From the Institute of Clinical Pharmacology and Toxicology and the § Institute of Pharmacology, Benjamin Franklin Medical Center, Freie Universität Berlin, Berlin 14195, Germany
Received for publication, March 5, 2001
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ABSTRACT |
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Proliferation and subsequent
dedifferentiation of vascular smooth muscle (VSM) cells contribute to
the pathogenesis of atherosclerosis and postangioplastic restenosis.
The dedifferentiation of VSM cells in vivo or in cell
culture is characterized by a loss of contractile proteins such as
smooth muscle-specific Fully differentiated, contractile vascular smooth muscle
(VSM)1 cells are major
determinants of blood pressure and flow. In chronic vascular diseases
such as hypertension and atherosclerosis, VSM cells proliferate and
undergo a phenotypic modulation characterized by local matrix
degradation and a loss of contractile function (1). In vivo,
dedifferentiated VSM cells can gradually revert toward a more
contractile phenotype (2). Interest in the underlying mechanisms and
participating signal transduction pathways leading to altered
phenotypes of VSM cells has led to extensive study of the VSM cell
phenotype both in vivo and in vitro (for review, see Ref. 3).
Differentiated VSM cells are characterized by high expression levels of
contractile proteins such as smooth muscle It has been shown that application of mechanical forces can actively
change the VSM phenotype (10). Depending on extracellular matrix
composition, cultured VSM cells can either proliferate or differentiate
in response to mechanical strain (11). These findings were corroborated
recently by applying mechanical forces to cultured whole vessels (12).
Interestingly, phenotypic modulation of VSM cells depends on the
activation of mitogen- activated protein kinases (MAP kinases)
in both experimental settings.
In many cellular systems, the receptor-mediated proliferation and
differentiation involves the extracellular signal-regulated kinase
(ERK) subfamily of MAP kinases (13, 14). ERKs are part of a multikinase
module through which a variety of extracellular stimuli (growth
factors, differentiation signals, and cellular stress) are transmitted
into the cell (15). Receptor tyrosine kinases, upon autophosphorylation
and activation of adaptor proteins, recruit Ras and subsequently engage
the Raf/MEK/ERK cascade. Alternatively, G protein-coupled receptors
have been shown to stimulate ERKs via the Gi,
Gq, or G12/13 subfamilies of heterotrimeric G
proteins. In addition, transactivation of receptor tyrosine kinases has been demonstrated to participate in signaling from G protein-coupled receptors to ERKs (16-18).
Receptor-mediated signaling pathways that alter the phenotype of VSM
cells are poorly defined. We therefore studied receptor-mediated pathways in neonatal rat VSM cells and in particular their
participation during phenotypic modulation. Our findings clearly
indicate that G Materials--
Culture media and trypsin were purchased from
Life Technologies, Inc. Fetal calf serum and phosphate-buffered saline
were obtained from Biochrom. Radiochemicals were from PerkinElmer Life Sciences. Maxiscript and RPA II kits from Ambion were used for RNase
protection assays. The anti-SM-1/SM-2 antiserum was kindly provided by
Berlex Pharmaceuticals. Pertussis toxin (PTX), recombinant platelet
derived growth factor (PDGF-BB), bisindolylmaleimide, and
phorbol-12-myristate-13-acetate were obtained from Calbiochem. Thrombin
receptor-activating peptide (TRAP, SFLLRNPNDKYEPF) was purchased from
Tocris. All other reagents were obtained from Sigma.
SM-MHC promoter-CAT construct was a generous gift from Cort S. Madsen,
Charlottesville, VA. Dominant negative Ras/Raf constructs were kindly
provided by Alan Hall, London. Constitutively active G Cell Culture, Transient Transfections, and Reporter
Assays--
Primary cultures of VSM cells from newborn rats were
established as described previously (19). Cells were grown in minimum Eagle's medium supplemented with 10% fetal calf serum (complete medium; CM), 2% tryptose phosphate broth, 50 units/ml penicillin, and
50 units/ml streptomycin. In all experiments, cells from passages 10-15 were used. Growth arrest was induced in a serum-free quiescent medium (QM) containing 1% (w/v) bovine serum albumin and 4 mg/ml transferrin instead of serum. Prior to agonist application, cells were
maintained in QM for 48-72 h. Where indicated, cells were pretreated
with 200 ng/ml PTX for 12-18 h.
The transcriptional regulation of SM-1/SM-2 was assessed with a
chloramphenicol acetyltransferase (CAT) reporter gene expressed under
the control of the MHC promoter (nucleotides Immunostaining--
VSM cells were grown to confluence on Nunc
chamber slides (Nalge Nunc International). After fixation in 1%
formaline in phosphate-buffered saline and methanol, a monoclonal
anti-proliferating cell nuclear antigen (anti-PCNA) antibody (1:100;
DAKO) was incubated for 1 h at 20 °C in phosphate-buffered
saline supplemented with 5% fetal calf serum. A secondary,
biotinylated anti-mouse IgG (Sigma) and streptavidin-conjugated Texas
Red (Amersham Pharmacia Biotech) were applied for detection. After the
PCNA staining, SM- Immunoblotting Methods--
VSM cells were lysed directly in
1 × Laemmli buffer containing 10 mM dithiothreitol.
