We have shown that lysophosphatidylcholine
(lyso-PC) increases endothelial nitric-oxide synthase (eNOS) expression
at the transcriptional level (Zembowicz, A., Tang, J.-L., and Wu,
K. K. (1995) J. Biol. Chem. 270, 17006-17010).
To elucidate the mechanism by which lyso-PC increases the eNOS
transcription, we identified Sp1 sites at
104 to
90 and PEA3 sites
at
40 to
24 as being involved in lyso-PC-induced promoter activity.
Site-directed mutagenesis of Sp1 sites resulted in a marked reduction
of basal and lyso-PC-induced activity whereas PEA3 site mutation
abrogated response to lyso-PC. Band shift assays revealed that lyso-PC
augmented Sp1 binding activity. Pretreatment of cells or nuclear
extracts with okadaic acid reduced the Sp1 binding activity.
Furthermore, okadaic acid treatment abrogated the lyso-PC induced
promoter augmentation. Lyso-PC increased the nuclear extract protein
phosphatase 2A (PP2A) activity, which was suppressed by okadaic acid
treatment. These results suggest that lyso-PC up-regulates eNOS
transcription by a PP2A-dependent increase in Sp1 binding
activity.
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INTRODUCTION |
Nitric oxide (NO)1 is an
important mediator of diverse physiological and pathological processes,
including vasodilation, cytotoxicity, and neurotransmission (1-4). Its
biosynthesis is catalyzed by nitric-oxide synthase (NOS). Three
isoforms of NOS, i.e. neuronal NOS (nNOS or NOS-I),
inducible-NOS (iNOS or NOS-II), and endothelial-NOS (eNOS or NOS-III)
have been identified and characterized (5-7). NOS-I and -III are
constitutively expressed. The inducible form is expressed by
stimulation with several inflammatory and mitogenic mediators (8, 9).
Altered endothelial NO productions have been implicated in several
important cardiovascular disorders: hypertension, atherosclerotic heart
disease, and diabetes. The mechanism by which NO synthesis is deranged
in these disorders remained unclear. Although NOS-III is considered to
be a housekeeping gene, recent studies have provided evidence to
suggest that NOS-III is induced by shear stress (10), physical exercise
(11), hypoxia (12), estrogen treatment (13), lysophosphatidylcholine
(lyso-PC or LPC), and low levels of oxidized low density lipoprotein
(14, 15). These findings imply that induction of NOS-III plays an important role in protecting vascular integrity under stress. Work from
our laboratory has shown that lyso-PC increases NOS-III expression at
the transcriptional level (14). However, the mechanism by which lyso-PC
and other inducing agents increase NOS-III gene transcription remains
to be elucidated.
The 5'-flanking region of human eNOS gene has been cloned and sequenced
(16). The region adjacent to the transcription initiation sites is
TATA-less and GC-rich. Functional analysis of the promoter activity
conferred by the 5'-flanking region has shown that a canonical Sp1 site
situated between
104 to
90 is required for the basal promoter
activity (17). However, it is unclear whether this site is involved in
lyso-PC-induced transcriptional activation. The purpose of this study
is to identify cis-acting elements and nuclear transcriptional
activators that are important in augmentation of eNOS transcription in
response to lyso-PC stimulation. Our results indicate that Sp1 site at
104 to
90 and PEA3 site at
40 to
24 are required for
lyso-PC-induced transcriptional activation. Lyso-PC increases the
promoter function by enhancing the Sp1 binding activity mediated by the
action of protein phosphatase 2A.
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EXPERIMENTAL PROCEDURES |
Reagents--
The following reagents were obtained from the
indicated sources: QIA filter Midi kit was from Qiagen; Wizard PCR
Preps DNA purification system, restriction endonucleases,
T4 polynucleotide kinase, Sp1 consensus oligonucleotide,
luciferase assay kit, serine/threonine phosphatase assay system, and
pGL3 plasmid were from Promega; Lipofectin reagent, Opti-MEM medium,
Klenow enzyme, and T4 DNA ligase were from Life
Technologies, Inc.; Taq DNA polymerase was from Fisher;
synthetic oligonucleotides were from Genosys;
1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC) was
from Avanti Polar Lipids; radionucleotides were from Amersham Pharmacia
Biotech; Bio-Spin chromatography column was from Bio-Rad; Coomassie
protein assay reagent was from Pierce; tissue culture reagents were
from Sigma, except endothelial cell mitogen was from Biomedical
Technologies. Other reagents were from Sigma.
