Mol Cell Biochem (2010) 342:171–181
DOI 10.1007/s11010-010-0481-y
oxHDL decreases the expression of CD36 on human macrophages
through PPARc and p38 MAP kinase dependent mechanisms
Jingyi Ren • Wenying Jin • Hong Chen
Received: 28 January 2010 / Accepted: 22 April 2010 / Published online: 11 May 2010
Ó Springer Science+Business Media, LLC. 2010
Abstract CD36, belongs to class B scavenger receptor
family, is a macrophage receptor for oxidized low-density
lipoprotein (oxLDL) and has been proven to play a critical
role in atherosclerotic foam cell formation. In addition,
CD36 expression is regulated by many factors including
oxLDL and HDL. A recent study suggests that CD36 can
also bind with oxidized high-density lipoprotein (oxHDL).
However, the direct role of oxHDL in atherosclerosis is
still not clear and it is not known whether oxHDL has any
influence on the expression of CD36 in macrophages. Here,
we performed experiments to investigate the effect of
oxHDL on the expression of CD36 on human peripheral
blood monocytes–macrophages and the possible mechanisms. Our results suggest that the uptake of oxHDL by
CD36 on macrophages accelerates foam cell formation. In
addition, oxHDL can down-regulate both the mRNA and
surface protein expression of CD36 on human peripheral
macrophages in vitro. oxHDL increased the mRNA
expression and protein phosphorylation of peroxisome
proliferators-activated receptor-c (PPARc). Using different
mitogen-activated protein kinase (MAPK) inhibitors, we
demonstrated that oxHDL regulated CD36 and PPARc
expression in a p38-MAP kinase dependent mechanism.
Keywords oxHDL CD36 PPARc MAP kinase Macrophages Atherosclerosis
Ren J and Jin W contributed equally to this project and considered
co-first authors.
J. Ren W. Jin H. Chen (&)
Department of cardiology, Peking University People’s Hospital,
Beijing, China
e-mail: [email protected];
[email protected]
Abbreviations
HDL
High-density lipoprotein
oxHDL Oxidized high-density lipoprotein
LDL
Low-density lipoprotein
oxLDL Oxidized low-density lipoprotein
PPARc Peroxisome proliferators-activated receptor-c
MAPK Mitogen-activated protein kinase
PBS
Phosphate-buffered saline
FACS
Fluorescence-activated cell sorting
SR
Scavenger receptor
LOX-1 Lectin-like oxidized low-density lipoprotein
receptor-1
PMSF
Phenylmethylsulfonil fluoride
TBARS Thiobarbituric acid reactive substances.
Introduction
The formation of lipid laden foam cells beneath the
endothelium of vascular wall is the hallmark of the atherosclerosis, which is the primary cause of coronary heart
disease and stroke. In the early stage of atherosclerosis
process, the activated endothelial cells, which could be
stimulated by many factors such as injury, shear stress, and
oxidative stress, can secrete many chemotactic factors and
adhesion molecules and thus induces the recruitment of
leukocytes [1]. Following activation, the monocytes
migrate into the subendothelial space where they differentiate into macrophages and ingest lipoproteins via scavenger receptors leading to the formation of foam cells.
Both high-density lipoprotein (HDL) and low-density
lipoprotein (LDL) are involved in the balance of lipid
homeostasis and both participate in the formation process
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172
of foam cells. Scavenger receptors on macrophage have
been proven to be critical for the foam cell development
due to their ability to bind and internalize modified lipoproteins. It has been demonstrated that LDL can undergo
chemical modifications including acetylation and oxidation. Scavenger receptor (SR) family members SR-A and
CD36 have been identified as the two major receptors
responsible for the lipoproteins uptake into macrophages.
Loss of SR-A and CD36 significantly impaired the ability of
macrophages to ingest modified LDL [2]. CD36, belonging
to class B scavenger receptor family, is an 88-kDa integral
membrane protein that expressed on a wide variety of cells
especially in monocytes-macrophages [3]. Prevailing
experimental evidence suggest CD36 is a major macrophage
scavenger receptor for oxidized LDL (oxLDL).
