(American Journal of Pathology. 2000;156:1875-1886.)
© 2000 American Society for Investigative Pathology
Increased Expression and Activation of Stress-Activated Protein Kinases/c-Jun NH2-Terminal Protein Kinases in Atherosclerotic Lesions Coincide with p53
Bernhard Metzler*,
Yanhua Hu
,
Hermann Dietrich
and
Qingbo Xu
From the Division of Cardiology,*
Department of Internal Medicine,
and the Institute for General and Experimental
Pathology,
University of Innsbruck Medical
School; and the Institute for Biomedical Aging
Research,
Austrian Academy of Sciences,
Innsbruck, Austria
 |
Abstract
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Hyperlipidemia alters gene expression of arterial endothelial and
smooth muscle cells (SMCs) and induces atherosclerotic lesions,
in which cell proliferation and apoptosis co-exist. The signal
transduction pathways that mediate these responses in the vessel wall
in vivo have yet to be identified. Stress-activated
protein kinases (SAPKs) or c-Jun NH2-terminal
protein kinases (JNKs) are thought to be crucial in transmitting
transmembrane signals required for cell differentiation and apoptosis
in vitro. In the present study, we investigated
the localization and activity of SAPK/JNK in atherosclerotic lesions of
cholesterol-fed rabbits. Immunofluorescence analysis revealed abundant
and heterogeneous distribution of pan-SAPK/JNK and phosphorylated
SAPK/JNK, which were mainly localized in cell nuclei of the
lesional cap and basal regions. Double staining of the lesions
demonstrated that a portion of
-actin+ SMCs and
RAM11+ macrophages contained abundant phosphorylated
SAPK/JNK proteins. SAPK/JNK protein levels in protein extracts from
atherosclerotic lesions were two- to threefold higher than the vessels
of chow-fed rabbits. SAPK/JNK activities were elevated three- to
fivefold higher than the normal vessels. Interestingly,
increased SAPK/JNK in lesions was co-localized or coincided with high
levels of transcription factor p53 as identified by double labeling and
immunoprecipitation. Abundant pro-apoptotic protein BAX and
BCL-XS were also observed. Furthermore, low-density
lipoprotein (LDL) and oxidized LDL stimulated SAPK/JNK activation in
cultured SMCs in a time- and dose-dependent manner. LDL also induced
SAPK/JNK activation in vascular SMCs derived from
LDL-receptor-deficient Watanabe rabbits, indicating a
LDL-receptor-independent process. Thus, SAPK/JNK persistently
hyperexpressed and activated in lesions may play a key role in
mediating cell differentiation and apoptosis during the development of
atherosclerosis via activation of transcription factor
p53.
 |
Introduction
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Mammalian cells respond to
extracellular stimuli by activating signal transduction pathways, which
culminate in changes in gene expression. A critical component of
signaling pathways is the activation of protein kinases that
phosphorylate a host of cellular substrates, such as transcription
factors controlling the induction of various genes. Stress-activated
protein kinases (SAPKs) or c-Jun NH2-terminal
protein kinases (JNKs) are highly activated in the cardiovascular
system in response to stresses,1-3
including acute
hypertension,4
angioplasty,5
mechanical
stress,6-8
ischemia/reperfusion,9
free
radicals,10
heat shock,11
and inflammatory
cytokines12
in vivo or in vitro. The
pathways leading to SAPK/JNK activation have been extensively studied
in cultured cells. Upstream activators of SAPKs/JNKs have been found
involving SEK1/MKK4, MEKK13, and the small GTP-binding proteins Rac1
and Cdc42.13-15
JNK was named based on its ability to
phosphorylate the c-Jun protein, leading to its enhanced
transcriptional activity.16-18
Recently, they have
also been shown to be capable of phosphorylating the transcription
factors ATF2, Elk1, and p53,19-23
which form heterodimers or homodimers binding to promoter regions
present in numbers of genes that play a crucial role in mediating cell
(de)differentiation and apoptosis.
The atherosclerotic lesion is defined by arterial intimal cell
proliferation, lipid accumulation, and connective tissue deposition.