Preparation of 5'-Promoter Region and Its 5'-Deletion
Mutants--
A 5'-flanking fragment at nucleotide positions from
1322 to +22 designated GH was obtained by polymerase chain reaction
(PCR), using genomic DNA as template and synthetic oligomers as
primers: EN1322G (5'-AAAGATCTTCCATCTCCCTCCTCCTG-3') and EN3H
(5'-GGGAAGCTTGTTACTGTGCGTCCACTCTG-3'). The PCR product was purified
from the agarose gel digested with BglII/HindIII
and cloned into the promoterless luciferase reporter vector pGL3.
5'-Deletion mutants of wild-type were also prepared by PCR and
constructed into pGL-3 by a similar procedure.
Site-directed Mutagenesis--
A 4-base mutation at the Sp1 site
at position
104/
90 was created by PCR using synthetic
oligonucleotides: 5'-GGGATAGGGTCAGTACGAGGGCCAG-3' (mutated at positions
100,
98,
96, and
95) and EN3H as sense and antisense primers,
respectively. The PCR product was purified and used with
oligonucleotide EN1322G to produce the mutated 1.3-kilobase GH
fragment, which was recloned into the pGL3 vector. Site-directed mutations in PEA-3 sites were constructed by similar procedures using
the following primers: M1 (5'-CCCCTCTTCGAATTCAACAGGCC-3') (mutated at
positions
34,
33,
31,
30,
29, and
26) and EN3H; M2
(5'-CCCCTCTTCCTAAGAATTCGGCC-3') (mutated at positions
29,
27,
26,
and
25) and EN3H. The PCR products were purified and used with
oligonucleotide EN1322G to produce the 1.3-kilobase mutant fragments
that were recloned into the pGL-3 vector. We also produced a S/M1
double mutant using BmpI restriction endonuclease and
T4 DNA ligase.
Cell Culture and Transient Expression--
Human umbilical vein
endothelial cells (HUVEC) were cultured in Medium 199 containing 20%
fetal bovine serum (FBS) and 50 µg/ml endothelium mitogen in six-well
plates. Passage 1 HUVEC were used in all experiments. Transient
expression by lipofection was performed as described (18). In brief,
HUVEC were incubated in serum-free medium containing a mixture of 10 µl of Lipofectin and 2 µg of promoter constructs at 37 °C for
5 h. Medium was removed, and cells were incubated with fresh
complete medium overnight. Cells were then washed and incubated with
Medium 199 containing 0.5% FBS for 16 h. The medium was removed
and replaced with medium containing 5% FBS in the presence or absence
of 100 µM lyso-PC at 37 °C for 6 h. The cells
were harvested and lysed with lysis buffer. The promoter activity was
determined by luciferase assay in a luminometer (Analytical
Luminescence Laboratories, Monolight model 2010) as described (19).
Construction of DNA Probe--
The eNOS promoter construct GH
was obtained by digesting with HindIII. The smaller fragment
of NOS-III promoter was obtained by PCR using primers: P3
(AAAGATCTGCGGCGTGGAGCTGAGGCTTTA) and EN3H. DNA was end-labeled with
[
-32P]ATP by Klenow polymerase. Unincorporated
nucleotides were removed by Bio-Spin chromatography columns. End
labeling oligonucleotide probes, Sp1 and PEA-3, were labeled with
[
-32P]ATP by T4 polynucleotide kinase.
Preparation of Nuclear Extract (NE)--
Cells were cultured in
complete medium (Medium 199, 20% FBS, 50 µg/ml endothelial mitogen).
