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INTRODUCTION |
Elevated plasma lipids are associated with a risk for
the development of atherosclerosis, a disease now believed to be an inflammatory one characterized by vascular lesions containing lipid,
macrophages, T cells, and fibrous components. Smooth muscle cell
migration from the medial layer of the vessel to the intima accompanied
by proliferation is critical to the development of the plaque (1). A
potential relationship between hyperlipidemia and disease progression
has been suggested in studies showing that atherogenic lipoproteins
such as low density lipoprotein (LDL)1 and oxidized LDL
induce smooth muscle cell proliferation (2-11). 
-migrating very low
density lipoprotein (VLDL) is a cholesteryl ester-rich atherogenic
lipoprotein that accumulates in the plasma of cholesterol-fed animals
and humans with type III hypercholesterolemia (12). Earlier we showed
that VLDL enhances smooth muscle cell growth potentiating activity
of monocytes/macrophages (13). Moreover, VLDL added directly to
cultured smooth muscle cells stimulates the rate of cell proliferation
(14, 15).
It has been shown that smooth muscle cell proliferation can be achieved
via the activation of mitogen-activated protein (MAP) kinases (16). The
importance of MAP kinase activation to smooth muscle cell proliferation
in vivo has been shown in balloon injury models (17-20).
LDL (21, 22) as well as minimally modified and oxidized LDL (11,
22-24) have been shown to stimulate MAP kinases in smooth muscle
cells, suggesting a potential link to their growth-promoting ability.
Treatment with LDL results in the activation of protein kinase C and
MAP kinase as well as the induction of the cell cycle-related genes
c-fos, c-myc (5), and early growth response
gene-1 (egr-1; Ref. 25). Interestingly, it has been shown
that the activation of MAP kinase can induce not only cell
proliferation but differentiation as well, highlighting the importance
of measuring biological responses downstream of MAP kinase activation
(26-30).
The MAP kinases ERK1/ERK2 (p44MAPK and p42MAPK,
respectively) are stimulated in pathways initiated by extracellular
stimuli that activate receptor tyrosine kinases such as the epidermal
growth factor (EGF) receptor or nontyrosine kinase receptors including
G protein-coupled receptors. Phosphorylation of the EGF receptor can
activate Ras, commencing the Raf-1
MAP kinase kinase (MEK)MAP
kinase cascade. Pertussis toxin-sensitive G protein-coupled receptors
such as those for lysophosphatidic acid (LPA), thrombin, and
2-adrenergic agonists stimulate the MAP kinase pathway by
Gi-mediated Ras activation as shown in Rat-1 cells (31). LPA-mediated
activation of MAP kinase via a pertussis toxin-sensitive
transactivation of the EGF receptor has also been described (32, 33),
and Ras-independent activation of MAP kinase through protein kinase C
has been noted (34).
Although lipoproteins have been shown to activate MAP kinases, the
molecular events upstream of MAP kinases leading to their activation
remain uncertain. Moreover, the possibility that the atherogenic
lipoprotein VLDL activates MAP kinases has not been explored. We
sought to determine whether VLDL activates MAP kinase and, if so,
what the targets upstream of MAP kinase might be. Interestingly, the
data show that VLDL activates the MAP kinases ERK1/ERK2 via a G
protein-coupled receptor that transactivates the EGF receptor, is Ras-
and Src-dependent, and involves protein kinase C. Additional experiments examining the effect of VLDL on the
proliferation of quiescent aortic smooth muscle cells showed that
despite its ability to activate MAP kinase, VLDL alone does not
stimulate quiescent cells to enter the S phase of the cell cycle. In
combination with EGF, however, serum-deprived aortic smooth muscle
cells enter S phase. Moreover, the stimulation of proliferation is
mediated through a sustained activation of MAP kinase. To our
knowledge, this is the first report demonstrating that a lipoprotein
can transactivate the EGF receptor via activation of a pertussis
toxin-sensitive G protein-coupled receptor.
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EXPERIMENTAL PROCEDURES |
Isolation of Aortic Smooth Muscle Cells--
Neonatal rabbit
aortic smooth muscle cells were isolated as described previously (14,
15). Experiments were performed by plating into first or second passage
at a density of 2 × 104/cm2 (except for
Ras expression studies in which cells were seeded at 0.52 × 104/cm2) in Dulbecco's modified Eagle's
Medium (J.R.H. Biosciences, Lenexa, KS) supplemented with 3.7 g/liter
NaHCO3, 100 units/ml penicillin, 100 µg/ml streptomycin,
0.1 mM minimum Eagle's medium nonessential amino acids
(Life Technologies, Inc.), and 1 mM minimum Eagle's medium sodium pyruvate solution (Life Technologies, Inc.) (DMEM) containing 10% fetal bovine serum (FBS; Sigma). After an overnight incubation, the cells were rendered quiescent by washing twice with
Puck's saline (140 mM NaCl, 5.4 mM KCl, 1.1 mM KH2PO4, 1.1 mM
Na2HPO4, and 6.1 mM glucose, pH
7.4) and incubating for 3 days in DMEM with 0.5% FBS.
Isolation and Characterization of VLDL--
VLDL was
isolated as described previously by density gradient
ultracentrifugation from serum of male New Zealand White rabbits fed a
diet rich in cholesterol and peanut oil (13, 14). Protein concentrations were determined as described previously (13, 14).
VLDL preparations were tested routinely for thiobarbituric acid-reacting substances as described previously (14).
Effect of VLDL on Activity of MAP Kinase--
Neonatal rabbit
aortic smooth muscle cells were seeded at a density of 2 × 104 cells/cm2 in DMEM containing 10% FBS.
After an overnight incubation, the cultures were washed twice with
Puck's saline and then incubated in DMEM containing 0.5-1% FBS for 3 days, at which time the cells were treated with VLDL, EGF (Sigma),
LPA (Sigma), basic fibroblast growth factor (bFGF; Life Technologies,
Inc.), phorbol-12-myristate-13-acetate (PMA; Sigma), or FBS as
indicated. Pretreatment with the following inhibitors was also
implemented: PD98059 (MEK inhibitor; Calbiochem), AG1478 (EGF receptor
tyrosine kinase inhibitor; Calbiochem), pertussis toxin (G
protein-coupled receptor inhibitor; List Biological Laboratories, Inc.,
Campbell, CA), bisindolylmaleimide I (protein kinase C inhibitor; Calbiochem), calphostin C (protein kinase C inhibitor; Calbiochem), or
PP2 (specific Src family tyrosine kinase inhibitor; Calbiochem). In
addition, the cells pretreated with chloroquine prior to the addition
of VLDL were studied (see below). Total cell protein was isolated
and subjected to Western blot analysis using antibodies directed
against the active phosphorylated forms of ERK1/ERK2 or total ERK1/ERK2
(see below). Immunoprecipitation was carried out with an antibody
against the EGF receptor followed by Western blot analysis using a
monoclonal antibody against phosphotyrosine.