Just like LDL, HDL is also susceptible to chemical
modification including oxidation [4]. Oxidized HDL
(oxHDL) has been proven to exist in the intima of atheromatous plaques [5, 6]. Oxidative modification of HDL not
only attenuates its beneficial properties, such as stimulation
of cholesterol efflux from foam cells, endothelium-dependent vaso-reactivity, and anti-oxidative activity, but also
demonstrates a direct pro-atherogenic effect [7]. HDL is an
important therapy target for the atherosclerosis diseases.
However, the cellular receptors for oxHDL are still poorly
defined. It is reported that SR-BI and LOX-1 on endothelial
cells can bind hypochlorite-modified HDL [8]. Recently,
Thorne et al. reported that CD36 on macrophage is also a
receptor for oxHDL [9]. Prevailing evidence suggest that
CD36 expression can be regulated by various factors,
including oxLDL and HDL [10, 11]. HDL is reported to
induce the phosphorylation of peroxisome proliferator
activated receptor-c (PPARc) and further down-regulate
CD36 expression on macrophages [11]. Here, we tested
whether CD36 can also be regulated by oxHDL.
Materials and methods
Human monocyte-derived macrophages
Buffy coats from the blood cells of healthy donors were
obtained from the Beijing Red Cross Blood Center. Mononuclear cells were isolated by density gradient centrifugation
by using lymphocyte separation solution. Cells were resuspended and cultured in RPMI-1640 medium (GIBCO, Grand
Island, NY) supplemented with 10% fetal bovine serum,
50 lg/ml each of penicillin and streptomycin and 2 mM
glutamine at 37°C in a 5% CO2 incubator. Mononuclear cells
were plated in six-well cell culture dishes (Corning-Costar
Corp., Cambridge, Massachusetts, USA) and incubated for
24 h at 37°C. Non-adherent cells were then removed by
washing the dishes twice with PBS (150 mmol/l NaCl and
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Mol Cell Biochem (2010) 342:171–181
10 mmol/l phosphate buffer, pH 7.2), and the remaining
adherent cells were grown in the culture medium. The
medium was replaced every 3 days. Before treatments,
macrophages were switched to serum-free medium for 3–5 h
and then given treatments in serum-free medium.
Isolation of HDL and preparation of oxHDL
High-density lipoprotein (HDL, d 1.063–1.210 g/ml) was
isolated from normal human plasma by sequential ultracentrifugation after removal of very low-density lipoprotein (\1.019 g/ml) and LDL (1.019–1.063 g/ml). HDL was
dialyzed against phosphate-buffered saline (PBS) containing 0.3 mM EDTA, sterilized by filtration through a
0.22-lm filter. Protein concentration was determined by
the method of Lowry et al. [12].
oxHDL was prepared by dialysis of HDL (500 lg/ml) in
PBS containing 5 lM CuSO4 for 10 h at 37°C, followed by
dialysis in PBS containing 0.3 mM EDTA for 2 9 12 h.
The degree of oxidation of HDL was determined by measuring the amount of thiobarbituric acid reactive substances
(TBARS). oxHDL had TBARS value of 3.5 nmol/ml,
while oxLDL had TBARS value of 16.3 nmol/ml.
RT-PCR analysis
Cells were harvested and total RNA was isolated using the
TRIZOL reagent (Invitrogen Corp., Carlsbad, CA). The
total RNA was purified and then subjected to RT-PCR
analysis. RNA was reverse transcribed using RevertAidTM
First Strand cDNA Synthesis Kit (MBI Fermentas, St.
Leonrod, Germany). The transcribed cDNA was then used
for PCR amplification to estimate the expression of CD36,
PPARc, and GAPDH. The primers used were as follows:
CD36 (50 -GAG AAC TGT TAT GGG GCT AT-30 )
(50 -TTC AAC TGG AGA GGC AAA CG-30 ); PPARc
(50 -GGA AAG ACA ACA GAC AAA TCA C-30 ) (50 -TGC
ATT GAA CTT CAC AGC AAA C-30 ); and GAPDH
(50 -TGC CAC TCA GAA GAC TGT GG-30 ) (50 -TTC AGC
TGT GGG ATG ACC TT-30 ). The amplified transcripts
were analyzed by gel electrophoresis, and the signal
intensity of the bands with the expected sizes (389 bp for
CD36, 414 bp for PPARc, and 128 bp for GAPDH) were
measured with a scanning densitometer (SX-300, Shanghai, China) and quantified using Sximage software. Briefly,
an electrophoresed gel or processed film was digitally
scanned and analyzed to produce transmission/reflection
density values accordingly.