16 h before experiment, the medium was changed to 0.5% FBS. Cells
were treated with 100 µM LPC (in 5% FBS), and after
3 h of incubation, cells were harvested. Harvested cells were
suspended in cold phosphate-buffered saline containing 0.5 mM PMSF, spun down at 400 × g for 5 min,
transferred to microcentrifuge tubes, spun down again and resuspended
in two package cell volumes of buffer A (10 mM HEPES, pH
7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 300 mM sucrose, 0.5% Nonidet P-40, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 0.5 mM PMSF), and
allowed to stay on ice for 10 min. Cells were centrifuged at 6500 rpm
for 20 s, washed with 3/4 package cell volume with buffer A
and spun down again. Extraction buffer B (20 mM HEPES, pH
7.9, 1.5 mM MgCl2, 420 mM NaCl, 1 mM DTT, 0.2 mM EDTA, pH 8.0, 25% glycerol, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 0.5 mM PMSF) was
added to the nuclei at
package cell volume. Nuclei were
passed 10 times through a 23-gauge needle and stirred on ice for 30 min. The nuclear debris was pelleted for 5 min at 12,000 rpm. The
supernatant was diluted isovolumetrically in buffer D (20 mM HEPES, pH 7.9, 100 mM KCl, 1 mM
DTT, 0.2 mM EDTA, pH 8.0, 20% glycerol, 1 µg/ml
leupeptin, 1 µg/ml aprotinin, 0.5 mM PMSF). Protein
concentrations were determined using Bradford assay (Coomassie protein
assay reagent: Pierce), using bovine serum albumin as a standard.
Electrophoretic Mobility Shift Assay (EMSA)--
5.0 µg of
nuclear extract in binding buffer containing 50 mM HEPES,
pH 7.9, 1 mM EDTA, 100 mM KCl, 20% glycerol,
2.5 mM MgCl2, 0.5 mM DTT, 0.5 mM PMSF, 1.5 µg of poly(dI-dC) was incubated with competing oligonucleotides for 15 min on ice. 32P-Labeled
probe (10,000 cpm) was added and incubated at room temperature for 10 min. The mixture was electrophoresed at 12.5 V/cm on 5% polyacrylamide
gel in 0.25 × TBE. The gel was vacuum-dried and autoradiographed
for 2 h at
70 °C with an intensifying screen.
Serine/Threonine Protein Phosphatase (PP) Assays--
Assays for
PP2A, PP2B, and PP2C were based on determining the amount of free
phosphate generated in reaction by measuring the absorbance of a
molybdate-malachite green-phosphate complex. 10 µg of nuclear extract
proteins obtained from cells treated with or without lyso-PC (100 µM) was incubated on 96-well plates in the presence or
absence of an inhibitor (5 nM OA), together with a peptide
substrate RRA(pT)VA and appropriate buffer for 30 min at 30 °C.
After incubation, the molybdate complex dye was added and incubated for
an additional 30 min at room temperature to allow for color
development. Reaction was read at 630 nm with a plate reader. This
assay system detects PP2A, PP2B, and PP2C activities by using different
buffers: for PP2A, 50 nM imidazole pH 7.2, 0.2 mM EGTA, 0.02%
-mercaptoethanol, 0.1 mg/ml bovine serum
albumin; for PP2B, 50 nM imidazole, pH 7.2, 0.2 mM EGTA, 0.02%
-mercaptoethanol, 10 mM
MgCl2, 0.4 mM CaCl2, 50 µg/ml
calmodulin; and for PP2C, 50 nM imidazole, pH 7.2, 0.2 mM EGTA, 0.02%
-mercaptoethanol, 5 mM
MgCl2, 0.1 mg/ml bovine serum albumin.
 |
RESULTS |
Involvement of Sp1 and PEA3 Sites in Lyso-PC-induced NOS-III
Promoter Activity--
We have shown previously that a 5'-flanking
fragment from nucleotide
165 to +22 confers basal eNOS promoter
activity (17). To determine lyso-PC-induced promoter activity, we
transfected into HUVEC a series of 5'-deletion mutants of the
1.3-kilobase GH fragment of eNOS gene and incubated the transfected
cells in medium containing 100 µM lyso-PC or vehicle in
the presence of 5% FBS. The results show that the region from
265 to
+22 that confers the basal activity is responsive to lyso-PC induction (Fig. 1). In this region, there is a GC
box located at
104 to
90 and PEA3 sites between
40 and
24 (Fig.