The major cellular components of the human atherosclerotic plaques are
smooth muscle cells (SMCs), which dominate the fibrous cap, and
macrophages, which are the most abundant cell type in the lipid-rich
core region.24,25
Although the pathogenic mechanism of
atherosclerosis is not fully understood, a high concentration of
circulating cholesterol or low-density lipoproteins (LDL) is believed
to be a major risk factor. LDL delivers cholesterol to vascular SMCs
and macrophages, stimulates cell proliferation, and induces gene
expression of platelet-derived growth factor (PDGF), PDGF receptors,
c-fos, and egr-125,26
in the
arterial wall. LDL or oxidized LDL stimulation can also evoke cell
apoptosis,27-29
which has been observed in
atherosclerotic lesions.30-33
In addition, p53 has been
identified to be highly expressed in atherosclerotic lesions and to be
involved in oxidized LDL-induced apoptosis.27
However, the
precise signal transduction pathways that link hypercholesterolemia or
LDL stimulation to quantitative changes in gene expression and, to
apoptosis in the pathogenesis of atherosclerosis, remain to be
elucidated. In the present study, we examined SAPK/JNK expression,
localization, and activation in atherosclerotic lesions of
cholesterol-fed rabbits and provide the first evidence of SAPK/JNK
overexpression and activation in lesions. Furthermore, we demonstrate
that LDL and oxidized LDL stimulate SAPK/JNK activation in cultured
SMCs independent of classic LDL receptors.
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Materials and Methods
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Rabbit Model for Atherosclerosis
New Zealand White male rabbits weighing between 1,800 and 2,200 g
were obtained from Charles River (Kissleg, Germany). All animals were
selected for serum cholesterol levels under 100 mg/dl, individually
housed in wire-bottomed cages at 22°C with a relative humidity of
55%. All received water ad libitum and were fed either a
normal standard chow diet (T775; Tagger & Co., Graz, Austria) or a
cholesterol-enriched diet (0.2% w/w) for 16 weeks, as described
previously.34,35
Animals were sacrificed by heart puncture
under ketamine (25 mg/kg) and xylazine (5 to 10 mg/kg) anesthesia.
Serum was collected for cholesterol assays and LDL isolation. The
aortas were carefully removed intact from the aortic arch to the iliac
bifurcation, immediately put into cold phosphate-buffered saline (PBS)
(4°C) and prepared for histological analysis, tissue culture, and
protein extractions. The total surface area of aortas covered by
lesions was evaluated. For conventional histology, tissue fragments
were fixed in 4% buffered (pH 7.2) formaldehyde, embedded in paraffin,
and sectioned for hematoxylin and eosin (H&E) staining. Serum
cholesterol values were measured every 2 or 4 weeks using an enzymatic
procedure (Sigma Chemical Co., St. Louis, MO).
Immunohistochemical and Immunofluorescence Double Staining
The procedure used for immunohistochemical staining was similar to
that described elsewhere.34,35
Briefly, serial
4-µm-thick frozen sections were overlaid with mouse monoclonal
antibodies against
-actin (Sigma), macrophages (RAM11; DAKO,
Copenhagen, Denmark), or T cells (L11/135; ATCC, Rockville, MD) and
incubated with rabbit anti-mouse immunoglobulin (Ig) conjugated with
peroxidase (DAKO) and developed for 20 minutes at room temperature
using a substrate solution.
For immunofluorescence staining, a monoclonal antibody against
SAPK/JNK1 (Transduction Lab., Lexington, KY) was added to the sections.
After three washes with PBS, the sections were incubated with a rabbit
anti-mouse Ig-tetramethylrhodamine B isothiocyanate (TRITC) conjugate
(DAKO) for 30 minutes, or sections were incubated with a monoclonal
antibody against phosphorylated SAPK/JNK conjugated with fluorescein
isothiocyanate (FITC) (p-JNK; Santa Cruz Biochem., Santa Cruz, CA),
mounted in glycerol/PBS, and examined in an epiillumination
immunofluorescence microscope equipped with appropriate filter
combinations for the three wavelength method (Leitz, Wetzlar, Germany).
For immunofluorescence double staining, frozen sections were incubated
with monoclonal antibodies against
-actin Cy3 conjugate (Sigma) or
RAM11 macrophages. After three washes with PBS, the sections were
incubated with a rabbit anti-mouse Ig-TRITC conjugate (DAKO) for 30
minutes, rinsed, blocked with normal mouse serum (1:5), and stained
with a mouse monoclonal antibody against phosphorylated-SAPK/JNK-FITC
conjugate (p-JNK). For p53 and JNK1 double staining, a mouse monoclonal
anti-p53 antibody and rabbit anti-JNK1 antibodies (Santa Cruz Biotech.
Inc.) were used and visualized with swine anti-mouse Ig-FITC and goat
anti-rabbit Ig-TRITC. For visualization of nuclei, sections were
counterstained with the DNA stain Hoechst 33258 (1 µg/ml PBS; Lambda
Probes, Graz, Austria) for 1 minute.
Western Blot Analysis
The procedure used for protein extracts was similar to that
described previously36
with a slight modification.
Briefly, the atherosclerotic-lesioned intima and media were dissected
on ice from the remaining adventitia with tweezers and scissors.