Proteins were separated on polyacrylamide gels and electroblotted onto
nitrocellulose membranes. SM myosin isoforms were separated on 4% gels
and detected with a polyclonal anti-SM-1/SM-2 antiserum (1:1,000). This
antiserum has been characterized previously (11). ERK1/2 were separated
on 10% gels and probed with affinity-purified polyclonal
anti-phospho-ERK1/2 or with anti-ERK1/2 antibodies (New England
Biolabs) to confirm equal loading of the gels. Primary antibodies were
detected with a horseradish peroxidase-coupled secondary antibody
(1:2,000, New England Biolabs) using a chemiluminescence substrate
(Lumiglo, New England Biolabs).
RNase Protection Assay--
RNA isolation, generation of DNA
templates, and hybridization conditions have been described previously
(11). In brief, 10 µg of total RNA was hybridized with a radiolabled
probe covering the alternatively spliced C-terminal exons of SM-1 and
SM-2 variants of rat SM-MHC. After overnight incubation at 42 °C,
nonhybridized fragments were digested with a diluted RNase A/T1
mixture. The remaining protected fragments (380 nucleotides for SM-2
and 261 nucleotides for SM-1) were separated by denaturing (8% urea)
polyacrylamide gel electrophoresis and exposed to Amersham Hyperfilm at
Single cell [Ca2+]i
Measurements--
Cells were seeded on 24-mm glass coverslips and
grown for 24 h prior to loading with 2-4 µM fura-2
in a buffer (Hepes-buffered saline) containing 135 mM NaCl,
6 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5.5 mM glucose, 10 mM Hepes pH 7.4, and 0.2% bovine serum albumin. Coverslips
were mounted in a monochromator-equipped (TILL-Photonics) inverted
microscope (Carl Zeiss). Fura-2 was excited alternately at 340 nm and
380 nm. Emitted light was filtered (505 nm long pass) and recorded with
a 12-bit CCD camera. After correction for background signals,
intracellular [Ca2+]i was
calculated as described (22). Rmax,
Rmin, and F Regulation of SM-MHC Expression by Serum Components--
The
presence of serum is essential to grow VSM cells in primary cell
culture. In addition to its mitogenic properties, we observed that
fetal calf serum enhanced the expression of contractile proteins in
neonatal rat VSM cells. Dual staining of PCNA and SM-specific
Because serum contains multiple growth factors, vasoactive peptides,
and other agonists mediating their responses through activation of
several signaling pathways, we analyzed which receptor-coupled pathways
are involved in increasing SM-MHC transcription. RNase protection
assays revealed that SM-1/SM-2 transcripts are 22 ± 1.2-fold more
abundant in serum-treated (CM) VSM cells compared with serum-starved
controls (QM). The relatively low expression levels of SM-1/SM-2 in
serum-starved VSM cells allowed us to study the effect of single
compounds on SM-1/SM-2 expression. Neither 1 µM
angiotensin II nor 10 ng/ml PDGF-BB altered the SM-1/SM-2 expression
significantly, whereas treatment with 10 ng/ml transforming growth
factor-
Because the inhomogeneous response to vasoactive peptides may rely on
the presence or absence of the corresponding receptors in cultured VSM
cell preparations, functional coupling of receptors in VSM cells was
characterized by single cell
[Ca2+]i analysis. 0.1 unit/ml
thrombin induced transient elevations of
[Ca2+]i in more than 95% of VSM
cells after a lag phase of 10-20 s (Fig.
3). To exclude unspecific,
receptor-independent effects of the serine protease thrombin, we
applied TRAP, which corresponds to the intramolecularly tethered ligand
of the PAR-1 receptor. Indeed, 80 µM TRAP induced
similar calcium transients without a typical protease lag phase (Fig.
3). Addition of lysophosphatidic acid (LPA) induced calcium responses
in about 60% of the cells. Only a few cells (less than 10%) were
activated by 1 µM angiotensin II. Challenging VSM cells
with PDGF-BB (10 ng/ml) elicited a delayed and more sustained elevation
of [Ca2+]i, characteristic for
ligands binding to tyrosine kinase receptors (Fig. 3). 100 µM carbachol failed to raise
[Ca2+]i in VSM cells, indicating
the absence of endothelial cell contaminations (data not shown). Using
single cell [Ca2+]i analysis, we
demonstrated that PAR receptors, endothelial differentiation
gene receptors, and PDGF receptors are present and functional in
the majority of VSM cells.
Serum, Thrombin, and TRAP Induce Biphasic ERK
Phosphorylation--
Because both proliferative and differentiating
signals can be transmitted via ERK1/2, depending on the cellular
context and the transient or sustained character of their activation,
we studied the kinetics of ERK phosphorylation in VSM cells. A rapid
and transient phosphorylation of ERK1/2 was elicited by serum, PDGF-BB, EGF, thrombin, TRAP, LPA, and to a lower extent by angiotensin II. The
early ERK1/2 phosphorylation was maximal after 3-5 min for all
agonists. Only serum, thrombin, TRAP, and LPA elicited a delayed second
phase ERK phosphorylation (Fig.
