Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1862-1871
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1862-1871.)
© 1999 American Heart Association, Inc.
LDL Stimulates Mitogen-Activated Protein Kinase Phosphatase-1 Expression, Independent of LDL Receptors, in Vascular Smooth Muscle Cells
Bernhard Metzler;
Chaohong Li;
Yanhua Hu;
Gertraud Sturm;
Nassim Ghaffari-Tabrizi;
Qingbo Xu
From the Institute for Biomedical Aging Research (B.M., C.L., Y.H., G.S.,
Q.X.), Austrian Academy of Sciences, and the Division of Cardiology (B.M.),
Department of Internal Medicine, University Hospital of Innsbruck, and the
Institute for Medical Biology and Human Genetics (N.G.-T.), University of
Innsbruck Medical School, Innsbruck, Austria.
Correspondence to Dr Qingbo Xu, Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, A-6020 Innsbruck, Austria. E-mail qingbo.xu{at}oeaw.ac.at
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Abstract
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AbstractLow density lipoprotein
(LDL) is a well-established
risk factor for
atherosclerosis, stimulating vascular smooth
muscle
cell (SMC) differentiation and proliferation, but the
signal
transduction pathways between LDL stimulation and cell
proliferation
are poorly understood. Because mitogen-activated
protein
kinases (MAPKs) play a crucial role in mediating cell
growth, we
studied the effect of LDL on the induction of MAPK
phosphatase-1
(MKP-1) in human SMCs and found that LDL stimulated
induction of MKP-1
mRNA and proteins in a time- and dose-dependent
manner. Heparin,
inhibiting LDL-receptor binding, did not influence
LDL-stimulated MKP-1
mRNA expression, and human LDL also induced
MKP-1 expression in rat
SMCs and fibroblasts derived from LDL
receptordeficient mice,
indicating an LDL receptorindependent
process. Pretreatment of SMCs
with pertussis toxin markedly
inhibited LDL-induced MKP-1 expression.
Depletion of protein
kinase C (PKC) by phorbol 12-myristate 13
acetate or inhibition
of PKC by calphostin C blocked MKP-1 induction,
but the phospholipase
C inhibitor U73122 had no effect.
Pretreatment of SMCs with
genistein or herbimycin A abrogated
LDL-stimulated MKP-1 induction.
The MAPK kinase inhibitor
PD98059 abolished LDL-stimulated activation
of extracellular
signalregulated protein kinases (ERKs)
but not MKP-1 induction.
Furthermore, constitutive expression
of MKP-1 in vivo reduced
LDL-induced expression of Elk-1dependent
reporter genes, and SMC
lines overexpressing recombinant MKP-1
exhibited decreased ERK
activities and retarded proliferation
in response to LDL. Our findings
demonstrate that LDL induces
MKP-1 expression in SMCs via activation of
PKC and tyrosine
kinases, independent of LDL receptors and ERK-MAPKs,
and that
MKP-1 plays an important role in the regulation of
LDL-initiated
signal transductions leading to SMC proliferation.
Key Words: LDL mitogen-activated protein kinase phosphatase-1 signaling mitogen-activated protein kinases smooth muscle cells
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Introduction
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Ahigh concentration of circulating LDLs is believed to be
a
major risk factor for atherosclerosis. The main
physiological
function of LDL is to deliver
cholesterol to peripheral cells.
LDL
specifically binds to apoB/E receptors, resulting in endocytosis
of the
ligand-receptor complex. The protein portion of LDL is
degraded into
amino acids, and cholesterol is used or deposited
within
the cell.
1 In addition to lipid transport, LDL can
effectively
stimulate vascular smooth muscle cell (SMC) proliferation,
a
key event in the development of
atherosclerosis.
2 3 4 There
is evidence
that LDL induces gene expression of platelet-derived
growth factor
and the platelet-derived growth factor receptors,
c
-fos
and
egr-1,
4 5 which play an essential
role in SMC proliferation.
However, the LDL-initiated signal
transduction pathways leading
to such gene expression and cell
proliferation have not been
fully elucidated.
Mitogen-activated protein kinases (MAPKs), a ubiquitous group
of serine/threonine kinases, are thought to play a crucial role in
transmitting transmembrane signals required for cell growth and
differentiation.6 7 8 MAPKs include extracellular
signalregulated kinases (ERKs), c-Jun
NH2-terminal protein kinases (JNKs) or
stress-activated protein kinases (SAPKs), and p38
MAPKs.6 7 8 9 10 11 The ERKs, SAPKs, and p38 MAPKs are
activated by the reversible, dual threonine and tyrosine
phosphorylation of a conserved TEY, TPY, and TGY motif,
respectively.6 7 8 9 10 11 They are highly activated in
the cardiovascular system in response to extracellular
stimuli,12 13 14 including LDL,15 16 17 18 acute
hypertension,19 angioplasty,20 mechanical
stress,21 22 23 and
ischemia/reperfusion24 in vivo or in vitro.
Recently, distinct and selective MAPK activators have been
cloned and characterized,6 7 8 9 10 11 but less is known about
negative regulation of these kinases.
