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INTRODUCTION |
Excessive or uncontrolled replication and migration of vascular
smooth muscle cells (VSMCs)1
are critical events involved in a number of vascular diseases including
atherosclerosis, hypertension, and restenosis after balloon angioplasty
(1-3). Morphologic studies of the sequencing events in the arterial
wall have revealed that macrophages are present in atherosclerotic
lesions (4, 5) and are involved in the production of several growth
factors such as platelet-derived growth factors (PDGFs), basic
fibroblast growth factor, tumor necrosis factor-
, and
transforming growth factor-
1 (3). Particularly, interleukin (IL)-1
is one of the major secretory products of activated macrophages and can induce proliferation and migration of
fibroblasts and VSMCs (6-8). Previous studies have demonstrated that
the mitogenic response of IL-1
for VSMCs is mediated by an indirect
pathway, causing the release of PDGF-AA, which specifically binds to the PDGF
-receptor (PDGF
R) subtype on the cell surface (9-11). In addition, IL-1
can also up-regulate PDGF
R expression itself in rat lung fibroblasts, thereby enhancing PDGF-mediated mitogenesis and chemotaxis of the cells (12). Previously, we have shown
that the PDGF
R promoter contains an enhancer core sequence for
CCAAT/enhancer-binding protein (C/EBP), and IL-1
-mediated induction of PDGF
R expression is mainly regulated by a specific nuclear factor, C/EBP
, in VSMCs (13).
Peroxisome proliferator-activated receptors (PPARs) belong to the
nuclear receptor superfamily of ligand-dependent
transcription factors, and cross-regulation between the C/EBP family
and the PPAR family is very important in maintaining adipocyte
differentiation. Especially, C/EBP
and
play a critical role in
the determination of pre-adipocyte development by activating expression
of C/EBP
and PPAR
genes (14, 15). Recent studies have shown that
in addition to its proposed roles in the regulation of adipocyte differentiation and glucometabolism, PPARs probably regulate
inflammatory responses by interaction with other transcription factors
in several types of cells (16, 17). However, expression and function of
PPAR
in VSMCs are somewhat controversial. In human VSMCs, Staels
et al. (16) observed faint expression of PPAR
that was not involved in the negative regulation of cytokine-induced IL-6 and
cyclooxygenase-2 expression; this effect was mediated by PPAR
. In
contrast, Marx et al. (18) demonstrated that human VSMCs expressed PPAR
that inhibited matrix metalloproteinase expression and cell migration. In particular, the latter observations suggest that
such a role of PPAR
may be to limit the arterial remodeling that
occurs in response to hypertension, atherosclerosis, and restenosis, or
even to counterbalance other vascular effects. Indeed, PPAR
activation can be shown to inhibit VSMC proliferation and migration in
a variety of assays (18-20).
In the present study, we demonstrated new roles of PPAR
activators,
troglitazone (TRO) and a naturally occurring ligand, 15-deoxy-
12,14-prostaglandin J2
(PGJ2), on IL-1
-induced expression of PDGF
R and
clarified the underlying molecular mechanism by demonstrating a
functionally important interaction between PPAR
and C/EBP
in rat
cultured VSMCs.
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EXPERIMENTAL PROCEDURES |
Materials--
TRO was provided from Sankyo, Co. (Tokyo, Japan).
Recombinant rat IL-1
was purchased from R & D Systems (Tokyo,
Japan). Prostaglandin F2
(PGF2
) and
PGJ2 were from Cayman (Ann Arbor, MI). Affinity-purified
antibodies for PDGF
R, PPAR
, C/EBP
, C/EBP
, and C/EBP
raised against peptidic epitopes corresponding with amino acid
sequences of human PDGF
R (951-1,089), rat C/EBP
(253), rat
C/EBP
(258), and rat C/EBP
(247) were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA).