4A). The second phase ERK
phosphorylation appeared ~45 min after agonist application and rose
continuously for another 2 h. In contrast, a delayed ERK
phosphorylation was absent in response to PDGF-BB, EGF, or angiotensin
II. The weak and monophasic ERK1/2 activation by angiotensin II may
rely on low AT1 receptor expression in our VSM cell
preparation (Fig. 3). The early ERK1/2 phosphorylation, but not the
late phase ERK activation, correlated closely with the ability of
agonists to raise [Ca2+]i.
Consistently, Ca2+ ionophores (1 µM ionomycin
or 1 µM A23187) induced early but not delayed ERK1/2
phosphorylation (Fig. 4B). Permanent activation of protein
kinases C by 100 nM phorbol 12-myristate-13-acetate resulted in a monophasic and sustained ERK1/2 activation (Fig. 4B). Thus, in VSM cells, three distinct temporal patterns of
ERK1/2 phosphorylation are elicited by different receptor ligands,
Ca2+ ionophores, or phorbol esters.
Activation of Ras/Raf/MEK/ERK Is a Prerequisite for SM-MHC Promoter
Activation by Serum and Thrombin--
Enhanced SM-1/SM-2 expression in
response to thrombin (Fig. 2) is indicative of a G protein-mediated
regulation of the SM-MHC promoter activity. To characterize signaling
pathways that control transcription of contractile proteins, we studied
the SM-MHC promoter activity by using a CAT reporter gene construct
expressed under the control of the
In serum-starved VSM cells transfected with pCAT-1346, the addition of
10 ng/ml PDGF-BB, 10 ng/ml EGF, 10 µM LPA, or 1 unit/ml thrombin resulted in a 1.1 ± 0.1-, 1.2 ± 0.1-, 2.1 ± 0.2-, and 2.0 ± 0.1-fold increase in CAT activity over control
cells incubated in serum-free QM (Fig. 5A). These data
confirm that increases in SM-1/SM-2 mRNA steady-state
concentrations (Fig. 2) indeed result from transcriptional activation.
Furthermore, the reporter gene assay allows for analysis of signaling
cascades by applying genetically encoded modulators. To define
participation of members of the Ras/Raf/MEK/ERK cascade, the reporter
gene construct pCAT-1346 was cotransfected with expression plasmids
encoding dominant negative N17-Ras or N17-Raf. In all cotransfection
experiments the total amount of transfected plasmid cDNA was kept
constant by adding cDNA encoding promoterless pCAT-basic. The
thrombin-stimulated CAT activity was abrogated by coexpression of
dominant negative N17-Ras or N17-Raf in a
concentration-dependent manner (Fig. 5B). Conversely, coexpression of the Raf C terminus increased CAT activity about 2-fold in the absence of agonists (data not shown). Additionally, the MEK inhibitor PD98059 largely reduced the thrombin-stimulated SM-MHC promoter activity (Fig. 5C). Because PD98059 was
dissolved in dimethyl sulfoxide, the effect of the solvent on the
SM-MHC promoter activity was assessed in parallel. Dimethyl sulfoxide (up to 0.5%) further increased the thrombin-stimulated CAT activity almost 1.8-fold, an effect that was also blocked by PD98059. The observed half-maximal inhibitory concentration of PD98059 (3-5 µM) is well in line with its described IC50
to inhibit ERK1/2 phosphorylation (23). Correspondingly, in VSM cells
the serum- or thrombin-mediated ERK1/2 phosphorylation was largely
reduced by 5-20 µM PD98059 and abolished by 50 µM MEK inhibitor (data not shown). These higher
concentrations, however, exhibited a toxic effect during long term
incubation of VSM, thereby precluding a subsequent determination of
SM-MHC promoter activity. Both modulations, expression of dominant
negative Ras/Raf and pretreatment of VSM cells with PD98059, also
impaired the serum-mediated up-regulation of the SM-MHC promoter (data
not shown). These data strongly suggest that the thrombin- and
serum-induced increase in SM-1/SM-2-expression depends on an intact
Ras/Raf/MEK/ERK signaling cascade. Furthermore, the ability of
different agonists to up-regulate the SM-MHC promoter activity
correlated closely with a biphasic and sustained ERK1/2 phosphorylation.
Differentiation of VSM Cells Requires Pertussis Toxin-sensitive G
Proteins--
The transient elevation of
[Ca2+]i and biphasic ERK1/2
phosphorylation induced by thrombin could be mimicked with the tethered
ligand of the PAR-1 receptor, TRAP. PAR-1 receptors couple to the
Gi, Gq, and G12/13 subfamilies of
heterotrimeric G proteins (24). The putative involvement of
G12/13 in the regulation of the SM-MHC promoter was tested
by overexpressing constitutively active (GTPase-deficient) mutants of
G
Because sustained ERK activation may be required for the regulation of
transcriptional activity, we tested whether PTX pretreatment affects
the ligand-induced SM-MHC promoter activity. In addition to the
modulation of ERK1/2 signaling, PTX treatment abolished the
thrombin-induced up-regulation of the SM-MHC promoter activity (Fig.
7A). Moreover, even the strong
induction of the SM-MHC promoter by serum was reverted completely in
PTX-pretreated VSM cells. This indicates that all serum components that
are involved in the up-regulation of the SM-MHC promoter depend on the
presence of functional Gi proteins.
