried out in a total volume
of 10 µL containing 5 to 10 µg nuclear extract, 1 µg
poly(dIdC)-poly(dIdC) (Sigma), and
32P-labeled oligonucleotide
probe (100 000 cpm) in 10 mmol/L Tris HCl, pH 7.5, 10 mmol/L
NaCl, 1 mmol/L EDTA, 5% glycerol, and 1 mmol/L
dithiothreitol at 25°C for 30 minutes. Nuclear extract was
preincubated with 1 µL of an affinity-purified anti-peptide antibody
(Santa Cruz Biotechnology) for 10 minutes before the addition of the
oligonucleotide probe. Nuclear
extractoligonucleotide mixtures were subjected to
electrophoresis through 5% (wt/vol) polyacrylamide gels
containing 10% glycerol. After they were dried, the gels were
autoradiographed and analyzed by Fuji Bioimage Analyzer
BAS2000 (Fuji Photo Film Co
Ltd).
| Results |
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Lyso-PCInduced Expression of Egr-1 mRNA
Depends on Gene Transcription
To examine whether lyso-PCinduced Egr-1 mRNA
expression depends on new RNA synthesis, we pretreated BAECs with 5
µg/mL actinomycin D for 30 minutes before exposure to lyso-PC.
Actinomycin D pretreatment abolished lyso-PCinduced expression of
Egr-1 mRNA as well as that induced by phorbol 12-myristate
13-acetate (PMA, data not shown). To obtain direct evidence that
lyso-PC activates transcription of the Egr-1 gene, a nuclear
runoff assay was carried out. As shown in
Figure 2
, 15 µmol/L lyso-PC treatment for 1 hour
significantly activated transcription of the Egr-1 but not the
GAPDH gene. These results indicate that increased levels of Egr-1 mRNA
induced by lyso-PC result primarily from gene
transcription.
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Induced Expression of Egr-1, but Not Sp-1, in
Nuclei of Lyso-PCTreated BAECs
Nuclear extracts of BAECs exposed to lyso-PC for 1 hour
were subjected to Western blot analysis to measure the amount
of Egr-1 protein in nuclei. Lyso-PC significantly increased nuclear
Egr-1 protein levels in a dose-dependent fashion; a maximal increase in
Egr-1 was observed at 15 µmol/L lyso-PC
(Figure 3A
). Egr-1 protein levels in nuclei were induced
within 30 minutes and remained elevated for 1 hour
(Figure 3B
). In contrast, nuclear levels of Sp-1 were not
significantly induced by lyso-PC
(Figure 3B
). We performed the same experiments in human
umbilical vein endothelial cells, and the results were
almost the same as those observed in BAECs (data not
shown).
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Lyso-PC Activates PDGF-A Chain Core
PromoterDependent Gene Transcription
To determine whether lyso-PC can activate the
PDGF-A chain core promoterdependent gene transcription,
oligonucleotides corresponding to the PDGF-A chain core
promoter (oligo A) were linked to the luciferase reporter gene and
transiently transfected into BAECs. After treatment with lyso-PC,
luciferase activities were measured. As shown in
Figure 4
, BAECs transfected with oligo A construct showed an
5-fold increase in the luciferase activity after exposure to
lyso-PC. In contrast, the luciferase activity was not significantly
induced by lyso-PC in BAECs transfected with the mutant (3 guanine
insertions) oligo A (oligo Am) construct
(Figure 4
).
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Lyso-PCInduced Egr-1 Binds to the Core
Promoter of PDGF-A Chain Gene
To determine whether lyso-PCinduced Egr-1 in nuclei
can bind to the core promoter of the PDGF-A chain, an electrophoretic
mobility shift assay (EMSA) was carried out with the use of
radiolabeled oligo A. As shown in
Figure 5A
, a nucleoprotein complex was induced by treatment
with lyso-PC for 1 hour, and this complex appeared identical to that
induced by PMA, a potent inducer of Egr-1 and PDGF-A chain gene
transcription. This complex declined after 2 hours
(Figure 5A
). Addition of a 100-fold excess amount of
unlabeled oligo A completely abolished the lyso-PCinduced shift band,
indicating that this binding is specific to oligo A
(Figure 5B
). The same molar excess of oligo Am slightly
reduced the appearance of the lyso-PCinduced shift band; however, the
effect was much less prominent compared with that of oligo
A(Figure 5B
). Inclusion of a polyclonal antibody directed to
Egr-1, but not c-Jun or c-Fos, eliminated the lyso-PCinduced shift
band without affecting the other bands
(Figure 5B
), indicating that Egr-1 binds to this site of the
promoter in lyso-PCtreated BAECs. An antiSp-1 antibody did not
affect the constitutive or lyso-PCinducible shift bands with
radiolabeled oligo A (data not shown). These results provide evidence
that Egr-1 induced by lyso-PC can specifically bind to the core
promoter of the PDGF-A chain gene.