Cell Culture--
VSMCs were isolated from thoracic aortas of
male Harlan Sprague-Dawley rats (Charles River Japan Inc., Kanagawa,
Japan) as described previously (21) and were maintained in Dulbecco's modified Eagle's medium with 10% heat-inactivated fetal calf serum at
37 °C in a humidified atmosphere of 95% air, 5% CO2.
In preparation for all experiments, subconfluent cells at 3-8 passages
from primary culture were made quiescent by placing them in Dulbecco's
modified Eagle's medium supplemented with 0.1% fetal calf serum for 2 days.
Protein Extraction and Western Blotting--
Whole-cell lysates
were extracted from VSMCs by the standard method (21), and nuclear
extracts were prepared according to the method described by Dignam
et al. (22). Western blotting was essentially carried out as
described previously (23).
RNA Extraction and Measurement of mRNA--
Total RNA was
extracted from VSMCs with the use of ISOGEN (Nippon Gene, Tokyo,
Japan). Measurement of mRNA was done by Northern blotting. Northern
hybridization, autoradiography, and densitometric analysis were
performed as described previously (24).
Measurement of BrdUrd Incorporation--
VSMCs were
seeded on 96-well plates (1 × 104 cells per
well) and were cultured in the presence or absence of TRO (10 µmol/liter) or PGJ2 (5 µmol/liter) for 24 h. Then,
cells were treated with IL-1
(10 ng/ml) for 4 h
and were stimulated with PDGF (20 ng/ml) for 12 h. BrdUrd
incorporation was finally determined by the Cell Proliferation
enzyme-linked immunosorbent assay system (Amersham Pharmacia Biotech).
Electromobility Shift Assay (EMSA), Supershift Assay,
and Plasmid Transfection--
The EMSA and supershift assay were
performed for the C/EBP-binding motif seen in rat PDGF
R gene
promoter as described previously (25). VSMCs (5 × 105
cells per dish) were seeded in 60-mm dishes 24 h before
transfection, and then the C/EBP
expression vector, designated
MSV-C/EBP
in our earlier studies (13, 25), was transfected (3 µg
per dish) onto the cells with the use of LipofectAMINE Plus (Life
Technologies, Inc.) according to the manufacturer's specifications.
Statistical Analysis--
Analysis of variance with
Boferroni-Dunn post hoc was used to analyze differences between two
experimental groups. All data are expressed as means + S.E., and
statistical significance was defined as p < 0.05.
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RESULTS |
TRO or PGJ2 Inhibits IL-1
-mediated PDGF
R
Expression--
We confirmed that rat cultured VSMCs used in this
study expressed PPAR
mRNA and protein (data not shown). VSMCs
were cultured in the presence or absence of a PPAR
activator, TRO
(0-10 µmol/liter) or PGJ2 (0-5 µmol/liter), for
24 h. Then, expression levels of PDGF
R were determined by
Northern (Fig. 1A) and Western
(Fig. 1B) blotting 12 h after treatment with or without
IL-1
(10 ng/ml). Previously, we have demonstrated that expression
levels of PDGF
R were increased by treatment with IL-1
at doses up
to 10 ng/ml in a dose-dependent manner (13). Therefore, we
used the dose of IL-1
at 10 ng/ml thereafter. In quiescent cells,
base-line levels of PDGF
R mRNA and protein were very low. On the
other hand, expression levels of PDGF
R were increased drastically by treatment with IL-1
in the absence of TRO or PGJ2,
whereas induction of PDGF
R expression was significantly reduced by
pretreatment with TRO or PGJ2 in a
dose-dependent manner.

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Fig. 1.
Inhibitory effect of TRO and PGJ2
on IL-1 -mediated
PDGF R expression. Quiescent VSMCs were
cultured in the presence or absence of a PPAR activator, TRO or
PGJ2, at the indicated concentrations for 24 h. After
stimulation with (+) or without IL-1 ( ) for
12 h, mRNA (A) and protein (B) levels of
PDGF R were determined by Northern or Western blotting, respectively.