Because either the
Finally, the Gi protein-dependent
redifferentiation in response to thrombin and serum was confirmed by
analyzing the expression of contractile proteins in untransfected
cells. In whole cell lysates from VSM cells stimulated with thrombin or
serum and normalized for protein content, an increased expression of
SM- In this study we describe a receptor-mediated signaling pathway
leading to differentiation of VSM cells. The thrombin-induced SM-MHC
expression is transmitted via the Ras/Raf signaling cascade and leads
to a biphasic temporal pattern of ERK1/2 phosphorylation. Pertussis
toxin abrogated both the second phase ERK1/2 phosphorylation and the
up-regulation of contractile proteins in response to serum, thrombin,
and LPA. Because coexpression of G A limited number of reports describe a phenotypic modulation of mature
VSM cells toward a more contractile phenotype. Vasoconstrictors such as
angiotensin II or vasopressin have been shown to increase levels of
steady-state mRNA and SM- Multiple upstream signaling pathways link receptor activation to
phosphorylation of ERK1/2. In VSM cells, the biphasic kinetic pattern
of ERK phosphorylation in response to serum, thrombin, or LPA suggests
that at least two independent pathways control the early and delayed
phases of ERK phosphorylation. Considering that strong Ca2+
signals result from thrombin stimulation of VSM cells, a
Ca2+- and PKC-dependent formation of Ras/Raf-1
complexes (39, 40) may engage ERKs. Alternatively,
Ca2+/calmodulin-dependent activation of Pyk2
(41) and subsequent Src activation may target Ras either including
(42-44) or bypassing transactivated receptor tyrosine kinases (45,
46). Because Ca2+ ionophores evoked large
[Ca2+]i signals but failed to
induce a long lived ERK phosphorylation in VSM cells, an isolate
Ca2+ elevation was not sufficient to mimic the effects of
serum components. In PTX-pretreated VSM cells stimulated with serum,
thrombin, or LPA, the remaining activation of Gq/11 and
G12/13 induced an early ERK activation but failed to
generate a sustained phospho-ERK signal. Because PTX pretreatment also
abolished the contractile protein expression in response to serum and
thrombin, we focused on signaling pathways that are initiated by either
G Receptors for endogenous vasoconstrictors such as endothelin-1,
angiotensin II, and vasopressin or serum components such as thrombin or
LPA activate the Gi, Gq, and G12/13
classes of heterotrimeric G proteins. The
G In summary, we have defined the Gi component of multiply
coupling receptors as a pivotal step in the receptor-mediated
expression of contractile proteins. Our data clearly indicate that
G-actin and myosin heavy chain (SM-MHC). Serum
increased the expression of contractile proteins in neonatal rat VSM
cells, indicating a redifferentiation process. RNase protection
assays defined thrombin as a serum component that increases the
abundance of SM-MHC transcripts. Additionally, serum and thrombin
transiently elevated cytosolic Ca2+ concentrations, led to
a biphasic extracellular signal-regulated kinase (ERK) phosphorylation,
up-regulated a transfected SM-MHC promoter construct, and induced
expression of the contractile proteins SM-MHC and
-actin. Pertussis
toxin, N17-Ras/Raf, and PD98059 prevented both the serum- and
thrombin-induced second phase ERK phosphorylation and SM-MHC promoter
activation. Constitutively active G
q, G
i,
G
12, and G
13 failed to up-regulate SM-MHC
transcription, whereas G
concentration-dependently
increased the SM-MHC promoter activity. Furthermore, the G
scavenger
-adrenergic receptor kinase 1 C-terminal peptide abolished
the serum-mediated differentiation. We conclude that receptor-mediated
differentiation of VSM cells requires G
and an intact
Ras/Raf/MEK/ERK signaling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin (SM-
-actin) and
smooth muscle myosin heavy chain (SM-MHC) (4). The expression of SM-MHC
isoforms SM-1 and SM-2 is restricted to smooth muscle cells (5, 6) and
is down-regulated in proliferating cells (7). High expression levels of
SM-1/2, therefore, are valuable markers for the differentiated
phenotype of VSM cells. Similar to pathological proliferation during
vascular disease, VSM cells down-regulate SM-1/2 expression in primary
culture. Although cultured VSM cells initially retain SM-1/2 expression when cultured on laminin or under serum-free conditions, they undergo
morphological changes toward a dedifferentiated phenotype within a few
days (8). Patterns of gene expression similar to those in cultured VSM
cells from neonatal rats have been observed in neointimal cells within
injured vessels (9). Neonatal VSM cells can, therefore, provide an
in vitro model for studying phenotypic modulation processes
in vascular disease. The mechanisms and signaling pathways that induce
a phenotypic modulation toward the contractile phenotype of VSM cells
are still largely elusive.
subunits released from the Gi
subfamily of heterotrimeric G proteins mediate enhanced expression
of contractile proteins in VSM cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
i,
G
q, and G
12/G
13 constructs
were from S. Offermanns, Heidelberg, Germany and M. I. Simon,
Pasadena, CA. G
expression plasmids were from L. Birnbaumer, Los
Angeles, CA and M. I. Simon.
ARK1ct was from R .J. Lefkowitz,
Durham, NC.
