This Article Abstract Full Text (PDF) All Versions of this Article: 92/2/212    most recent 01.RES.0000053386.46813.E9v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chinetti, G. Articles by Staels, B. Search for Related Content PubMed PubMed Citation Articles by Chinetti, G. Articles by Staels, B. Related Collections Cell signalling/signal transduction Gene regulation Lipid and lipoprotein metabolism

(Circulation Research. 2003;92:212.)
© 2003 American Heart Association, Inc.

Molecular Medicine

Peroxisome Proliferator-Activated Receptor Reduces Cholesterol Esterification in Macrophages

G. Chinetti, S. Lestavel, J.-C. Fruchart, V. Clavey, B. Staels

From the UR 545 INSERM, Institut Pasteur de Lille and Université de Lille 2, Lille, France.

Correspondence to Giulia Chinetti, UR 545 INSERM, Institut Pasteur de Lille, 1, rue Calmette BP245, 59019 Lille, France. E-mail giulia.chinetti{at}pasteur-lille.fr

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Peroxisome proliferator-activated receptor (PPAR) is a nuclear receptor activated by fatty acid derivatives and hypolipidemic drugs of the fibrate class. PPAR is expressed in monocytes, macrophages, and foam cells, suggesting a role for this receptor in macrophage lipid homeostasis with consequences for atherosclerosis development. Recently, it was shown that PPAR activation promotes cholesterol efflux from macrophages via induction of the ABCA1 pathway. In the present study, the influence of PPAR activators on intracellular cholesterol homeostasis was investigated. In human macrophages and foam cells, treatment with fibrates, synthetic PPAR activators, led to a decrease in the cholesteryl ester (CE):free cholesterol (FC) ratio. In these cells, PPAR activation reduced cholesterol esterification rates and Acyl-CoA:cholesterol acyltransferase-1 (ACAT1) activity. However, PPAR activation did not alter ACAT1 gene expression, whereas mRNA levels of carnitine palmitoyltransferase type 1 (CPT-1), a key enzyme in mitochondrial fatty acid catabolism, were induced. Finally, PPAR activation blocked CE formation induced by TNF-, possibly due to the inhibition of neutral sphingomyelinase activation by TNF-. In conclusion, our results identify a role for PPAR in the control of cholesterol esterification in macrophages, resulting in an enhanced availability of FC for efflux through the ABCA1 pathway.

Key Words: nuclear receptor • atherosclerosis • gene regulation • cholesterol homeostasis

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that regulate the expression of genes controlling lipid and glucose metabolism.¹ PPAR, which is activated by fibrates, fatty acids, and eicosanoids,² is highly expressed in liver, heart, muscle, and kidney, but also present in cells of the arterial wall including monocytes, macrophages, macrophage foam cells, smooth muscle, and endothelial cells.² Recent observations suggest a role for PPAR in macrophage lipid homeostasis and cholesterol efflux, the first step of the reverse cholesterol transport pathway. In these cells, PPAR activators induce the expression of the scavenger receptor CLA-1/SR-BI, which binds HDL with high affinity.³ In addition, in differentiated macrophages and macrophage-derived foam cells PPAR activators induce ABCA1 gene expression and promote apoAI-mediated cholesterol efflux.⁴

In cholesterol-loaded macrophages, cholesterol efflux is closely related to the rate of cholesteryl ester (CE) turnover.⁵ Within cells, modified LDL-derived cholesteryl esters are hydrolyzed in lysosomes to free cholesterol (FC). This FC is probably initially transported to the plasma membrane where it integrates in the cell membrane.⁵ Excess membrane cholesterol is transported back to the endoplasmic reticulum where it is re-esterified by acyl-CoA:cholesterol acyltransferase 1 (ACAT1) with fatty acids (FAs) and stored as lipid droplets.⁶ Thus, the cholesterol esterification rate also controlled by FA availability depends partially on their catabolism by enzymes such as carnitine palmitoyltransferase 1 CPT-1, a key enzyme regulating mitochondrial FA entry.⁷ On the other hand, FC can be effluxed from cells to extracellular acceptors such as apoAI through the ABCA1 pathway and as such, enter into the reverse cholesterol transport pathway. This mechanism limits CE accumulation in vascular macrophages, thus preventing foam cell formation.⁵ ACAT1 enzyme activity as well as cholesterol esterification rates are controlled by different factors.^8,9 For instance, TNF- has been reported to stimulate cholesteryl ester formation in human fibroblasts by activating the neutral sphingomyelinase (N-SMase) pathway.¹⁰ As such, TNF- treatment probably leads to a decrease in plasma membrane levels of sphingomyelin, which functions as a trap for FC. In this way, FC could move from the plasma membrane, thus reaching the esterification site and providing more substrate for ACAT1 in the endoplasmic reticulum.⁵ Because PPAR is expressed in differentiated macrophages and macrophage-foam cells where it controls free cholesterol removal through activation of ABCA1,⁴ we decided to study the role of PPAR in processes upstream of cholesterol efflux. Our results demonstrate that, without influencing total cholesterol accumulation in macrophages,⁴ PPAR activation reduces the CE:FC ratio in macrophages and macrophage-derived foam cells by inhibiting cellular cholesteryl ester formation activity. Moreover, PPAR activation blocks TNF-–stimulated cholesteryl ester formation activity possibly by negatively interfering with the N-SMase pathway. Our results further expand the role for PPAR in the control of cholesterol homeostasis in macrophages. Along with the induction of ABCA1 expression, a decrease in CE:FC ratio may contribute to an enhanced liberation and efflux of free cholesterol to extracellular acceptors, the first step of the reverse cholesterol transport pathway, after PPAR activation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Culture
Mononuclear cells isolated from blood of healthy normolipidemic donors by Ficoll gradient centrifugation were suspended in RPMI 1640 medium containing gentamycin (40 mg/mL), glutamine (0.05%), and 10% pooled human serum.¹¹ Differentiation of monocytes into macrophages occurs spontaneously by adhesion of the cells to the culture dishes. Mature monocyte-derived macrophages were used for experiments after 10 days of culture. For experiments, medium was changed to medium without serum but supplemented with 1% Nutridoma HU (Boehringer Mannheim).