28S, ethidium bromide staining of 28 S ribosomal RNA.
Similar results were obtained from three independent experiments.
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PGF2
Diminishes the Inhibitory Effect of TRO or
PGJ2 on IL-1
-mediated PDGF
R Expression--
To
further gain direct evidence that the inhibitory effect of TRO or
PGJ2 on IL-1
-mediated PDGF
R expression is actually caused by PPAR
-specific activation, we examined the action of PGF2
, an agent inactivating PPAR
by causing
phosphorylation (Fig. 2). Incubation with
PGF2
(200 nmol/liter) diminished the inhibitory effect
of TRO (10 µmol/liter) (Fig. 2, lane 3 versus lane 4) or PGJ2 (5 µmol/liter) (lane 5 versus lane 6) on IL-1
-mediated induction of PDGF
R
expression in VSMCs.

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Fig. 2.
Effect of
PPAR -specific inactivation by
PGF2 on
IL-1- -mediated PDGF R
expression. VSMCs were pretreated with (lanes 4 and
6) or without (lanes 1-3 and 5)
PGF2 (200 nmol/liter) for 12 h. Then,
cells were treated with (+) or without IL-1
( ) for 12 h in the presence (+) or absence
( ) of TRO (10 µmol/liter) or PGJ2 (5 µmol/liter), and whole-cell lysates obtained from each treated VSMC
were analyzed by Western blotting for PDGF R. Similar results were
obtained from three independent experiments.
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TRO or PGJ2 Suppresses PDGF-mediated VSMC
Proliferation--
In Fig. 3, VSMCs were
cultured in the presence and absence of TRO (10 µmol/liter) or
PGJ2 (5 µmol/liter) and were stimulated with or without
IL-1
. Then, cell proliferation activity was determined as BrdUrd
incorporation 12 h after treatment with PDGF (20 ng/ml). In
quiescent cells, pretreatment with TRO or PGJ2 did not
affect base-line levels of cell proliferation activity. On the other hand, IL-1
increased (by 1.8-fold) cell proliferation activity, and
TRO or PGJ2 suppressed this effect of IL-1
almost
completely. Furthermore, PDGF-AA or -BB showed an additive effect in
the cell proliferation activity compared with IL-1
alone, the extent
of activation being on the order of 1.4- or 2.2-fold, respectively. This additive effect of PDGFs was significantly suppressed by pretreatment with TRO or PGJ2.

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Fig. 3.
Suppressive effect of TRO or PGJ2
on PDGF-mediated VSMC proliferation. VSMCs were cultured in the
absence (open bars) or presence (closed bars) of
TRO (10 µmol/liter) or PGJ2 (5 µmol/liter)
(hatched bars) for 24 h and then stimulated
with (+) or without ( ) IL-1 for 4 h.
Then, VSMC proliferation activity was measured as BrdUrd incorporation
12 h after treatment with (+) or without
( ) 20 ng/ml of PDGF-AA or -BB and was finally presented as
a fold activation in reference to the control activity seen in
quiescent VSMCs without PPAR activators. All data are expressed as
means + S.E. of six separate assays. *, p < 0.05, significant difference versus control activity; and
, p < 0.05, significant difference among the three
treatment groups represented by open, closed, and
hatched bars.
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TRO or PGJ2 Inhibits C/EBP Binding
Activity--
Recently, we have demonstrated that induction of
PDGF
R expression is mediated by a specific transcription factor,
C/EBP, in VSMCs (13, 25). To clarify the effect of PPAR
activation on DNA binding of C/EBP members, EMSA and supershift assays were performed using a labeled C/EBP probe containing a consensus sequence seen in the rat PDGF
R promoter region (Fig.
