© 1999 American Heart Association, Inc.
Molecular Medicine |
Angiotensin II Stimulates Platelet-Derived Growth Factor-B Chain Expression in Newborn Rat Vascular Smooth Muscle Cells and Neointimal Cells Through Ras, Extracellular Signal–Regulated Protein Kinase, and c-Jun N-Terminal Protein Kinase Mechanisms
From the Departments of Molecular and Cellular Physiology (J.D.), Surgery (J.D., M.M.), and Internal Medicine (T.N.), Graduate School of Medicine, University of Tokyo, Japan; Department of Physiology, Kanazawa University School of Medicine (Y.T.), Kanazawa, Japan; and Vascular Research Division, Department of Pathology, Brigham and Women's Hospital (T.C.), Boston, Mass.
Correspondence to Yoh Takuwa, MD, Department of Physiology, Kanazawa University School of Medicine, 13-1 Takara-machi, Kanazawa 920-8640, Japan. E-mail ytakuwa{at}med.kanazawa-u.ac.jp
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Abstract—Platelet-derived growth factors (PDGFs) have been implicated in the pathogenesis of vascular proliferative disorders. Vascular smooth muscle cells (VSMCs) are one of the cell types that produce PDGF-B chain in proliferative lesions, although the mechanism of regulation of PDGF-B chain production in these cells is not well understood. In the present study, we demonstrate that angiotensin II (Ang II), which is also implicated in vascular stenosis after angioplasty and atherosclerosis, markedly stimulates PDGF-B chain mRNA expression in cultured newborn rat medial VSMCs and neointimal VSMCs via an AT1, but not in adult rat VSMCs. In newborn rat VSMCs, Ang II activates extracellular signal–regulated protein kinase (ERK), c-Jun N-terminal protein kinase (JNK), and p38 mitogen–activated protein kinase. The mitogen-activated protein/ERK (MEK) inhibitor PD98059, but not the p38 inhibitor SB203580, abrogates Ang II–induced PDGF-B mRNA expression. Transient transfection analysis using a PDGF-B promoter-luciferase gene reporter construct reveals that Ang II induces transcriptional activation of PDGF-B chain gene, which is abolished by the expression of a dominant negative form of either ERK or JNK, but not of p38. The expression of a dominant negative form of Ras abolishes the stimulatory effects of Ang II on ERK activity and PDGF-B mRNA expression. In adult rat VSMCs, Ang II activates ERK and JNK, but weakly induces Egr-1, a transcription factor implicated in PDGF-B chain gene expression, compared with newborn VSMCs. These data indicate that Ang II activates PDGF-B chain gene expression in VSMCs through mechanisms involving Ras-ERK and JNK.
Key Words: angiotensin II • platelet-derived growth factor B chain • vascular smooth muscle • Ras • mitogen-activated protein kinase
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Introduction |
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Platelet-derived growth factors (PDGFs) are potent mitogens and chemoattractants for vascular smooth muscle cells (VSMCs).1 2 These growth factors consist of homo- or heterodimers of 2 polypeptides, A and B chains.1 The receptors for the PDGFs consist of
- and ß-isoforms. The
-receptor binds either PDGF-A chain or -B chain, and the
ß-receptor binds only B chain. PDGF-B chain is generally more potent
than PDGF-A chain for VSMCs,3 indicating that activation
of the ß-receptor rather than the -receptor leads to more
pronounced activation of VSMCs. It is believed that PDGFs are involved
in the pathogenesis of atherosclerosis and
stenosis after balloon angioplasty.4 5 In a rat
carotid balloon injury model, procedures to suppress the action of
PDGFs, such as repeated injections of neutralizing anti-PDGF
antibody6 and local application of antisense
oligonucleotides to PDGF ß-receptors,7
suppress neointima formation. In situ hybridization
analysis reveals that transcripts of PDGF ß-receptor and
PDGF-B chain are both detected in the
neointima.8 9 These findings suggest a
paracrine/autocrine role for PDGF in lesion formation. In addition, we
have recently observed that the extent of PDGF ß-receptor tyrosine
phosphorylation is increased in balloon-injured rat
carotid arteries.10 These observations are
consistent with an essential role for PDGF-B chain and its
receptor in neointima formation in balloon-injured
arteries. Despite the potential importance of PDGF-B chain in the
formation of occlusive lesions in injured arteries, relatively little
is known about the regulation of PDGF-B chain expression in
neointimal cells. Angiotensin II (Ang II) has also been implicated in the pathogenesis of neointima formation after balloon injury, especially in the rat model, as demonstrated by the observations that angiotensin-converting enzyme inhibitors11 and antagonists for AT112 effectively suppress neointima formation. Several in vitro studies using cultured VSMCs demonstrate that Ang II induces expression of growth factors including transforming growth factor-ß,13 14 PDGF-A,15 16 and basic fibroblast growth factor,17 suggesting the possibility that Ang II may stimulate growth of VSMCs via an autocrine mechanism. We recently showed that the administration of an AT1 antagonist abolished balloon injury-induced stimulation of PDGF ß-receptor tyrosine phosphorylation, with a reduction in PDGF-B mRNA expression, in rat carotid arteries.10 These observations raise the interesting possibility that Ang II may be involved in PDGF-B gene expression in vivo in VSMCs, especially in neointimal VSMCs, of injured blood vessels.