Western Blot Analysis--
Cells were washed twice with cold
PBS and then lysed in protein extraction buffer (25 mM Tris Cl, pH 7.4, 50 mM NaCl, 0.5% sodium
deoxycholate, 2% Nonidet P-40, 0.2% SDS, 1 mM EDTA, 0.2 mM sodium vanadate, 1 mM phenylmethylsulfonyl
fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). The extracts
were incubated on ice for 15 min and centrifuged at 13,000 rpm
(Eppendorf centrifuge) at 4 °C to remove insoluble material. Protein
concentrations were determined by the BCA method (Pierce) according to
manufacturer instructions using bovine serum albumin as a standard.
Equal amounts of protein (~30 µg/lane) were subjected to
SDS-polyacrylamide gel electrophoresis according to Laemmli (35).
Stacking gels contained 4% acrylamide/bisacrylamide (29:1) and
separating gels contained either 8% (for immunoprecipitated EGF
receptor) or 12% (all other Westerns) acrylamide/bisacrylamide.
Electrophoresis was carried out at 200 V in running buffer (25 mM Tris, 250 mM glycine, and 0.1% SDS). The
samples were electrophoretically transferred to nitrocellulose
membranes (Schleicher & Schüll) using transfer buffer (48 mM Tris, pH 8.3, 39 mM glycine, and 20%
methanol) at 100 V for 1 h at 4 °C with constant stirring.
After transfer, nitrocellulose membranes were equilibrated in TBST (25 mM Tris, pH 8.0, 125 mM NaCl, and 0.1% Tween
20) for 10 min at room temperature and blocked with TBST containing 4%
nonfat dry milk (Nestle Foods, Glendale, CA) for 1 h. The
nitrocellulose membranes were incubated for 1 h at room
temperature with the primary antibodies that were diluted in TBST
containing 4% milk. After washing twice with TBST containing 4% milk
for 10 min, horseradish peroxidase anti-mouse-conjugated secondary
antibody (Sigma) diluted in TBST containing 4% milk (1:1000) was
applied for 1 h. The blots were washed once with TBST containing
4% milk for 10 min and then twice with TBST for 10 min. Bound
horseradish peroxidase was detected using ECL reagents (Amersham
Pharmacia Biotech) according to manufacturer instructions. The primary
antibodies (diluted 1:1000) used included mouse monoclonal antibodies
against the active forms of ERK1/ERK2 (New England Biolabs) as well as
phosphotyrosine, Ras, cyclin D1, and p27KIP1 antibodies
from Transduction Laboratories (Lexington, KY) and a rabbit
immunoaffinity-purified IgG that recognizes all forms (active as well
as inactive) of ERK1/ERK2 (Upstate Biotechnology, Lake Placid, NY).
Immunoprecipitation--
Cells were washed twice with cold PBS
and incubated on ice for 10 min with 1 ml of radioimmune precipitation
buffer (20 mM Tris, pH 7.5, 0.15 M NaCl, 1%
Triton X-100, 1 mM Na3VO4, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml leupeptin, and 2 µg/ml pepstatin).
Lysates were centrifuged at 13,000 rpm (Eppendorf centrifuge, 4 °C
for 10 min) to remove insoluble material. The protein concentration was
determined by the BCA method. Five hundred micrograms of protein were
incubated with 1 µg of rabbit polyclonal antibody against the C
terminus of the EGF receptor (Santa Cruz Biotechnology, Inc., Santa
Cruz, CA) for 1 h at 4 °C and then with protein A-conjugated
agarose beads overnight with constant shaking at 4 °C. The beads
were washed three times with radioimmune precipitation buffer, and half
of each sample was subjected to Western blot analysis using a
monoclonal antibody against phosphotyrosine.
[14C]Oleic Acid Incorporation into Cholesteryl
Ester--
Quiescent smooth muscle cells were incubated with VLDL
in the presence or absence of chloroquine. All cells received
[14C]oleic acid complexed to albumin (0.43 µCi/ml) and
were incubated for 24 h. Lipids were extracted and
[14C]cholesteryl oleate accumulation was analyzed by thin
layer chromatography essentially as described (14). The data are
expressed as nmol of [14C]cholesteryl oleate/mg of protein.
Construction of a Dominant Negative Ras Expression
Vector--
DNA restriction endonucleases, DNA polymerase I large
fragment (Klenow), and T4 DNA ligase were purchased from New England Biolabs. The dominant negative ras mutant
(ras-17N prepared by Feig and Cooper (36) was kindly
provided by Dr. Debabrata Mukhopadhyay, Beth Israel Hospital, Boston,
MA) was precut with EcoRI and BamHI and ligated
into pBPST-4 (see below) that was digested with EcoRI and
BamHI. lacZ was also ligated into pBPST-4. The
ras-17N- and -galactosidase-expressing vectors are
referred to as pBPST-Ras-17N and pBPST-lacZ, respectively.
The pBST-4 expression plasmid was generated originally to make stable
transfectants and incorporates a tetracycline (tet)-inducible system as
shown previously by Paulus et al. (37). Preliminary studies
showed that expression levels in transient transfections were so high
that experiments were performed on transiently transfected cultures,
and thus the inducible nature of the vector was not taken advantage of
in these studies. A tet-inducible retroviral vector was constructed
from the tet-inducible expression system containing two plasmids,
pUHD15-1 and pUHD10-3 (38), and the puromycin resistance gene
(puro), which was isolated from pBabe-puro (39) with
HindIII and ClaI, and then blunt-ended with the
Klenow enzyme. The puro fragment was inserted in a sense orientation by
blunt-end ligation into the pBabe-puro that was precut with BamHI and SalI to make plasmid pBabe-puro-puro.
The puro gene in pBabe-puro-puro was removed with
HindIII and ClaI and replaced with the following
fragment containing HindIII, SnaBI, and
ClaI restriction sites to create plasmid
pBPS-SnaBI: 5'-AGCTTTACGTAAT-3' and 3'-AATGCATTAGC-5'.
The pBPS-SnaBI was cut with SnaBI,
dephosphorylated with calf intestinal phosphatase, and then ligated
with the tet-regulated transcription activator (tTA) gene,
which was isolated from pUHD15-1 with EcoRI and
BamHI and blunt-ended with Klenow enzyme. The new plasmid
was named pBPST. To generate pBPST-4, a 0.88-kb fragment containing the
tet-inducible promoter tetO-CMV was extracted from pUHD10-3 with
HinpI I and inserted in reverse orientation to the internal
SV40 promoter into pBPST, which was digested with ClaI.