4). Nuclear extracts were prepared from
the IL-1
-stimulated VSMCs after pretreatment with or without TRO (10 µmol/liter) or PGJ2 (5 µmol/liter). Intensities of two
specifically retarded bands (B1 and B2) were increased by treatment
with IL-1
(Fig. 4, lane 1 versus lane
2), whereas both band intensities were reduced by pretreatment
with TRO (lane 3) or PGJ2 (lane 4)
almost completely. Competition experiments indicated that the retarded
bands were competed out exclusively by adding a 100-fold molar excess
of unlabeled C/EBP probe (Fig. 4, lane 6) but not of
an unrelated probe for a nuclear factor-
B consensus sequence
(lane 7). To determine the nature of C/EBP members that
actually contribute to the DNA binding, a supershift assay was
performed using specific antibodies against three major members of the
C/EBP family: C/EBP
,
, and
(Fig. 4, lanes
8-10). The retarded bands (B1 and B2) seen in EMSA were
supershifted by preincubation with antibodies against C/EBP
(Fig.
4, lane 9) or C/EBP
(lane 10) but not
C/EBP
(lane 8).

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Fig. 4.
Effect of TRO or PGJ2 on DNA
binding activity of C/EBP. Nuclear extracts prepared from either
quiescent (lane 1) or IL-1 -treated VSMC for 2 h
(lanes 2-10) after pretreatment with TRO (10 µmol/liter)
(lane 3) or PGJ2 (5 µmol/liter) (lane
4) or without a PPAR activator (lanes 1,
2, and 5-10). Competition experiments were
carried out by adding a 100-fold molar excess of unlabeled C/EBP probe
(lane 6) or unlabeled nuclear factor- B probe
(lane 7). Supershift assay was performed using antibodies
against three major members of the C/EBP family: C/EBP (lane
8), C/EBP (lane 9), or C/EBP (lane
10). B1 and B2, specifically retarded bands;
S, supershifted band.
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TRO or PGJ2 Suppresses IL-1
-mediated C/EBP
Expression--
In our earlier observations, we have shown that
C/EBP
and C/EBP
act as the major transcriptional activator and
the suppressor, respectively, of PDGF
R expression in VSMCs
(25). Therefore, we investigated a direct effect of PPAR
activation
on C/EBP
expression (Fig. 5).
Base-line levels of C/EBP
mRNA (Fig. 5A) and protein
(Fig. 5B) were very low in quiescent VSMCs, whereas both
levels were markedly increased by treatment with IL-1
. This IL-1
-mediated induction of C/EBP
expression was significantly suppressed by pretreatment with TRO or PGJ2 in a
dose-dependent manner.

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Fig. 5.
Suppressive effect of TRO or PGJ2
on IL-1 -mediated C/EBP
expression. Quiescent VSMCs were cultured in the presence or
absence of a PPAR activator, TRO or PGJ2, at the
indicated concentrations for 24 h. After stimulation with
(+) or without ( ) IL-1 for 12 h,
mRNA (A) and protein (B) levels of C/EBP
were determined by Northern or Western blotting, respectively.
28S, ethidium bromide staining of 28 S ribosomal RNA.
Similar results were obtained from three independent experiments.
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C/EBP
Overexpression Neutralizes the Inhibitory Effect of TRO on
PDGF
R Expression--
To determine whether C/EBP
can directly
modulate the inhibitory effect of PPAR
activation on PDGF
R
expression, transient transfection experiments were performed using a
C/EBP
expression vector, MSV-C/EBP
(Fig.
6). Mock DNA plasmid (Fig. 6, lanes
1-3) or MSV-C/EBP
(lanes 4-6) was
transfected onto VSMCs. Transfected cells were treated with or without
TRO (10 µmol/liter) for 24 h and were stimulated with or without
IL-1
for 12 h. Then, whole-cell lysates were extracted from the
cells, and protein levels of PDGF
R were determined by Western
blotting. In the mock DNA-transfected cells (Fig. 6, lanes
1-3), protein levels of PDGF
R were markedly increased by
treatment with IL-1
(lane 1 versus lane 2),
whereas TRO suppressed this enhanced effect almost completely
(lane 3). In the MSV-C/EBP
-transfected VSMCs, C/EBP
overexpression caused a significant induction of PDGF
R protein
expression (Fig. 6, lane 1 versus lane 4),
and an additive effect on PDGF
R expression was observed in the cells
treated with IL-1
(lane 4 versus lane 5). In
addition, the suppressive effect of TRO on IL-1
-mediated induction
of PDGF
R expression (Fig. 6, lane 2 versus
lane 3) was completely abolished by C/EBP
overexpression
(lane 5 versus lane 6).

