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Introduction
Massive clustering of macrophage-derived foam cells in the subendothelial spaces of arterial walls is one of the
characteristic features of the early stages of atherosclerotic
lesions[1]. Macrophages take up oxidized low-density lipoprotein
(ox-LDL) through the scavenger receptor pathways and transform into foam
cells[2]. Foam cells produce various bioactive
molecules, such as cytokines and growth factors, and are believed to play an important role in the development and
progression of atherosclerosis[1].
Multiple intracellular signal pathways, including peroxisome proliferator-activated receptor
g (PPARg), have been reported to be involved in macrophage-derived foam cell
formation[3]. PPARg is a member of a nuclear hormone superfamily that
heterodimerizes with the retinoid X receptor. These proteins are transcriptional regulators of genes that encode proteins
involved in adipogenesis and lipid
metabolism[4]. 15-deoxy-D12,14 prostaglandin
J2 (15d-PGJ2) and the thiazolidinedione
(TZD) class of antidiabetic drugs is nature and synthesis ligand of
PPARg, respectively[5]. Components of ox-LDL, including
9-hydroxyoctadecadienoic acid (9-HODE) and 13-HODE also activate
PPARg and subsequently induce the expression of the
CD36 scavenger receptor, a key mediator for uptake of ox-LDL in
macrophage[6]. This observation suggested that
PPARg ligand might promote the formation of foam cells. But Chinetti et al have shown that the treatment of human macrophages
with PPARg agonists did not facilitate foam cell formation because they induced the expression of ATP-binding cassette
transporter, class A1 (ABCA1), a transporter that controls apoAI-mediated cholesterol efflux from macrophages. These
effects are likely to be caused by the enhanced expression of liver-x-receptor alpha
(LXRa), an oxysterol-activated nuclear receptor that induces ABCA1 transcription. In fact, Chinetti et al showed that PPARg activators increased apoAI-induced
cholesterol efflux from macrophage-derived foam
cells[7]. Thus, the effects of ox-LDL uptake in response to increased
macrophage CD36 expression following PPARg activation is balanced by
LXRa activation and ABCA1-mediated cholesterol efflux. Because previous studies implicated
PPARg in both proatherogenic and antiatherogenic pathways mediated by
components of ox-LDL and synthesis PPARg agonist, respectively, we hypothesized that, in addition to activating
PPARg in a ligand-dependent manner, other components of ox-LDL might have a negative regulatory effect on
PPARg activity through unidentified mechanisms.
Several studies have shown that
PPARg is a phospho-protein. Multiple kinase pathways, such as cAMP-dependent
protein kinase (PKA), AMP-activated protein kinase (AMPK), and mitogen-activated protein kinase (MAPK), have been
implicated in the regulation of PPARg
phosphorylation[8]. Phosphorylation significantly inhibits both ligand-independent
and ligand-dependent transcriptional activation by
PPARg[9]. The implications of the post-translational modification of
PPARg activity through phosphorylation might be the pathway by which various growth factors and cytokines could affect
the transcription of numerous genes involved in lipid metabolism as well as lipid homeostasis in the macrophage-derived
foam cells induced by ox-LDL.
The present study was designed to study the role of
PPARg phosphorylation in macrophage-derived foam cell formation
induced by ox-LDL. We found that ox-LDL evaluated
PPARg phosphorylation status during foam cell formation.
ox-LDL-induced PPARg phosphorylation was mediated by c-Jun N-terminal kinase
(JNK)-MAPK activation. Treatment of JNK inhibitor suppressed
PPARg phosphorylation and subsequently prevented ox-LDL-induced foam cell formation. These
observations demonstrate that PPARg phosphorylation mediated by MAPK facilitates foam cell formation from macrophages
exposed to ox-LDL.
Materials and methods
Cell culture The human monocytes line THP-1 was obtained from the cell bank in Shanghai Institute for Biological
Sciences, Chinese Academy of Sciences. THP-1 cells were cultured in RPMI-1640 medium supplemented with 10%
(v/v) fetal bovine serum, 100 U/mL penicillin, 100 µg/mL strepto-mycin, 2 mmol/L glutamine, and 12 mmol/L sodium carbonate. Cell
cultures were maintained and incubated in a humidified atmosphere containing 5%
(v/v) CO2 at 37 °C. Differentiation of
THP-1 monocytes into macrophages was induced by culturing the cells at a density of
1.0×106 cells/well in a 6-well plate in the
presence of phorbol 12-myristate 13-acetate (PMA) 160 nmol/L for 24 h. Cells were then cultured for another 48 h without
PMA, washed with serum-free medium or buffer to remove non-adherent cells, and then incubated with the respective stimuli
for various periods in serum-free medium.