Tissues were homogenized in buffer A (20 mmol Hepes, pH 7.4, 50 mmol/L
ß-glycerophosphate, 2 mmol/L EGTA, 1 mmol/L dithiothreitol, 1
mmol/L Na3VO4, 1% Triton X-100, 10% glycerol,
1 µg/ml leupeptin, 100 µmol/L phenylmethyl sulfonyl fluoride
(PMSF), and 1 µg/ml aprotinin), and proteins were extracted. Proteins
(50 µg/lane) were separated by electrophoresis through a 10%
SDS-polyacrylamide gel and transferred to an immobilon-p transfer
membrane. The membranes were processed with monoclonal antibodies
against SAPK/JNK1, actin, p53, BAX, and BCL-XS/L
(Santa Cruz Biotech. Inc.), respectively, and specific antigen-antibody
complexes were detected with the ECL Western Blot Detection Kit
(Amersham Co., Little Chalfont, UK).
Immunoprecipitation and Kinase Assay
One-half milliliter of the supernatant containing 0.5 mg proteins
was incubated with 10 µl of rabbit anti-SAPK/JNK1 antibodies and 40
µl of protein A-agarose suspension (Santa Cruz Biochem.). The
immunocomplexes were precipitated by centrifugation and washed 2 times
with buffer A, buffer B (500 mmol/L LiCl, 100 mmol/L Tris, 1 mmol/L
dithiothreitol, 0.1% Triton X-100, pH 7.6), and buffer C (20 mmol/L
Mops, 2 mmol/L EGTA, 10 mmol/L MgCl2, 1 mmol/L
dithiothreitol, 0.1% Triton X-100, pH 7.2), respectively. The
activities of SAPK/JNK in the immunocomplexes were measured as
described previously.11,16
Briefly, immunocomplexes were
incubated with 35 µl of buffer C supplemented with glutathione
S-transferase (GST)-c-Jun (5 µg),
-32P-ATP (Amersham, Little Chalfont, UK), and
MgCl2 (50 mmol/L) for 20 minutes at 37°C with
vortexing every 5 minutes. The GST-c-Jun fusion protein (plasmid
provided by Dr. Woodgett, Toronto, Canada) was produced in
Escherichia coli and isolated using glutathione Sepharose 4B
RediPack Columns (Pharmacia Biotech Inc., Piscataway, NJ) according to
the manufacturers protocol. 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 SDS-polyacrylamide gel
electrophoresis (12% gel) and subjected to autoradiography.
For immunoprecipitation of SAPK/JNK1 and p53 Western blot analysis, 1
ml of the supernatant containing 2 mg of protein was incubated with 50
µl of rabbit anti-SAPK/JNK1 antibodies and 100 µl of protein
A-agarose suspension. The immunocomplexes were precipitated as
described for the kinase assay and resolved in SDS-polyacrylamide gel
electrophoresis (15% gel).
Cell Cultures
Human and rabbit vascular SMCs were cultivated from arteries using
the procedure described by Ross and Kariya37
with a slight
modification.38
In short, specimens of human carotid
arteries were obtained from the Department of Vascular Surgery,
University Hospital of Innsbruck. Intima and inner layer of media were
dissected from the arteries and cut into pieces, digested with
collagenase and elastase, and cultured in Dulbeccos modified
essential medium (DMEM; Life Technologies, Inc., Grand Island,
NY). Thoracic aortas of chow- and cholesterol-fed rabbits were removed,
and lesioned intima and normal media were carefully dissected from the
vessel, cut into pieces (~1 mm3), and implanted
onto a gelatin-coated (0.02%) plastic bottle (Becton Dickinson,
Oxnard, CA). The bottle was incubated up-side-down at 37°C in
a humidified atmosphere of 95% air/5% CO2 for 3
hours, and then medium supplemented with 20% fetal calf serum,
penicillin (100 U/ml), and streptomycin (100 µg/ml) was slowly added.
Cells were passaged by treatment with 0.05% trypsin/0.02%
ethylenediaminetetraacetic acid (EDTA) solution. Experiments were
conducted on SMCs that had just achieved confluence. Similarly, aortic
SMCs from Watanabe rabbits (La Roche, Basel, Switzerland) were
cultivated and used for SAPK/JNK kinase assays.
LDL Isolation and Oxidation
EDTA plasma was pooled from normolipemic, fasting (12 to 14 hours)
male and female donors, aged 20 to 30 years. Lipoproteins were prepared
by differential centrifugation using solid KBr to adjust the
density, as described previously.39,40
LDL were obtained
in fraction between 1.020 to 1.050 g/ml. Concentrations of LDL were
determined gravimetrically by aliquot weight after drying, and
quantities of lipoproteins were expressed as total
weights.39,40
LDL oxidation was performed by incubation of
LDL (1 mg/ml PBS) with 10 µmol/L CuCl2 at
37°C for 18 hours.41
The extent of oxidation was
assessed by measurement of thiobarbituric acid reactive substances
(TBARS) (9.8 ± 1.3 nmol/mg).42
Statistical Analysis
The Mann-Whitney U test was used for comparison between
two groups. P < 0.05 was considered statistically
significant.