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Fig. 6.
Effect of C/EBP
overexpression on TRO-mediated suppression of
PDGF R expression. VSMCs were seeded in
60-mm dishes (5 × 105 cells per dish) 24 h
before transfection. At ~70-80% confluence, 3 µg of mock DNA
plasmid (lanes 1-3) or MSV-C/EBP vector (lanes
4-6) was transfected onto cells with the use of LipofectAMINE
Plus. One day after transfection, cells were treated with TRO (10 µmol/liter) (lanes 3 and 6) for 24 h and
then stimulated with IL-1 for 12 h (lanes 2,
3, 5, and 6). Whole-cell lysates were
prepared from each treated VSMC and were analyzed by Western blotting
for PDGF R.
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DISCUSSION |
In the present study, we have produced several findings of
importance, which include the following. (1) PPAR
activators, TRO and PGJ2, suppress the induction of PDGF
R expression
and cell proliferation activity induced by PDGFs in a
ligand-dependent manner. (2) This suppressive effect of
PPAR
activators is caused by a decrease in the DNA binding activity
of C/EBP
and C/EBP
(but not C/EBP
). (3) PPAR
activation
causes a relevant inhibition of C/EBP
and an ensuing suppression of
PDGF
R gene expression, (4) C/EBP
overexpression can neutralize
the inhibitory effect of TRO on PDGF
R expression almost completely.
These results reveal a new role of PPAR
activators on VSMC growth
and proliferation and also provide us with important information to
understand the underlying mechanism in the pathogenesis and progression
of atherosclerosis and restenosis.
Previous studies (9-11) have shown that the mitogenic response of
IL-1
for fibroblasts and VSMCs is mediated by an indirect pathway,
causing the release of endogenous PDGFs, especially PDGF-AA, via an
autocrine or paracrine loop. Because the action of PDGF-AA is mediated
by its specific receptor, PDGF
R, there is a possibility that it
becomes a therapeutic target to control PDGF
R expression in the
proliferative VSMCs of atherosclerotic lesions. Recently, we have
reported that the rat PDGF
R promoter region contains an enhancer
core sequence for C/EBP (23), and an enhanced effect of PDGFs on VSMC
proliferation activity is caused mainly by a high level of C/EBP
expression (13, 25). These results strongly suggest that the C/EBP
family, particularly C/EBP
, becomes a novel candidate gene that
regulates vascular growth and development and also plays an important
role in the pathogenesis of vascular remodeling and atherosclerosis.
Some recent studies have demonstrated that one of the PPAR
activators, TRO, inhibits VSMC migration and proliferation induced by
PDGFs (18-20) and suppresses neointimal formation of the arterial wall
after balloon injury (19). In the present study, we showed that VSMC
proliferation activity was increased by IL-1
alone. Moreover, both
PDGF-AA and -BB enhanced the effect of IL-1
on cell proliferation
activity (Fig. 3). Because PDGF-BB can bind not only PDGF-
receptor but also PDGF
R, the enhanced effect of PDGF-BB is
mediated by the action through both subtypes of PDGF receptors.
Furthermore, pretreatment with TRO or PGJ2 significantly reduced IL-1
-mediated induction of PDGF
R expression in a
dose-dependent manner (Fig. 1) and suppressed cell
proliferation activity after treatment with PDGFs (Fig. 3). Because TRO
or PGJ2 did not alter PDGF-
receptor expression in VSMCs
(data not shown), the suppressive effect of PPAR
activators on VSMC
proliferation was mainly caused by down-regulation of PDGF
R expression.