LDL isolation and oxidization Human LDL (1.019-1.063 g/mL) were prepared from different human healthy donors by
density gradient ultracentrifugation in the presence of 1 mg/mL EDTA (pH 7.4). The isolated LDL was dialyzed to remove
EDTA and filtered (0.22 µm pore size), and stored at 4 °C. The LDL was analyzed for protein content by the Bradford method,
using bovine serum albumin as standard. The purity and charge of the lipoproteins were evaluated by examining
electrophoretic mobility in an agarose gel. Oxidation of LDL was carried out with copper sulfate (final concentration of 10
mmol/L) at 37 °C for 12 h. The degree of oxid-ation was determined by measuring the amount of thiobar-bituric acid-reactive
substances (TBARS). ox-LDL had TBARS of 18 nmol/mg. ox-LDL was then dialyzed against PBS containing EDTA 0.01% for 24
h at 4 °C and sterile filtered.
Oil red O staining In parallel experiments, THP-1-derived macrophages were plated at a density of
1.0×106 cells/well in a 6-well plate containing glass coverslips and incubated in serum-free RPMI-1640 medium in the presence of ox-LDL 100
µg/mL at 37 °C for 48 h. Cells were washed three times with PBS, fixed by 10% formalin in PBS for 1 h at room temperature, and
then stained with 0.1 mL/mL Oil red O solution for 2 h, washed three times with water, and vaporized of all water (at 32 °C for
45 min). Cells were viewed in situ in 35-mm diameter tissue culture plates under a bright-field microscope in 100×fields using
a microscope (Olympus IX70, Tokyo, Japan).
Measurements of free and total cholesterol THP-1-derived macrophages (5×105 cells/mL) were added to each well of a 24-well plate with ox-LDL (100
µg/mL). The incubation at 37 °C lasted for 48 h. The THP-1 cells were washed three times in PBS,
then 1 mL isopropyl alcohol was added, and the cells were
sonicated for 30 s. Total cholesterol and free cholesterol in extracts
were determined by the cholesterol oxidase enzymatic method using a commercial kit by a Hitachi 7020 autoanalyser (Tokyo,
Japan). Lipid-extracted cells were dissolved in 0.1% sodium dodecyl sulfate-0.1 mol/L NaOH for 30 min, and total cell protein
was determined with a protein assay kit. Esterified cholesterol was calculated from (total cholesterol)-(free cholesterol)
values. Results were expressed in mg/g protein.
Western blot analysis of
PPARg/phosphorylated PPARg After treatment, cells were washed twice with PBS and then
resuspended in 400 µL of cold buffer A (10 mmol/L HEPES, pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1
mmol/L DTT, 0.5 mmol/L PMSF). After 15-min incubation on ice, 25 µL of 10% NP-40 was added to the cell suspension, which
was subjected to a vortex for 10 s. The supernatant was removed after being spun for 30 s at 13
150×g. The pellet was resuspended in 100 µL of cold buffer C (20
mmol/L HEPES, pH 7.91, 400 mmol/L KCl, 1 mmol/L EDTA, 1 mmol/L EGTA, freshly
added 1 mmol/L DTT, 1 mmol/L PMSF, 1 µg/mL pepstatin A, 1 µg/mL leupeptin, 0.1 mmol/L P-aminobenza-midine, and 10
µg/mL aprotinin) and kept for 15 min at 4 °C. The mixture was spun for 5 min at 13
150×g. and the supernatant was collected as
nuclear proteins. Nuclear proteins (500 µg) from each sample were incubated with an antibody to mouse
PPARg antibody (Sigma Chemical Co, St Louis, MO, USA). Immunoabsorbed proteins were separated by SDS-PAGE and transferred onto
nylon-enhanced nitrocellulose membrane, then analyzed by Western blot for phosphorylated
PPARg (PPARg-Pi) by incubation with anti-phosphoserine antibodies (Sigma). The nuclear proteins were also used to analyze
PPARg protein expression by SDS-PAGE/Western blot.