Flow cytometric analysis of CD36 expression
Immunofluorescence flow cytometric analysis (FACS) was
performed by using PE-conjugated mouse monoclonal
Mol Cell Biochem (2010) 342:171–181
antibodies against human CD36 (Biolegend, CA, USA).
After treatment, cells were removed with trypsin and
washed twice with PBS. Cells were incubated with antiCD36 labeled with PE for 1 h at 4°C. Then, the labeled
cells were washed twice before being assayed with a
FACScan flow cytometer (Becton Dickinson, San Jose,
California, USA).
Oil Red O staining
After treatments, cells was washed with PBS twice and
then fixed with 2% paraformaldehyde at room temperature
for 15 min followed by washing with PBS briefly. Cells
were stained with Oil Red O solution for 30 min. Cells
were washed three times with PBS and the nuclei were
counterstained with hematoxylin. After washing, cells were
examined by light microscopy (magnification, 9200;
ULWCD 0.30, Olympus Optical, Tokyo, Japan). Foam
cells were defined as the cells with more than 10 Oil Red
O-positive droplets or the cells with areas of droplets
bigger than the nuclei.
Western blot analysis
Cells were lysed using a Tris–glycine buffer (0.25 M Tris,
0.173 M glycine) containing 3% SDS and 1 mM PMSF
(phenylmethylsulfonil fluoride). Aliquots of the samples
were diluted in a 2% b-mercaptoethanol buffer containing
glycerol and bromophenol blue and electrophoresed on 8%
SDS-polyacrylamide gels, and then the proteins were
electrotransferred to nitrocellulose membranes. Membranes were blocked with TBS-T buffer (20 mM Tris base,
150 mM NaCl, 0.1% Tween 20, pH7.6) containing 5% fatfree milk for 2 h at room temperature and then incubated
with primary antibodies overnight at 4°C. The blots were
rinsed three times (3 9 10 min) with TBS-T buffer and
then incubated with horseradish peroxidase-conjugated
goat anti-rabbit IgG for another 1 h at room temperature.
After washing three times with TBS-T buffer, the membranes were incubated for 2 min in a mixture of equal
volumes of western blot enhanced chemiluminescence
reagents 1 and 2 (Pierce Biotechnology, Rockford, IL). The
membranes were then exposed to film before development.
The primary antibodies were as follows: 1:200 for rabbit
polyclonal anti-PPARc and anti-phospho PPARc antibody
(Abcam, Cambridge, United Kingdom); 1:500 for rabbit
polyclonal anti-p38 and anti-phospho p38 antibody (Cell
Signaling Technology Inc., MA,USA); 1:500 for rabbit
polyclonal anti-p44/42 and anti-phospho p44/42 antibody
(Cell Signaling Technology Inc., MA,USA); 1:1,000
for rabbit polyclonal anti-actin antibody (Santa Cruz,
Biotechnology Inc., CA, USA).
173
Results
oxHDL decreases CD36 expression
Thorne et al. reported that CD36 can act as a receptor for
oxidized HDL [9]. Since CD36 expression can be regulated
by many factors including its own ligand oxLDL, we
first set out to investigate the effect of oxHDL on CD36
expression on human monocytes-derived macrophages.
Cells cultured in serum-free RPMI 1640 medium
were incubated with various concentrations of oxHDL
(0–100 lg/ml) for 24 h. RT-PCR analysis was performed
to determine mRNA expression of CD36. The result
showed that oxHDL decreased CD36 mRNA expression in
a dose-dependent manner. Approximately 50–100 lg/ml
final concentration of oxHDL caused a 40% reduction of
CD36 expression. In addition, when we used oxHDL
(50 lg/ml) to treat cells for different times, it showed that
oxHDL also decreased the expression of CD36 in human
macrophages in a time-dependent way (Fig. 1).
We further investigated the effect of oxHDL on surface
protein expression of CD36 by flow cytometric analysis.