1346 to +25, pCAT-1346)
(20). For transient transfection assays, cells were seeded into
six-well plates at a density of 7.5 × 104 cells/well
(60-80% confluence) and growth arrested in QM for 48 h prior to
transfection. Transient transfections were performed in triplicate with
1 µg of plasmid DNA and 10 µl/well Superfect transfection reagent
(Qiagen) for 5 h. After 48 h, cell lysates were prepared
using the CAT enzyme assay system (Promega). CAT activities were
normalized to the protein concentration of each sample as measured by
the BCA assay. Transfection of a promoterless CAT construct served as a
base-line indicator, allowing all other promoter constructs to be
expressed relative to promoterless activity. All CAT activities
(means ± S.E.) represent at least three independent transfection
experiments with each setting tested in triplicate per experiment.
Cotransfection of a viral promoter/
-galactosidase or LacZ construct
to control for transfection efficiency was discontinued because
variations in transfection efficiency among independent experimental
samples are small (
10%). Furthermore, it has been shown that such
constructs interfere with SM-specific promoters, presumably because of
competition for common transcription factors (21).
-actin was detected by using a monoclonal antibody
(1:150; Sigma) and a fluorescein isothiocyanate-conjugated goat
anti-mouse (1:40, Dianova). Representative visual fields were exposed
sequentially by applying appropriate filter sets.
80 °C for 2-24 h. Bands were excised and counted in a liquid
scintillation counter. Equal loading was controlled by hybridization of
a second aliquot with a rat glutaraldehyde phosphate
dehydrogenase-radiolabled probe.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin
or SM-MHC revealed that after short term (24 h) exposure to serum, PCNA
expression was positive, whereas SM-
-actin was poorly detectable
(Fig. 1A). Continuous
culturing in the presence of serum for an additional 4 days markedly
enhanced the expression of SM-
-actin (Fig. 1B) and SM-MHC
(data not shown). Consistently, the expression of the SM-1 and SM-2
splice variants of SM-MHC were up-regulated by serum within 2-3 days
as detected by immunoblotting of whole cell lysates normalized for
their protein content (Fig. 1C). Serum withdrawal reduced
SM-
-actin and SM-1/SM-2 expression again within 48 h to about
20% of the expression levels in the presence of serum, demonstrating
that alterations in contractile protein expression are bidirectional
(data not shown). Thus, serum contains factors capable of inducing
in vitro redifferentiation of rat neonatal VSM cells.
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Fig. 1.
Effects of serum on the expression of
contractile proteins in vascular smooth muscle cells. Rat neonatal
VSM cells were maintained in serum-free QM for 48 h and then
reexposed to serum for 24 h (panel A) or 120 h
(panel B). Filamentous SM- -actin and proliferating cell
nuclear antigen reflecting replicating nuclei were identified with
immunofluorescence applying appropriate first and secondary antibodies
(see "Experimental Procedures"). Epifluorescence microphotographs
were taken sequentially and then superimposed. The images represent
typical viewing fields of six independent SM-
-actin staining
experiments showing similar results. White bars depict a
50-µm scale. Panel C, cell lysates were prepared from VSM
cells cultured without serum (QM) and then reexposed to serum (10%)
for the indicated number of days. Whole cell lysates (20 µg of
protein/lane) were separated by 4% SDS-polyacrylamide gel
electrophoresis, electroblotted, and probed with a polyclonal antiserum
detecting both SM-1 (204 kDa) and SM-2 (200 kDa) isoforms of
SM-MHC.
resulted in a further reduction of SM-1/SM-2 steady-state expression (Fig. 2). In contrast, 1 unit/ml thrombin increased SM-1/SM-2 expression by the 10 ± 0.9-fold. The substantial increase in SM-1/SM-2 steady-state expression
most likely represents an up-regulation of the transcriptional
activity, although changes in RNA stability and/or turnover cannot be
ruled out.
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Fig. 2.
Serum and receptor ligands up-regulate
transcription of the SM-1/SM-2 genes in VSM cells. VSM cells were
cultured either in serum-containing CM, in serum-free QM, or in QM
supplemented with 10 ng/ml PDGF-BB, 1 ng/ml transforming growth
factor- (TGF-
), 1 unit/ml thrombin, and 1 µM
angiotensin II (Ang-II) for the indicated times. RNase protection
assays were performed with 10 µg of total RNA/lane. The lengths of
protected fragments correspond to the expected sizes for the SM-1 and
SM-2 splice variants of SM-MHC (P, full-length probe). The
abundance of a control transcript was unaltered as demonstrated by
reprobing equal aliquots of total RNA with a glutaraldehyde phosphate
dehydrogenase probe (data not shown).
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Fig. 3.
Agonist-induced transient elevation of
[Ca2+]i in single VSM cells. VSM cells
were subcultured on glass coverslips, loaded with the fluorescent
Ca2+ indicator fura-2, and washed in a Hepes-buffered
medium containing 1 mM Ca2+. Coverslips were
mounted in a monochromator-equipped fluorescence imaging system built
around an inverted microscope, and fura-2 was excited alternatively at
340 and 380 nm every 0.5 s. Emitted light was recorded with a
cooled CCD camera. Cells were stimulated with 0.1 unit/ml thrombin, 80 µM TRAP, 10 µM LPA, 1 µM
angiotensin II (Ang-II), or 10 ng/ml PDGF-BB, as indicated.