Transient Transfection--
Transfection was performed using
Effectene transfection reagent according to manufacturer instructions
(Qiagen). Briefly, smooth muscle cells were seeded at a density of
5.2 × 103 cells/well. After an overnight incubation,
the cells were washed and incubated in 0.9 ml of DMEM with 10% FBS for
2-4 h. For each transfection, 0.8 µg of DNA (pBPST-Ras-17N or
pBPST-lacZ) were added and incubated at 37 °C for 16-24 h, at which
time the medium was removed, and the cells were washed with Puck's
saline and incubated in serum-free DMEM for 48 h. The cells were
then control-treated or treated with bFGF, EGF, LPA, or VLDL for 5 min. Total cell protein was isolated and subjected to Western blot
analysis using antibodies against Ras and the active forms of
ERK1/ERK2. To examine the transfection efficiency, the cells were fixed
in 0.5% glutaraldehyde and incubated in 50 mM
ferrocyanide, 2 mM MgCl2, and 25 mM
5-bromo-4-chloro-3-indoyl--D-galactoside (Sigma) in PBS
at 37 °C overnight. The percentage of stained
(-galactosidase-positive) cells was evaluated.
Effect of VLDL and EGF on Cell Proliferation--
Quiescent
smooth muscle cells were treated with VLDL, EGF (Sigma), VLDL
plus EGF, or FBS for the indicated time. Total RNA was isolated and
subjected to Northern blot analysis to measure the steady-state levels
of expression of egr-1, c-fos, and histone H2B (see below). DNA synthesis was measured by labeling
cells with [3H]thymidine and evaluating its incorporation
into DNA and the percentage of radiolabeled nuclei (see below). In some
cases, cells were pretreated in the absence or presence of the
following inhibitors prior to the addition of reagents: PD98059,
bisindolylmaleimide I, AG1478, or pertussis toxin.
Northern Blot Analysis--
Total cellular RNA was extracted,
and Northern blot analysis was performed essentially as described
previously (15). Equal amounts of total RNA were loaded onto each lane.
Equal loading was evaluated by ethidium bromide staining (data not
shown). After transfer, the blots were hybridized with a denatured DNA
probe that was radiolabeled with [-32P]dCTP (6 × 103 Ci/mmol; DuPont) by the random-primed method using DNA
polymerase I Klenow fragment according to manufacturer instructions
(Roche Molecular Biochemicals). Blots were exposed to film with
intensifying screens at 
80 °C. Blots were probed for histone
H2B, rat egr-1, which was provided kindly by Dr.
Vikas Sukhatme of Harvard Medical School (Boston, MA) (40), and
mouse c-fos cDNA, which was provided kindly by Dr.
Michael Karin of the University of California San Diego (La Jolla, CA)
(41).
Analysis of [3H]Thymidine Incorporation into
DNA--
Cells plated into 96-well flat-bottom tissue culture plates
were treated as described above; once rendered quiescent, the cells
were incubated with reagents for 20 h, at which time the medium
was removed, and the cells were washed with serum-free DMEM. The cells
were radiolabeled for 4 h with 100 µl of
[methyl-3H]thymidine (20 µCi/ml, 5 Ci/mmol;
PerkinElmer Life Sciences) in serum-free DMEM. Cells were
harvested, and [3H]thymidine incorporation into DNA was
assessed as described previously (15). Alternatively, cells similarly
treated in 6-well trays were evaluated for the percentage of
radiolabeled nuclei as follows: the
[3H]thymidine-containing medium was removed, and the
cells were washed three times with cold PBS and then fixed for 2 min
with 100% methanol. The cells were exposed to a photographic emulsion (1:1 Kodak NBT-2/H2O; Eastman Kodak Co.) for 3 days at room
temperature in the dark, at which time the radiolabeled nuclei were
visualized after counterstaining of the cells with hematoxylin. The
percentage of radiolabeled nuclei was determined by counting both
positive and negative nuclei and expressing the number of positives as a percentage of the total.
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RESULTS |
VLDL Activates the MAP Kinase Pathway--
To determine whether
VLDL activates MAP kinase, the phosphorylation of MAP kinase
isoforms ERK1/ERK2 was monitored in neonatal rabbit aortic smooth
muscle cells. Fig. 1a shows
that VLDL activated ERK1/ERK2 in a dose-responsive fashion at
concentrations ranging from 3-100 µg/ml VLDL. Total levels of
ERK1/ERK2 were similar in control as well as VLDL-treated samples
(Fig. 1b). At a concentration of 30 µg/ml, VLDL induced
a time-dependent activation of ERK1/ERK2 with a maximal
response noted after 5 min in the presence of the lipoprotein (Fig.
1c). Phosphorylated ERK1/ERK2 began to decrease at 15 min
and returned to baseline more than 6 h later. To determine whether
VLDL-induced MAP kinase activation is mediated through the classical
MAP kinase cascade, PD98059, a MEK-specific inhibitor, was used. The
data in Fig. 1d show that pretreatment of the cells with
PD98059 (20 µM) for 30 min blocked baseline activity and completely blocked the VLDL-induced phosphorylation of
ERK1/ERK2.
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Fig. 1.
VLDL activates MAP kinase.
a, smooth muscle cells were treated with increasing
concentrations of VLDL (3-100 µg/ml) for 5 min. Total cell
protein was extracted and subjected to Western blot analysis using an
antibody against phosphorylated ERK1/ERK2. b, smooth muscle
cells were treated as described for a. The samples were
subjected to Western blot analysis using an antibody against total
ERK1/ERK2. c, smooth muscle cells were treated with VLDL
(30 µg/ml) for various times (5 min-15 h). Phosphorylated ERK1/ERK2
was measured as described for a. d, smooth muscle
cells were preincubated in the absence () or presence (+) of PD98059
(20 µM) for 30 min and then incubated in the
absence () or presence (+) of VLDL (30 µg/ml) for 5 min.
Phosphorylated ERK1/ERK2 was measured as described for
a.
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VLDL Activates a Pertussis Toxin-sensitive G Protein-coupled
Receptor That Transactivates the EGF Receptor--
To analyze the
contribution of pertussis toxin-sensitive G proteins to the
VLDL-induced MAP kinase activation, the effect of pertussis toxin on
the phosphorylation of ERK1/ERK2 by VLDL was examined. The data in
Fig. 2a show that pretreatment
with pertussis toxin (100 ng/ml) for 24 h inactivated pertussis
toxin-sensitive G protein-coupled receptors as evidenced by a loss of
LPA-induced MAP kinase activation. As expected, the activation by EGF
was unaffected by pertussis toxin treatment. Interestingly, the data clearly show that the VLDL-induced activation of MAP kinase is mediated by G protein-coupled receptors, because its effect was diminished by the addition by pertussis toxin.