Previous studies have demonstrated that neointimal VSMCs are phenotypically distinct from medial VSMCs isolated from adult animals, but rather resemble newborn rat medial VSMCs.18 19 20 21 22 For example, adult rat medial VSMCs do not express a detectable level of PDGF-B chain, whereas newborn rat medial VSMCs, like neointimal VSMCs, express a readily detectable level of PDGF-B mRNA.19 20 22 23 In the present study, we demonstrate for the first time that Ang II potently stimulates PDGF-B chain mRNA expression in both neointimal VSMCs and newborn rat VSMCs. We also studied the signaling mechanisms underlying Ang II–induced PDGF-B gene expression and found that it is mediated via the AT1 in a manner dependent on Ras, extracellular signal–regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). Thus, the present results indicate that PDGF-B chain gene is one of the target genes regulated by the Ras–mitogen-activated protein kinase (MAPK) signaling pathway in VSMCs.
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Materials and Methods |
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Materials
Polyclonal rabbit anti-ERK2 antibody (C-14), polyclonal rabbit anti-JNK1 antibody (C-17), and polyclonal goat anti-p38 MAPK antibody (C-20) were purchased from Santa Cruz Biotechnology. Ang II and myelin basic protein were bought from Sigma. Glutathione-Sepharose and PD98059 were purchased from Pharmacia Biotechnology and Calbiochem, respectively. [
-32P]ATP and
[-32P]dCTP were purchased from DuPont-NEN.
pGL2-Basic and pSV-ßgal were purchased from Promega. pGEX-cJun(5-89),
pactEF–dominant negative (DN) ERK, pGEM-ATF2, pMT-Asn17-H ·
Ras, pBS-rPDGF-B, human Egr-1 cDNA, human Sp1 cDNA, and AxCALacZ were
provided by Dr A.S. Kraft (University of Alabama School of Medicine,
Tuscaloosa, Ala), Dr K. Okazaki (Kurume University Institute of Life
Sciences, Kurume, Japan), Dr M.R. Green (University of Massachusetts
Howard Hughes Medical Institute. Worcester, Mass), Dr J.M. Cooper
(Harvard Medical School, Cambridge, Mass), Dr D. Katayose (Tohoku
University Medical School, Sendai, Japan), RIKEN Gene Bank (Tsukuba,
Japan), Dr R. Tjian (University of California, Berkeley), and Dr
I. Saito (University of Tokyo Institute of Medical Sciences, Tokyo,
Japan), respectively. Monoclonal anti-Ras antibody, monoclonal anti-ERK
antibody, polyclonal anti-phospho-JNK antibody, and monoclonal
anti-calponin antibody were purchased from Transduction Laboratory,
Zymed, Promega, and Sigma, respectively. Specific polyclonal anti-ERK2
antibody, anti-JNK1 antibody, and anti-p38 antibody were purchased from
Santa Cruz Biotechnology. Monoclonal anti-PDGF-B–specific antibody was
a kind gift from Mochida Pharmacy. CV11974 (Candesartan) and PD123319
were kindly donated by Takeda Chemical Industries and Parke-Davis,
respectively.