As shown in Fig. 2, both oxHDL and HDL caused a significant reduction of CD36 protein expression on cell surface, which is consistent with the change of mRNA
expression.
oxHDL accelerates foam cell formation
Given the fact that uptake of oxLDL by CD36 induces
foam cell formation, we next examined the effect of oxHDL on this process. Cells were treated with oxHDL or
HDL (both at 50 lg/ml) for 24 h and then subjected to Oil
Red O staining. Foam cells were defined as the cells with
more than 10 Oil Red O-positive droplets or the cells with
areas of droplets bigger than the nuclei. We quantified the
percentage of foam cells and the result suggested that
compared with HDL, oxHDL significantly accelerated
foam cell formation. Whereas blocking CD36 receptor can
partially inhibit the effect of oxHDL. When cells were
treated by oxHDL together with CD36 antibody, foam cell
formation rate was decreased to a similar level to HDLtreated cells, suggesting that the uptake of oxHDL into
macrophages through CD36 contributes to foam cell formation (Fig. 3).
oxLDL can reverse the inhibitory effect of oxHDL
on CD36 expression
In view of evidence that both oxHDL and oxLDL exist in
vivo and CD36 is the major scavenger receptor for oxLDL,
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Mol Cell Biochem (2010) 342:171–181
0
oxHDL
A
12 .5
25
50
100
12 h
24h
48 h
(µg/m l)
CD36
GAPDH
B
oxHDL
0h
(100µg/ml)
6h
CD36
GAPDH
D
CD36/GAPDH mRNA
CD36/GAPDH mRNA
C
1.2
0.8
*
*
0.4
**
0
0
12 .5
25
50
*
12h
24 h
**
0.4
0
0h
6h
48 h
28 h) and mRNA expression were evaluated by RT-PCR analysis. c
and d Quantification data from multiple experiments. Bar graph
shows the relative band intensities of CD36 mRNA after densitometry
and normalized to GAPDH mRNA expression. The values are
mean ± S.E. of three independent experiments performed in duplicate. * P \ 0.05, ** P \ 0.01, compared with control
B
CD36 (+) cell (%)
Red -oxHDL
Green -control
C
*
0.8
A
100
*
80
blue -HDL
Green -control
**
60
40
20
0
Control
oxHDL
we next performed experiments to investigate what will be
the consequences when cells were treated with both
oxHDL and oxLDL. As shown in Fig. 4, oxLDL
123
1.2
100 ( µg/ml)
Fig. 1 oxHDL inhibits mRNA expression of CD36 in macrophages.
a Human monocyte-derived macrophages were incubated with
various concentrations of oxHDL (0, 12.5, 25, 50, 100 lg/ml) for
24 h. RT-PCR analysis was performed to examine the mRNA
expression. b Human monocytes-derived macrophages were incubated with 100 lg/ml of oxHDL for different times (0, 6, 12, 24,
Fig. 2 oxHDL decreases CD36
surface protein expression in
macrophages. a and b human
monocyte-derived macrophages
were treated with 50 lg/ml of
oxHDL or HDL as indicated for
24 h. CD36 surface protein was
analyzed by FACS assay with
PE-conjugated anti-human
CD36 antibody. c The relative
expression of CD36 was
quantified by comparing mean
fluorescence intensities of each
population with the control
sample. The values are
mean ± S.D. of three
independent experiments.
* P \ 0.05, ** P \ 0.01,
compared with control
1.6
HDL (50µg/ml)
significantly reversed the inhibitory effect of oxHDL on
CD36 expression. Co-incubation of 5 lg/ml of oxLDL
partially rescued CD36 expression with 50 lg/ml of
Mol Cell Biochem (2010) 342:171–181
Fig. 3 Uptake of oxHDL
through CD36 accelerates foam
cell formation. a Human
monocyte-derived macrophages
were incubated with PBS buffer,
50 lg/ml of oxHDL, 50 lg/ml
HDL and oxHDL plus antihuman CD36 antibody (1:100)
respectively for 24 h. Oil Red O
staining was performed to
examine foam cell formation.
Foam cells were defined as the
cells with more than 10 Oil Red
O-positive droplets or the cells
with areas of droplets bigger
than the nuclei. b Percentage
of foam cells was quantified
from multiple experiments. The
values are mean ± S.D. of three
independent experiments
performed in duplicate.
* P \ 0.05, ** P \ 0.01.