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Fig. 2.
Pertussis toxin-sensitive G proteins
mediate VLDL-induced EGF receptor
transactivation. a, smooth muscle cells were
preincubated in the absence () or presence (+) of pertussis toxin
(100 ng/ml) for 24 h and then control-treated or treated with EGF
(20 ng/ml), LPA (25 µM), or VLDL (30 µg/ml) for 5 min. Phosphorylated ERK1/ERK2 was measured as described in the Fig.
1a legend. b, smooth muscle cells were
preincubated in the absence () or presence (+) of AG1478 (1 µM) for 30 min and then control-treated or treated with
bFGF (20 ng/ml), EGF (20 ng/ml), LPA (25 µM), or VLDL
(30 µg/ml) for 5 min. Phosphorylated ERK1/ERK2 was measured as
described in the Fig. 1a legend. c, smooth muscle
cells were preincubated in the absence () or presence (+) of
pertussis toxin (100 ng/ml) for 24 h and then control-treated or
treated with EGF (20 ng/ml) or VLDL (30 µg/ml) for 5 min. Cell
lysates were immunoprecipitated with an antibody against the EGF
receptor. The immunoprecipitates were subjected to Western blot
analysis using an antibody against phosphotyrosine. d,
smooth muscle cells were preincubated in the absence () or presence
(+) of pertussis toxin (100 ng/ml) for 24 h and/or AG1478 (1 µM) for 30 min and then control-treated or treated with
VLDL (30 µg/ml) for 5 min. Phosphorylated ERK1/ERK2 was measured
as described in the Fig. 1a legend.
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In a preliminary study, Western blot analysis of VLDL-treated cells
was performed using an antibody against phosphotyrosine residues. The
data (not shown) suggested that a 170-kDa protein was phosphorylated in
response to VLDL. The EGF receptor is 170 kDa and has been shown to
be transactivated by LPA (32). To determine directly whether VLDL
activated the EGF receptor, AG1478, a specific EGF receptor tyrosine
kinase inhibitor, was used. The cells were pretreated with the
inhibitor, and then bFGF, EGF, LPA or VLDL, was added for 5 min, at
which time MAP kinase activity was assessed. The data in Fig.
2b show that low levels of MAP kinase activity in control
cultures were inhibited by AG1478, suggesting that baseline activity
results from activation of the EGF receptor. As expected, bFGF-induced
activation of MAP kinase was unaffected by AG1478. The inhibitor was
shown to be effective in that MAP kinase activation by EGF was
completely abolished in its presence. LPA-induced activation of smooth
muscle cell MAP kinase was partially mediated by EGF receptor tyrosine
kinase activity, as shown previously in Rat-1 fibroblasts (31).
Finally, the data show that AG1478 partially inhibited the ability
of VLDL to activate MAP kinase, furthering the hypothesis that it
transactivates the EGF receptor.
To pursue the possibility that pertussis toxin-sensitive G proteins
mediate the VLDL-induced EGF receptor transactivation, smooth muscle
cells were treated with pertussis toxin for 24 h and then with EGF
or VLDL for 5 min. The EGF receptor was immunoprecipitated, and then
Western blot analysis was performed with an antibody against
phosphotyrosine residues to visualize activated receptors. The data in
Fig. 2c show that VLDL induced tyrosine phosphorylation of the EGF receptor. As expected, phosphorylation of the EGF receptor by EGF was unaffected by pertussis toxin. Confirmation of the activation of the EGF receptor was achieved by the demonstration that
pertussis toxin completely blocked the EGF receptor tyrosine phosphorylation that was induced by VLDL.
To determine whether the MAP kinase activation by a pertussis
toxin-sensitive G protein-coupled receptor is entirely mediated through
transactivation of the EGF receptor, the cells were pretreated either
with pertussis toxin, AG1478, or pertussis toxin plus AG1478, and then
VLDL was added and MAP kinase activation was measured. The data
(Fig. 2d) show that pertussis toxin had no additional effect
on the partial inhibition by AG1478, suggesting that the pathway to
VLDL-mediated MAP kinase activation by a G protein-coupled receptor
was entirely through transactivation of the EGF receptor.
VLDL Activates Src Kinase--
To determine whether Src is
involved in VLDL-induced MAP kinase activation, smooth muscle cells
were pretreated with the specific Src family tyrosine kinase inhibitor
PP2. The data in Fig. 3 show that PP2
inhibited MAP kinase activation by LPA but had no effect on EGF-induced
activation of MAP kinase at low doses, as expected. Furthermore, the
data show that pretreatment with the inhibitor significantly decreased
the activation of MAP kinase by VLDL.
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Fig. 3.
VLDL-induced MAP kinase
activation is Src-dependent. Smooth muscle cells were
preincubated in the absence () or presence (+) of PP2 (5 µM) for 30 min and then control-treated or treated with
LPA (25 µM), VLDL (30 µg/ml), or EGF (20 ng/ml) for
5 min. Phosphorylated ERK1/ERK2 was measured as described in the Fig.
1a legend.
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Protein Kinase C Mediates VLDL-induced MAP Kinase
Activation--
Protein kinase C was shown to be activated by LDL (5).
To determine whether protein kinase C is involved in VLDL-induced MAP kinase activation, smooth muscle cells were pretreated with a
protein kinase C inhibitor, bisindolylmaleimide I, for 30 min and then
treated with EGF, PMA, or VLDL. The data in Fig.
4a show that as expected, the
inhibitor did not affect EGF-induced MAP kinase activation but
completely blocked PMA-induced MAP kinase activation (42, 43).
Interestingly, VLDL-induced MAP kinase activation was substantially
blocked by pretreatment with the inhibitor. To further substantiate the
role of protein kinase C and to see if the activation of MAP kinase
partially or fully depended on protein kinase C, the cells were
pretreated with either increasing doses of bisindolylmaleimide I, with
the protein kinase C inhibitor calphostin C, or with PMA and then
treated with VLDL. The data in Fig. 4b show that although
bisindolylmaleimide I only partially inhibited the VLDL-induced
activation of MAP kinase (except with very high doses of the
inhibitor), pretreatment with calphostin C at doses known to
specifically inactivate protein kinase C completely blocked the
VLDL-induced activation of MAP kinase. Likewise, cells that were
depleted of protein kinase C by overnight treatment with PMA were
virtually devoid of the active forms of MAP kinase.
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Fig. 4.