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Results
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Atherosclerotic Lesions in Hypercholesterolemic Rabbits
Animals in the control group (chow diet) had blood cholesterol
levels <100 mg/dl, whereas blood cholesterol levels in rabbits
receiving the 0.2% cholesterol diet were significantly elevated and
reached 500 mg/dl at 4 weeks, and ~700 mg/dl at 16 weeks. Rabbit
aortas from both groups were examined morphologically and
immunohistologically 16 weeks after chow or cholesterol feeding. Fifty
percent to 80% of aortic intima was covered by atherosclerotic
lesions, including fatty streaks and plaques in cholesterol-fed
rabbits. Aortic lesions of rabbits fed with a cholesterol-enriched diet
were characterized by cell proliferation, foam cell accumulation, and
lipid deposition in the intima. Atherosclerotic lesions displayed great
variations in cell type and distribution. The overlying endothelium was
intact to variable degrees, and
-actin-positive SMCs appeared in
various stages of lesions, most frequently in advanced lesions (Figure 1)
. Cells expressing the macrophage
antigen identified by the RAM11 antibody were observed in all
atherosclerotic lesions. T lymphocytes identified by monoclonal
antibody L11/135 were also found in lesions (Figure 1)
.

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Figure 1. Cells in atherosclerotic lesions. A and B:
H&E-stained sections from animals receiving chow
(A) or
cholesterol-enriched diets
(B) for 16
weeks. CF: Cryostat sections from aortic segments of
rabbits fed a 0.2% cholesterol diet for 16 weeks were labeled with
normal mouse Ig as a negative control
(C), or
monoclonal antibodies against -actin
(D),
macrophages (E;
RAM11), or T lymphocytes
(F; L11/135)
and visualized with the peroxidase system. Note the presence of
positive staining (dark)
in lesions. Arrowheads indicate internal elastic lamina, and
arrows point to examples of positive-stained cells. Original
magnification, x250.
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SAPK/JNK Activation in Atherosclerotic Lesions
SAPK/JNK kinases are activated by dual phosphorylation of tyrosine
and threonine residues in response to stress stimuli or
mitogens.13-15
After activation, they translocate from
cytoplasm into the nucleus in cultured cells. We performed an
immunofluorescent analysis of atherosclerotic lesions using the
antibody recognizing the pan-SAPK/JNK and phosphorylated SAPK/JNK,
respectively. Intimal endothelium, media, and adventitia of normal
aortas showed very weak SAPK/JNK staining (Figure 2, a and d)
, while the lesion-covered
areas in intima from rabbits receiving a cholesterol-rich diet showed
increased immunostaining intensity (Figure 2, c, e, and f)
. Nonspecific
reactivity was minimal in the negative controls stained with a normal
mouse Ig (Figure 2b)
. The pattern of pan-SAPL/JNK and phosphorylated
SAPK/JNK staining in the lesioned aortas is shown in Figure 2, c, e, and f
. Heterogeneity of pan-SAPK/JNK and phosphorylated SAPK/JNK
staining became more evident in atherosclerotic plaques. Sites of
increased pan-SAPK/JNK and phosphorylated SAPK/JNK were mainly within
the cap and base regions of the atherosclerotic plaque (Figure 2, c, e, and f)
. Fatty streaks displayed elevated SAPK/JNK content in
subendothelial regions.

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Figure 2. SAPK/JNK staining in atherosclerotic lesions. Cryostat sections from
aortic segments of rabbits receiving a standard-chow diet
(a and
d) or a 0.2% cholesterol-diet
(b, c, e, and
f) were labeled with normal mouse Ig
as a negative control
(b), or a
monoclonal antibody against pan-SAPK/JNK
(a and
c) visualized with rabbit anti-mouse
Ig-TRITC, or a monoclonal antibody against phosphorylated SAPK/JNK
conjugated with FITC
(df). Note
the presence of positive staining in the cap
(c and
e) and base
(f) regions of
lesions. Arrows indicate the surface of intimal lesions, and
thick arrows point to examples of positive-stained cells.
Co, core region of the lesion. Original magnification, x250.
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Next, we performed immunofluorescence double staining using the
antibody against phosphorylated-SAPK/JNK to identify the cells
responsible for the elevated SAPK/JNK expression. Figure 3, ad
, shows data representing double
immunostaining with antibodies against phosphorylated-SAPK/JNK (a and
c; green),
-actin (b; red), and RAM11 macrophages (d; red).