RNA Extraction and Analysis
Total cellular RNA was extracted from 10-day–cultured primary human macrophages or macrophage-derived foam cells treated or not with the PPAR ligands Wy14643 (25, 50, and 100 µmol/L), ciprofibrate (50 µmol/L), bezafibrate (50 µmol/L), fenofibric acid (50 µmol/L), and GW647 (600 nmol/L) for 24 hours using Trizol (Life Technologies, France). For Northern blot analysis, membranes containing 10 µg of total RNA were hybridized with radiolabeled ACAT1¹² or 36B4 cDNA probes.

For quantitative PCR, total RNA were reverse transcribed using random hexameric primers and Superscript reverse transcriptase (Life Technologies, France). cDNAs were quantified by real-time PCR on a MX 4000 (Stratagene), using specific primers CPT-1: 5'-ACAGTCGGTGAGGCCTCTTATGAA-3' and 5'-TCTTGCTG-CCTGAATGTGAGTTGG-3' and cyclophilin: 5'-GCATACGGGT-CCTGGCATCTTGTCC-3' and 5'-ATGGTGATCTTCTTGCTGG-TCTTGC-3'. PCR amplification was performed in a volume of 25 µL containing 100 nmol/L of each primer, 4 mmol/L MgCl₂, the Brilliant Quantitative PCR Core Reagent Kit mix as recommended by the manufacturer (Stratagene) and SYBR Green 0.33X (Sigma-Aldrich). The conditions were 95°C for 10 minutes, followed by 40 cycles of 30 seconds at 95°C, 30 seconds at 55°C, and 30 seconds at 72°C. CPT-1 mRNA levels were subsequently normalized to cyclophilin mRNA.

Cellular Cholesterol Measurement
Ten-day–cultured human macrophages were pretreated for 24 hours and thereafter every 24 hours with the PPAR activators Wy14643 (25, 50 and 100 µmol/L), ciprofibrate (50 µmol/L), bezafibrate (50 µmol/L), fenofibric acid (50 µmol/L), and GW647 (600 nmol/L) and cholesterol loaded by incubation with AcLDL (50 µg/mL, containing or not [³H]cholesterol)¹³ in RPMI 1640 medium supplemented with 1% Nutridoma for 48 hours. Intracellular lipids were extracted by hexane/isopropanol, and total cholesterol (TC) and free cholesterol (FC) were measured by enzymatic assay (Boehringer). Cholesteryl esters (CEs) were calculated as the difference between TC and FC. In other experiments, intracellular lipids were separated by thin layer chromatography (TLC) in Petroleum ether/Diethyl ether/Acetic acid (180:20:10, vol:vol:vol). Spots corresponding to CE and FC were scraped and radioactivity measured by scintillation counting.

Measurement of Cholesteryl Ester Formation
Cholesteryl ester formation within the cells was assessed by measuring the incorporation of [¹⁴C]oleate into cholesteryl esters. Ten-day–cultured human macrophages were cholesterol loaded by incubation with AcLDL (50 µg/mL) in RPMI 1640 medium supplemented with 1% Nutridoma for 48 hours. The PPAR activators Wy14643 (25, 50, and 100 µmol/L), bezafibrate (50 µmol/L), fenofibric acid (50 µmol/L), and GW647 (600 nmol/L) were added to the culture medium 24 hours before cholesterol loading and thereafter every 24 hours. After the cholesterol-loading period, cells were incubated for 2 hours at 37°C with 1 µCi [¹⁴C]oleic acid per mL of medium. Oleic acid was presented to the cells as a BSA-sodium oleate complex, as described.¹⁴ In the experiments with TNF-, cells were washed and subsequently incubated for 1 hour in fresh RPMI 1640 medium containing or not TNF- (60 ng/mL) and Wy14643 (50 µmol/L). At the end of the assay, cells were washed and lipids extracted with hexane/isopropanol. [¹⁴C]oleic acid incorporation into cholesteryl esters was measured on lipid extracts after separation by TLC in hexane/diethyl ether/acetic acid (90:10:1, vol:vol:vol). Spots corresponding to cholesteryl oleate and oleic acid were scraped and radioactivity measured by scintillation counting.