Compared with control
175
A
control
HDL
oxHDL
oxHDL+Ab
**
B
CD36 (+) cell (%)
*
*
*
60%
50%
40%
30%
20%
10%
0%
control
oxLDL totally reversing the effect of oxHDL with the
expression of CD36 remaining at basal levels.
oxHDL increases PPARc mRNA expression
and protein phosphorylation
It has been shown that PPARc is essential for the basal
regulation of CD36 and many factors regulate CD36
expression through a PPARc dependent mechanism [3]. In
order to determine the mechanism by which oxHDL
decreased expression of CD36, we next investigated the
effect of oxHDL on PPARc expression. As shown in Fig. 5a
and b, oxHDL significantly increased PPARc mRNA
expression in human macrophages. In addition, PPARc
protein levels and PPARc phosphorylation were evaluated
by western blot analysis (Fig. 5c, d). Treatment with oxHDL
significantly increased the phosphorylated form of PPARc
HDL
oxHDL
OH+Ab
protein expression. PPAR total protein levels also showed an
increase but this trend was not significant.
p38-MAPK pathways is involved in the regulation
of CD36 by oxHDL
Previous studies have demonstrated that PPARc can be
phosphorylated by mitogen-activated protein kinase
(MAPK) and phosphorylation inhibits PPARc activity, thus
induces the decreased expression of CD36 [11]. We suspected that MAPK pathways may also be involved in
oxHDL-mediated regulation of the expression of CD36 and
PPARc. We examined the impacts of MAP kinase inhibitors on the expression of CD36 and PPARc in oxHDLtreated human macrophages. As shown in Fig. 6, in presence of the p38-MAPK inhibitor SB203580 (10 lM) the
negative regulatory effect of oxHDL on CD36 mRNA
expression was abolished. Similarly, the increase in mRNA
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Mol Cell Biochem (2010) 342:171–181
Fig. 4 oxLDL can reverse the
inhibitory effect of oxHDL on
CD36 expression. a Human
monocyte-derived macrophages
were incubated with 50 lg/ml
of oxHDL in presence of
different concentrations of
oxLDL (0, 5,10, 25, 50 lg/ml)
for 24 h. CD36 mRNA
expression was determined by
RT-PCR analysis. b
Quantification data from
multiple experiments. Bar
graph shows the relative band
intensities of CD36 mRNA after
densitometry and normalized to
GAPDH mRNA expression.
The values are mean ± S.E. of
three independent experiments
performed in duplicate.
* P \ 0.05, compared with
control; à P \ 0.05, compared
with oxHDL group
A
-
oxHDL (50µg/ml)
oxLDL (µg/ml)
+
-
+
+
+
+
5
10
25
50
CD36
GAPDH
CD36/GAPDH mRNA
B
2.4
‡
2
*
*
‡
*‡
+
+
+
+
5
10
25
50
‡
*
1.6
1.2
0.8
0.4
0
oxHDL (50µg/ml) oxLDL (µg/ml)
+
-
-
B
oxHDL
0
25
/GAPDH mRNA
A
50 (µg/m l)
PPAR
PPAR
GAPDH
1.6
0.8
0.4
0
OH-0
0
25
50 (µg/m l)
PPAR
actin
PPAR -Pi
OH-25
OH-50 (µg/ml)
D
protein/control
oxHDL
PPAR
C
*
*
1.2
3.5
*
3
*
2.5
2
1.5
1
0.5
0
OH-0
OH-25
OH-50 (µg/ml)
Fig. 5 oxHDL increases PPARc mRNA expression and protein
phosphorylation. a Human monocyte-derived macrophages were
incubated with 25 and 50 lg/ml of oxHDL respectively for 24 h.
PPARc mRNA expression was determined by RT-PCR analysis. b
Quantification data from multiple experiments. Bar graph shows the
relative band intensities of PPARc mRNA after densitometry and
normalized to GAPDH mRNA expression. The values are
mean ± S.D. of three independent experiments performed in duplicate. * P \ 0.05, compared with control. c Human monocyte-derived
macrophages were incubated with 25 and 50 lg/ml of oxHDL
respectively for 24 h. Cells were harvested and total protein was
resolved by SDS-PAGE and immuno-blotted with anti-PPARc, antiphospho PPARc and anti-actin antibodies as described in experimental procedure. d Quantification data from multiple experiments. Bar
graph shows the relative band intensities of PPARc protein and
phosphorylated PPARc protein after densitometry and normalized to
control group. The values are mean ± S.D. of three separate
experiments. * P \0.05, compared with control
expression of PPARc by oxHDL was also blocked by
SB203580. However, MEK1/2 inhibitor U0126 (10 lM)
and JNK inhibitor SP600125 (10 lM) showed little effect
(Fig. 6). Statins have been showed to regulate CD36
expression [13, 14], but our results suggested lovastatin did
not change the effect of oxHDL.