2). Further 5'-deletion of this region to
remove the putative Sp1 binding sites resulted in a marked diminution
of basal and lyso-PC-induced promoter activity. To evaluate the
potential involvement of Sp1 and PEA3 sites in basal and
lyso-PC-induced promoter activity, we constructed a Sp1 mutant and two
PEA3 mutants in fragment
1322 to +22 by site-directed mutagenesis:
the Sp1 mutant (Sp1-M or S) contains a four-nucleotide mutation, PEA3
mutant 1 (M1) contains a six-nucleotide mutation located both in the
sense and antisense PEA3 motifs, and PEA3 mutant 2 (M2) contains a
four-nucleotide alteration in the sense motif (Fig. 2). These mutants
were inserted into the promoterless luciferase expression vector pGL3
and transiently expressed in HUVEC by Lipofectin. Results from 3-5
experiments are shown in Fig. 3. Sp1
mutation reduced basal and lyso-PC-induced promoter activities more
than 95% (Fig. 3). Residual activities were noted in basal and
lyso-PC-treated cells, and the activity in the lyso-PC-treated cells
was about twice that of the basal activity (Fig. 3). PEA3 M1 and M2
mutations decreased the basal promoter activity to 68 and 53% of the
wild-type activity, respectively, and severely curtailed the
lyso-PC-induced promoter activity (Fig. 3). Both basal and
lyso-PC-induced promoter activities were almost entirely abolished by
combined S and M1 mutations (Fig. 3). These results indicate that
activation of transcription by lyso-PC requires Sp1 sites at
104 to
90 and PEA3 sites at
40 to
24.

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Fig. 1.
Functional analysis of the basal and
lyso-PC-induced promoter activity conferred by 5'-flanking DNA
fragments of eNOS gene. A, serial 5'-deletion mutants
were constructed into pGL3 and expressed in cultured human umbilical
vein endothelial cells. C denotes negative control, and
C+ denotes positive control. B, luciferase
activity conferred by GH and its 5'-deletion mutants. The data are
mean ± S.D. of three to five experiments. , LPC; ,
+LPC.
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Fig. 2.
Nucleotide sequence of wild-type
(WT), mutated Sp1 (Sp-1 M or S),
two site-directed mutants of PEA3 sites M1 and M2, and double mutant
S/M1. This figure shows only nucleotides from 110 to 20 of
fragment 1322 to +22 at the 5'-flanking region of eNOS gene.
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Fig. 3.
Functional analysis of promoter
activity. DNA fragment ( 1322/+22) of WT, S, M1, M2, and S/M1
were constructed into pGL3 vector and expressed in HUVEC. The results
are mean ± S.D. of three to five experiments. , LPC; ,
+LPC.
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Lyso-PC Increased Binding of Sp1-related Proteins--
Binding of
Sp1-related proteins to this minimal promoter region was determined by
EMSA, using [
-32P]dATP-labeled fragment
165 to +22
as a probe. The binding activity of nuclear extracts obtained from
resting and lyso-PC-stimulated HUVEC is shown in Fig.
4. Multiple retarded bands were noted
when basal and lyso-PC NE were incubated with the labeled probe. A main
slow migrating band was competed out by a 25- or 50-fold molar excess
of a 22-mer Sp1 consensus oligonucleotide but not by a PEA3 consensus
oligonucleotide (Fig. 4). This Sp1-DNA complex band was enhanced by
lyso-PC stimulation as illustrated in Fig. 4 (compare lane 2 with lane 7). A slightly faster migrating band was competed
out by a consensus PEA3 oligonucleotide but not by a Sp1
oligonucleotide (Fig. 4). This Ets-DNA complex band was slightly
reduced by lyso-PC treatment as illustrated in Fig. 4 (compare
lane 2 with lane 7). To further evaluate the Sp1
binding activity in the lyso-PC stimulated NE, the Sp1 consensus
sequence was labeled and incubated with NE from resting and
lyso-PC-treated cells. A single Sp1-DNA complex band was noted, and
this band was enhanced by lyso-PC (Fig.