Cell Culture
Aortic VSMCs were isolated by the explant method from 1-week-old
and 18-week-old Wistar male rats as described.10 24 To
obtain neointimal VSMCs, left carotid arteries were removed
from an 18-week-old male Wistar rat at 14 days after
deendothelializing balloon injury10 and
cut open longitudinally. Fine pieces (width <0.5 mm) of the
neointima were excised by using fine forceps and scissors
under a microscope and transferred to tissue culture dishes for the
explant culture. VSMCs were maintained in DMEM supplemented with 10%
FCS (Equitech-Bio), 105 U/L penicillin G, and
137 µmol/L streptomycin under an atmosphere of 95% air plus 5%
CO2 at 37°C. Before each experiment, confluent
cells were serum-deprived by incubation in serum-free DMEM for 48
hours. Cells between the 5th and 20th passages were used in the
present study. VSMCs from an 18-week rat media, which were
spindle-shaped and grew multilayered with characteristic
hill-and-valley formation, were positive for anti–smooth muscle
actin staining, expressed a detectable amount of calponin as evaluated
with Western blotting, and were responsive to vasoactive peptides
including Ang II and endothelin-1 as evaluated with the intracellular
Ca2+ measurement. In contrast to adult VSMCs,
VSMCs obtained from a neonate and neointimal cells were
polygonal in shape and grew to a monolayer at confluence, as described
in previous reports.18 19 20 21 22 Western analysis using
anti-calponin antibody did not detect expression of calponin in newborn
VSMCs. These cells also responded to Ang II with an increase in the
[Ca2+]i. Both newborn rat
VSMCs and neointimal VSMCs were negative for von
Willebrand factor as evaluated by immunocytochemical staining
using anti–von Willebrand antibody (Dako), which excludes the
possibility that VSMC cultures were contaminated with
endothelial cells.
Northern Blot Analysis
Ten micrograms of total RNA, isolated from VSMCs by the
acid-guanidinium isothiocyanate/phenol/chloroform method, was separated
by formaldehyde-1.0% agarose gel electrophoresis and transferred onto
a nylon membrane (Hybond N, Amersham) as described.24
Blots were hybridized with cDNA probes labeled with
[-32P]dCTP by the random priming method.
PDGF-B probe is a 530-bp rat cDNA fragment from pBS-rPDGF-B encoding
exons 1 to 5.25 Mouse AT1 cDNA was
obtained by polymerase chain reaction (PCR).24 A membrane
was rehybridized with 32P-labeled GAPDH cDNA
probe. The radioactivity of corresponding bands was quantified by a
Fuji BAS 2000 Bio-Image Analyzer (Fuji Film).10 24
mRNA levels were corrected for GAPDH mRNA level by calculating the
ratio of PDGF-B mRNA or other mRNAs/GAPDH mRNA radioactivity for each
sample.
Production of the Recombinant PDGF ß-Receptor
Extracellular Domain (XR) Protein
The cDNA of the extracellular region (corresponding to amino
acids 1 to 531) of human PDGF ß-receptor, with a stop codon TAG
followed by an EcoRI site at the 3' end and an
EcoRI site at the 5' end, was obtained by PCR using phPDGF-R
(a gift from Dr H. Okazaki, Kirin Brewery, Yokohama, Japan) as a
template and was ligated onto pVL1393 (PharMingen) at the
EcoRI site downstream of the polyhedron promoter to
create pVL1393-XR. Sf9 cells were cotransfected with pVL1393-XR and
Baculogold baculovirus DNA (PharMingen) by the lipofection method, and
the recombinant baculovirus encoding the PDGFXR cDNA was recovered.