MAPK activity assay JNK, p38, and extracellular signal-regulated kinase (ERK) activities were detected using a
stress-activated protein kinase (SAPK)/JNK, p38, and p44/42 MAP Kinase Assay Kit, respectively, according to the manufacturer's
instructions (Cell Signaling, Beverly, USA). For the p38 and ERK assays, aliquots of 200 µg of protein were incubated with
immobilized phospho-specific p38 or ERK MAPK monoclonal antibody. After washing with lysis and kinase buffer, pellets
were suspended in kinase buffer with 200 µg ATP and 2 µg ATF-2 or Elk-1 fusion proteins and incubated at 30 °C for 30 min.
For the JNK kinase assay, 250 µg of protein was incubated with 2 µg c-Jun fusion protein beads. After washing, pellets were
suspended in kinase buffer with 100 µg ATP and incubated at 30 °C for 30 min. The reaction was terminated with SDS sample
buffer, and boiled samples were analyzed by Western blotting using corresponding phospho-specific antibodies.
RT-PCR Total RNA was isolated using Trizol reagent. Total RNA content was determined by measuring the optical
absorbance ratio at 260/280 nm after the sample was dissolved in diethylpirocarbonate-treated water. RNA was then stored
at -70 °C before two-step RT-PCR protocol using 2 µg of total RNA. RNA was treated with DNase I, reverse transcribed, and
amplified for ABCA1 and GAPDH using PCR enzymes and reagents according to the following conditions: 10
min 95 °C, then 34 cycles of 1 min 95 °C, 1 min 60 °C, and 1 min 72 °C, and then a final annealing step at
72 °C for 10 min. PCR amplification was performed using ABCA1 (306 bp) primers (forward:
5'-GCTGCTGAAGCCA-GGGCATGGG-3' and reverse: 5'-GTGGGGCAGTGGCCATA-CTCC-3') and GAPDH (697 bp) primers (forward:
5'-TCACCA-TCTTCCAGGAGCCGAG-3', reverse: 5'-TGTCGCTGTTGAA-GTCAGAG-3'). PCR products were separated on 1.5% agarose
gel containing ethidium bromide. Densitometric quantita-tion of the intensity of GAPDH and ABCA1 products was
determined using the "Quantity One" quantitation analysis software (Bio-Rad Laboratories, Hercules, CA, USA). The relative
abundance of ABCA1 was expressed as the ratio of ABCA1 to GAPDH product.
Statistical analysis Data were expressed as mean±SD. Statistical significance of the data was evaluated by analysis of
variance and q test. P<0.05 was considered significant. All experiments were performed a minimum of three times. Results
ox-LDL increases PPARg phosphorylation After the cells were incubated with
15d-PGJ2 (20 µmol/L), troglitazone (5
mmol/L) or ox-LDL (25, 50, 100 µg/mL) for 12 h, total and phosphorylated
PPARg were determined by Western blot. As shown in
Figure 1A, no significant change of total and phosphorylated
PPARg was observed after incubation with
15d-PGJ2 and troglitazone, which indicated nature or synthesis
PPARg ligand was not involved in transcriptional and post-transcriptional
regulation of PPARg. In contrast, when macrophages were incubated with
ox-LDL, both total and phosphorylated PPARg were increased in a dose-dependent manner.
It is noteworthy that the ratio of phosphorylated/total
PPARg was also elevated
d by ox-LDL. Thus, our
results indicated that ox-LDL induced PPARg phosphorylation in THP-1 cells. Previous studies have demonstrated that
phosphorylation of PPARg inhibited both its
ligand-dependent and ligand-independent transcriptional
activity[9]. Therefore, we subsequently studied the effects of these compounds on the expression of ABCA1, a well-known
PPARg target gene involved in cholesterol efflux. As shown in
Figure 1B, treatment of THP-1 cells with the three compounds for 12 h all increased ABCA1 mRNA level. However, we found
that the elevation of ABC1 mRNA induced by ox-LDL did not occur in a dose-dependent manner. Instead, the mRNA level of
ABCA1 induced by higher concentration of ox-LDL was less than the lower (although it lacks statistical significance). These
observations clearly demonstrated that PPARg phosphorylation status was negatively correlated with
PPARg target gene expression.