Protein kinase C mediates
VLDL-induced MAP kinase activation.
a, smooth muscle cells were preincubated in the absence ()
or presence (+) of bisindolylmaleimide I (1 µM) for 30 min and then control-treated or treated with EGF (20 ng/ml), PMA (100 ng/ml), or VLDL (30 µg/ml) for 5 min. Phosphorylated ERK1/ERK2 was
measured as described in the Fig. 1a legend. b,
smooth muscle cells were preincubated with various concentrations of
bisindolylmaleimide I (5, 10, and 20 µM) for 30 min or
with calphostin C (0.5 and 2.5 µM) or PMA (100 and 500 ng/ml) overnight and then control-treated or treated with VLDL (30 µg/ml) for 5 min. Phosphorylated ERK1/ERK2 was measured as described
in the Fig. 1a legend.
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Dominant Negative Ras Mutant Blocks VLDL-induced MAP
Kinase Activation--
To determine whether Ras is required for
VLDL-induced MAP kinase activation, a dominant negative mutant form
of Ras with an asparagine substituted for serine at position 17 (36)
was expressed as described under "Experimental Procedures." Ras
expression was determined by Western blotting. Controls were provided
by cells transfected with the same plasmid expressing -galactosidase
(pBPST-lacZ) instead of ras-17N. Initial experiments were
performed with pBPST-lacZ to determine transfection efficiency;
although it did vary somewhat from experiment to experiment, it was
never lower than 20% and was sometimes as high as 60%. Most
importantly, in cultures transfected with pBPST-Ras-17N, the levels of
Ras were much higher than those of endogenous Ras (Fig.
5a), suggesting that this
approach could be used to determine whether VLDL activates Ras (the
additional lanes show that the expression of ras-17N was
equivalent in untreated and bFGF-, EGF-, LPA-, or VLDL-treated
cultures). To determine the effect of loss of Ras activity on
VLDL-induced MAP kinase activation, the levels of phosphorylated
ERK1/ERK2 were evaluated in cells transiently transfected with
pBPST-Ras-17N and pBPST-lacZ. The data in Fig. 5b show that
the dominant negative mutant inhibited baseline MAP kinase activity and
that the function of the Ras mutant was displayed by the inhibition of
MAP kinase activation by bFGF, EGF, and LPA, all previously shown to
activate MAP kinase in a Ras-dependent fashion (31, 44).
Finally, the data clearly show that VLDL-induced stimulation of MAP
kinase was inhibited by expression of the Ras mutant, demonstrating
that it is a Ras-dependent activation.
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Fig. 5.
Dominant negative mutant ras-17N
blocks VLDL-induced MAP kinase
activation. a, smooth muscle cells seeded at low
density were transfected with pBPST-Ras-17N or pBPST-lacZ and then
incubated in serum-free medium for 2 days. The cells were then
control-treated or treated with bFGF (20 ng/ml), EGF (20 ng/ml), LPA
(25 µM), or VLDL (30 µg/ml) for 5 min. Total cell
protein was extracted and subjected to Western blot analysis using an
antibody against Ras. b, smooth muscle cells were treated as
described for a. Phosphorylated ERK1/ERK2 was measured as
described in the Fig. 1a legend.
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VLDL-induced MAP Kinase Activation Is Independent of its
Degradation and Internalization--
VLDL binds to LDL receptors
and/or LDL receptor-related protein (LRP) and undergoes
receptor-mediated endocytosis, subsequently being degraded in
lysosomes. Free cholesterol released from lysosomes is esterified into
cholesteryl ester in the endoplasmic reticulum (45, 46). To determine
whether degradation of VLDL is critical to its ability to activate
MAP kinase, chloroquine was used to inhibit lysosomal function.
Chloroquine (10 µM) inhibited the VLDL-induced
esterification of cholesterol (Fig.
6a). It had no effect on
VLDL-induced MAP kinase activation, however, suggesting that
lysosomal degradation of the lipoprotein is not required (Fig.
6b).
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Fig. 6.
VLDL-induced MAP kinase
activation is independent of its lysosomal degradation.
a, smooth muscle cells were incubated for 24 h in the
absence () or presence (+) of chloroquine (10 µM) and
VLDL (0, 20, and 40 µg/ml). All cultures received
[14C]oleic acid complexed to albumin (0.43 µCi/ml).
Cultures were harvested, and [14C]cholesteryl ester
levels were determined. b, smooth muscle cells were
preincubated in the absence (0) or presence (10 and 20 µM) of chloroquine for 30 min and then incubated in the
absence () or presence (+) of VLDL (30 µg/ml) for 5 min.
Phosphorylated ERK1/ERK2 was measured as described in the Fig.
1a legend.
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Induction of egr-1 and c-fos by VLDL Is Sensitive to Inhibition
of the MAP Kinase Cascade--
Because the activation of MAP kinase
has been associated with cell proliferation and differentiation, it is
important to measure biological responses downstream of MAP kinase
activation (26-30). To determine the potential role of the signaling
cascade induced by VLDL, its contribution to smooth muscle cell
proliferation was assessed. The induction of immediate early genes such
as egr-1 and c-fos is crucial for growth
factor-stimulated cell proliferation, and we first determined whether
VLDL can induce egr-1 and c-fos expression.
Cultures were treated with 30 µg/ml VLDL, and the expression of
egr-1 and c-fos was examined by Northern blot
analysis. As shown in Fig. 7a,
VLDL induced a rapid increase in both egr-1 and
c-fos mRNA, with a maximal level achieved at 30 min.
Fig. 7b shows that the induction of egr-1 and
c-fos was dose-dependent, with maximal
expression occurring at a lipoprotein concentration of 50-100 µg/ml
VLDL. The role of the MAP kinase cascade in the induction of
egr-1 and c-fos by VLDL was examined by
determining the effect of PD98059 on expression of the genes.
Pretreatment of smooth muscle cells with PD98059 (20 µM)
for 30 min dramatically decreased the induction of both genes by
VLDL (Fig. 7c).
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Fig. 7.
VLDL induces smooth muscle cell
expression of egr-1 and c-fos
mRNA. a, quiescent smooth muscle cells were
treated with VLDL (30 µg/ml) for the time indicated. Total RNA was
harvested, and steady-state levels of egr-1 and
c-fos mRNA were assessed by Northern blot analysis.
b, quiescent smooth muscle cells were treated with the
indicated concentration of VLDL for 30 min, at which time total RNA
was harvested, and steady-state levels of egr-1 and
c-fos mRNA were assessed by Northern blot analysis.
c, quiescent smooth muscle cells were preincubated in the
absence () or presence (+) of the MEK inhibitor PD98059 (20 µM) for 30 min and then incubated in the absence () or
presence (+) of VLDL (30 µg/ml) for 30 min. Total RNA was
harvested, and steady-state levels of egr-1 and
c-fos mRNA were assessed by Northern blot analysis (10 µg/lane).