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Mol Cell Biochem (2010) 342:171–181
177
oxHDL
A
control
oxHDL
U0126
SP600125
SB203580
Lov
CD36
PPAR
GAPDH
CD36/GAPDH mRNA
*
0.8
*
*
*
0.6
0.4
0.2
0
oxHDL
U0126
SP60012 5
SB203580
Lov
/GAPDH mRNA
C
1
1.4
PPAR
B
0.4
*
*
*
+
-
+
+
-
+
+
-
*
1.2
1
0.8
0.6
0.2
0
-
+
-
+
+
-
+
+
-
+
+
-
+
+
oxHDL
U0126
SP60012 5
SB203580
Lov
-
+
+
-
+
+
Fig. 6 Effects of different kinase inhibitors and lovastatin on oxHDL
mediated regulation of mRNA expression of CD36 and PPARc.
a Human monocyte-derived macrophages were incubated with
50 lg/ml of oxHDL in presence of different kinase inhibitors
(10 lM of U0126, SP600125, SB203580 and Lovastatin respectively)
for 24 h. CD36 and PPARc mRNA expression was determined by
RT-PCR analysis. b and c Quantification data from multiple
experiments. Bar graphs show the relative band intensities of CD36
or PPARc mRNA after densitometry and normalized to GAPDH
mRNA expression. The values are mean ± S.D. of three independent
experiments performed in duplicate. * P \ 0.05, compared with
control
In addition, we further investigated the impact of MAPK
inhibitors on PPARc protein expression and its activation.
As shown in Fig. 7, western blot analysis suggested that
p38-MAPK inhibitor SB203580 abolished the increased
phosphorylation of PPARc mediated by oxHDL, whereas
U0126, SP600125, and lovastatin showed little effect.
Based on the above results, we next performed western
blot analysis to examine the direct effects of oxHDL on
p38 and p44/42 MAP kinases activities in macrophages. As
shown in Fig. 8, oxHDL treatment significantly increased
phosphorylation of p38 MAP kinase, but not p44/42 MAP
kinase. These results suggest that p38-MAPK signal pathways are involved in oxHDL-mediated regulation of CD36
expression.
atherosclerosis. Our results suggest that the uptake of
oxHDL mediated by CD36 accelerated the formation of
foam cells. But in contrast to oxLDL which increases
CD36 expression, oxHDL displayed an inhibitory effect on
CD36 expression on macrophages. Phosphorylation of
PPARc by p38-MAPK pathway may be involved in it.
CD36, a class B scavenger receptor, is expressed on
various tissues including platelets, monocytes/macrophages, microvascular endothelial cells, retinal pigment
epithelium, striated and smooth muscle, and adipose tissue.
Like other scavenger receptors, CD36 recognizes a broad
variety of ligands including oxLDL, anionic phospholipids,
long-chain fatty acids, apoptotic cells, thrombospondin,
collagen, plasmodium falciparum-infected erythrocytes,
and so on [15]. CD36 may also bind HDL, but SR-BI
mediates uptake of HDL with much greater efficiency than
CD36 [16]. On macrophages, CD36 is a major scavenger
receptor for oxLDL that accounts for as much as 40% of
oxLDL uptake by human macrophages [17]. Extensive
evidence point to a significant role of CD36 in atherosclerosis and suggest it could be an important target for
therapeutic treatment. Loss of CD36 resulted in significant
Discussion
The formation of lipid laden foam cells is an important
pathophysiological process in atherosclerosis. The expression and regulation of CD36, a major scavenger receptor
for oxLDL, is critical for the development of
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Mol Cell Biochem (2010) 342:171–181
Fig. 7 Effects of different
kinase inhibitors and lovastatin
on oxHDL mediated regulation
of protein expression of PPARc.
a Human monocytes-derived
macrophages were incubated
with 50 lg/ml of oxHDL in
presence of different kinase
inhibitors (10 lM of U0126,
SP600125, SB203580 and
Lovastatin respectively) for
24 h. PPARc protein level and
phosphorylated protein level
were determined by western
blot analysis. b Quantification
data from multiple experiments.