ox-LDL activates MAPK Recent investigations have demonstrated that the MAPK are activated by ox-LDL
stimulation[10,11]. Because previous studies have demonstrated that
PPARg is phosphorylated by the MAPK family
members[8], we hypothesized that ox-LDL-induced MAPK activation may regulate
PPARg phosphorylation in macrophage-derived foam cells. We first explored whether ox-LDL is
able to activate MAPK in the human monocytic cell line
THP-1. The application of ox-LDL resulted in increased activities of all three MAPK limbs. The increases of ERK- and p38-MAPK
activity peaked at 24 h followed by failing to basal level at 48
h. In contrast, increased JNK activity maintained up to 72 h. The different kinetics,
however, of the three MAPK suggested that they might play different roles in ox-LDL-induced macrophage foam cell
formation (Figure 2).
ox-LDL-induced phosphorylation of
PPARg is blocked by a JNK-MAPK inhibitor To investigate whether
ox-LDL-induced MAPK activation regulates PPARg phosphorylation in THP-1 macrophages, we used inhibitors that are selective for
each MAPK cascade (PD98059, an inhibitor for ERK; SP600125 for JNK;
SB203580 for p38) to evaluate their effects on
PPARg phosphorylation induced by ox-LDL. When ox-LDL-treated macrophages were incubated with
PD98059 (20 µmol/L) or SB203580 (20 µmol/L) for 12 h,
PPARg phosphorylation status did not change. In contrast, treatment with
SP600125 (20 µmol/L) significantly inhibited
ox-LDL-induced PPARg phosphorylation. These observations demonstrate that JNK
may be predominantly responsible for ox-LDL-induced
PPARg phosphorylation during macrophage foam cell formation (Figure 3).
Foam cell formation induced by
ox-LDL is attenuated by inhibition of PPARg phosphorylation To determine the impact of MAPK activation and
PPARg phosphorylation on ox-LDL-induced foam cell formation, we used SP600125, a specific JNK
inhibitor, to evaluate its effect on cholesterol accumulation by staining with Oil red O. Cholesterol accumulation was greatly
increased in cells incubated with ox-LDL (100 µg/mL) for 48 h. When THP-1 cells were incubated with ox-LDL in the presence
of different concentrations of SP600125 (5, 10, and 20 µmol/L), cholesterol accumulation decreased in a dose-dependent
manner (Figure 4). These results were consistent with the morphological features identified by Oil red O staining. Thus, these
data suggested that the JNK pathway was involved in
PPARg phosphorylation and macrophage foam cell formation induced
by ox-LDL.
Discussion
In this study, we have observed an effect of
ox-LDL on PPARg phosphorylation in THP-1-derived macrophage.
We found that ox-LDL evaluated PPARg phosphorylation during foam cell formation. ox-LDL-induced
PPARg phosphorylation was mediated by MAPK activation.
Using pharmaceutical inhibitors, we found that activation of the JNK pathway, but not the
ERK or p38 pathway, was responsible for PPARg phosphorylation in THP-1-derived macrophage. This data illustrated the
complexity of regulation of PPARg activity and provided a new insight into the mechanism of macrophage foam cell
formation induced by ox-LDL.
In recent years the detection of
PPARg in lesion macro-phages, coupled with its identification as the molecular
target of antidiabetic agents, has raised significant interest in developing models of
PPARg function and its role as a therapeutic target for coronary artery
disease[12]. In particular, TZD, synthesis ligands of
PPARg, are widely used in
patients with diabetes, who also have a high risk of cardiovascular
disease[13]. Several groups have evaluated the effects of
TZD on the foam cell formation and showed that there was no significant difference in cholesterol accumulation in
TZD-treated cells[14,15]. To determine the overall impact of TZD on the development of atherosclerosis, several groups have
recently evaluated their effects in
vivo[16,17]. Studies in LDL receptor-deficient or apolipoprotein E-deficient mice have
consistently demonstrated protective effects of TZD on the development of diet-induced atherosclerosis. These
observations indicated that PPARg activation mediated by ligand-dependent manner was involved in antiathero-genic pathways.