PDGFXR protein in serum-free conditioned medium of Sf9 cells infected
with the baculovirus carrying the PDGFXR cDNA was purified by wheat
germ agglutinin Sepharose 6 MB column as
described.26 27
Activation of ERK, JNK, and p38
Each MAPK was immunoprecipitated by using respective specific
polyclonal antibodies, and the MAPK activities were assayed in vitro by
using myelin basic protein, glutathione
S-transferase–cJun(5-89) and glutathione
S-transferase–ATF2(1-109) as substrates for ERK, JNK, and
p38, respectively, as described previously.28 The
radioactivity in the bands corresponding to substrate proteins was
measured by using a Fuji BAS 2000 BioImage Analyzer, as
described.28
Transient Transfection Assay Using the PDGF-B Promoter-Luciferase
Reporter Plasmid
A 1.0-kb fragment (–956 to +45 as indicated as the number of
base pairs upstream of the TATA box) of the mouse PDGF-B
promoter29 was isolated by PCR and cloned in sense
orientation into the NheI-HindIII sites of
pGL2-Basic to create Sis-Luc. Human full-length p38 MAPK cDNA was
cloned by PCR from the HEL cell cDNA library, and point mutations were
created by a PCR-based method.25 28 The
nucleotide sequences of the cDNAs obtained by the PCR
method were confirmed by sequencing with an ALFred DNA sequencer
(Pharmacia Biotechnology). VSMCs were cotransfected by using
LipofectAMINE (GIBCO) with Sis-Luc and either of the vectors encoding
DN-ERK (pactEF-ERK-D170A),30 DN-JNK1(pME18S-JNK1-T183A,
Y185F),30 or DN-p38(pME18S-p38-T180A,
Y182F)31 or an empty vector (pME18S or pactEF). Cells were
allowed to recover after transfection for 3 hours in DMEM containing
10% FCS and then serum-deprived for 48 hours. Cell lysates were
prepared, and luciferase activity was measured with a Lumat LB95001
luminometer (Berthold) using the luciferase assay system (Promega), as
described.30 Luciferase activity was normalized for
ß-galactosidase activity measured in parallel cultures cotransfected
with the ß-galactosidase expression plasmid pSV-ßgal and either of
the expression vectors for DN forms of the MAPKs or empty vectors.
Western Blot Analysis of PDGF-B Protein in the
Conditioned Media
Serum-deprived VSMCs were incubated with or without Ang II (100
nmol/L) for 36 hours. The conditioned media (8 mL) were collected,
concentrated to 1 mL with a centrifugal concentrator (Centriprep
10; Amicon), and acid-precipitated with trichloroacetic acid (a
final concentration of 10%) for 1 hour at 4°C. The precipitates were
solubilized in Laemmli SDS sample buffer and separated on SDS–17.5%
PAGE under reducing conditions, followed by electrotransfer onto
Immobilon-P membrane (Millipore). Separated proteins were probed by
monoclonal anti-PDGF-B–specific antibody, which was raised against
amino acids 73 to 97 of human PDGF-B chain peptide and reactive for rat
PDGF-B chain.
Construction of an Adenovirus and Gene Transfer
We constructed a recombinant replication-deficient adenovirus
containing each of the genes of DN Ras (Asn17-H · Ras) and DN
Rac (Asn17-Rac1), driven by the CAG promoter that consists of the
cytomegalovirus 1E enhancer and chicken ß-actin promoter, by use of
homologous recombination, as described in detail
previously.32 VSMCs were infected with adenoviruses at a
multiplicity of infection of 
50, allowed to recover in DMEM with
10% FCS for 3 hours, and then serum-deprived for 48 hours before
experiments.
Statistics
The data are presented as mean±SE of 3 or more
determinations or means of 2 determinations. The statistical
significance of differences between the 2 groups was determined by
Student t test, whereas multiple comparisons were
analyzed by Scheffé test.
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Results |
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Ang II Stimulates Expression of PDGF-B mRNA and Protein in Newborn Rat VSMCs and Neointimal VSMCs
VSMCs from a newborn rat expressed a low level of PDGF-B mRNA at quiescent states (Figure 1A
). Stimulation
of VSMCs with Ang II (10 nmol/L) induced a marked increase in PDGF-B
mRNA, which peaked at 4 hours and then gradually declined to an
unstimulated level by 24 hours (Figure 1A). Ang II stimulation
did not change GAPDH gene expression. Neointimal VSMC also
responded to Ang II with an induction of PDGF-B mRNA, which became
detectable at 2 hours, peaked at 6 hours, and then gradually declined,
but was still at a detectable level at 24 hours (Figure 1B). In
adult rat medial VSMCs, by contrast, PDGF-B mRNA was not at all
detectable under the basal unstimulated conditions, and Ang II failed
to induce PDGF-B gene expression (Figure 1C). The stimulatory
effect of Ang II on PDGF-B mRNA in newborn rat VSMCs increased dose
dependently with half-maximally and maximally effective concentration
values of 700 pmol/L and 10 nmol/L, respectively (Figure 2A and 2B). This stimulatory effect of
Ang II was abolished by the addition of the AT1
receptor-specific antagonist CV11974,10 but
not of the AT2 antagonist PD123319
(Figure 3), indicating that the
AT1 receptor mediates Ang II–induced PDGF-B gene
expression.