Bar graph shows the relative
band intensities of PPARc
protein and phosphorylated
PPARc protein after
densitometry and normalized to
control group. The values are
mean ± S.D. of three separate
experiments. * P \ 0.05,
** P \ 0.01, compared with
control
A
oxHDL
oxHDL
A
control
oxHDL
U0126
SP600125
SB203580
Lov
PPAR -Pi
PPAR
actin
PPAR
protein/control
B
2
1.6
25
*
*
*
0.8
0.4
0
oxHDL
U0126
SP600125
SB203580
Lov
0
** *
1.2
-
50 (µg/ml)
+
-
+
+
-
oxHDL
0
p38-Pi
p44/42-Pi
p38
P44/42
+
+
-
25
+
+
-
+
+
50 (µg/ml)
B
Phospho-protein/control
actin
1.6
1.2
**
0.8
0.4
0
OH-0
OH-25
Fig. 8 Effects of oxHDL treatment on protein phosphorylation of
MAP kinases. a Human monocyte-derived macrophages were incubated with 25 and 50 lg/ml of oxHDL respectively for 24 h. Total
protein form and phosphorylated protein form of p38-MAP kinase
and p44/42 MAP kinase were evaluated by western blot analysis.
123
*
OH-50 (µg/ml)
b Quantification data from multiple experiments. Bar graph shows
the relative band intensities of phosphorylated p38 and p44/42 MAP
kinase protein after densitometry and normalized to control group.
The values are mean ± S.D. of three separate experiments. * P\0.05,
** P \ 0.01, compared with control
Mol Cell Biochem (2010) 342:171–181
decreased ability of monocytes-derived macrophages to
accumulate oxLDL and cholesteryl ester in human or animals [17, 18]. Inhibition of CD36 expression has been
shown to reduce the development of atherosclerosis in
mice [19].
A number of cytokines or agents have been identified to
be able to regulate CD36 expression: oxLDL, phorbol
myristate acetate, IL-4, and M-CSF increase the mRNA and
protein levels of CD36, whereas HDL, lipopolysaccharide,
and dexamethasone decrease monocyte CD36 expression
[20]. It is demonstrated that oxLDL can stimulate its own
uptake by induction of CD36 gene expression [10]. In our
study, oxHDL can down-regulate the expression of CD36 in
human monocytes in both a time-dependent and dosedependent way, which is similar to HDL.
Although SR-A and CD36 is the major receptor for oxLDL, double-knockout of CD36 and SR-A in Apo E null
mice failed to eliminate foam cell formation, suggesting
there should be other ways for monocytes to uptake lipid
[21]. The cellular receptors for oxHDL are poorly defined,
and only SR-BI and LOX-1 on endothelial cells are reported
to bind hypochlorite-modified HDL [8]. Thorne et al. demonstrated that CD36 is also a receptor for oxHDL on macrophages [9]. CD36 selectively elicited lipid uptake from
Cu2?-oxidized HDL but not from native HDL or LDL.
CD36-mediated uptake of oxHDL by macrophage may
contribute to atheroma formation. Our results suggest that
oxHDL uptake through CD36 induced the formation of foam
cells. But in contrast to oxLDL which increases CD36
expression, oxHDL resulted in a reduction of CD36
expression on monocytes. This effect is similar to HDL but
seems conflict with the pro-atherogenic role for oxHDL.
There are several possible explanations: first, after uptake
into cells, oxHDL may activate some signal transduction
pathways to evoke foam cell formation, which cannot be
reversed by its inhibitory role on CD36 expression level.
Second, oxHDL increases foam cell formation through other
ways or receptors. Furthermore, oxHDL activates p38MAPK pathway and induces cell apoptosis. This apoptotic
process inhibits the differentiation from monocytes to
macrophages and induces the decreased expression of
CD36. Finally, in certain circumstances, oxHDL acts like
HDL and performs protective functions in atherosclerosis. It
has been proven that oxHDL enhances the depletion of
cellular cholesterol in many cell types, including human or
animal monocytes, human fibroblasts, and human aortic
smooth muscle cells [7, 22]. In our study, mixing oxLDL
with oxHDL prevented the decrease in CD36 expression
seen with oxHDL, suggesting that oxLDL is a much stronger
regulator for CD36 expression. Therefore, in foam cell
formation, the effect of oxHDL may be masked by oxLDL.