MAPK phosphatase-1 (MKP-1) is a member of the rapidly growing
dual-specificity tyrosine phosphatase family, which includes MKP-2,
MKP-3, its human homolog CL100, and the lymphocyte-specific PAC-1
protein.25 26 27 28 29 30 In the arterial wall, it has
been demonstrated that an elevation in blood pressure induces MKP-1
gene expression.31 MKP-1 has been shown to
dephosphorylate phosphothreonine and phosphotyrosine
residues of both ERKs and SAPKs, resulting in their inactivation,
although MKP-1 exhibits cell-type specificity.25 26 27 28 29 30 It is
believed that the balance between MAPK activation and MKP-1 induction
is an important issue in determining the fate of cells stimulated by
environmental insults.32
To investigate the potential effects of LDL on MKP-1 induction,
vascular SMCs cultivated from human and rat arteries were stimulated
with human LDL, and kinase assays and Western and Northern blot
analyses were performed. We demonstrate herein that LDL
stimulation results in MKP-1 mRNA expression, followed by increased
protein induction. The mechanism appears to involve LDL-stimulated
tyrosine kinase and protein kinase C (PKC) activation, which is
independent of both classic LDL receptors and MAPK kinase
(MEK1/2)-ERK1/2 signal pathways. Moreover, MKP-1 overexpression in SMCs
inhibits ERK activation and Elk-1mediated gene expression and cell
proliferation stimulated by LDL.
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Methods
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Materials
Rat MKP-1 cDNA was isolated from a rat lung cDNA library by
Liu
et al.
29 Plasmids expressing sense and antisense
recombinant
(r) MKP-1 were propagated in
Escherichia coli,
and cDNA was
obtained by cutting the plasmids with
EcoRI.
The Elk-1 PathDetect
trans-reporting systems were purchased from
Stratagene. Plasmid
pRL-TK expressing
Renilla luciferase and
the dual-luciferase
reporter assay system were purchased from Promega.
SuperFect
reagent for transfection was obtained from Qiagen. Polyclonal
antibodies
against MKP-1, mammalian ERK2, and JNK1/SAPK and mouse
monoclonal
antibodies against phosphorylated ERK1/2
were obtained from
Santa Cruz Biochemicals. Heparin, pertussis toxin,
genistein,
herbimycin A, and
N-acetylcysteine were from
Calbiochem-Novaniochem
International. Calphostin C, phorbol
12-myristate 13 acetate
(PMA), C2-ceramide, sphingomyelinase,
and U73122 were obtained
from Biomol Research Laboratories,
Inc.
Cell Culture
SMCs were isolated by enzymatic digestion according to the
procedure of Ross and Kariya,33 with a slight
modification. In brief, specimens of human carotid arteries were
obtained from the Department of Vascular Surgery, University Hospital
of Innsbruck. Use of such human material was approved by the
appropriate institutional committee. Aortic tissues were also obtained
from male 12-week-old Fischer 344 rats (Charles River, Sulzfeld,
Germany). The intima and inner layer of the media were dissected from
the arteries and cut into pieces (
1 mm3),
digested with collagenase and elastase, and cultured in
RPMI 1640 (Gibco) supplemented with 20% FCS, penicillin (100 U/mL),
and streptomycin (100 µg/mL). Cells were incubated at 37°C with 5%
CO2. The medium was changed every 3 days, and
cells were passaged by treatment with a 0.05% trypsin0.02% EDTA
solution. Experiments were conducted on SMCs that had just achieved
confluence. The purity of SMCs was routinely confirmed by
immunostaining with antibodies against
-actin.
All animal experiments were performed according to protocols approved
by the Institutional Committee for the Use and Care of Laboratory
Animals. All animals were housed in cages at 22°C with a relative
humidity of 55%, drank water ad libitum, and were fed a normal
standard chow diet. The rats were killed under anesthesia
with pentobarbital sodium (50 mg/kg body weight IP).
Fibroblasts were isolated by enzymatic digestion of lung tissues from
LDL receptordeficient mice (see Reference 34; The Jackson
Laboratory. Bar Harbor, Me) according to established
procedures35 and cultured in RPMI 1640 supplemented with
10% FCS, penicillin (100 U/mL), and streptomycin (100 µg/mL).
Experiments were conducted on fibroblasts from the second passage after
a 24-hour culture in serum-free medium. After exposure to LDL or
reagents, the cells were harvested for protein extracts.
LDL Isolation
EDTA-plasma was pooled from normolipemic, fasting (12 to 14
hours) male and female donors, aged 20 to 35 years. Lipoproteins were
prepared by differential centrifugation with the
addition of solid KBr to adjust the density as described by Havel et
al.36 LDLs were obtained in the fractions between
1.020 and 1.050 g/mL. During preparation, LDL was protected from
oxidation by EDTA (1 mmol/L). The sample was dialyzed against
150 mmol/L NaCl with 0.1 mmol/L EDTA, sterilized on a
0.2-µm Millipore membrane, and stored at 4°C for up to 3 weeks. No
oxidation of LDL was observed at least 3 weeks after LDL isolation, as
determined by measurement of malondialdehyde by the thiobarbituric acid
method.37 The endotoxin contents of freshly isolated LDL
and of LDL after 3 weeks of storage at 4°C were both below the
detection limit (<1 ng/mL; endotoxin kit, Sigma). Concentrations of
LDL were determined gravimetrically by aliquot weight after drying, and
quantities of lipoproteins were expressed as total weights.