Therefore, the implication of PPARg in proatherogenic pathway mediated by ox-LDL suggested that, in addition to activating
PPARg via 9-HODE and 13-HODE, another interaction might exist between ox-LDL and
PPARg, through which ox-LDL facilitates macrophage foam cell formation.
Growth factors, such as epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), have been shown to
phosphorylate PPARg via MAPK signaling pathway and to decrease
PPARg transcriptional activity[18]. The
NH2-
terminal domain of PPARg contains a consensus MAPK site
in a region conserved between PPARg1 and
PPARg2 isoforms[9]. PPARg proteins migrate on immunoblots as closely spaced doublets, a pattern suggestive of phosphorylation. A putative
MAPK site is phosphorylated by ERK and JNK kinase. Phosphorylation significantly inhibits both ligand-independent and
ligand-dependent transcriptional activation by
PPARg[18]. This repression is mediated by MAPK phosphorylation of Ser82
on PPARg. Mutation of the phosphorylated residue (Ser82) prevents
PPARg phosphorylation as well as the growth
factor-mediated repression of PPARg dependent transcription. Previously, Han et al showed that TGF-b decreased the expression
of CD36 in THP-1-derived macrophage by phosphorylation of MAPK, subsequent MAPK phosphorylation of
PPARg, and decreased CD36 transcription by phosphorylated
PPARg[19]. Although phosphorylation of
PPARg has been implicated in macrophage lipid homeostasis, whether it is involved in ox-LDL-induced foam cell formation is unclear. Our study found that
ox-LDL-
induced MAPK activation led to phosphorylation and subsequent deactivation of
PPARg. This observation indicated that unknown component of ox-LDL might negative regulated
PPARg activity through MAPK-mediated phosphorylation pathway,
which in turn promote macrophage foam cell formation. In contrast to the prevailing notion that ox-LDL is a positive regulator
for PPARg, our results demonstrated that ox-LDL also inhibited
PPARg transcriptional activity via phosphorylation pathway.
We assume that the consequence of interaction between ox-LDL and
PPARg may depend on the stage of macrophage-derived foam cell formation. Feature studies are needed to clarify this assumption.
MAPK play an important role in many cellular processes,
such as proliferation, apoptosis, and adaptation to changes
in the extracellular environment[20]. At least three major groups of MAPK have been identified in mammalian cells so far:
(i) ERK, (ii) JNK or SAPK, and (iii) p38 MAPK. The ERK pathway is preferentially activated by growth-related stimuli, while
the JNK and p38 pathways are often linked with cellular stress. MAPK can, however, be activated by oxidative stress in a
variety of cells. Both ERK- and p38-MAPK members have been shown to be activated by ox-LDL in smooth muscle
cells[10], and recently Zhao et
al have reported a similar effect on p38-MAPK in the murine macrophage cell line,
J774[21]. Napolitano et al reported that the activation of ERK-, but not p38-MAPK was involved in the induction of cholesterol esterification by
acetylated LDL in human monocyte-derived
macrophages[22]. These findings, therefore, suggested that MAPK might play
an important role in the regulation of macrophage foam cell formation induced by modified LDL and the development of
atherosclerosis. Consistent with previous studies, our results also demonstrated that ox-LDL induced activation of the p38-,
JNK-, and ERK-MAPK. However, the three MAPK had different kinetics of activation. The different kinetics of the three
MAPK suggest that their role may be different in macrophage foam cell formation.
Using pharmaceutical inhibitors, we demonstrated that the activation of JNK pathway, but not ERK or p38 pathway, was
necessary and sufficient to phosphorylate PPARg and subsequently facilitated macrophage foam cell formation. We do not
know at present whether ox-LDL activates JNK directly, or if ox-LDL activates other cellular kinase pathways, such as PKA
and AMPK, which in turn may activate MAPK. We still do not know which component of
ox-LDL is responsible for MAPK activation and subsequent
PPARg phos-phorylation. But it is clear that post-translational regulation of
PPARg via the phosphorylation pathway is crucial for macrophage foam cell formation induced by ox-LDL. A future challenge will be to
clarify those problems in order to develop new strategies for the prevention and treatment of atherosclerosis through
modulation of PPARg phosphorylation status.
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