Measurement of N-SMase Activity
To determine N-SMase activity, 10-day–cultured human macrophages were treated for 24 hours and thereafter every 24 hours with Wy14643 (50 µmol/L) and labeled with [³H]choline (2.5 µCi/mL) for 48 hours.¹⁰ After this period, cells were washed and subsequently incubated for 1 hour in fresh RPMI 1640 medium containing or not TNF- (60 ng/mL) and Wy14643 (50 µmol/L). Medium was subsequently removed, cells were washed in PBS and intracellular lipids were extracted by hexane:isopropanol, as described above. The different phospholipids present were separated by TLC using chloroform/methanol/formic acid (65:25:4, vol:vol:vol) as solvent.¹⁰ After chromatography, the gel area corresponding to sphingomyelin was scraped and the radioactivity measured. Results are expressed as the difference of [³H] radioactivity associated to sphingomyelin per mg of cellular protein, between values obtained in the presence or in the absence of TNF-.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
PPAR Activation Decreases the Cholesteryl Ester:Free Cholesterol Ratio in Cholesterol-Loaded Macrophages
To determine the influence of PPAR activation on the cholesterol distribution between FC and CE, primary human monocyte-derived differentiated macrophages were loaded with AcLDL (50 µg/mL) for 48 hours and treated with the specific synthetic PPAR ligand Wy14643 (50 µmol/L) added 24 hours before cholesterol loading and thereafter every 24 hours. As previously shown,⁴ PPAR activation by Wy14643 did not modify macrophage AcLDL-induced total cholesterol accumulation. However, the cholesterol distribution changed with a significant decrease of the CE fraction (*P<0.05) (Figure 1A). A similar effect was also observed in unloaded macrophages (Figure 1A). In order to demonstrate that the variation of intracellular cholesterol distribution was not due to the action of the PPAR ligand on de novo cholesterol synthesis, the distribution of radiolabeled [³H]cholesterol between FC and CE was analyzed by TLC analysis after [³H]cholesterol-AcLDL loading (Figure 1B). Treatment with Wy14643 at different concentrations (25, 50, and 100 µmol/L) significantly decreased the amount of [³H]cholesterol present in the cholesteryl ester fraction (Figure 1B), indicating that this effect is not due to an action on endogenous cholesterol synthesis and is dependent on the concentration of activator used.

View larger version (23K): [in this window] [in a new window]   Figure 1. PPAR decreases cholesteryl ester levels in human macrophages and foam cells. A, Human macrophages were cholesterol-loaded with [3H]cholesterol-containing AcLDL (50 µg/mL) for 48 hours. Wy14643 (50 µmol/L) was added 24 hours before cholesterol-loading and thereafter every 24 hours. Intracellular total cholesterol (TC) and free cholesterol (FC) were enzymatically determined. Cholesteryl esters (CE) were calculated as the difference between TC and FC. Results are the mean±SD of triplicate determinations, representative of 5 independent experiments. B, Human macrophages were treated as described in A using different concentrations of Wy14643 (25, 50, and 100 µmol/L). Lipids were extracted and separated by TLC. Spots corresponding to CE and FC were scraped and radioactivity measured by scintillation counting. Results are expressed as cholesteryl ester percent of TC±SD of triplicate determinations of 2 independent cell preparations. Statistically significant differences between treatments are indicated (ANOVA followed by t test; ***P<0.001).

Similar effects were observed with other specific PPAR ligands, such as ciprofibrate (50 µmol/L), bezafibrate (50 µmol/L), fenofibric acid (50 µmol/L), and GW647 (600 nmol/L) at concentrations specifically activating human PPAR^15,16 (Figure 2). Under these conditions, no net increase in total cholesterol accumulation was observed with any of the drugs tested (data not shown). These data suggest a role for PPAR in the control of macrophage cholesterol esterification.

View larger version (23K): [in this window] [in a new window]   Figure 2. Different PPAR activators decrease cholesteryl ester content in human macrophage-foam cells. Human macrophages were treated as described in Figure 1B with Wy14643 (50 µmol/L), ciprofibrate (50 µmol/L), bezafibrate (50 µmol/L), fenofibric acid (50 µmol/L), and GW647 (600 nmol/L). Lipids were extracted and separated by TLC. Spots corresponding to CE and FC were scraped and radioactivity measured by scintillation counting. Results are expressed relative to untreated cells set as 100%. Statistically significant differences between treatments are indicated (ANOVA followed by t test; **P<0.01, ***P<0.001).

PPAR Activation Decreases Cholesteryl Ester Formation in Human Macrophages
Because PPAR activation reduces the amount of CE in macrophages, the influence of PPAR activators on cholesterol esterification was assessed in macrophage foam cells. Treatment of AcLDL-loaded macrophages with different PPAR activators, including Wy14643 (50 µmol/L), bezafibrate (50 µmol/L), fenofibric acid (50 µmol/L), and GW647 (600 nmol/L), resulted in a reduction of cholesteryl ester formation as measured by a decreased incorporation of [¹⁴C]oleic acid into cholesteryl esters (Figure 3). To determine whether these compounds act as direct ACAT inhibitor, their effects on in vitro ACAT activity in microsomal preparation were tested. Our results demonstrate that none of these PPAR activators are competitive ACAT inhibitors (data not shown).