5, compare lane 2 with
lane 7). Competition with molar excesses of unlabeled
oligonucleotides showed that the binding activity of lyso-PC-treated NE
is about 5-fold higher than the binding activity of basal NE. By
contrast, there was no apparent change in binding of NE proteins to
PEA3 consensus sequence by lyso-PC treatment (data not shown). These data suggest that lyso-PC causes a change in the binding activity of
Sp1-related proteins in NE.

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Fig. 4.
Determination of binding of nuclear extract
proteins to [ -32P]dATP-labeled probe ( 165/+22) by
EMSA. Lane 1 is a free probe. Lanes 2-6
show the binding activity of NE from non-lyso-PC-treated cells;
lanes 7-11, lyso-PC-treated cells. Competitors Sp1 and PEA3
denote pretreatment of NE with a 25-50-fold molar excess of Sp1 or
PEA3 consensus oligonucleotides. The arrow refers to the
major Sp1-DNA complex, and the bracket refers to two Ets-DNA
complexes.
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Fig. 5.
Binding of nuclear extract proteins to
labeled Sp1 oligonucleotides in the presence or absence of unlabeled
oligonucleotides in a 1-25-fold molar excess. Control
denotes nuclear extracts obtained from cells not treated with lyso-PC,
whereas LPC denotes nuclear extract obtained from cells
treated with lyso-PC.
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Identification of Sp and Ets Binding Proteins--
Four Sp
proteins have been characterized. Specific antibodies against each
class of proteins were used in a supershift assay to determine the
exact Sp protein that is responsible for the binding activity detected
in NE. The Sp-DNA complex band was supershifted only with antibodies
against Sp1 (Fig. 6A,
lanes 4 and 5). Antibodies to Sp2, Sp3, and Sp4
did not cause any shift of the band (Fig. 6A, lanes
6-11). The supershifted band of lyso-PC-stimulated NE was denser
than that of basal NE consistent with an enhancement of Sp1 binding to
its canonical Sp1 sequence on the promoter region. Supershift assays
were also performed to identify the Ets protein that binds to labeled
PEA3 consensus oligonucleotides. Pretreatment of NE obtained from
control and lyso-PC-treated cells with antibodies to Ets 1+2, erg 1+2,
Elk1, or PEA3 resulted in a marked reduction of the Ets-DNA complex
compared with the control (Fig. 6B). As has been reported
previously (20), antibodies to Ets proteins did not cause a shift of
the complex, probably due to the interference of these antibodies with
the binding of Ets proteins to DNA. These results suggest that the PEA3
sites on the promoter region were promiscuous for binding by a broad
spectrum of Ets family proteins.

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Fig. 6.
Supershift assay for Sp (A) and
Ets (B) using labeled consensus oligonucleotides as the
probes. In A, Sp1 was supershifted by Sp1-specific
antibodies. In B, various Ets-DNA complexes were markedly
reduced by selective antibodies.
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Effect of Okadaic Acid and Sodium Orthovanadate on Sp1 Binding
Activity and Lyso-PC-induced eNOS Promoter Activity--
To determine
whether PP are involved in modifying Sp1 binding activity in
lyso-PC-treated cells, NEs obtained from these cells were pretreated at
room temperature for 30 min with okadaic acid, a serine PP inhibitor,
or vanadate, a tyrosine PP inhibitor, and the Sp1 binding activity in
NE was evaluated by EMSA using labeled Sp1 consensus oligonucleotides
as the probe. Okadaic acid reduced the NE Sp1 binding activity in
lyso-PC-treated cells in a concentration-related manner, and the
maximal suppression was noted at 50 nM okadaic acid (Fig.
7A). By contrast, vanadate at
50 µM had no apparent effect on blocking lyso-PC-induced
Sp1 binding activity (Fig. 7B). To ascertain that the
okadaic acid effect occurs in vivo, HUVEC were incubated in
medium containing 50 nM okadaic acid in the presence or
absence of lyso-PC for 3 h. Nuclear extracts from these cells were
prepared and used in EMSA. The results indicate that okadaic acid
reduced the lyso-PC-induced Sp1 binding activity to the basal level
(Fig. 7C, compare lane 4 with lanes 2 and 1). Okadaic acid had no effect on the basal Sp1 binding
activity.