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Lyso-PC Induces Egr-1 in BAECs via Activation
of the MEK-ERK Pathway
To explore whether the MAP kinase kinase (MEK)-ERK
pathway is involved in lyso-PCinduced Egr-1 expression, the effects
of PD98059, a specific inhibitor for MEK1, were examined.
As shown in
Figure 6
, PD98059, but not the p38 MAP kinase
inhibitor SB202190, dose-dependently inhibited
lyso-PCinduced Egr-1 expression. PMA-induced expression of Egr-1 was
also blocked by PD98059, but not by SB202190, as previously
shown.39 These results thus
indicate that the MEK-ERK pathway, but not p38 MAP kinase, is involved
in lyso-PCinduced expression of
Egr-1.
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| Discussion |
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The data in the present study indicate that Egr-1
appears to be involved in lyso-PCinduced PDGF-A chain expression;
however, we do not know, at present, whether nuclear expression of
Egr-1 alone is sufficient for lyso-PCinduced gene expression. In
fact, previous reports have shown that lyso-PC can activate
NF-
B23 and
AP-1.20 22
Furthermore, our recent studies have demonstrated that lyso-PC can also
activate Jun2 12-O-tetradecanoylphorbol 13-acetate
response element.47
Therefore, other transcriptional regulatory mechanisms may also be
involved in lyso-PCinduced gene transcription. In addition, because
NF-
B can act in cooperation with other transcription factors, such
as high-mobility group protein
I(Y),48
Sp-1,49 activating
transcription factor-2,50 and
AP-1,51 Egr-1 might also
interact with other transcription factors. These points remain to be
clarified.
In the present study, we were unable to demonstrate constitutive bindings of Sp-1 to the core promoter of the PDGF-A chain, although we used the antiSp-1 antibody from the same commercial source as the previous studies.36 37 We do not know the exact reason; however, this might result from the difference in the experimental conditions in EMSA, in the quality of the antibody, or in the basal culture conditions. In addition, previous studies with the ecNOS promoter have shown that protein phosphatase 2Adependent enhancement of Sp-1 binding is involved in lyso-PCinduced ecNOS transcription.25 In contrast to lyso-PCinduced ecNOS transcription,26 our preliminary results have shown that okadaic acid, which inhibits the activities of phosphatases, does not affect lyso-PCinduced PDGF-A chain expression (data not shown). Therefore, lyso-PC may stimulate multiple transcriptional regulatory mechanisms, some of which remain to be elucidated.
Lyso-PC appears to activate the transcription of
Egr-1, because inhibition of de novo RNA synthesis blocked
lyso-PCinduced Egr-1 mRNA expression (data not shown). Direct
evidence that lyso-PC stimulates Egr-1 gene transcription was
demonstrated by nuclear runoff assay
(Figure 2
). A recent report has indicated that induced
expression of Egr-1 by cyclic strain depends on the Ras-ERK pathway but
not on Rac-JNK.52 In cultured
vascular smooth muscle cells, Egr-1 expression by phorbol ester also
depends on ERK1 but not p38 MAP
kinase.39 Our previous
studies have shown that lyso-PC can activate
ERK19 as well as
JNK19 20 ;
therefore, lyso-PCinduced transcription of Egr-1 may involve the
Ras-ERK pathway. In fact, a specific inhibitor of MEK1
suppressed lyso-PCinduced Egr-1 expression in the present study
(Figure 6
).
In summary, the present study demonstrates that lyso-PC can induce the expression of Egr-1 in nuclei and activate the core promoter of the PDGF-A chain. Transcriptional regulation of Egr-1 by this atherogenic lipid may be one of the common molecular mechanisms involved in atherogenesis, because Egr-1 expression is also implicated in the transcriptional regulation of basic transcription element binding protein-2,39 which plays a key role in phenotypic modulation of vascular smooth muscle cells in atherogenesis.53 Further studies related to the roles of Egr-1 and its regulation by atherogenic lipids in atherogenesis may provide new insight into the pathogenesis of this complex disease.
| Acknowledgments |
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Received April 12, 2000; accepted January 16, 2001.
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