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To examine whether Ang II stimulates the production of PDGF-B
protein, the conditioned media from Ang II-stimulated and nonstimulated
newborn rat VSMCs were analyzed for PDGF-B protein by Western
blotting (Figure 4). An
anti-PDGF-B–reactive band with a molecular mass of 16 kDa was detected
in both nonstimulated and stimulated conditioned media. The amount of
the anti-PDGF-B–reactive protein was much larger in the stimulated
conditioned medium than in the nonstimulated medium. Preincubation of
anti-PDGF-B antibody with PDGF-B abolished this band, which indicates
that the observed anti-PDGF-B–reactive protein represents
PDGF-B peptide.
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Blocking PDGF-B Action Partially Inhibits Ang II–Induced DNA
Synthesis in Newborn VSMCs
Ang II stimulates DNA synthesis of serum-deprived newborn VSMCs
2-fold at 48 hours, whereas Ang II does not affect DNA synthesis of
adult VSMCs at either 24 or 48 hours (Table). The addition of
recombinant extracellular domain of PDGF ß-receptor (XR), which has
the activity to bind to and block the action of PDGF-B
chain,26 27 significantly attenuates Ang II–induced DNA
synthesis in newborn VSMCs. Both VSMC types show the comparable degrees
of mitogenic responses to PDGF-BB (0.4 nmol/L) and to
epidermal growth factor (0.2 nmol/L) (3.8- and 4.3-fold stimulation for
PDGF-BB and 3.9- and 3.3-fold stimulation in newborn and adult VSMCs,
respectively). The addition of XR abolishes DNA synthesis induced by
PDGF-BB, but not by epidermal growth factor, in either cell type.
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Ang II–Induced PDGF-B Gene Expression Is Dependent on ERK and
JNK
Stimulation of newborn rat VSMCs with Ang II (100 nmol/L)
activated ERK/MAPK in a time-dependent manner, with a maximal
7-fold stimulation at 7 minutes (Figure 5A and 5B). Ang II also activated
JNK and p38, the other 2 members of the MAPK family, although less
strongly than ERK. Ang II–induced activation of ERK, JNK, and p38 are
all abrogated by the addition of the AT1
antagonist CV11974, but not of the
AT2 antagonist PD123319 (data not
shown). PD98059 (30 µmol/L), which is an inhibitor
of mitogen-activated protein/ERK (MEK, the upstream activating
kinase of ERK), nearly totally abolished Ang II–induced PDGF-B mRNA
expression (Figure 6A). PD98059 at the
same dose also strongly inhibited Ang II–induced ERK activation
(Figure 6B). By contrast, the p38-specific inhibitor
SB203580 (10 µmol/L) did not inhibit Ang II–induced PDGF-B gene
expression (Figure 6A). This dose of SB203580 inhibits a variety
of p38-mediated responses in many cell types.33
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We next studied whether Ang II regulates PDGF-B gene expression at the
level of transcription, and if so, whether the MAPKs mediate this, by
examining the effects of expression of DN forms of the MAPKs and the
addition of the chemical MAPK inhibitors on the promoter
activity of PDGF-B gene. We constructed a PDGF-B gene
promoter–luciferase fusion vector (Sis-Luc). We transfected newborn
rat VSMCs with Sis-Luc and either an expression vector for a DN form of
each MAPK or an empty vector, stimulated VSMCs with Ang II (100 nmol/L)
for 4 hours, and determined luciferase activity in cell lysates. Ang II
induced 2-fold stimulation of the luciferase activity (Figure 7A), indicating that Ang II
activates the transcription of PDGF-B gene. The expression of a
DN form of ERK abolishes Ang II–induced stimulation of luciferase
activity. Similarly, the expression of a DN form of JNK abolishes Ang
II–induced stimulation of luciferase activity. By contrast, the
expression of a DN form of p38 is without effect on Ang II–induced
stimulation of the luciferase activity. Consistent with the
above results, treatment of the cells with PD98059, but not with
SB203580, inhibits Ang II–induced stimulation of luciferase activity
(Figure 7B). All of these results indicate that ERK and JNK, but
not p38, are involved in Ang II–induced transcriptional activation of
PDGF-B gene.