[Ca2+]i was calibrated as
described (22). Time courses of
[Ca2+]i in individual single cells
(gray lines) are shown to indicate the number of cells
responding to the respective agonist. Mean
[Ca2+]i (bold black
lines) was calculated from all cells selected in the
experiment.
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Fig. 4.
Time course of receptor-mediated ERK1/2
phosphorylation. Panel A, serum-starved VSM cells were
stimulated with 10% fetal calf serum, 10 ng/ml PDGF-BB, 10 ng/ml EGF,
1 µM angiotensin II, 1 unit/ml thrombin (Thr.), 80 µg/ml TRAP, and 1 µM LPA for the indicated times. Whole
cell lysates were subjected to 10% SDS-polyacrylamide gel
electrophoresis and electroblotted. Activated ERK was detected with a
phospho-specific anti-ERK1/2 antiserum. Exposure times were optimized
to maximize differences in band intensity within one experiment. Shown
are representative experiments of three independent experiments with
similar results. Each blot was reprobed with antibodies detecting total
ERK1/2 demonstrating equal loading of the lanes (data not shown).
Panel B, the effects of calcium elevation and of protein
kinases C were assessed by 1 µM ionomycin, 1 µM calcimycin (A23187), or 100 ng/ml phorbol
12-myristate-13-acetate (PMA).
1346 nucleotide promoter region of
the SM-MHC gene (pCAT-1346). In the absence of serum, CAT activities in
VSM cells transfected with pCAT-1346 were ~4-6-fold higher compared with cells transfected with a promoterless pCAT-basic vector. Serum
treatment further increased the CAT activity by 5.1 ± 0.5-fold (Fig. 5A). Expression of CAT
driven by an SV40 promoter (pCAT-control) was about 4-fold higher
compared with the SM-MHC promoter in the presence of serum (data not
shown). Transfection of pCAT-1346 in Swiss 3T3 fibroblasts did not
significantly induce CAT activity irrespective of the absence or
presence of serum (data not shown).
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Fig. 5.
Receptor-mediated SM-MHC promoter activation
requires the Ras/Raf/MEK/ERK signaling cascade. Panel
A, VSM cells, serum-starved for 48 h, were transfected with a
1346-nucleotide SM-MHC promoter-CAT fusion construct (pCAT-1346) and
then incubated in the presence of serum free medium (QM) or in QM
supplemented with 10 ng/ml PDGF, 10 ng/ml EGF, 10 µM LPA,
1 unit/ml thrombin (Thr.), or 10% serum for another 48 h. Cells
were lysed and assayed for CAT activity. Depicted CAT activities were
normalized for protein concentrations and compared with the CAT
activity of cells transfected with a reporter gene construct lacking
the SM-MHC promoter. Bars represent the means ± S.E.
of at least five independent transfection experiments. Panel
B, to test for participation of Ras/Raf in the thrombin-mediated
SM-MHC promoter induction, VSM cells were cotransfected with 0.5 µg/well pCAT-1346 and the indicated amounts (in µg) of dominant
negative N17-Ras or N17-Raf expression constructs. The total amount of
plasmid DNA was kept constant (1 µg/well) with promoterless
pCAT-basic. Panel C, VSM cells transfected with 1 µg/ml
pCAT-1346 were preincubated for 30 min with different concentrations of
the MEK inhibitor PD98059 (black bars) and then stimulated
with 1 unit/ml thrombin. Because dimethyl sulfoxide (the solvent of
PD98059) further enhanced thrombin-stimulated SM-MHC promoter
activities, controls were incubated in equivalent concentrations of the
solvent (open bars). All values were normalized to the
thrombin-mediated CAT activity without PD98059.
12 (pCIS/G
12 Q229L) and G
13 (pCIS/G
13 Q226L). Both constructs and
their combination failed to induce SM-MHC promoter activity
significantly over a wide range of transfected cDNA concentrations
(data not shown). The biological activity of these constructs has been
demonstrated previously by their ability to induce contraction of VSM
cells (25). Gq and Gi proteins couple to
phospholipases C
to release [Ca2+]i from inositol
1,4,5-trisphosphate-sensitive stores. The possible role of the
Gi class of heterotrimeric G proteins was assessed by
pretreating cells with 200 ng/ml PTX for at least 18 h.
Inactivation of Gi proteins by this protocol was
demonstrated by a more than 80% reduction of
[Ca2+]i signals in response to LPA
(Fig. 6A). PTX reduced the
peak [Ca2+]i after thrombin
stimulation by about 40% (Fig. 6A). The partial block of
thrombin-induced [Ca2+]i
transients in PTX-pretreated VSM cells reflects coupling to both
Gi and Gq/11. To test further whether the
Gi subfamily also participates in the prolonged ERK1/2
activation, serum-starved VSM cells were pretreated with PTX.
Subsequent addition of serum, thrombin, and LPA left early ERK1/2
phosphorylation almost unaltered, whereas the second phase of ERK1/2
activation was completely abrogated in PTX-pretreated cells (Fig.
6B).
View larger version (40K):
[in a new window]
Fig. 6.