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Fig. 7.
A, effect of OA on NE binding to a
labeled Sp1 consensus oligonucleotides. NE were preincubated with OA at
0 (lanes 2 and 3), 10 nM (lanes
4 and 5), 50 nM (lanes 6 and
7), and 100 nM (lanes 8 and
9) before EMSA. Lanes 10 and 11 are NE
pretreated with a 25-fold molar excess of unlabeled Sp1
oligonucleotides. C denotes control NE, and L
denotes lyso-PC-treated cells. B, effect of sodium
orthovanadate on NE binding to labeled Sp1 consensus oligonucleotides.
Lane 1, free probe; lanes 2 and 3, NE
from cells treated without and with lyso-PC, respectively; lanes
4 and 5, NE from basal or lyso-PC-stimulated cells
pretreated with 50 µM monosodium orthovanadate.
C, cells were pretreated with or without 50 nM
OA, and their NE binding to a labeled Sp1 oligonucleotides was
determined by EMSA.
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The effect of okadaic acid on lyso-PC-induced eNOS promoter activity
was evaluated by preincubating HUVEC transfected with eNOS promoter GH
with 50 nM okadaic acid, and 1 h later, lyso-PC was
added and incubated for 6 h. Okadaic acid reduced the
lyso-PC-induced promoter to the basal promoter activity (Fig.
8). It has been shown that okadaic acid
at low concentrations (10-50 nM) selectively inhibits PP2A
activity (21). Our results are consistent with a role of PP2A in
controlling lyso-PC-induced Sp1 binding activity and eNOS promoter
activity.

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Fig. 8.
Effect of okadaic acid on lyso-PC-induced
promoter activity. HUVEC transfected with WT GH were treated with
50 nM OA prior to lyso-PC treatment. Each bar
represents mean ± S.D. of three experiments. C denotes
control, and L denotes lyso-PC-treated cells. , OA;
, +OA.
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Increase in Nuclear Extract PP2A Activity by Lyso-PC--
The
effect of lyso-PC on PP2A activity was evaluated by assaying PP2A as
well as PP2B and PP2C activities in nuclear extracts of HUVEC treated
with and without lyso-PC (100 µM). Lyso-PC significantly increased the PP2A activity (Fig.
9A), which was suppressed by OA (Fig. 9A). By contrast, lyso-PC had no effect on PP2B or
PP2C activity (Fig. 9B).

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Fig. 9.
Influence of lyso-PC on PP2A, PP2B, and PP2C
activities. A, lyso-PC increased PP2A activity, which
was suppressed by OA (5 nM). B, lyso-PC had no
apparent effect on PP2B or PP2C activity. , untreated; ,
LPC.
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 |
DISCUSSION |
Lyso-PC is a main component of oxidized low density lipoprotein
(22). The level of lyso-PC is highly elevated in human atherosclerotic tissues (23). It has been shown that lyso-PC increases chemotaxis for
monocytes and induces endothelial adhesive molecules such as
intercellular adhesive molecule-1 and vascular cell adhesive molecule-1, which mediate monocyte and neutrophil adhesion to vascular
endothelium (24). Hence, lyso-PC has been considered to play an
important role in monocyte accumulation in the arterial walls.