We have previously demonstrated that PDGF
R gene transcription is
regulated mainly by C/EBP
through a C/EBP-binding motif identified
in a promoter region of the PDGF
R gene (13, 25). Therefore, we
further determined the effect of PPAR
activators on PDGF
R and
C/EBP
expression. EMSA and supershift assay using a C/EBP probe
clearly demonstrated that PPAR
activators reduced DNA binding of
C/EBP
and C/EBP
(but not C/EBP
) (Fig. 4). Furthermore, PPAR
activators significantly suppressed IL-1
-mediated C/EBP
expression (Fig. 5). Overexpression studies demonstrated that exogenous
C/EBP
expression modulated the inhibitory effect of TRO on PDGF
R
expression (Fig. 6). These results strongly suggest that the effect of
PPAR
activators on PDGF
R gene transcription is mainly mediated by
suppression of C/EBP
expression in VSMCs.
Interaction between PPAR
and C/EBPs has already been investigated in
several other types of cells (14, 15). However, detailed mechanisms of
PPAR
activation on C/EBP
modification in VSMCs are unknown. Our
previous study has demonstrated that a core promoter region of the rat
C/EBP
gene does not contain any obvious peroxisome proliferator
response element motifs (26). This observation suggests that C/EBP
gene transcription is not regulated directly by PPAR
through
peroxisome proliferator response element motifs, but presumably through
an indirect pathway, i.e. the interaction between PPAR
and other inflammatory transcription factors. Indeed, several recent
studies have indicated that PPARs repress gene transcription by
interfering with signal transducers and activators of
transcription, AP-1, and nuclear factor-
B signaling pathways in a peroxisome proliferator response element-independent fashion (17). These findings suggest that other transcriptional factors
such as nuclear factor-
B and AP-1 may also contribute to the
inhibitory effect of PPAR
activation on the IL-1
-mediated PDGF
R gene transcription in VSMCs. However, further detailed studies
are necessary to clarify the involvement of other factors.
The expression of C/EBP
is undetectable or minor in normal tissues;
however, it is induced rapidly and drastically by treatment with inflammatory cytokines such as IL-1
, IL-6, and tumor necrosis factor-
. Recently, these cytokines have been called
"adipocytokines," and have been thought to be major factors
relating to atherosclerosis and insulin resistance. TRO was originally
identified as a PPAR
activator to improve insulin resistance and is
known to normalize the gene expression of tumor necrosis factor-
or
leptin by regulating adipocyte differentiation (27). As well as the
PDGF
R gene, many other genes including tumor necrosis factor-
,
leptin, cyclooxygenase-2, Na+/H+ exchanger-1,
and IL-6 are also regulated by members of the C/EBP family (28-32).
Therefore, C/EBPs are supposed to regulate various target genes and
play a pivotal role in the pathological conditions of many types of
cells including adipocytes and VSMCs. We have previously demonstrated
that gene expression of both C/EBP
and PDGF
R is markedly elevated
in cultured VSMCs prepared from spontaneously hypertensive rats but not
from normotensive rat strains such as Harlan Sprague-Dawley, Wistar,
and Wistar-Kyoto rats (23). These results strongly suggest the
possibility that C/EBPs play important roles in the pathogenesis of
atherosclerosis, insulin resistance, and hypertension.
The results obtained herein show evidence for new roles of the two
transcriptional factors PPAR
and C/EBP
in regulating IL-1
-induced PDGF
R gene activation and in controlling
opposing biological effects in VSMC proliferation. The functionally
important interaction between C/EBP
and PPAR
is probably involved
in the regulation of inflammatory responses in the early process of
vascular remodeling and resultant atherosclerosis and in the
success of maintaining homeostasis in the arterial wall.