View larger version (22K): [in this window] [in a new window]   Figure 3. PPAR activation decreases cholesteryl ester formation in human macrophage-foam cells. Human macrophages were cholesterol-loaded by incubation with AcLDL for 48 hours. Wy14643 (50 µmol/L), bezafibrate (50 µmol/L), fenofibric acid (50 µmol/L), and GW647 (600 nmol/L) was added for 24 hours before cholesterol-loading and thereafter every 24 hours and cholesteryl ester formation was measured by incubation with [14C]oleic acid complexed to BSA. Intracellular lipids were then extracted and separated by TLC. Spots corresponding to cholesteryl oleate and oleic acid were scraped and radioactivity measured by scintillation counting. Cholesteryl ester formation was calculated as percentage of [14C]oleate incorporated into cholesterol. Results are expressed relative to untreated cells set as 1. Statistically significant differences between treatments are indicated (ANOVA followed by t test; *P<0.05, **P<0.01, ***P<0.001).

ACAT1 Gene Expression Is Not Decreased by PPAR Activation in Human Macrophages and Macrophage-Foam Cells
To determine whether PPAR decreases cholesteryl ester formation through regulation of ACAT1 gene expression, Northern blot analysis was performed using RNA from human macrophages and foam cells incubated with Wy14643. As previously reported,¹² 4 different ACAT1 transcripts were observed in differentiated macrophages as well as in macrophage-derived foam cells (Figure 4A). PPAR activation did not affect ACAT1 mRNA levels in either cell types (Figure 4B), thus excluding a role for PPAR action via changes in ACAT1 gene expression.

View larger version (42K): [in this window] [in a new window]   Figure 4. PPAR activation does not influence ACAT1 gene expression in human macrophages and cholesterol-loaded macrophages. A, Northern blot analysis of ACAT1 mRNA from macrophages (unloaded) or macrophage foam cells (AcLDL-loaded) treated with Wy14643 (50 µmol/L) for 24 hours. 36B4 probe was used as internal control. B, Quantification by optical densitometry of ACAT1 mRNA levels in unloaded and AcLDL-loaded macrophages. Values are normalized to internal control 36B4 mRNA. Results presented are from experiments performed on 2 independent cell preparations.

PPAR Activation Increases CPT-1 Expression in Primary Human Macrophages
In order to evaluate whether PPAR may control FA availability for cholesterol esterification by ACAT1, the effect of PPAR ligands was studied on the expression of CPT-1 (the CPT-1 isoform that is highly expressed in liver), a key enzyme in mitochondrial fatty acid catabolism. Treatment with different PPAR activators significantly induced the expression of CPT-1 mRNA levels in primary human monocyte-derived macrophages (Figure 5A). This induction occurred in a dose-dependent manner, as shown for Wy14643 (Figure 5B). The effects on CPT-1 induction by Wy14643 correlated with the observed reduction of cholesteryl ester levels (Figure 1B). These observations provide one potential mechanism by which PPAR controls cholesteryl ester accumulation.

View larger version (17K): [in this window] [in a new window]   Figure 5. PPAR activation increases CPT-1 gene expression in human macrophages. A, Q-PCR analysis of CPT-1 and cyclophilin was performed on RNA isolated from human macrophages treated or not with ciprofibrate (50 µmol/L), bezafibrate (50 µmol/L), fenofibric acid (50 µmol/L), or GW647 (600 nmol/L) for 24 hours. CPT-1 mRNA levels were normalized to cyclophilin mRNA and are expressed relative to the levels in untreated cells set as 1. Results are expressed as mean±SD CPT-1 level determinations obtained from 4 independent macrophage preparations. B, Induction of CPT-1 expression in human macrophages treated for 24 hours with increasing concentrations of Wy14643 (25, 50, and 100 µmol/L). Statistically significant differences between treatments are indicated (ANOVA followed by t test; **P<0.01, ***P<0.001).

PPAR Decreases TNF-–Induced Cholesteryl Ester Formation in Human Macrophage-Foam Cells
To identify other potential biochemical mechanisms contributing to the effects of PPAR activation on cholesteryl ester formation, it was analyzed whether PPAR activation interferes with cholesteryl ester formation stimulated by TNF-.¹⁰ Incubation of cholesterol-loaded macrophages with TNF- (60 ng/mL) increased cholesteryl ester formation as demonstrated by enhanced incorporation of [¹⁴C]oleic acid into cholesterol (Figure 6A). The presence of Wy14643 blocked the inductive effect of TNF- on cholesterol esterification (Figure 6A). These data suggest the existence of a negative cross-talk between the TNF- pathway and PPAR in the control of cholesterol esterification.