LDL Lipid Extracts
The procedure for lipid extraction was similar to that described
elsewhere.16 In brief, LDL lipids were extracted by
chloroform and methanol. Chloroform extracts were dried under
N2 gas, dissolved in dimethyl sulfoxide, and
added to the culture.
RNA Isolation and Northern Blot Analysis
Total RNA was isolated by following a standard
protocol.38 RNA (10 µg per lane) was denatured with
formaldehyde (Merck), electrophoresed in a 1% agarose gel, transferred
onto a nylon membrane (Zeta Probe, Bio-Rad Laboratories), and
UVcross-linked in a UV Stratalinker (Stratagene Inc). Hybridizations
were performed with a fluorescein-labeled (Amersham Co)
cDNA probe for MKP-1. The membranes were then washed, detected with
anti-fluorescein alkaline phosphatase conjugate (1:5000,
Amersham), and exposed to enhanced chemiluminescence films (Amersham).
Graphs of the blots were obtained in the linear range of detection.
Accuracy of loading and transfer as well RNA integrity was confirmed by
quantitative analysis of the 28S and 18S RNAs.
Protein Extractions and Western Blot Analysis
SMCs and fibroblasts were serum-starved for 2 (human and mouse)
or 3 (rat) days and incubated with LDL with or without
inhibitors at 37°C for the times indicated in the figure
legends. After 2 washes with cold (4°C) PBS (pH 7.4), the cells were
harvested on ice in buffer A, containing 20 mmol/L HEPES (pH 7.4),
2 mmol/L EDTA, 50 mmol/L ß-glycerophosphate, 1 mmol/L
DTT, 1 mmol/L Na3VO4,
1% Triton X-100, 10% glycerol, 1 µg/mL leupeptin, 1 µg/mL
aprotinin, and 100 µmol/L PMSF. The suspension was incubated on
ice for 20 minutes with vortexing every 5 minutes. Cellular debris was
then pelleted by centrifugation for 30 minutes at
13 000 rpm (Eppendorf centrifuge) at 4°C, supernatants were
collected, and protein concentrations were measured by the Bio-Rad
assay (Bio-Rad Laboratories). The procedure used for Western blot
analysis was similar to that described
previously.39 In brief, 50 or 100 µg of total cell
proteins was separated by electrophoresis through a 10%
SDS-polyacrylamide gel and transferred onto nitrocellulose
membranes. The blots were probed with affinity-purified polyclonal
antibodies against MKP-1 or phosphorylated-ERK1/2, and
specific antibody-antigen complexes were detected with the enhanced
chemiluminescence Western blot detection kit. Graphs of the blots were
obtained in the linear range of detection and quantified for specific
induction or inhibition by scanning laser densitometry (Power-Look II,
UMAX Data System Inc) of the graphs.
Kinase Assays
For kinase assays, 0.5 mL of the supernatant containing 0.5 mg
protein was incubated with 10 µL of antibodies against mammalian ERK2
or JNK1/SAPK for 2 hours at 4°C with rotation. Subsequently, 40 µL
of protein Gagarose suspension (Santa Cruz Biochemicals) was added,
and rotation was continued for 1 hour at 4°C. Immunocomplexes were
precipitated by centrifugation and washed 2 times with
buffers A, B (500 mmol/L LiCl, 100 mmol/L Tris, 1 mmol/L
DTT, and 0.1% Triton X-100; pH 7.6), and C (20 mmol/L MOPS,
2 mmol/L EGTA, 10 mmol/L MgCl2, 1
mmol/L DTT, and 0.1% Triton X-100; pH 7.2), respectively.
ERK2 activities in the immunocomplexes were measured as described
previously.40 41 In brief, immunocomplexes were incubated
with 35 µL of buffer C supplemented with 6 µg of myelin basic
protein (Upstate Biotechnology Inc),
[
-32P]ATP (5 µCi),
MgCl2 (50 mmol/L), and ATP (30
µmol/L) for 20 minutes at 37°C with vortexing every 3 minutes. To
stop the reaction, 15 µL of 4x Laemmli buffer was added and the
mixture was boiled for 5 minutes. Proteins in the kinase reaction were
resolved by SDSpolyacrylamide gel electrophoresis (PAGE, 15%
gel) and subjected to autoradiography.
The JNK/SAPK assay was performed as described above by using
glutathione S-transferasec-Jun as the substrate (the
plasmid was provided by Dr J. Woodgett, Ontario Cancer Center,
Toronto, Canada) produced in E coli and isolated
with glutathioneSepharose 4B RediPack Columns (Pharmacia Biotech Inc)
as per the manufacturer's protocol. Proteins in the kinase reaction
were resolved by SDS-PAGE (12% gel) and subjected to
autoradiography.40 41
Cotransfection and Luciferase Activity Assays
Human SMCs were seeded at a concentration of
8x105 per 60-mm dish 1 day before transfection.
Plasmids pRL-TKRenilla luciferase (1 µg) and
Elk-1luciferase (1 µg), along with 5 µg of either pSG5 (vector)
or constructs expressing sense (pSG5rMKP-1) or antisense
(pSG5rMKP-1 as) were mixed with SuperFect reagent in a 1:2 ratio
(wt/wt). The mixture was added to SMCs in the medium (3 mL per dish)
containing 10% FCS and incubated at 37°C for 20 hours. The cells
were serum-starved for 12 hours and then treated with LDL for 16 hours.