PTX blocks late phase ERK
phosphorylation. Panel A, time courses of 0.1 unit/ml
thrombin- and 1 µM LPA-induced
[Ca2+]i transients in controls and
in PTX-pretreated VSM cells (+PTX, 200 ng/ml for 18 h). The
mean ± S.E. [Ca2+]i was
computed from three independent experiments, each comprising at least
100 single cells. Panel B, effects of 10% serum, 1 unit/ml
thrombin, and 1 µM LSA on ERK1/2 phosphorylation in
control and PTX-pretreated (+PTX) VSM cells. Phosphorylated ERK1/2 was
detected as described in Fig. 4. Blots were reprobed with anti-total
ERK1/2 demonstrating equal loading of the lanes (data not shown).
View larger version (18K):
[in a new window]
Fig. 7.
G
released from Gi proteins induce the SM-MHC
promoter. Panel A, transfected VSM cells (pCAT-1346)
were either left untreated or were pretreated with PTX (200 ng/ml for
18 h) and then incubated in serum-free medium (QM) or stimulated
with 1 unit/ml thrombin or 10% serum in QM for 48 h. PTX was
either present or absent throughout the whole experiment. Panel
B, CAT activities were determined in unstimulated cells
transfected with 0.5 µg/well pCAT-1346 and the indicated amounts (in
µg) of expression plasmids encoding either constitutively active
G
i (Q205L-mutant) or equal amounts of wild-type
G
1 and G
2. Total plasmid DNA
concentrations were adjusted to 1 µg/well with pCAT-basic.
Panel C, cells were transfected with 0.5 µg/well
pCAT-1346, an expression plasmid encoding the G
scavenger
ARK1ct as indicated, and adjusted to 1 µg/well with pCAT-basic.
CAT activities were assayed in either unstimulated (open
bar) or in serum-stimulated (black bars) VSM cells.
Mean CAT activities and S.E. were calculated from at least six
independent transfection experiments.
or the
subunits may transmit the signal
that results in SM-MHC expression, we coexpressed the constitutively active G
i (Q205L) together with pCAT-1346.
G
i (Q205L), however, even reduced the SM-MHC promoter
activity below base-line values (Fig. 7B). On the contrary,
coexpression of G
1 and G
2 mimicked the
receptor-mediated up-regulation of the SM-MHC promoter in a
concentration-dependent fashion. Expression of neither
G
1 nor G
2 alone was sufficient to
increase the activity of the cotransfected CAT reporter. Consistent
with an essential role of G
, coexpression of the
G
-scavenging C-terminal peptide of the
-adrenergic receptor kinase 1 (
ARK1ct) concentration-dependently reverted the
serum-induced activation of the SM-MHC promoter (Fig.
7C).
-actin and of SM-MHC was detected (Fig.
8). When incubated in the continuous presence of PTX, thrombin and serum failed to increase the expression of both contractile proteins (Fig. 8). Hence, these data demonstrate that G
released from Gi proteins link proximal
signaling to the Ras/Raf/MEK/ERK cascade to mediate the in
vitro redifferentiation of vascular smooth muscle cells shown in
Fig. 1.
View larger version (68K):
[in a new window]
Fig. 8.
PTX-sensitive induction of contractile
protein expression by thrombin and serum. VSM cells were
pretreated with (+PTX, 200 ng/ml for 18 h) or not treated and then
cultured in serum-free medium (QM). 1 unit/ml thrombin or 10% serum
was added to the medium for the indicated number of days in the
continuous presence or absence of PTX. Medium was changed every 24 h. Whole cell lysates were normalized for their protein content (10 µg/lane), subjected to SDS-polyacrylamide gel electrophoresis, and
probed with monoclonal antibodies detecting either SM- -actin or
SM-MHC.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits mimicked and
ARK1ct abrogated the activation of the SM-MHC promoter in response
to serum components, we conclude that G
mediate the agonist-induced differentiation of VSM cells.
-actin protein expression (26-29). In
both cases, CC(A/T)6GG cis-elements (CArG boxes)
within the SM-
-actin promoter are essential for the ligand-induced
promoter regulation. In addition, the same serum response
factor-binding cis-elements positively regulate the SM-MHC
promoter activity in VSM cells (20, 30). The upstream signaling
pathways that regulate the receptor-induced expression of contractile
proteins in VSM cells are poorly defined. The findings presented herein demonstrate that serum components activate the Ras/Raf/MEK/ERK cascade
in VSM cells. Because dominant negative N17-Ras, N17-Raf, or the MEK
inhibitor PD98059 prevented the SM-MHC promoter regulation by serum and
thrombin, the entire Ras/Raf/MEK/ERK cascade appears to be required.
Activated ERKs may then translocate into the nucleus to phosphorylate
Elk-1 or related transcription factors (31). This signaling cascade may
therefore provide a comprehensible model for activation of serum
response factors in response to G protein-coupled receptors in VSM
cells. Alternatively, Garat et al. (32) described that the
vasopressin-mediated transcriptional regulation of SM-
-actin is
completely blocked by pharmacological inhibition of p38 and partially
sensitive to overexpressed dominant negative JNKs. The upstream
signaling molecules that link the vasopressin-induced receptor
activation to the MAP kinase branches p38 and c-Jun N-terminal kinase,
however, remain unresolved. Moreover, RhoA-mediated cytoskeletal
rearrangements have been implicated in the regulation of the
transcriptional regulation of SM-22
and SM-
-actin (33). Because
the authors observed an activation of these promoters by either
pharmacological disruption or polymerization of actin stress fibers,
RhoA-promoted cytoskeletal assembly cannot serve as the only mechanism
for regulation of contractile protein expression in VSM cells.