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) with 100 nmol/L Ang II or left unstimulated
(
) for 4 hours, and the luciferase activity in cell
lysate was measured as described in Materials and Methods. Each value
represents a mean±SE of 4
determinations. * and § denote
statistical significance (P<0.05) compared with no
stimulation and Ang II stimulation of the "Vector" group or the
nonpretreated group, respectively. The experiments were repeated 3
times with similar results.
Ang II–Induced PDGF-B mRNA Expression and ERK Activation Are
Dependent on Ras
To explore the Ras dependence of Ang II–induced MAPK activation
and PDGF-B gene expression, we studied the effect of expression of
DN-Ras (Asn17-H · Ras) by using an adenoviral vector. We
infected VSMCs with an adenovirus encoding DN-Ras or a control
adenovirus encoding Escherichia coli ß-galactosidase
(LacZ). In newborn VSMCs infected with an adenovirus containing DN-Ras,
a prominent band with a molecular mass of 21 kDa corresponding to
DN-Ras protein was detected by Western analysis (Figure 8A). A faint band detected in VSMCs
infected with an adenovirus containing LacZ represents
endogenous Ras. In VSMCs infected with the control
adenovirus containing LacZ, Ang II stimulated ERK and JNK (Figure 8C) and PDGF-B mRNA expression (Figure 8B), as in
noninfected VSMCs. In VSMCs infected with an adenovirus encoding
DN-Ras, Ang II–induced PDGF-B mRNA expression (Figure 8B) and
ERK activation (Figure 8C) are strongly inhibited. By contrast,
JNK activation by Ang II is not inhibited by the expression of DN-Ras
(Figure 8C). Some studies34 previously
demonstrated that G protein–coupled receptor agonist–induced JNK
activation is dependent on the small G protein Rac. However, the
expression of DN-Rac does not inhibit Ang II–induced JNK activation
(data not shown).
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Adult VSMCs Show Expression of AT1 mRNA and Activation
of ERK and JNK in Response to Ang II
Unlike newborn VSMCs, adult VSMCs do not respond to Ang II with an
induction of PDGF-B chain mRNA (Figure 1C
). We examined the Ang
II–signaling pathway in adult VSMCs. Adult VSMCs as well as newborn
VSMCs expressed AT1 mRNA, as evaluated with
Northern analysis (Figure 9A).
Adult VSMCs responded to Ang II with the activation of ERK and JNK
(Figure 9B). Adult VSMCs also responded to Ang II with an
increase in the [Ca2+]i
(data not shown). Thus, like newborn VSMCs, adult VSMCs express
functional AT1.
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Ang II Induces a Greater Stimulation of Egr-1 Expression in Newborn
VSMCs Than in Adult VSMCs
To explore a mechanism underlying the difference in PDGF-B mRNA
response to Ang II between newborn VSMCs and adult VSMCs, we examined
possible differences in the signaling events downstream of the MAPKs.
We compared the expression of the immediate-early transcription factor
Egr-1 and the zinc finger transcription factor Sp1, which are
implicated in inducible and basal PDGF-B gene expression,
respectively.23 36 37 38 The basal expression level of Egr-1
was similar in both cell types, but Ang II–induced Egr-1 expression
was greater in newborn VSMCs (the maximal 5.8-fold stimulation over
basal) than in adult VSMCs (the maximal 2.5-fold stimulation) (Figure 10). Compared with Egr-1, Sp1 is
expressed at a slightly higher level in newborn VSMCs than in adult
VSMCs and is not so prominently induced in either cell type.