Protein extracts were prepared from treated and untreated SMCs and
assayed for luciferase activities by using a dual-luciferase reporter
assay kit according to the manufacture's instructions. Luciferase
activities were measured with the Beta-Jet-Luminometer (Wallac). Elk-1
luciferase activity was normalized with respect to Renilla
luciferase activity.
SMC Lines Stably Transfected With MKP-1
Rat vascular SMCs were transfected stably with MKP-1 plasmid by
using the SuperFect reagent as described above. Transfected cells were
selected in the presence of G418 (500 µg/mL, Sigma) for 5 weeks, and
individual cell colonies were expanded and maintained in culture medium
supplemented with 200 µg/mL G418. MKP-1transfected SMC lines were
identified by Western blotting analysis.
SMC Proliferation Assays
Transfected SMCs (103), cultured in
96-well plates in medium containing 20% FCS at 37°C for 24 hours,
were serum-starved for 4 days. LDL in 2% serum was added, and
incubation was continued at 37°C for 24 hours.
[3H]Thymidine was added 6 hours before cell
harvest. Radiation activities were measured with a radiation counter
and recorded as counts per minute.
Statistical Analysis
ANOVA was performed when >2 groups were compared. An unpaired
Student's t test was used to assess differences between 2
groups. A value of P<0.05 was considered significant.
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Results
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LDL-Stimulated MKP-1 Induction
MKP-1 mRNA levels in LDL-treated SMCs were
analyzed by Northern
blots. As shown in Figure 1A

, LDL (100 µg/mL) treatment
resulted
in significantly increased
MKP-1 mRNA in SMCs; kinetic
analysis
indicated that this response occurred as early as 10
minutes
(Figure 1A

), with maximum levels (10-fold greater than
untreated
controls) being achieved 30 minutes after treatment and
returning
to basal levels by 3 hours. The lower panel of Figure 1A

shows
the amounts of 28S and 18S RNAs from the corresponding
blot.
Likewise, growth-arrested SMCs were exposed to 100 µg/mL
LDL
for various times, and equal amounts of proteins from control
and
experimental samples were used in Western blots to test
MKP-1
induction. As shown in Figure 1B

, exposure of cells to
LDL
resulted in a time-dependent induction of MKP-1 proteins,
being evident
at 15 minutes, peaking at 30 minutes, and declining
thereafter. Figure 1C

summarizes MKP-1 protein induction as
determined by
quantification of optical densities from autoradiograms
of
3 experiments. Exposure of cells to LDL produced 5- to 8-fold
changes
in MKP-1 proteins in protein extracts of SMCs. MKP-1 proteins
did
not return to baseline by 6 hours after treatment.

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Figure 1. Time course of MKP-1 induction in LDL-treated
SMCs. Vascular SMCs were dissociated from human carotid arteries by
collagenase and cultivated in RPMI 1640 medium. LDLs were
isolated from human EDTA-plasma by density
ultracentrifugation. Subconfluent cells were
serum-starved for 2 days and incubated with LDL (100 µg/mL) at 37°C
for the indicated times. A, Northern blot analysis. Total RNA
was extracted from treated SMCs, and 10 µg of total RNA per lane was
electrophoresed on 1% agarose and transferred to the membrane.
Integrity and quantity of RNA were verified by analysis of 18S
and 28S RNAs (lower panel). Northern blots were hybridized with
MKP-1 cDNA probes. Data are examples of 2 independent
experiments. B, Western blot analysis. After incubation, cells
were lysed in the buffer, and protein extracts were separated on 10%
SDS-polyacrylamide gel, transferred to membranes, and probed
using an antibody to protein MKP-1. C, Statistical data for MKP-1
protein induction from SMCs treated with LDL. *Significant difference
from control at P<0.05.
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To further establish the relationship between LDL treatment and
MKP-1 expression, we performed a dose-response
analysis of LDL-induced MKP-1 mRNA accumulation. As
shown in Figure 2A
, MKP-1 mRNA
levels increased in a dose-dependent manner between 50 and 400 µg/mL
and decreased at the concentration of 500 µg/mL. Figure 2B
summarizes MKP-1 mRNA induction as determined by
quantification of optical densities from autoradiograms
of 2 experiments. Exposure of cells to LDL produced 5- to 7-fold
changes in MKP-1 mRNA levels of human SMCs.

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Figure 2. Concentration-dependent MKP-1 stimulation.
LDL-induced MKP-1 mRNA expression. A is an example of a
Northern blot, and B summarizes results of 2 independent experiments.
The procedures were similar to those described in the legend to Figure 1 and in Methods.
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LDL ReceptorIndependent MKP-1 Induction
LDLs can specifically bind to their receptors to deliver
cholesterol to the cell, but whether the receptor binding
initiates mitogenic signaling pathways stimulated by LDL
remains to be clarified. We performed several experiments to verify
receptor involvement in LDL-induced MKP-1 expression. Although heparin
has been shown to block LDL-receptor binding, pretreatment of LDL with
heparin did not inhibit LDL-stimulated MKP-1 mRNA expression
(Figure 3A
). When rat SMCs were
stimulated with human LDL, increased MKP-1 induction was observed
(Figure 3B
), and LDL stimulated MKP-1 expression in primary
cultured fibroblasts from LDL receptorknockout mice (Figure 3C
), indicating LDL receptorindependent induction.