Nonspecific staining was minimal in the corresponding negative controls
stained with a normal mouse Ig. Typical double-positive cells are
indicated by arrows, demonstrating a portion of SMCs and macrophages in
lesions overexpressing SAPKs/JNKs.

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Figure 3. Immunofluorescence double labeling of phosphorylated SAPK/JNK and cell
markers in atherosclerotic lesions. Cryostat sections from rabbit
aortic tissue 16 weeks after administration of a cholesterol-rich diet
were incubated with a mouse monoclonal antibody against phosphorylated
SAPK/JNK conjugated with FITC
(a) for 30
minutes at room temperature. After washing, sections were incubated
with a monoclonal antibody against -actin conjugated with Cy3
(b).
c and d: Results of immunofluorescent double
staining for phosphorylated SAPK/JNK and RAM11+
macrophages. Arrow indicates internal elastic lamina, and
thick arrows point to an example of double-positive cells.
Original magnification, x250.
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Elevated Protein Levels and Activities of SAPKs/JNKs
Because of the increased intensity of immunostaining for
lesions, it would be interesting to determine SAPK/JNK1 protein levels
of atherosclerotic lesions. Protein extracts from tissues of normal
intima/media, lesioned intima, and media were analyzed by Western blot
analysis. Abundant SAPK/JNK1 proteins in atherosclerotic lesions were
observed (Figure 4A)
. SAPK/JNK proteins
in lesioned intima were significantly higher than intima/media of
control animals and media of cholesterol-fed rabbits after
normalization to the respect of actin contents (Figure 4C)
. To further
demonstrate higher SAPK/JNK1 protein levels associated with increased
SAPK/JNK1 activity, a kinase assay was performed using c-Jun as a
substrate. Figure
4B shows the results of an experiment examining
SAPK/JNK1 activities in the vessel wall, indicating elevated
SAPK/JNK1 activities in atherosclerotic lesions. When immunocomplexes,
obtained similarly as for the kinase assay, were subjected to Western
blot analysis, the results shown in Figure 4B
(lower panel)
demonstrated equal efficacy of the immunoprecipitation from protein
extracts of both normal vessels and atherosclerotic lesions and higher
levels of SAPK/JNK proteins in the lesions. Figure 4C
summarizes data
from two independent experiments, implicating that SAPK/JNK activities
were at low levels in control vessels and media of cholesterol-fed
rabbits, but increased three- to fivefold in atherosclerotic lesions.

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Figure 4. Elevated SAPK/JNK proteins and activities in atherosclerotic lesions.
Intima and media (I/M)
from chow-fed rabbits and media
(M) and
atherosclerotic-lesioned intima
(AS) from cholesterol-fed
rabbits (16-week diet)
were dissected on ice from the remaining adventitia with a tweezers and
scissors. Tissues were frozen in liquid nitrogen and homogenized with a
polytron homogenizer. Protein extracts (50
µg/lane) were separated on 10%
SDS-polyacrylamide gel, transferred onto membrane, and probed with the
antibody against SAPK/JNK1
(A) and
anti-actin antibodies after stripping
(A, lower
panel). Immunocomplexes were visualized by
a Western blot detection kit. For the kinase assay, SAPK/JNK1 proteins
were immunoprecipitated from the protein extractions at 0.5 mg for the
kinase assay (B, upper
panel) and 0.2 mg for Western blot
(B, lower
panel) and their kinase activity
(B) measured
based on phosphorylation of GST-c-Jun substrate. SAPK/JNK1 in
immunoprecipitant (IP)
was identified by Western blot analysis
(blot). Each lane
represents an individual animal. C: Graph of means ±
SD obtained from five rabbits per group. *, Significant difference from
the intima/media, P < 0.05.
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Expression of p53, BAX, and BCL-XS
There is evidence that transcription factor p53 accumulates in
human atherosclerotic tissues.43,44
Immunofluorescence
staining confirmed high levels of p53 in the lesions induced by the
cholesterol-diet in rabbits (Figure 5)
.