View larger version (32K): [in this window] [in a new window]   Figure 6. PPAR activation blocks TNF-–induced cholesterol esterification by inhibiting the activation of the N-SMase pathway. A, Cholesterol-loaded human macrophages were treated with Wy14643 (50 µmol/L) in the presence or not of TNF- (60 ng/mL) for 1 hour and cholesteryl ester formation was measured as described under Materials and Methods. Lipids were extracted and separated by TLC. Spots corresponding to cholesteryl oleate and oleic acid were scraped off and radioactivity measured by scintillation counting. Cholesteryl ester formation was calculated as percentage of [14C]oleate incorporated into cholesteryl esters. Results are expressed relative to untreated cells set as 1. B, [3H]Choline-labeled human macrophages were treated or not with Wy14643 (50 µmol/L) and subsequently incubated in RPMI 1640 medium containing or not TNF- (60 ng/mL) and Wy14643 (50 µmol/L) for 1 hour. Lipid were extracted and separated by TLC. N-SMase activity was calculated as the difference of [3H] radioactivity associated to sphingomyelin per mg of cellular protein between values obtained in the absence or in the presence of TNF-. Statistically significant differences between treatments are indicated (ANOVA followed by t test; *P<0.05, ***P<0.001).

PPAR Decreases TNF-–Induced N-SMase Activity in Primary Human Macrophages
The stimulatory effect of TNF- on cholesteryl ester formation may be mediated via the activation of cell membrane–associated neutral sphingomyelinase (N-SMase).¹⁰ Treatment with TNF- resulted in a stimulation of N-SMase activity, as determined by a decrease in [³H]choline incorporation into sphingomyelin (Figure 6B). TNF-–induced N-SMase activity was blocked in the presence of Wy14643 (Figure 6B). However, basal macrophage N-SMase gene expression was not affected by PPAR activation (data not shown). These findings indicating that PPAR activation interferes negatively with the N-SMase pathway may thus constitute another mechanism contributing to the decreased cholesteryl ester formation activity due to a reduced supply of free cholesterol substrate for the ACAT1 enzyme.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
PPAR is a lipid-activated transcription factor that regulates genes involved in lipid and glucose metabolism and inflammation control.¹ PPAR is expressed in human endothelial cells, smooth muscle cells, and macrophages in vitro as well as in resident atherosclerotic lesion macrophages where it plays an important role in the control of vascular cholesterol homeostasis and inflammation.² In macrophages, PPAR ligands enhance ABCA1 expression as well as apoAI-mediated cholesterol efflux,⁴ indicating a role of PPAR in the control of macrophage cholesterol homeostasis. In this study, we show that PPAR also regulates the balance between free cholesterol and cholesteryl esters, a process upstream of and closely related to cholesterol removal, in human macrophages and foam cells.

PPAR activation did not affect lipid accumulation after AcLDL loading, thus confirming our previous observations⁴ that PPAR activation does not promote foam cell transformation of primary human macrophages. Moreover, in the present study, we demonstrate that, in human macrophages as well as in macrophage-derived foam cells, PPAR activation reduces cholesteryl ester levels. These effects are not due to a PPAR effect on endogenous cholesterol synthesis, because esterification of exogenously added radiolabeled cholesterol was also reduced. Instead, PPAR activation led to a decrease of cholesteryl ester formation. These actions of PPAR are not due to a decreased expression of the ACAT1 gene, the enzyme responsible for cholesteryl ester formation in macrophages, because ACAT1 mRNA levels do not change. By contrast, PPAR agonists increase the expression of CPT-1,^17,18 an enzyme located in the mitochondrial outer membrane catalyzes the entry of long chain FAs into the mitochondria, thus determining the flux of FAs into mitochondria for ß-oxidation.⁷ Increased expression of CPT-1 mRNA may thus result in a reduced availability of long chain FAs as substrate for ACAT1, thus leading to decreased cholesterol esterification. The potential role of CPT-1 in the control of CE levels in macrophages is supported by previous studies showing that CPT-1 inhibition by etomoxir results in a 3.5-fold induction of cholesterol incorporation into CE.¹⁹ Our data add CPT-1 to the list of genes that are upregulated by PPAR through a PPRE-dependent mechanism in macrophages, along with LPL²⁰ and the nuclear receptor LXR.⁴ Moreover, to determine if the decrease in cholesteryl ester content on PPAR activation could lead to an increased triglyceride synthesis in human macrophages, the triglyceride cellular content was determined. Our data demonstrated that Wy14643 did not affect the triglyceride levels in macrophages and AcLDL-loaded macrophages (triglyceride content; untreated cells, 73.7±5.4 µg/mg protein; Wy14643, 69.7±6.4; AcLDL, 89.1±4.1; AcLDL+Wy14643, 92.9±5.6). Interestingly, PPAR also inhibits cholesterol esterification in human macrophage-foam cells induced by TNF-, a factor known to stimulate cholesteryl ester formation in other cell types, such as human fibroblasts.¹⁰ Although, TNF- may also induce cellular death by apoptosis,²¹ these experiments were performed using TNF- at lower concentrations and shorter incubation times than those reported to induce macrophage apoptosis.¹¹ This negative regulation of TNF- activity is another example of the antiinflammatory action of PPAR in macrophages. A defect in the delivery of cholesterol substrate to ACAT1, by trapping it within phospholipid-containing pools in cell membranes, could indeed result in the inhibition of CE formation. Thus, PPAR may inhibit cholesteryl ester formation activity via two complementary mechanisms both leading to decreased substrate availability: on the one hand, by inhibiting membrane cholesterol mobilization due to inhibition of TNF- activity and, on the other hand, by increasing FA oxidation due to CPT-1 gene induction. PPAR activation could also influence the cholesteryl ester hydrolysis by affecting neutral cholesteryl ester hydrolase (NCEH) activity. However, PPAR activators rather reduced cholesteryl ester hydrolysis activity in the macrophages (data not shown), thus indicating that the effects of PPAR on CE:FC ratio occurs rather via inhibition cholesterol esterification than by stimulation of cholesteryl ester hydrolysis. Further studies are required to determine whether PPAR also controls other steps of intracellular cholesterol homeostasis, such as cholesterol trafficking.