Furthermore, pretreatment of LDL with sphingomyelinase markedly
enhanced MKP-1 induction (Figure 3D
), and exogenous ceramide
stimulated MKP-1 production (Figure 3E
). To further
exclude LDL receptor involvement, lipid extracts from LDL were used to
stimulate SMCs, and MKP-1 was also induced by such treatment (Figure 3F
).

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Figure 3. LDL receptorindependent MKP-1 expression. A,
Effect of heparin on MKP-1 mRNA expression. LDLs were
preincubated with heparin (2 mg/mL) for 40 minutes before addition to
SMCs. Total RNA was isolated and Northern blots were performed using
MKP-1 cDNA as the probe. B, Human LDLstimulated MKP-1 protein
induction in rat SMCs. Rat SMCs were isolated from their aortas by
enzymatic digestion, as described previously.20 C, Human
LDLstimulated MKP-1 protein induction in LDL receptordeficient
fibroblasts of mice. Lung tissues from LDL receptorknockout mice were
dissociated with collagenase and cultivated in RPMI 1640
supplemented with 10% FCS. Cells of the second passage were used for
experiments. D, Sphingomyelinase-enhanced MKP-1 expression. Human LDL
(1 mg/mL) was incubated with sphingomyelinase (SMase, 1 U/mL) at 37°C
for 30 minutes and then added to rat SMCs. E, Ceramide-stimulated MKP-1
induction. C2-ceramide (50 µmol/L) was added to serum-starved
rat SMCs. After a 1-hour incubation (B through E), cells were lysed in
the buffer, and protein extracts were separated on 10%
SDS-polyacrylamide gel, transferred to membranes, and probed
using an antibody to protein MKP-1. F, Lipid extracts (60 µg/mL) from
LDL were added to stimulate SMCs for 30 minutes. Dimethyl sulfoxide
(0.4%) was used in negative controls (Ctl), and a Northern blot
analysis was performed. Data are examples of 2 experiments.
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Inhibition of LDL-Induced MKP-1 by Pertussis Toxin
It has been demonstrated that SMCs pretreated with pertussis toxin
exhibit decreased ERK activation in response to LDL.17 We
were interested in whether such a pathway is also involved in
LDL-induced MKP-1 expression. Figure 4
data indicate that pertussis toxin treatment significantly inhibited
MKP-1 induction by LDL. These results suggest a similar signal pathway
as described by Sachinidis et al,17 mediated by pertussis
toxinsensitive G proteins that may be involved during MKP-1 gene
expression.

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Figure 4. Effects of pertussis toxin on LDL-induced MKP-1
expression. Quiescent SMCs were pretreated for 18 hours with pertussis
toxin (PTX, 100 ng/mL). Cells were exposed to LDL for 30 minutes and
harvested for RNA isolation as described in Methods. A is an example of
results, and B summarizes data from 2 independent experiments.
*Significant difference from LDL-treated group at
P<0.05. Ctl indicates control.
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LDL-Induced MKP-1 via Tyrosine Kinase Activation
There is evidence that LDL stimulation results in activation of
inositol phospholipid catabolism and calcium
mobilization,17 42 in which tyrosine kinases have been
shown to be involved. Therefore, we pretreated SMCs with genistein or
herbimycin A to inhibit different types of tyrosine kinases. As shown
in Figure 5
, genistein treatment blocked
LDL-stimulated MKP-1 mRNA induction in human SMCs, and
herbimycin A significantly inhibited this induction. Our data support
the involvement of genistein- and herbimycin-sensitive tyrosine kinases
in LDL-induced MKP-1 expression. Because it has been demonstrated that
LDL stimulates superoxide generation in a concentration-dependent
manner in neutrophils,43 we assessed whether
LDL-induced MKP-1 expression was mediated by released free radicals via
activation of NADPH oxidase. The antioxidant
N-acetylcysteine, shown to be a superoxide anion scavenger,
blocked H2O2-induced ERK
activation in HeLa cells.44 Addition of
N-acetylcysteine to the culture with the pH adjusted did not
influence LDL-stimulated MKP-1 production (Figure 6
).

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Figure 5. Genistein and herbimycin A prevent LDLstimulated
MKP-1 induction. Quiescent SMCs were pretreated for 1 hour with
genistein (100 µmol/L) or for 16 hours with herbimycin A (1
µmol/L). Cells were exposed to LDL for 30 minutes and harvested for
RNA isolation as described in Methods. A and B, Examples of results; C
summarizes data from 3 independent experiments. S indicates FCS
treatment without LDL. *Significant difference from the other 3 groups
at P<0.001.
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Figure 6. Oxidative stress is not involved in LDL-stimulated
MKP-1 expression. Quiescent SMCs were pretreated for 45 minutes with
N-acetylcysteine (NAC, 20 mmol/L). Cells were
exposed to LDL for 30 minutes and harvested as described in Methods. A
presents the results of Northern blot analysis, and B
summarizes data from 2 experiments. Note no significant difference
between NAC-pretreated and untreated SMCs stimulated by LDL.