Interestingly, the most p53+ cells showed an
elevation in pan-SAPK/JNK1 proteins, ie, co-localized in
atherosclerotic lesions (Figure 5, b and c)
. The distribution pattern
of p53 was similar to pan-SAPK/JNK1. In addition, both p53 (Figure 5d)
and SAPK/JNK1 (Figure 5e)
proteins in cells of arterial walls from
control rabbits were expressed at very low levels, if any. To further
verify p53 levels in lesions, protein extracts were analyzed by Western
blot using anti-p53 antibodies. Data shown in Figure 6
indicated that p53 protein content in
the lesions was higher than proteins from the normal intima and media
(Figure 6A)
. When SAPK/JNK1 was immunoprecipitated and analyzed with
Western blot, a 53-kd band was identified by anti-p53 antibody (Figure 6B)
, indicating that a complex of SAPK/JNK1 and p53 is present in
atherosclerotic lesions. These findings are concomitant with the
results obtained from in vitro studies using cultured
cells.22,23

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Figure 5. Immunofluorescence double labeling of p53 and pan-SAPK/JNK1 in
atherosclerotic lesions. Cryostat sections from rabbit aortic tissues
16 weeks after administration of a cholesterol-rich diet
(ac) or
standard-chow diet (d and
e) were incubated with normal mouse
serum (a), a
mouse monoclonal antibody against p53
(b and
d), and visualized with a swine
anti-mouse Ig-FITC (a, b,
and d). After washing and blocking
with normal mouse serum
(1:5), sections
b and d were labeled with rabbit
anti-pan-SAPK/JNK1 antibodies and visualized with goat anti-rabbit Ig
conjugated with TRITC (c and
e). Open arrow indicates
internal elastic lamina and filled arrows point to examples
of double-positive cells. Original magnification, x250.
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Figure 6. Elevated p53 proteins coincided with SAPK/JNK1. A: Western
blot analysis. Intima and media
(I/M) from chow-fed
rabbits and media (M) and
atherosclerotic-lesioned intima
(AS) from cholesterol-fed
rabbits (16-week diet)
were dissected on ice from the remaining adventitia with a tweezers and
scissors. Tissues were frozen in liquid nitrogen and homogenized with a
polytron homogenizer. Protein extracts (50
µg/lane) were separated on 15%
SDS-polyacrylamide gel, transferred onto membrane, and probed with the
antibody against p53 and anti-actin antibodies after stripping
(A, lower
panel). Immunocomplexes were visualized by
a Western blot detection kit. Each lane represents an individual
animal. B: Immunoprecipitation
(IP) and Western blot
analysis. SAPK/JNK1 proteins were immunoprecipitated from the protein
extractions (2 mg). The
immunoprecipitants were separated on 15% SDS-polyacrylamide gel,
transferred onto membrane, and probed with the antibody against p53 and
SAPK/JNK1 after stripping. Immunocomplexes were visualized by a Western
blot detection kit. Each lane represents proteins derived from three
animals.
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BAX and BCL-XS are considered to be pro-apoptotic
proteins which are primarily mediated by transcription factor
p53.44,45
Western blot analysis of protein extracts from
the vessel wall revealed marked increases in both BAX and
BCL-XS proteins from atherosclerotic lesions, but
not BCL-XL (Figure 7)
.

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Figure 7. Elevated protein levels of BAX and BCL-XS in
atherosclerotic lesions. Protein extracts from intima and media
(I/M) from chow-fed
rabbits and atherosclerotic-lesioned intima
(AS) and media
(M) from cholesterol-fed
rabbits (16-week diet)
were prepared as described in the legend to Figure 4
. Western blot
analysis was performed with antibodies against BAX
(A) or
BCL-XS/L
(B) and
reprobed with anti-actin antibodies (lower
panel) after stripping. Immunocomplexes
were visualized by a Western blot detection kit. Each lane represents
an individual animal. Data are from two independent experiments.
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LDL and Oxidized LDL-Activated SAPK/JNK in SMCs
LDL is a main lipoprotein carrying and transporting
cholesterol in hypercholesterolemic rabbits induced by cholesterol
feeding. LDL can be oxidized in the arterial wall. To test whether LDL
and oxidized LDL can induce SAPK/JNK activation, vascular SMCs from
humans and rabbits were stimulated with human LDL and oxidized LDL and
the kinase assay was performed. As shown in Figure 8, A and B
, LDL and oxidized LDL (100
µg/ml) treatment resulted in significantly increased SAPK/JNK
activation in SMCs; kinetic analysis indicated that this response
occurred as early as 10 minutes after treatment (Figure 8, A and B)
,
with maximum levels (five- to eightfold greater than untreated control)
achieved 20 minutes after treatment, almost returning to basal levels
by 4 hours. To further establish the relationship between LDL or
oxidized LDL treatment and SAPK/JNK activity, we performed a
dose-response analysis of LDL- or oxidized LDL-induced SAPK/JNK
activation. As shown in Figure 8, C and D
, SAPK/JNK activities
increased in a dose-dependent manner between 5 and 400 µg/ml (LDL),
and reached higher levels at concentrations between 100 to 400 µg/ml.
To verify the efficacy of immunoprecipitation and the phosphorylation
of SAPK/JNK1 stimulated by LDL, the immunocomplexes, obtained similarly
as for kinase assays, were subjected to electrophoreses and blotted
with antibodies against phosphorylated SAPK/JNK (Figure 8E
, upper
panel) and pan-SAPK/JNK1 (Figure 8E
, lower panel). The results indicate
that oxidized LDL stimulated SAPK/JNK1 phosphorylation and did not
alter protein levels (Figure 8E)
. Taken together, these findings
demonstrate that LDL and oxidized LDL induced SAPK/JNK activation and
phosphorylation, which were strongly potentiated by oxidized LDL
compared to native LDL.