The inhibition of cholesteryl ester formation by PPAR agonists could have broader implications than to regulate cholesterol efflux. First, macrophage cholesteryl esters appear to be an important stimulus for the secretion of matrix metalloproteinases (MMPs) in advanced lesions.²² PPAR activators reduce the secretion of MMP9 in human monocytic THP1 cells.²³ The reduced secretion of MMPs after PPAR activation could be mediated, in part, via the control of cholesteryl ester formation by PPAR in advanced lesion macrophages. Second, biochemical analysis has shown that large pools of cholesteryl ester derivatives can predispose plaques to rupture.²⁴ PPAR activators could thus influence plaque instability by controlling the amounts of cholesteryl esters in plaques. In vivo studies performed using the apoE^-/- mouse showed that treatment with the specific PPAR activator fenofibrate, results in a reduction of the cholesterol content of the plaque, an effect that is predominantly due to a decrease in cholesteryl ester accumulation.²⁵ These in vivo observations extend our results obtained in human macrophages in vitro.

In conclusion, our results demonstrate a novel role for PPAR in the control of the balance between free cholesterol and cholesteryl esters, an effect which, associated with the induction of ABCA1, may contribute to enhanced liberation and efflux of free cholesterol and stimulation of the initial step of the reverse cholesterol transport pathway.

	Acknowledgments

 
This work was supported by grants from Fondation Leducq, the European Community (grant QLRT-1999-01007), and from the FEDER and Conseil Régional Région Nord/Pas-de-Calais (Genopole project No. 01360124). We thank D. Junquero and A. Delhon (Pierre Fabre, Castres) for the measurement of in vitro ACAT activity and P. Brown (Glaxo Smith Kline) for providing us the GW647 compound. B. Noel, R. Barbeau, and L. Thumerel are acknowledged for the technical help.

Received July 26, 2002; revision received November 22, 2002; accepted December 11, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Pineda Torra I, Gervois P, Staels B. Peroxisome proliferator-activated receptor alpha in metabolic disease, inflammation, atherosclerosis and aging. Curr Opin Lipidol. 1999; 10: 151–157.[CrossRef][Medline] [Order article via Infotrieve]

2. Chinetti G, Fruchart JC, Staels B. Peroxisome proliferator-activated receptors (PPARs): nuclear receptors at the crossroads between lipid metabolism and inflammation. Inflamm Res. 2000; 49: 497–505.[CrossRef][Medline] [Order article via Infotrieve]

3. Chinetti G, Gbaguidi GF, Griglio S, Mallat Z, Antonucci M, Poulain P, Chapman J, Fruchart JC, Tedgui A, Najib-Fruchart J, Staels B. CLA-1/SR-BI is expressed in atherosclerotic lesion macrophages and regulated by activators of peroxisome proliferator-activated receptors. Circulation. 2000; 101: 2411–2417.[Abstract/Free Full Text]

4. Chinetti G, Lestavel S, Bocher V, Remaley AT, Neve B, Pineda Torra I, Teissier E, Minnich A, Jaye M, Duverger N, Brewer BH, Fruchart JC, Clavey V, Staels B. PPAR and PPAR activators induce cholesterol removal from human macrophage foam cells through stimulation of the ABCA1 pathway. Nat Med. 2001; 7: 53–58.[CrossRef][Medline] [Order article via Infotrieve]

5. Tabas I. Cholesterol and phospholipid metabolism in macrophages. Biochim Biophys Acta. 2000; 1529: 164–174.[Medline] [Order article via Infotrieve]

6. Buhman KF, Accad M, Farese RV. Mammalian acyl-CoA: cholesterol acyltransferases. Biochim Biophys Acta. 2000; 1529: 142–154.[Medline] [Order article via Infotrieve]

7. McGarry JD, Brown NF. The mitochondrial carnitine palmitoyltransferase system: from concept to molecular analysis. Eur J Biochem. 1997; 244: 1–14.[Medline] [Order article via Infotrieve]

8. Panousis CG, Zuckerman SH. Regulation of cholesterol distribution in macrophage-derived foam cells by interferon-gamma. J Lipid Res. 2000; 41: 75–83.[Abstract/Free Full Text]