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MKP-1 Induction Is Dependent on PKC
LDL and oxidized-LDL have been shown to activate
PKC,42 45 46 and the PKC agonist PMA induces MKP-1 gene
expression in several cell types. We confirmed MKP-1 expression induced
by low concentrations of PMA in human vascular SMCs (Figure 7A
). To determine whether PKC mediates
LDL-stimulated MKP-1 induction in SMCs, PKC was either depleted by
exposing SMCs to 1 µmol/L PMA for 24 hours or inhibited by
incubating the cells with calphostin C before LDL stimulation. Whereas
PMA pretreatment for 24 hours significantly reduced MKP-1
mRNA levels, PMA alone slightly stimulated MKP-1 expression
(Figure 7B
). Similar findings were obtained with the
PKC-specific inhibitor calphostin C, ie, significant (80%)
inhibition of LDL-induced expression (Figure 7C
). These results
indicate that LDL-stimulated MKP-1 expression is dependent on PKC. We
also determined the effects of the phospholipase C
inhibitor U73122 on MKP-1 expression, because phospholipase
C has been shown to be involved in LDL-stimulated signaling. The data
shown in Figure 7D
indicate no marked influence on MKP-1
induction by this inhibitor, and Figure 7E
summarizes data from 2 or 3 independent experiments, demonstrating a
PKC-dependent and phospholipase Cindependent MKP-1 induction.

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Figure 7. PKC-dependent MKP-1 expression. Quiescent SMCs
were incubated with PMA for 1 hour (A) or pretreated for 24 hours with
PMA at a concentration of 1 µmol/L (B), with calphostin C (500
nmol/L) for 3 hours (C), or with U73122 (10 µmol/L) for 3 hours
(D). Cells were harvested (A) for Western blot analysis or
exposed to LDL for 30 minutes and then harvested (B through D) for RNA
isolation as described in Methods. B, C, and D present the results
of MKP-1 mRNA expression, and E shows statistical data
for 2 or 3 independent experiments. S indicates serum treatment without
LDL. *Significant difference from inhibitor-treated
groups.
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ERK-Independent MKP-1 Induction
It has been shown that ERK-MAPKs can be activated by LDL
and oxidized-LDL.15 16 17 18 To investigate whether ERK kinases
are involved in LDL-induced MKP-1 expression, activities of both ERK
and JNK/SAPK kinases and MKP-1 induction were
simultaneously determined in SMCs pretreated with PD98059,
a specific MEK1/2 inhibitor. We confirmed LDL-stimulated
ERK activation and demonstrated that LDL-activated ERK1/2 was
inhibited by PD98059 in a concentration-dependent manner (Figure 8A
). Kinase assays indicated that 50
µmol/L PD98059 completely blocked ERK2 activation induced by LDL
(Figure 8A
and 8B
) but not JNK/SAPK activation (Figure 8C
). MKP-1 induction was not influenced by this
inhibitor (Figure 8D
). These results support the
concept that LDL-induced MKP-1 production is independent of ERK
activation.

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Figure 8. PD98059 inhibited ERK activation but not MKP-1
induction. Quiescent SMCs were pretreated with PD98059 for 1 hour.
Cells were exposed to LDL for 8 minutes (A and B), 20 minutes (C), or 1
hour (D) and harvested for protein extracts. A shows the results of
Western blot analysis for phosphorylated-ERK1/2
inhibited by PD98059 in a concentration-dependent manner, and B and C,
the results of ERK and JNK/SAPK kinase assays. D shows the results of
Western blot analysis for MKP-1. For the kinase assay, ERK2 and
JNK/SAPK proteins were immunoprecipitated from the protein extracts and
their kinase activities measured on the basis of
phosphorylation of myelin basic protein (MBP) or
glutathione S-transferasec-Jun (GSTC-Jun) fusion
protein substrates, respectively. Data are an example of similar
results of 2 independent experiments. S indicates FCS treatment without
LDL.
|
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MKP-1Inhibited Elk-Mediated Gene Expression During LDL
Stimulation
Elk-1, an essential transcription factor for cell growth, has been
shown to be activated by both MAPKs, ERK and
JNK/SAPK.47 48 It has been demonstrated that MKP-1
inactivates ERK in several cell types,25 26 27 28 29 30
including SMCs,49 but whether MKP-1 inhibits
Elk-1mediated gene expression in SMCs induced by LDL stimulation
remains unclear. To address this question, human SMCs were
cotransfected with Elk-1 luciferase plasmids and constructs expressing
rMKP-1 in the sense or antisense orientation. Transfected SMCs were
subsequently treated with LDL, and Elk-1mediated gene expression was
determined on the basis of luciferase activity. Figure 9
indicates that MKP-1 expression
inhibited Elk-1 luciferase activity, LDL stimulation increased Elk-1
luciferase activity 5-fold in the absence of rMKP-1, and rMKP-1 had
little effect on the basal levels of Elk-1 luciferase but significantly
(>60%) inhibited induction in response to LDL stimulation. In
contrast, the construct expressing antisense rMKP-1 enhanced Elk-1
luciferase expression (Figure 9
). These results suggest that
MKP-1 expression blocks Elk-1mediated gene transcription, possibly
via inactivation of both ERK and JNK/SAPK in SMCs stimulated by
LDL.