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VLDL Plus EGF Synergistically Promote Smooth Muscle Cell
Proliferation--
The effect of VLDL on the proliferation of
quiescent smooth muscle cells was examined. Quiescent cells were
treated with either VLDL or EGF (20 ng/ml), VLDL plus EGF, or
increasing concentrations of FBS (v/v). The cells were incubated for
20 h, at which time [3H]thymidine was added as
described under "Experimental Procedures." Four hours later, the
cells were harvested, and the level of [3H]thymidine
incorporation into DNA was determined. The data in Fig.
8 show that [3H]thymidine
incorporation in the control cultures was very low and that the cells
responded with a proliferative burst to the addition of FBS.
(Additional experimentation demonstrated that EGF was effective in the
5-25 ng/ml range (data not shown). Moreover, Fig. 8 shows that the
addition of VLDL or EGF alone elicited only small increases in
[3H]thymidine incorporation. The stimulation of
[3H]thymidine incorporation into DNA induced by treatment
of the cells with both 100 µg/ml VLDL and EGF was robust. To
further verify the proliferative capacity of this regimen, its effect on histone expression was determined by Northern blot analysis of total
cellular RNA. The data in Fig. 9 show
that as expected, the addition of 10% FBS resulted in release of the
cells from growth arrest as demonstrated by the increase in
steady-state levels of mRNA expression of the S phase-specific
histone H2B. Likewise, treatment with VLDL plus EGF
resulted in an increase in the expression of steady-state levels of
histone mRNA, although either agent alone was not sufficient to
alter its expression. Similar experiments in which the percentage of
[3H]thymidine-labeled nuclei was examined substantiated
these results as shown in Table I,
i.e. initially very few of the nuclei were radiolabeled
(1.3%) confirming that the cells were quiescent, and the addition of
10% FBS or the simultaneous addition of VLDL plus EGF stimulated a
substantial increase in that number (31.0 and 24.1%,
respectively).
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Fig. 8.
VLDL plus EGF stimulate DNA
synthesis. Quiescent smooth muscle cells were control-treated or
treated with VLDL (100 µg/ml), EGF (20 ng/ml), VLDL plus EGF.
Inset, quiescent smooth muscle cells were treated with
increasing concentrations of FBS. After incubation for 20 h, the
cells were radiolabeled with [3H]thymidine (20 µCi/ml)
for an additional 4 h and harvested for determination of the
incorporation of [3H]thymidine into DNA as described
under "Experimental Procedures." The data are expressed as mean
cpm/well ± S.D. (n = 5).
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Fig. 9.
VLDL plus EGF induce expression
of histone mRNA. Quiescent smooth muscle cells were
control-treated or treated with VLDL (100 µg/ml), EGF (13 ng/ml),
VLDL plus EGF, or 10% FBS. After incubation for 24 h, total
RNA was harvested, and steady-state levels of histone H2B
mRNA were assessed by Northern blot analysis (15 µg/lane).
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VLDL Plus EGF Synergistically Induce Cyclin D1 Expression and
Down-regulate p27KIP1 Expression--
To determine whether
VLDL plus EGF induce cyclin D1 expression, quiescent cells were
treated, and the expression of cyclin D1 was examined by Western blot
analysis. The data in Fig.
10a show that VLDL plus
EGF synergistically induced cyclin D1 expression; however, either agent
alone was ineffective. The effect of VLDL plus EGF on
p27KIP1 expression was examined by Western blot analysis,
which shows that similar to 10% FBS, VLDL plus EGF down-regulated
the expression of p27KIP1 protein; however, VLDL alone
or EGF alone had no effect on this parameter (Fig. 10b). The
data on [3H]thymidine incorporation, histone mRNA,
cyclin D1, and p27KIP1 expression show that neither VLDL
nor EGF alone are capable of inducing quiescent smooth muscle cells
into S phase.
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Fig. 10.
Sustained activation of ERK1/ERK2 by
VLDL plus EGF is associated with an induction of
cyclin D1 expression and a decrease of p27KIP1
expression. a, quiescent smooth muscle cells were
control-treated or treated with VLDL (100 µg/ml), EGF (20 ng/ml),
VLDL plus EGF, or 10% FBS for the indicated time. Total cell
protein was extracted and subjected to Western blot analysis using an
antibody against cyclin D1. b, smooth muscle cells were
treated as described for a. Total cell protein was extracted
and subjected to Western blot analysis using a mouse monoclonal
antibody against p27KIP1. c, quiescent smooth
muscle cells were control-treated or treated with VLDL (100 µg/ml), EGF (20 ng/ml), VLDL plus EGF, or 10% FBS for the
indicated time. Phosphorylated ERK1/ERK2 was measured as described in
the Fig. 1a legend.
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It has been suggested that cell proliferation is induced only when MAP
kinase activation is sustained. Because VLDL or EGF alone do
activate MAP kinase but do not induce the cells to enter S phase, it
was of interest to determine the levels of activation of MAP kinase by
each agent alone as well as in combination. As shown in Fig.
10c, VLDL strongly activated the MAP kinases ERK1/ERK2 after 5 min of treatment as reported above. MAP kinases were strongly activated after 5 min of treatment with EGF as well, but by 7 h
after the addition of either agent alone, the levels of phosphorylated ERK1/ERK2 were dramatically decreased, and by 17 h they were close to baseline levels. Interestingly, the response to the addition of
VLDL plus EGF was also highest at early time points; however the
activation of MAP kinase was still evident even at 17 h at levels
equivalent to those induced by 10% FBS.
Pertussis Toxin-sensitive G Proteins, the EGF Receptor, and Protein
Kinase C-mediated MAP Kinase Activation Are Responsible for the
Mitogenic Effect of VLDL Plus EGF--
To determine whether MAP
kinases and their upstream signaling molecules are critical for
VLDL-induced DNA synthesis, quiescent smooth muscle cells were
pretreated with PD98059, pertussis toxin, AG1478, or
bisindolylmaleimide I and then treated with 100 µg/ml VLDL, 20 ng/ml EGF, or VLDL plus EGF for 20 h, at which time the
incorporation of [3H]thymidine into DNA was examined. The
data in Fig. 11 show that the
inhibition of MEK, pertussis toxin-sensitive G proteins, the EGF
receptor, or protein kinase C significantly blocked VLDL plus
EGF-induced DNA synthesis, indicating a critical role of MAP kinase
activation on the mitogenic effect of VLDL.
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Fig. 11.