Nonetheless, these findings emphasize the importance of cytoskeletal
structures for the maintenance of SM-
-actin expression in cultured
VSM cells. Whether receptor-dependent signaling utilizes
cytoskeletal dynamics to regulate the expression of contractile
proteins remains to be clarified. Our data outline that the
serum-induced expression of SM-MHC is transmitted by the
Ras/Raf/MEK/ERK cascade. ERKs therefore either exclusively or in
concert with other MAP kinases promote the expression of contractile
proteins. Because thrombin stimulation activated ERKs, but neither p38
nor c-Jun N-terminal kinases in VSM
cells,2 activation of ERKs
appears to be both sufficient and necessary for receptor-mediated
differentiation of VSM cells. The ERK pathway regulates two mutually
opposing processes, cellular proliferation and differentiation,
depending on the duration of activation and the cellular context (15,
34, 35). Our data indicate that a sustained rather than a short lived
ERK phosphorylation is a requirement for the differentiation of VSM
cells. Several other cellular models including megakaryocytes
(36), thymocytes (37), and PC-12 cells (38) support the idea
that the short versus long term ERK phosphorylation
determines the proliferative or differentiating outcome, respectively.
i or G
subunits released from activated
Gi proteins. The inhibition of adenylyl cyclases by
G
i lowers cAMP concentrations and subsequently protein
kinase A activity. Signaling via the Ras/Raf/MEK/ERK cascade is
counterregulated by protein kinase A-dependent
phosphorylation and inactivation of Raf-1 (47, 48). Indeed, forskolin
treatment further reduced the basal ERK phosphorylation in
serum-starved VSM cells.2 Thus, a disinhibition of Ras/Raf
signaling by further reducing the cAMP concentrations in quiescent
cells might result in an increased activity of the c-Raf kinase as has
been shown for another cell system (49). However, expression of
constitutively active G
i (Q205L) failed to increase the
SM-MHC promoter activity. Most strikingly, coexpression of G
subunits mimicked while the G
scavenger
ARK1ct attenuated the
effects of thrombin or LPA. Within the multiple G
effector
systems, G
-sensitive PLC isoforms, phosphoinositide 3-kinases, or
further unknown effectors bear the potential to feed into the
Ras/Raf/MEK/ERK cascade. Although the molecular mechanisms are
currently poorly defined, a growing body of evidence points to a role
of G
in initiating the assembly of a multiprotein complex
including
-arrestin and c-Src in clathrin-coated pits (50, 51).
Within these microdomains, a ligand-independent transactivation of
receptor tyrosine kinases such as the EGF receptor may link G
signaling to Ras. Other concepts favor a direct association of G
with Raf-1 (53) or the activation of a Ras-guanine nucleotide exchange
factor other than Sos1 (53). Our preliminary data
demonstrating that the tyrphostin AG1478 prevents the thrombin-induced
ERK phosphorylation in VSM cells point to a crucial role of an EGF
receptor transactivation.
q-dependent pathway, via phospholipase
C
-catalyzed formation of inositol 1,4,5-trisphosphate, increases
[Ca2+]i to activate the
calmodulin-dependent myosin light chain kinase. In parallel,
activated G12/13, via RhoA and Rho-kinase, inhibits a
myosin phosphatase (25). Both pathways synergistically control the
contraction of VSM cells by increasing the myosin light
chain20 phosphorylation. The additional coupling to
Gi proteins does not contribute to the acute regulation of
contraction (25). It is, therefore, tempting to speculate that G
released from Gi proteins function to maintain the
contractile phenotype by enhancing the expression of contractile proteins.
subunits induce a sustained ERK phosphorylation that is
critical for the differentiation of VSM cells. In the past, substantial
data have been accumulated regarding the serum
factor-dependent promoter regulation of contractile
proteins. The addition of our data, demonstrating how receptor-mediated
differentiation signals may be transmitted to the nucleus, may converge
to a more clearly defined step-by-step model describing the regulation
of contractile protein expression in VSM cells.
![]() |
FOOTNOTES |
---|
* This study was supported by the Sonderforschungsbereich 366 of the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Institut für
Klinische Pharmakologie und Toxikologie, Freie Universität
Berlin, Garystr. 5, 14195 Berlin, Germany. Fax: 49-30-8445-1761;
E-mail: reusch@medizin.fu-berlin.de.
Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.M101963200
2 H. P. Reusch, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
VSM, vascular smooth
muscle;
SM, smooth muscle;
MHC, myosin heavy chain;
MAP kinase, mitogen
activated protein kinase;
ERK, extracellular signal-regulated kinase;
MEK, MAP kinase/ERK;
PTX, pertussis toxin;
PDGF-BB, recombinant
platelet-derived growth factor;
TRAP, thrombin receptor-activating
peptide;
CM, complete medium;
QM, quiescent medium;
CAT, chloramphenicol acetyltransferase;
PCNA, proliferating cell nuclear
antigen;
[Ca2+]i, cytosolic
Ca2+ concentration;
LPA, lysophosphatidic acid;
EGF, epidermal growth factor;
ARK1ct,
-adrenergic receptor kinase 1 C-terminal peptide;
PAR, proteinase-activated receptor.
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