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Figure 9. RMKP-1 expression inhibits Elk-1mediated gene
induction. Human SMCs were cotransfected with plasmids Elk-1 luciferase
(1 µg), pRL-TKRenilla luciferase (1 µg), and 5
µg of either pSG5 (vector) or constructs expressing either the sense
(pSG5rMKP-1) or antisense (pSG5rMKP-1 as) direction by using the
SuperFect reagent in a 1:2 ratio (wt/wt). The transfected cells were
serum-starved for 12 hours and treated with LDL for 16 hours. Protein
extracts were prepared and assayed for luciferase activities by using a
dual-luciferase reporter assay kit according to the manufacture's
instructions. Results (mean±SD from 3 separate experiments) are
presented as percent of maximum luciferase expression seen for
LDL-treated SMCs cotransfected with Elk-1 luciferase and pSG5 vector.
*Significant difference from groups of pSG5 vector and pSG5rMKP-1 as
at P<0.05.
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MKP-1Inhibited ERK Activation and SMC Proliferation
To directly determine the influence of MKP-1 on MAPK inactivation
and the effects of MKP-1 on SMC proliferation, we established stably
transfected-SMC lines overexpressing MKP-1 and determined ERK
phosphorylation and DNA synthesis in the transfected
cell lines in response to LDL. Because the antibody recognizes both
endogenous and overexpressed rMKP-1, MKP-1 proteins of
transfected SMCs were detected with Western blot analysis after
stimulation with a low concentration of serum (2%). Figure 10A
data indicate that MKP-1 is
overexpressed in MKP-1transfected cells and that such treatment did
not significantly induce endogenous MKP-1 expression
implicated in the pSG5 vectortransfected cells. In comparison with
cells transfected with vector, rMKP-1transfected SMCs showed a 40%
to 60% reduction in ERK phosphorylation when
stimulated with LDL or serum (Figure 10B
). To determine the
effects of MKP-1 on DNA synthesis induced by LDL, rMKP-1 or
vector-transfected cell lines were treated with LDL.
[3H]Thymidine incorporation in
rMKP-1transfected SMCs was significantly lower than in
vector-transfected cells (Figure 10C
), indicating the role of
MKP-1 in the inhibition of SMC growth.

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Figure 10. SMCs stably transfected with rMKP-1. Rat vascular
SMCs were transfected with plasmid pSG5 (vector, 10 µg per dish) or
constructs expressing rMKP-1 by using the SuperFect reagent in a 1:2
ratio (wt/wt). The transfected cells were cultured overnight, divided
1:5, and placed in culture medium supplemented with 500 µg/mL G418.
A, Overexpression of rMKP-1 in transfected SMCs. rMKP-1 (M) and
vector- (V) transfected SMCs were serum-starved for 4 days and treated
with 2% serum for 30 minutes. Western blot analysis was
performed with the antiMKP-1 antibody. B, Inhibition of ERK1/2
activation. Transfected SMCs were serum-starved for 4 days and treated
with LDL (100 µg/mL) or 20% serum for 10 minutes. Western blot
analysis was performed with the
antiphosphorylated ERK1/2 antibody. C, Reduced growth
in rMKP-1transfected cells. Transfected SMCs were serum-starved for 4
days, LDL (100 µg/mL) was added to the culture medium, and incubation
was continued for 24 hours. [3H]Thymidine was added 4
hours before harvest. Radiation activities were measured. Data are
mean±SD of triplicates from 2 independent experiments. M1 and M2
represent 2 rMKP-1transfected cell lines. *Significant
difference from vector-transfected cells.
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 |
Discussion
|
|---|
Proliferation of vascular SMCs is a hallmark in the pathogenesis
of
atherosclerotic lesions. LDLs are mitogenic to cultured
SMCs
and have been demonstrated to activate MAPK-ERK signal
pathways.
15 16 17 18 In the present study, we have provided
the first evidence
that LDLs can stimulate MKP-1 expression, which is
crucial in
the regulation of MAPK activities in SMCs. MKP-1 serves as a
negative
regulator, controlling cell growth via inactivation of both
MAPKs,
because LDL-stimulated ERK and JNK/SAPK activation appears to
be
a component common to signaling pathways initiated by a wide
range of
growth-stimulating factors, including mitogens and
hormones.
50 51 52 Thus, our findings could significantly
advance our understanding
of the possible role of MKP-1 under
physiological conditions
or during pathological
changes, ie, inhibition of SMC proliferation
stimulated by LDL.
It is well known that LDL specifically binds to apoB/E receptors to
deliver cholesterol to SMCs,1 but
LDL-initiated signal transduction pathways leading to SMC proliferation
are not fully understood. In the present study, we have
demonstrated that LDL-induced MKP-1 expression is mediated by tyrosine
kinases and PKC, independent of LDL receptors and ERK-MAPKs.