Inhibition of MAP kinase activation blocks
DNA synthesis induced by EGF plus VLDL.
Quiescent smooth muscle cells were preincubated in the absence
(no inhibitor) or presence of PD98059 (50 µM),
AG1478 (1 µM), or bisindolylmaleimide I (1 µM) for 30 min or pertussis toxin (100 ng/ml) for 24 h. The cells were then control-treated or treated with VLDL (100 µg/ml), EGF (20 ng/ml), or VLDL plus EGF. Inset,
quiescent smooth muscle cells were treated with increasing
concentrations of FBS. After incubation for 20 h, the cells were
radiolabeled with [3H]thymidine (20 µCi/ml) for an
additional 4 h and harvested for determination of the
incorporation of [3H]thymidine into DNA as described
under "Experimental Procedures." The data are expressed as mean
cpm/well ± S.D. (n = 5).
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DISCUSSION |
These experiments show that VLDL activates smooth muscle cell
MAP kinase, and the activation is mediated by a G protein-coupled receptor that transactivates the EGF receptor. VLDL-induced
activation of MAP kinase is mediated by protein kinase C and is
Ras-dependent. Previous workers noted that native and
oxidized LDL stimulate smooth muscle cell MAP kinase in a protein
kinase C-dependent manner (22). Interestingly, MAP kinase
was stimulated by LDL and oxidized LDL in U-937 macrophage-like cells;
however, G protein-coupled receptors or protein kinase C were not
involved (47). Others showed that oxidized LDL-induced proliferation of
macrophages, however, resulted from the activation of protein kinase C
via pertussis toxin-sensitive G proteins and the internalization of lysophosphatidylcholine (48). Sachinidis et al. (21)
demonstrated that LDL-stimulated smooth muscle cell proliferation
involves the activation of MAP kinase via a G protein-coupled receptor, which seems to be different from the classical lipoprotein receptor. Our results with chloroquine suggest that the VLDL-induced MAP kinase pathway does not involve lysosomal processing of the lipoprotein.
Cross-talk of signaling pathways was suggested by studies in which
transactivation of the EGF receptor by tumor necrosis factor- was
noted in human endothelial cells (49). EGF receptor transactivation by
thrombin, endothelin and LPA receptors (32, 33), and the m1 muscarinic
acetylcholine receptor (50) as well as the angiotensin II
type I receptor (51) has been demonstrated. A novel finding of the
present study is that the VLDL-induced activation of MAP kinase can
be significantly inhibited by AG1478, a specific EGF receptor tyrosine
kinase inhibitor. Moreover, pertussis toxin inhibited VLDL-induced
tyrosine phosphorylation of the EGF receptor, indicating that pertussis
toxin-sensitive G proteins mediate EGF receptor transactivation,
thereby leading to MAP kinase activation. To our knowledge, this is the
only report of lipoprotein-induced transactivation of the EGF receptor
via activation of a G protein-coupled receptor. Our data also show that
LPA-induced MAP kinase activation required transactivation of the EGF
receptor in smooth muscle cells as was shown in Rat-1 cells (32).
Interestingly, EGF receptor transactivation by LPA in GD25 fibroblast
cells (52) and PC12 cells (53) was not shown, suggesting that
involvement of the EGF receptor in G protein-coupled receptor-induced
MAP kinase activation is cell type-dependent.
Incomplete inhibition of VLDL-induced MAP kinase activation by
AG1478 and pertussis toxin indicates that the MAP kinase activation may
not be exclusively mediated via the G protein-coupled receptor-mediated transactivation of the EGF receptor pathway and must involve other upstream transducers. Because the activation is completely inhibited by
calphostin C, it is likely that another pathway that goes through protein kinase C is responsible.
A potential intermediate in the G protein-coupled receptor-mediated
transactivation of the EGF receptor could be the Src family kinases.
Src has been shown to be recruited to a complex consisting of G protein
subunit, -arrestin, and a G protein-coupled receptor (54)
where it is tyrosine-phosphorylated and activated after stimulation of
a G protein-coupled receptor in Cos-7 cells (53, 55). A constitutively
active Src mutant stimulated tyrosine phosphorylation of the EGF
receptor (56); however, there is conflicting evidence that Src is not
required for LPA-induced MAP kinase activation in Rat-1 or Cos-7 cells
(57). Our data using the Src kinase inhibitor PP2 demonstrates that the
activation of MAP kinase by VLDL depends on the activity of Src.
Agonist-stimulated MAP kinase can be Ras-dependent or
Ras-independent. For example, it was noted that activation of MAP
kinase by angiotensin II differed in cardiac fibroblasts and cardiac myocytes such that the activation was pertussis toxin-sensitive and
Ras-dependent in fibroblasts, whereas MAP kinase activation in myocytes was Ras-independent and insensitive to treatment with pertussis toxin but strongly repressed by inhibitors of protein kinase
C activity (58). The present study demonstrates that Ras is at least
partially responsible for VLDL-induced MAP kinase activation in
aortic smooth muscle cells, because a dominant negative Ras mutant
blocked activation. Our results strongly suggest that VLDL induces
the RasRaf-1MEKMAP kinase cascade. Oxidized LDL stimulated
smooth muscle cell Ras-GTP formation and MAP kinase (23); however, the
relationship between the two remains to be investigated. Sachinidis
et al. (21) found LDL-stimulated MAP kinase activation to be
independent of Raf-1 phosphorylation in smooth muscle cells, and our
data provide no direct evidence for Raf-1 involvement. Angiotensin II
was shown to stimulate MAP kinase by a Ras-independent pathway in rat
smooth muscle cells (59), whereas other workers showed that angiotensin
II-mediated activation of MAP kinase in rat smooth muscle cells was
pertussis toxin-insensitive, Ras-dependent, and mediated by
phospholipase C (60). Takahashi et al. (59) proffer the
explanation that phenotypic differences between smooth muscle cell
cultures may be responsible for the variations in results. This
highlights the importance of characterizing the cells under study and
the recognition that receptor expression as well as availability of
downstream targets are likely to vary not only with cell type but with
changes in phenotype. The cells examined in this report were deprived
of serum prior to the addition of VLDL, rendering the cells
quiescent. It would be of interest to determine whether changes in the
proliferative/differentiation state of the cells contribute to the
ability of VLDL to activate MAP kinase and/or the pathway mediating
such activation.