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Figure 8. LDL-activated and oxidized LDL-activated SAPK/JNK in SMCs. Vascular
SMCs were dissociated from human carotid arteries by collagenase and
cultivated in DMEM medium. LDL were isolated from human EDTA-plasma by
density ultracentrifugation and oxidized by incubation with
Cu+2. Subconfluent cells were serum-starved for 3 days and
incubated with LDL
(A) or
oxidized LDL
(B)
(100 µg/ml) at 37°C
for the indicated times or for 20 minutes in the presence of LDL
(C) or
oxidized LDL
(D) in the
indicated concentrations. For kinase assay, SAPK/JNK1 proteins were
immunoprecipitated from the protein extracts
(0.5 mg) and their kinase
activities measured based on phosphorylation of GST-c-Jun substrate.
For Western blot analysis
(E),
immunoprecipitated SAPK/JNK1 from 0.1-mg protein extracts was probed
with phosphorylated-SAPK/JNK and SAPK/JNK1 after stripping. Data are
from two independent experiments. Ctl, negative control; S, fetal calf
serum treatment without LDL addition.
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LDL can specifically bind to their receptors to deliver cholesterol to
the cell, but whether the receptor binding initiates signaling pathways
stimulated by LDL remains to be clarified. We performed several
experiments to verify receptor involvement in LDL-induced SAPK/JNK
activation. SMCs from normal or Watanabe rabbits lacking the classic
LDL receptors were incubated with human LDL for 20 minutes, and kinase
activities measured as described above. Figure 9A
indicates that LDL stimulated SAPK/JNK
activation in LDL receptor-deficient SMCs. No significant difference
between the two types of SMCs was found. It has been established that
heparin blocks LDL-receptor binding, but pretreatment of LDL with
heparin did not inhibit LDL-stimulated SAPK/JNK activation (Figure 9B)
.
When LDL binds to the receptors, the complexes are internalized and
degraded, which can be inhibited by pretreatment of cells with
chloroquine or NH4Cl. To further exclude the
involvement of LDL receptors, SMCs were pretreated with both reagents
and stimulated with LDL, again with no apparent influence (Figure 9C)
,
indicating LDL receptor-independent activation.

View larger version (38K):
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|
Figure 9. LDL-receptor-independent SAPK/JNK activation. Arterial SMCs from normal
or LDL receptor-deficient
(Watanabe) rabbits
(A) or humans
(B and
C) were cultivated in DMEM medium
supplemented with 20% FCS. SMCs were serum-starved for 3 days, and
preincubated with chloroquine
(Chl) or
NH4Cl
(C) for 30
minutes. LDL were directly added (A
and C) or preincubated with heparin
(2 mg/ml; B)
for 40 minutes before addition to SMCs. SAPK/JNK1 proteins were
immunoprecipitated from the protein extractions and their kinase
activities measured based on phosphorylation of GST-c-Jun substrate.
Data are from two independent experiments. -, negative control of
Watanable rabbit SMCs, which have similar levels of kinase activities
as those of normal rabbit SMCs; S, fetal calf serum treatment without
LDL addition.
|
|
 |
Discussion
|
|---|
Atherosclerosis is the leading cause of death among persons with a
Western life style. Elevated plasma LDL levels are clearly major risk
factors for cardiovascular disease, but the underlying mechanisms are
not fully understood. LDL and oxidized LDL effectively stimulate SMCs
and macrophages to express a large number of genes, such as TGF, FGF,
PDGF, PDGF receptors, c-fos, and
egr-1,25,26,46-48
which are important in the
development of atherosclerotic lesions. In the present study, we
provide the first evidence of increased SAPK/JNK activities in intimal
lesions of cholesterol-fed rabbits, which coincide with high levels of
transcription factor p53, pro-apoptotic protein BAX, and
BCL-XS. These observations could be important for
understanding the mechanisms of signal transduction pathways between
hypercholesterolemia and altered gene expression and/or apoptosis of
SMCs and macrophages during atherogenesis.
It is well known that LDL specifically binds to apoB/E receptors to
deliver cholesterol to SMCs,49
but LDL-initiated signal
transduction pathways leading to SMC gene expression are not fully
elucidated. Recently, several groups reported that LDL and oxidized LDL
activate extracellular signal-regulated kinases in cultured SMCs and
macrophages.46,50-53
In the present study, we
demonstrated, for the first time, that LDL and oxidized LDL markedly
stimulate SAPK/JNK activation, and do not influence SAPK/JNK protein
levels in cultured SMCs. LDL-induced SAPK/JNK activation was observed
in both normal and Watanabe rabbit SMCs, which are LDL-receptor
deficient, and this induction was not influenced by heparin, an
inhibitor of LDL-receptor binding, indicating a process independent of
classic LDL receptors.