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Discussion |
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The present study demonstrates for the first time that Ang II induces PDGF-B mRNA expression in neointimal VSMCs and newborn rat medial VSMCs. Both PDGF-B chain and Ang II have been implicated in atherosclerosis and stenosis after balloon injury.4 5 6 7 11 12 39 The present results are consistent with our recent observation10 that an AT1 antagonist inhibits PDGF ß-receptor activation in vivo in balloon-injured carotid arteries. Collectively, these findings reveal a direct link between Ang II and PDGF-B and suggest that Ang II may stimulate the local production of PDGF-B chain found at sites of vascular proliferative lesions.
A number of studies suggest a role for Ang II in VSMC growth that
occurs as a consequence of balloon vascular injury,11 12
hypertension,40 and
atherosclerosis.39 Despite our recent
observation implicating PDGF-B as a mediator of the Ang II action in
vivo,10 previous in vitro studies15 16 failed
to demonstrate stimulation of PDGF-B gene expression in response to Ang
II. These previous studies used VSMCs derived from adult rats. It is
well established that the phenotype of adult rat VSMCs in
culture differs from that of neointimal
cells.20 21 22 It is also known that neointimal
VSMCs phenotypically resemble newborn rat medial VSMCs, in terms of
both cell morphology and gene expression.18 19 20 21 22 It was
demonstrated that enhanced expression of tropoelastin and
1-procollagen genes in vivo in the neointima compared
with underlying media was maintained in cultures of
neointimal cells.22 Similar phenotypes
were also observed in newborn rat aorta and cultures of newborn rat
medial VSMCs.22 These observations suggested that
neointimal cells and newborn medial VSMCs in culture
expressed phenotypes characteristic of earlier stages of artery
wall development.22 Both newborn rat VSMCs and
neointimal VSMCs express PDGF-B gene, as well as PDGF-A
gene, and secrete increased amounts of PDGF activities compared with
adult rat VSMCs.19 20 21 22 The present results reveal an
additional, important property (ie, responsiveness to Ang II with
induction of PDGF-B gene expression), which is shared by both
neointimal VSMCs and newborn rat VSMCs. We recently
observed that an AT1 antagonist
reduced the expression level of PDGF-B chain mRNA in balloon-injured
rat carotid arteries.10 In situ hybridization
analysis revealed that a portion of neointimal
cells in balloon-injured carotid arteries expressed PDGF-B
chain.9 The present results together with these
previous observations suggest that Ang II is involved in PDGF-B gene
expression in the neointima in vivo of injured arteries. A
number of recent studies provide evidence for the activation of the
local renin-angiotensin system in the rat carotid
balloon-injury model. It was demonstrated previously that mRNA
expression of renin, angiotensinogen,
angiotensin-converting enzyme, and
AT1 mRNA were upregulated in rat balloon-injured
arteries.41 42 43 Thus, these observations suggest that
enhancement of the local renin-angiotensin system augments
the AT1-mediated signaling in injured arteries,
leading to upregulation of PDGF-B gene expression in the
neointima.
In addition to the suggested pathological role for the Ang II–PDGF-B chain axis in the formation of vascular proliferative lesions, high constitutive23 and induced expression of PDGF-B chain (the present study) in newborn VSMCs may also suggest that PDGF-B chain plays a developmental role in blood vessels, because phenotypes of newborn VSMCs and neointimal cells in culture are shown to be similar to those of arterial VSMCs in vivo at earlier stages of blood vessel development.22 44 The observation of a thinner wall of aorta in PDGF-B chain–null mice lends support to a role for PDGF-B chain in blood vessel development.45 It is an interesting possibility that Ang II might be involved in PDGF-B expression in the vessel wall during prenatal and postnatal early developmental stages.