Furthermore, lyso-PC induces the expression platelet-derived growth
factor A and B subunits and heparin-binding epidermal growth factor
from endothelial cells (25, 26). These growth factors promote smooth
muscle cell migration and proliferation. These findings have led to the
conclusion that lyso-PC is a key mediator of atherosclerosis (22). Work
from this laboratory provides evidence to indicate that the expression
of NOS-III and cyclooxygenase-2 are stimulated by lyso-PC (27),
thereby increasing the synthesis of NO and prostacyclin, which act
synergistically to block monocyte adhesion and smooth muscle cell
proliferation; platelet activation and aggregation; and pathological
vasoconstriction. This leads to a postulate that vascular injurious
agents such as lyso-PC creates a yin-yan situation in which it induces
the expression of vasoprotective genes to counteract the vascular
damaging genes. It is interesting to note that lyso-PC induces the
expressions of those diverse genes with two distinct types of kinetics:
1) rapid, transient as in heparin-binding epidermal growth factor and
cyclooxygenase-2 gene induction and 2) delayed, sustained as in eNOS
and intercellular adhesive molecule-1 gene induction. It is unclear
whether these two types of genes are transcriptionally regulated by a
common mechanism. In fact, the mechanism by which lyso-PC induces any
of these genes had not been reported previously. This report is the
first to shed light on the promoter regulation of eNOS gene by lyso-PC.
Our results indicate that lyso-PC enhances the transcriptional
activation of this housekeeping gene primarily by increasing the Sp1
binding activity via a PP2A-dependent reaction. Ets binding
to its cognate site on the promoter region is also involved in basal
and lyso-PC-induced eNOS transcription.
Sp1 is a ubiquitous transcriptional activator mediating basal and
regulated gene expression. Sp1 binding to its cognate motif depends not
only on nuclear Sp1 levels but also on posttranslational modification
of the Sp1 molecule. Recent studies indicate that Sp1 binding activity
is influenced by phosphorylation: phosphorylation decreases, whereas
dephosphorylation by the action of phosphatases increases Sp1 binding
activity (28, 29). A recent study on the acetyl-CoA carboxylase gene
indicates that glucose induces this gene transcription by a mechanism
involving dephosphorylation of Sp1 by protein phosphatase 1 and 2A
(28). Our data, which show suppression of lyso-PC-induced Sp1 binding
activity by okadaic acid, but not vanadate, and a selective increase in
PP2A activity by lyso-PC, which was inhibited by okadaic acid, are
consistent with a role of PP2A in modifying Sp1 binding activity.
Results from this study led us to postulate that the Sp1 is
phosphorylated at the basal cellular state, and lyso-PC activates PP2A,
which in turn dephosphorylate Sp1, resulting in an increased Sp1
binding activity.
Signal pathways for lyso-PC-induced transcriptional activation have not
been fully established. A recent study suggests the involvement of
stress-activated protein kinase/c-Jun amino-terminal kinase pathway
that leads to AP1 activation and binding to its cognate site (30). It
is unclear whether JNK and/or other mitogen-activated protein kinase
pathways are involved in increased Sp1 binding activity. It is possible
that lyso-PC may increase Sp1 binding activity by activating protein
phosphatases via the mitogen-activated protein kinase or other kinase
pathways. This is being investigated in our laboratory.
Our results indicate that a PEA3/Ets sequence in the promoter/enhancer
region of eNOS gene is involved in basal and lyso-PC-induced promoter
activity. Nuclear Ets-related proteins contain a large family of
polypeptides sharing an ETS domain (31). They bind to purine-rich
sequences with a GGA core. The 5'- and 3'-flanking sequences of the GGA
core confer relative specificity for Ets family proteins. The
GGA-containing sequences on eNOS lack a specificity for any given class
of Ets-related proteins. This is in agreement with our experimental
data showing promiscuity of binding for several representatives of Ets
family proteins. Ets proteins are involved in cellular response to
external stresses. They generally serve as co-activators for
transcriptional activators including Sp1 (32, 33). Several reports have
suggested that PEA3 and Sp1 sequences are often colocalized on the
promoter/enhancer region and binding of Ets and Sp1 to their respective
sites causes a synergistic activation of a number of genes (33). As
regards the activation of eNOS gene, our data are in agreement with the proposition that Ets serves as a co-activator for Sp1. Binding of Ets
alone resulted in minimal promoter activation, whereas it augmented
Sp1-mediated transcriptional activation. It is possible that Ets
interacts with components of the general transcription factors in the
preinitiation complex through which it may enhance recruitment of the
preinitiation complex to the promoter region.
We thank Zhifei Zu for technical assistance
and Susan Mitterling for excellent assistance in preparing this
manuscript.