LDL-stimulated MKP-1 expression was observed in either human or rat
SMCs or in LDL receptordeficient fibroblasts, and such induction was
not influenced by heparin, an inhibitor of LDL-receptor
binding.53 The mechanism by which LDL initiates signaling
in LDL-stimulated MKP-1 induction is presently unknown, and we
hypothesize that 2 pathways may be responsible for the induction: (1)
An atypical LDL binding site on human SMCs,54
characterized as being independent of the classic receptors, may
mediate LDL (lipid)-induced tyrosine kinase activation and MKP-1
expression. (2) Neutral sphingomyelinase present on cell membranes
might directly catabolize the sphingomyelin of LDL to generate
ceramide, which serves as a second messenger between LDL stimulation
and tyrosine kinase activation. This concept is supported by the fact
that sphingomyelinase-treated LDL markedly enhanced MKP-1 induction and
that ceramide treatment mimicked MKP-1 induction in SMCs (Figure 3D
and 3E
). In addition, it has been shown that HDL stimulates
MAPK activation in human skin fibroblasts.55 We have also
observed that HDL moderately induces MKP-1 expression in SMCs (data not
shown) and that arachidonic acid stimulation results in
MKP-1 induction.56 Our findings together with other
reports further support the role of the lipid moiety of lipoproteins in
the regulation of expression of this gene.
Recently, Suc et al57 reported that oxidized LDL and
native LDL (to a lesser extent) directly stimulated
endothelial growth factor receptor
phosphorylation or activation and subsequently
activated phospholipase C and inositol trisphosphate kinases.
In the present experiment, we did not find any involvement of
phospholipase C in LDL-induced MKP-1 expression (Figure 7
).
Interestingly, pertussis toxin significantly inhibited MKP-1 induction,
indicating that pertussis toxinsensitive G-protein signal pathways
may be involved. In agreement with our findings, Sachinidis et
al17 demonstrated that LDL stimulates
Ca2+ elevation and ERK activation via a pertussis
toxinsensitive G-protein pathways. LDL might also elicit G
proteincoupled receptor conformation or activation, by which LDL
initiates signals leading to cell growth. It would be interesting to
clarify whether the atypical LDL binding site described by Tkachuk et
al54 is a G proteincoupled receptor.
PKC, a large and diverse family of protein kinases, plays important
signaling roles in cell growth, differentiation, and
homeostasis58 59 and can be activated by
LDL.42 45 46 Tyrosine kinases, such as Pyk2 and
Src-related kinases, have been shown to be essential intermediates
linking upstream kinases and MKP-1 gene expression. In our system, PKC
and tyrosine kinase inhibitors significantly blocked LDL
stimulation of MKP-1, indicating that both PKC and tyrosine kinases
play important roles during signaling. Both PKC and tyrosine kinases
activate MAPK pathways via phosphorylation and
activation of Ras, or c-Raf kinases and MEK.6 7 8 9 10 11 60 These
pathways may be important in LDL-induced signaling, because LDL
simultaneously activates both
ERK15 16 17 18 and JNK/SAPK (Figure 8
), which share a
common point during activation via Ras.6 7 8 9 10 11
Interestingly, Bokemeyer et al61 recently demonstrated
that JNK/SAPK activation is responsible for MKP-1 gene expression, at
least in fibroblasts. Our data, together with the other noted
observations, support the role of JNK/SAPK in LDL-stimulated MKP-1
induction.
As described above, atherosclerosis and its
complications, including myocardial infarction and stroke, are the most
prevalent cause of morbidity and mortality in Western countries. During
atherogenesis, early lesions spread progressively and form
atherosclerotic plaques. In this process, SMC proliferation plays a key
role.4 Although multiple factors, including growth
factors, cytokines, mechanical stress, neurotransmitters, and
hormones, are believed to contribute to the process leading to SMC
growth,4 62 LDL levels in the blood, strongly predictive
of coronary heart disease, play an important role in
atherogenesis. In addition to cholesterol transport, LDLs
are mitogenic to vascular SMCs.2 3 4 LDL
stimulates SMCs to generate both positive (ERKElk-1 pathway) and
negative (MKP-1) signals. MKP-1 may inactivate both MAPKs,
ERK and JNK/SAPK, and block Elk-1 transcription factor activation
during LDL stimulation. The balance between MKP-1 and MAPK
levels/activities stimulated by LDL in SMCs is critical for maintaining
homeostasis of the arterial wall. If LDL-initiated signals
leading to MAPK activation and MKP-1 induction can be arbitrarily
dissected, ie, switched on for MKP-1 or switched off for ERK in vivo,
new strategies could be developed for the prevention or treatment of
atherosclerosis.
 |
Acknowledgments
|
|---|
This work was supported by grants P12568-MED and P13099-BIO
(to
Q.X.) from the Austrian Science Fund and 6286 (to Q.X.)
from the
Jubiläumsfonds of the Austrian National Bank.
We thank Drs Nikki
J. Holbrook and Yusen Liu, National Institute
on Aging, National
Institutes of Health, Baltimore, Md, for
providing MKP-1 plasmids;
Gottfried Baier, Institute for Medical
Biology and Human Genetics,
University of Innsbruck Medical
School, Innsbruck, Austria, and Georg
Wick for critical reading
of the manuscript and valuable discussion; J.
Woodgett, Ontario
Cancer Institute, Toronto, Canada, for
providing the glutathione
S-transferasec-Jun expression
vector; and T. Öttl
for preparation of the photographs.
Received September 1, 1998;
accepted January 19, 1999.
 |
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