The present study also defined the signaling mechanisms underlying
Ang II–induced PDGF-B mRNA expression. Ang II activates all 3
members of the MAPKs (ie, ERK, JNK, and p38) in newborn rat VSMCs
(Figure 6), as in adult VSMCs. The MAPK cascades are important
signaling systems that convey signals into the nucleus to initiate
cellular responses, including gene expression.46
Therefore, we examined whether the MAPK cascades are involved in Ang
II–induced PDGF-B gene expression, by using expression vectors for a
DN form of each MAPK, as well as known specific inhibitors
for the MAPK cascades. The present results demonstrate that both
ERK and JNK, but not p38, are involved in Ang II–induced PDGF-B mRNA
expression by positively regulating transcription of PDGF-B gene
(Figures 7 and 8). We do not know at present whether
or not the activity of the MAPK cascades influences the stability of
PDGF-B mRNA. Recent studies22 35 36 37 reveal that the
interaction of the immediate-early gene product Egr-1 with a
cis-acting element in the proximal PDGF-B promoter is
essential for phobol ester- or injury-induced transcriptional
activation of PDGF-B gene in vascular endothelial
cells. We demonstrate in the present study that Ang II induces
Egr-1 mRNA expression in VSMCs (Figure 10). Because Ang II and
a phorbol ester induce activation of ERK, it is likely that ERK
mediates Egr-1 expression, which contributes to the subsequent
activation of the secondary responsive PDGF-B gene. We, in fact,
observed that Ang II induces Egr-1 mRNA expression in VSMCs in a
PD98059-sensitive manner (J.D., Y.T., unpublished data, 1998).
It is presently unknown whether the JNK cascade converges onto
Egr-1 gene expression or exists as a parallel, independent pathway
necessary for activating PDGF-B gene transcription.
Several recent studies47 48 49 demonstrate that Ang II
activates Ras, which is a known activator of the
Raf-ERK cascade. In the present study, we tested a role for Ras in
Ang II–induced MAPK activation and PDGF-B gene expression. The
expression of a DN-Ras mutant by using an adenoviral vector efficiently
suppresses Ang II–induced ERK activation, indicating that Ras is
required for Ang II–induced ERK activation (Figure 9C). This is
the first direct demonstration of Ras dependence of Ang II–induced ERK
activation in VSMCs. Consistent with the requirement of ERK for
Ang II–induced PDGF-B gene expression, the expression of a DN-Ras
abolishes Ang II–induced PDGF-B gene expression (Figure 9B).
Thus, PDGF-B gene is the target gene that is under the control of the
Ras-MAPK signaling pathway in VSMCs. In contrast to the ERK pathway,
Ang II–induced JNK activation is not dependent on Ras (Figure 9C).
Like newborn VSMCs, adult VSMCs express AT1 and
display Ang II–induced activation of ERK and JNK (Figure 9A and 9B), which are signals necessary for Ang II–induced PDGF-B chain gene
expression (Figures 6 and 7). We found a difference in
the Ang II–induced signaling at a site downstream of ERK; Egr-1
induction is much greater in newborn cells compared with adult cells
(Figure 10). We also observed that the Egr-1 response to Ang II
was enhanced in neointimal cells (unpublished data,
1999). These observations, together with the recent knowledge
that Egr-1 plays an important stimulatory role in PDGF-B gene
transcription,23 36 37 38 implicate a role for Egr-1 in Ang
II–induced PDGF-B chain gene expression in newborn VSMCs and
neointimal cells. However, adult VSMCs, which exhibit a
less prominent but significant Egr-1 mRNA response to ANG II, do not at
all show an induction of PDGF-B chain mRNA. This suggests that some
additional signals besides the activation of ERK-Egr1 and JNK are
indispensable for Ang II–induced PDGF-B chain gene expression in
newborn VSMCs. Newborn VSMCs generally show reduced levels of
expression of various smooth muscle–specific proteins, including
calponin and smooth muscle -actin,20 compared with
adult VSMCs. On the other hand, they express higher levels of PDGF-B
chain, tropoelastin, and osteopontin than adult VSMCs. It is not yet
known whether Egr-1 contributes to upregulation of tropoelastin and
osteopontin expression and also to downregulation of smooth
muscle–specific protein expression indirectly via other
transcriptional regulators. An especially interesting avenue to be
explored is how these features of gene expression in newborn cells,
many of which are shown to be shared by neointimal
cells,22 are determined.
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Acknowledgments |
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This work was supported by grants from the Ministry of Education, Science and Culture of Japan and by the Japan Society for the Promotion of Science "Research for the Future" Program. We thank Rieko Suzuki, Masako Katoh, and Nobuko Yamaguchi for preparing the manuscript.
Received September 28, 1998; accepted July 26, 1999.
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References |
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