9. Maung K, Miyazaki A, Nomiyama H, Chang CC, Chang TY, Horiuchi S. Induction of acyl-coenzyme A: cholesterol acyltransferase-1 by 1,25-dihydroxyvitamin D(3) or 9-cis-retinoic acid in undifferentiated THP-1 cells. J Lipid Res. 2001; 42: 181–187.[Abstract/Free Full Text]

10. Chatterjee S. Neutral sphingomyelinase action stimulates signal transduction of tumor necrosis factor-alpha in the synthesis of cholesteryl esters in human fibroblasts. J Biol Chem. 1994; 269: 879–882.[Abstract/Free Full Text]

11. Chinetti G, Griglio S, Antonucci M, Pineda Torra I, Delerive P, Majd Z, Fruchart JC, Chapman J, Najib J, Staels B. Activation of peroxisome proliferator-activated receptors and induces apoptosis of human monocyte-derived macrophages. J Biol Chem. 1998; 273: 25573–25580.[Abstract/Free Full Text]

12. Wang H, Germain SJ, Benfield PP, Gillies PJ. Gene expression of acyl-coenzyme-A: cholesterol-acyltransferase is upregulated in human monocytes during differentiation and foam cell formation. Arterioscler Throm Vasc Biol. 1996; 16: 809–814.[Abstract/Free Full Text]

13. Basu SK, Goldstein JL, Anderson GW, Brown MS. Degradation of cationized low density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts. Proc Natl Acad Sci U S A. 1976; 73: 3178–3182.[Abstract/Free Full Text]

14. Schmitz G, Niemann R, Brennhausen B, Krause R, Assmann G. Regulation of high density lipoprotein receptors in cultured macrophages: role of acyl-CoA: cholesterol acyltransferase. EMBO J. 1985; 4: 2773–2779.[Medline] [Order article via Infotrieve]

15. Willson TM, Brown PJ, Sternbach DD, Henke BR. The PPARs: from orphan receptors to drug discovery. J Med Chem. 2000; 43: 527–550.[CrossRef][Medline] [Order article via Infotrieve]

16. Brown PJ, Stuart LW, Hurley KP, Lewis MC, Winegar DA, Wilson JG, Wilkison WO, Ittoop OR, Willson TM. Identification of a subtype selective human PPAR agonist through parallel-array synthesis. Bioorg Med Chem Lett. 2001; 11: 1225–1227.[CrossRef][Medline] [Order article via Infotrieve]

17. Mascaro C, Acosta E, Ortiz J, Marrero P, Hegardt F, Haro D. Control of human muscle-type carnitine palmitoyltransferase I gene transcription by peroxisome proliferator-activated receptor. J Biol Chem. 1998; 273: 8560–8563.[Abstract/Free Full Text]

18. Yu GS, Lu YC, Gulick T. Co-regulation of tissue-specific alternative human carnitine palmitoyltransferase Iß gene promoters by fatty acid enzyme substrate. J Biol Chem. 1998; 273: 32901–32909.[Abstract/Free Full Text]

19. Moinat M, Kossovsky M, Chevey JM, Giacobino JP. Balance between fatty acid degradation and lipid accumulation in cultured smooth muscle cells and IC-21 macrophages exposed to oleic acid. Comp Biochem Physiol B. 1991; 98: 147–150.[CrossRef][Medline] [Order article via Infotrieve]

20. Gbaguidi GF, Chinetti G, Milosavljevic D, Teissier E, Chapman J, Olivecrona G, Fruchart JC, Griglio S, Fruchart-Najib J, Staels B. Peroxisome proliferator-activated receptor (PPAR) agonists decrease lipoprotein lipase secretion and glycated LDL uptake by human macrophages. FEBS Lett. 2002; 512: 85–90.[CrossRef][Medline] [Order article via Infotrieve]

21. Hsu H, Shu HB, Pan MG, Goeddel DV. TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways. Cell. 1996; 84: 299–308.[CrossRef][Medline] [Order article via Infotrieve]

22. Galis ZS, Sukhova GK, Kranzhofer R, Clark S, Libby P. Macrophage foam cells from experimental atheroma constitutively produce matrix-degrading proteinases. Proc Natl Acad Sci U S A. 1995; 92: 402–406.[Abstract/Free Full Text]

23. Shu H, Wong B, Zhou G, Li Y, Berger J, Woofds JW, Wright SD, Cai T-C. Activation of PPAR or reduces secretion of matrix metalloproteinase 9 but not interleukin 8 from human monocytic THP-1 cells. Biochem Biophys Res Commun. 2000; 267: 345–349.[CrossRef][Medline] [Order article via Infotrieve]

24. Libby P, Aikawa M, Schonbeck U. Cholesterol and atherosclerosis. Biochim Biophys Acta. 2000; 1529: 299–309.[Medline] [Order article via Infotrieve]

25. Duez H, Chao YS, Hernandez M, Torpier G, Poulain P, Mundt S, Mallat Z, Teissier E, Burton CA, Tedgui A, Fruchart JC, Fievet C, Wright SD, Staels B. Reduction of atherosclerosis by the PPAR agonist fenofibrate in mice. J Biol Chem. 2002; 277: 48051–48057.[Abstract/Free Full Text]