Compelling evidence has proven that the circulating
HDL level is inversely related to the incidence of
179
cardiovascular disease. HDL is believed to play a protective function against the development of cardiovascular
disease due to its role in mediating the reverse cholesterol
transport and other multiple beneficial effects such as
preventing the oxidative modification of LDL and cell
signaling mediated by oxLDL, and inhibiting several
adverse biological effects including cytotoxicity and
inflammatory response triggered by cytokines, oxLDL, or
oxidants [23]. HDL is susceptible to damaging structural
modification including oxidation. Studies of animal and
human have demonstrated the existence of oxidized HDL
in plasma and atherosclerotic lesion [5, 6, 24]. Although
the central role of oxLDL in atherogenesis has been widely
accepted, the role of oxHDL remains contested. Evidence
has suggested that after oxidation, HDL shows a pro-atherogenic role. Oxidation markedly impaired the function of
HDL to promote reverse cholesterol transport and oxHDL
displayed decreased ability in protecting endothelium,
inhibiting oxLDL oxidation and so on [7]. However, there
is also evidence suggests that mild oxidized HDL showed
some protective effect, such as promoting reverse cholesterol transport. Mild oxidative modification of HDL (tyrosyl radical or Cu2? mediated oxidation) improves its
function as cholesterol acceptor, increasing reverse cholesterol transport, but highly oxidative HDL cannot [7, 22].
Administration of tyrosyl radical-oxidized HDL inhibited
the development of atherosclerosis in Apo E-deficient mice
[25]. Thus, the direct role of HDL in atherosclerosis is
needed to be investigated, and more importantly, the exact
role of oxHDL in vivo is likely to be more complicated. It
is hypothesized that mild oxidation of HDL, by oxidants
that generate apoprotein crosslinks without large amounts
of lipid peroxidation products, might play beneficial roles
in vivo [26]. In our study, the degree of oxidation should be
low due to the TBARS content was only 3.5 nmol/ml
protein HDL.
PPARc is a ligand-activated transcription factor that has
been demonstrated to regulate glucose and lipid metabolism [27]. Studies showed that PPARc can be phosphorylated in the NH2-terminal domain via the MAP kinase
signaling pathway, which in turn will decrease the transcriptional activity of PPARc. CD36 is an important
PPARc-responsive genes and it has been proven that
PPARc activation by its ligands, such as oxLDL and
thiazolidinediones, can increase the expression of CD36
[20]. CD36 is barely detectable in the absence of PPARc,
suggesting that PPARc is essential for the basal regulation
of CD36 [28]. Han et al. reported that HDL can decrease
the expression of macrophage CD36 through MAP kinasemediated phosphorylation of PPARc [11]. Our results
suggest that oxHDL also down-regulate CD36 expression
through the phosphorylation of PPARc, which is mediated
by p38-MAPK pathway. The effects of oxHDL on PPARc-
123
180
mediated signaling are inhibited by p38-MAPK blocker
SB203580, whereas other MAPKs inhibitors showed little
effect. p38, but not p44/42 MAP kinase is phosphorylated
in response to incubation with oxHDL. Therefore, we
propose that oxHDL increases p38 MAP kinase activity
and PPARc phosphorylation. Since the phosphorylated
form of PPARc is a negative regulator of transcription, it
will further inhibit PPARc-dependent CD36 expression. It
is also reported that oxHDL can increase the expression of
plasminogen activator inhibitor-1 in endothelial cells
through p38-MAPK-mediated pathway [29].
In summary, our data demonstrate that macrophage
uptake of oxHDL by CD36 can accelerate foam cell formation. oxHDL can inhibit the expression of CD36 on
macrophages through a p38-MAPK-mediated phosphorylation of PPARc way. Thus, oxHDL has both its good side
and bad side. One hand, it reduces the expression level of
CD36, which may inhibit the uptake of oxLDL into macrophages. Other hand, oxHDL increases the rate of the
formation of foam cells, which may in the long run be more
deleterious. The exact responsible mechanisms remain to
be established. Further studies will be needed to resolve
this issue. Unraveling the signaling events initiated by
binding of oxHDL to macrophages may help in understanding the atherosclerotic pathogenesis.
Acknowledgments This study was supported by the National Natural Sciences Fund Committee (Grant no. 30570712) in China. We
thank Ms. P.Y. He and Y.J. Liu for their expert technical assistance,
Dr. H. L. Rui and Dr. J.J. Cui for their kind help with chemicals and
discussions.
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