As described above, oxidized LDL accumulation in the vascular wall is a
characteristic feature of atherosclerosis.54,55
Oxidized
LDL can be taken up into monocytes/macrophages to form foam cells via
scavenger receptors, eg, CD36.56
CD36 has been
demonstrated to be abundantly expressed in lipid-laden macrophages
around the core region of atherosclerotic lesions.57
Oxidized LDL-CD36 interaction leads to increased expression of CD11b,
CD18, cytokines such as IL-1 and TNF, and PDGF.58
Although
the mechanisms that transduce signals from oxidized LDL-CD36 binding to
the nucleus are not well defined, previous studies have suggested that
CD36 mediates nuclear factor
B activation in response to oxidized
LDL.59
We found that LDL and oxidized LDL strongly
stimulate SAPK/JNK activation, independent of classic LDL receptors,
implicating the involvement of scavenger receptors. Therefore, CD36
activation may be involved in oxidized LDL-activated SAPK/JNK signal
pathways.
In addition to LDL, SAPK/JNK can be activated by cytokines, mechanical
force, oxidative stress, and growth factors present in high
concentrations in atherosclerotic lesions or involved in the
pathogenesis of atherosclerosis.1-12
These factors may be
partially responsible for elevated SAPK/JNK activities in lesions
observed here. After activation, SAPK/JNK kinases can phosphorylate or
activate several transcription factors, including c-Jun,
ATF2, Elk, and p53.16-23
ATF2 can dimerize not only with c-Jun, but also
with itself and some other members of the activation transcription
factor family, including ATF3, CREB, and nuclear
factor
B. Elk-1, together with serum response factor, controls
transcription from the serum response element. These transcription
factors regulate gene expression, including matrix metalloproteinases,
adhesion molecule E-selectin, NO synthase, IL-8, and proliferating cell
nuclear antigen.1-3,60
These genes have been demonstrated
to play a key role in cell growth and cellular homeostasis in the
development of atherosclerosis. Thus, our findings of SAPK/JNK
activation in atherosclerotic lesions could be important to
understanding the mechanism controlling expression of these genes
during atherogenesis.
Apoptosis of SMCs and macrophages has recently been demonstrated in
atherosclerotic lesions of humans as well as animal
models.30-33,61-63
Oxidized LDL induces apoptosis of
endothelial cells, SMCs, and macrophages depending on
p53.27-29
Interestingly, isolated SMCs from human
atherosclerotic plaques were shown to have a higher propensity for both
spontaneous and induced apoptosis compared with SMCs from normal
vessels.31
On the other hand, activation of MEKK1 (a
SAPK/JNK upstream kinase) SAPK/JNK pathways is implicated in the
initiation of apoptosis in other cell types in response to stress
stimuli.64,65
In our study, we demonstrated coincidence of
p53, pro-apoptotic protein, BAX, and BCL-XS with
selective activation of SAPK/JNK in tissues of atherosclerotic lesions,
indicating the involvement of SAPK/JNK in mediating apoptosis in
vivo in response to hypercholesterolemia. It is conceivable that
some cells in the lesions with higher SAPK/JNK activities and p53
proteins are subjected to apoptosis, during which, SAPK/JNK-mediated
signal transduction pathways, ie, SAPK/JNK-activated p53, could be
important in vivo.
As described above, LDL and/or oxidized LDL stimulate SMC proliferation
as well as apoptosis, and both cell proliferation and apoptosis
co-exist in atherosclerotic lesions. We believe that LDL and oxidized
LDL can initiate several signaling pathways leading to
(de)differentiation, proliferation, or apoptosis, and that the balance
between the positive and negative signals in vascular SMCs is critical
for maintaining homeostasis of the arterial wall. Further understanding
of the mechanisms regulating SAPK/JNK activities could lead to new
strategies for the prevention or treatment of atherosclerosis.
 |
Acknowledgements
|
|---|
We thank Dr. Georg Wick for his continued support and critical
reading of the manuscript and A. Jenewein for excellent technical
assistance.
 |
Footnotes
|
|---|
Address reprint requests 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
Supported by grants P-12568-MED and P-13099-BIO (to Q. X.) from the Austrian Science Fund and 7919 (to Q. X.) from the Jubiläumsfonds of the Austrian National Bank. Dr. Hu is a recipient of an APART Award from the Austrian Academy of Sciences.
Accepted for publication March 3, 2000.
 |
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