Deciphering the contribution of atherogenic lipoproteins to smooth
muscle cell proliferation is important to understand the development of
the atherosclerotic plaque as well as restenosis after balloon
angioplasty. The effect of atherogenic lipoproteins on smooth muscle
cell proliferation was the subject of a number of past investigations
(4, 5, 61). Our laboratory showed that VLDL increased the
proliferative rate of neonatal aortic smooth muscle cells that were
actively proliferating in media containing lipid-deficient serum (14,
15). Others studying high density lipoprotein (HDL)- and LDL-induced
human smooth muscle cell proliferation noted synergy with other growth
factors including platelet-derived growth factor (PDGF), EGF,
insulin-like growth factor-I (IGF), and bFGF (5, 10), although Resink
et al. (10) found that LDL and high density lipoprotein
stimulated proliferation even in the absence of additional growth
factors. Our data extend this work to show that although the
atherogenic lipoprotein VLDL does not stimulate proliferation of
quiescent smooth muscle cells alone, when added in combination with EGF this lipoprotein increased both total [3H]thymidine
incorporation into DNA as well as the percentage of [3H]thymidine-labeled nuclei. Moreover, the expression of
histone mRNA confirmed that VLDL plus EGF stimulated entry of
the cells into the S phase of the cell cycle. The dependence of the
induction of cell proliferation on the pathway activating MAP kinase
was shown by inhibiting G protein-coupled receptors, the EGF receptor, protein kinase C, or MEK, which resulted in a dramatic decrease in
VLDL plus EGF-induced cell proliferation.
[3H]thymidine incorporation into DNA was inhibited
~80% by pertussis toxin, making it likely that the synergistic
action of VLDL plus EGF on smooth muscle cell proliferation largely
depends on activation of a G protein-coupled receptor by VLDL.
Cell cycle progression results from processes leading to the binding of
AP-1 transcription factors (consisting of dimers of the Fos and Jun
families), leading to the activation of target genes such as cyclin D1,
which is the regulatory subunit of cyclin-dependent kinases
essential for G1 progression (62, 63). Chatterjee et
al. (23) found that oxidized LDL but not native LDL
stimulated Ras, MAP kinase, and c-fos expression in human
aortic smooth muscle cells. Somewhat different results were noted by
Kusuhara et al. (22), who found that native as well as
oxidized LDL preparations stimulated MAP kinase in smooth muscle cells;
however, neither LDL nor oxidized LDL induced c-fos
expression. Our results with VLDL suggest that the native
lipoprotein stimulates MAP kinase and a transient expression of
c-fos and egr-1; however, this was unable to
sustain cell cycle progression in the absence of EGF. We showed that
cyclin D1 expression and ultimately G1 progression depended
on the synergistic action of VLDL plus EGF. It has been suggested
that the activation of MAP kinases is essential for growth
factor-stimulated induction of cyclin D1 and cell cycle progression
(64). That sustained activation of MAP kinase is required for
progression of cells to S phase has been suggested in a number of
studies. Auge et al. (11) demonstrated that oxidized LDL-induced smooth muscle cell proliferation was mediated by an activation of the sphingomyelin-ceramide pathway, which induces a
sustained activation of MAP kinase. Both LDL and minimally modified LDL
induced mesangial cell phosphorylation of platelet-derived growth
factor and EGF receptors, activated Ras, and resulted in sustained (up
to 24 h) activation of MAP kinase and cell proliferation (65). A
sustained activation of MAP kinase by LPA in Rat-1 cells seems
essential for the induction of cell proliferation (66, 67). These
authors noted that nonmitogenic doses of LPA stimulated a transient
activation of MAP kinase, which induced c-fos expression; however, mitogenic doses were necessary for sustained MAP kinase activation and robust expression of Fra-1, Fra-2, c-Jun and JunB, leading to cell proliferation. Although c-fos expression was
induced in a MAP kinase-dependent manner by nonmitogenic
stimuli in CCL39 cells, only agents that were able to sustain
activation of MAP kinase induce late AP-1 genes and cyclin D1
expression, leading to cell cycle entry (64, 68). Our results support
the notion of sustained MAP kinase activation leading to cell
proliferation in our system in that the activation of MAP kinase by
VLDL plus EGF was sustained for a longer period of time than was
evident when either agent was used alone. Sustained activation of MAP kinase correlated with an increase in cyclin D1 expression and decrease
in p27KIP1, the latter previously shown to be involved in
the regulation of smooth muscle cell proliferation (69-73). One must
be cautious, however, in assigning categorical significance to the time
of activation of MAP kinase, because it represents a measure of the convergence of multiple signaling pathways and is also a point of
divergence of additional myriad of downstream signals. It is important
to measure cell functionality, e.g. cell proliferation rather than MAP kinase activation, to be certain of the role of a
particular agonist in cell/organ functionality. Bornfeldt et al. (30) found that platelet-derived growth factor-induced MAP kinase activation stimulated cell proliferation in
cyclooxygenase-2-negative human smooth muscle cells; however, the
opposite effect on proliferation was noted when cells expressing
cyclooxygenase-2 were treated with platelet-derived growth factor
despite a similar increase in MAP kinase activation. These authors
proffer the explanation that target availability at the time of
stimulation dictates among varying functional responses potentially
elicited by MAP kinase activation. In this manner, a signal pathway can
serve multiple functions, and therefore it is important to study
functional responses downstream of MAP kinase activation.
The contribution of oxidation to the proliferative capacity of LDL
remains controversial. Heery et al. (8) found that only oxidized LDL stimulated smooth muscle cell proliferation. Resink et al. (10) found that native and oxidized LDL were equally effective in stimulating [3H]thymidine incorporation into
DNA, and Kusuhara et al. (22) noted that both native as well
as oxidized LDL preparations stimulated MAP kinase in a protein kinase
C-dependent manner; however, oxidized LDL was more
effective. Although it seemed that a lipoprotein-associated lipid
moiety was responsible for the activation, interestingly, neither
thiobarbituric acid-reacting substances nor lipid peroxide content
correlated with MAP kinase activation, suggesting that neither
aldehydes nor lipid peroxides are responsible. Although the VLDL
preparations used in this study were protected from oxidation as
reported previously (14), one cannot rule out the possibility that some
oxidation has occurred.
In summary, the data show that VLDL activates the MAP kinase
ERK1/ERK2 in smooth muscle cells. The activation is mediated by a G
protein-coupled receptor that transactivates the EGF receptor and
depends on the activation of Src and Ras as well as protein kinase C. Moreover, VLDL stimulates egr-1 and c-fos
expression in quiescent smooth muscle cells, and the stimulation is
sensitive to inhibition of the MAP kinase cascade. The addition of
VLDL plus EGF activated a sustained MAP kinase response and induced cell proliferation, which depends on the activation of pertussis toxin-sensitive G protein-coupled receptors, EGF receptors, protein kinase C, and the classical MAP kinase cascade.
-Migrating Very Low Density Lipoprotein (
TOP
ABSTRACT
INTRODUCTION