This article has been cited by other articles:

				L. Lei, Y. Xiong, J. Chen, J.-B. Yang, Y. Wang, X.-Y. Yang, C. C. Y. Chang, B.-L. Song, T.-Y. Chang, and B.-L. Li TNF-alpha stimulates the ACAT1 expression in differentiating monocytes to promote the CE-laden cell formation J. Lipid Res., June 1, 2009; 50(6): 1057 - 1067. [Abstract] [Full Text] [PDF]

				E. Rigamonti, G. Chinetti-Gbaguidi, and B. Staels Regulation of Macrophage Functions by PPAR-{alpha}, PPAR-{gamma}, and LXRs in Mice and Men Arterioscler Thromb Vasc Biol, June 1, 2008; 28(6): 1050 - 1059. [Abstract] [Full Text] [PDF]

				V. R. Babaev, H. Ishiguro, L. Ding, P. G. Yancey, D. E. Dove, W. J. Kovacs, C. F. Semenkovich, S. Fazio, and M. F. Linton Macrophage Expression of Peroxisome Proliferator Activated Receptor-{alpha} Reduces Atherosclerosis in Low-Density Lipoprotein Receptor Deficient Mice Circulation, September 18, 2007; 116(12): 1404 - 1412. [Abstract] [Full Text] [PDF]

				R. Paumelle, C. Blanquart, O. Briand, O. Barbier, C. Duhem, G. Woerly, F. Percevault, J.-C. Fruchart, D. Dombrowicz, C. Glineur, et al. Acute Antiinflammatory Properties of Statins Involve Peroxisome Proliferator-Activated Receptor-{alpha} via Inhibition of the Protein Kinase C Signaling Pathway Circ. Res., February 17, 2006; 98(3): 361 - 369. [Abstract] [Full Text] [PDF]

				F. Blaschke, Y. Takata, E. Caglayan, R. E. Law, and W. A. Hsueh Obesity, Peroxisome Proliferator-Activated Receptor, and Atherosclerosis in Type 2 Diabetes Arterioscler Thromb Vasc Biol, January 1, 2006; 26(1): 28 - 40. [Abstract] [Full Text] [PDF]

				X. Fang, S. Hu, B. Xu, G. D. Snyder, S. Harmon, J. Yao, Y. Liu, B. Sangras, J. R. Falck, N. L. Weintraub, et al. 14,15-Dihydroxyeicosatrienoic acid activates peroxisome proliferator-activated receptor-{alpha} Am J Physiol Heart Circ Physiol, January 1, 2006; 290(1): H55 - H63. [Abstract] [Full Text] [PDF]

				G. Chinetti-Gbaguidi, E. Rigamonti, L. Helin, A. L. Mutka, M. Lepore, J. C. Fruchart, V. Clavey, E. Ikonen, S. Lestavel, and B. Staels Peroxisome proliferator-activated receptor {alpha} controls cellular cholesterol trafficking in macrophages J. Lipid Res., December 1, 2005; 46(12): 2717 - 2725. [Abstract] [Full Text] [PDF]

				M. Xia, M. Hou, H. Zhu, J. Ma, Z. Tang, Q. Wang, Y. Li, D. Chi, X. Yu, T. Zhao, et al. Anthocyanins Induce Cholesterol Efflux from Mouse Peritoneal Macrophages: THE ROLE OF THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR {gamma}-LIVER X RECEPTOR {alpha}-ABCA1 PATHWAY J. Biol. Chem., November 4, 2005; 280(44): 36792 - 36801. [Abstract] [Full Text] [PDF]

				X. Fang, S. Hu, T. Watanabe, N. L. Weintraub, G. D. Snyder, J. Yao, Y. Liu, J. Y.-J. Shyy, B. D. Hammock, and A. A. Spector Activation of Peroxisome Proliferator-Activated Receptor {alpha} by Substituted Urea-Derived Soluble Epoxide Hydrolase Inhibitors J. Pharmacol. Exp. Ther., July 1, 2005; 314(1): 260 - 270. [Abstract] [Full Text] [PDF]

				P. R. Moreno and V. Fuster The year in atherothrombosis J. Am. Coll. Cardiol., December 7, 2004; 44(11): 2099 - 2110. [Full Text] [PDF]

				A. C. Li and C. K. Glass PPAR- and LXR-dependent pathways controlling lipid metabolism and the development of atherosclerosis J. Lipid Res., December 1, 2004; 45(12): 2161 - 2173. [Abstract] [Full Text] [PDF]

				S. S. T. Lee, W.-Y. Chan, C. K. C. Lo, D. C. C. Wan, D. S. C. Tsang, and W.-T. Cheung Requirement of PPAR{alpha} in maintaining phospholipid and triacylglycerol homeostasis during energy deprivation J. Lipid Res., November 1, 2004; 45(11): 2025 - 2037. [Abstract] [Full Text] [PDF]

				N. Marx, H. Duez, J.-C. Fruchart, and B. Staels Peroxisome Proliferator-Activated Receptors and Atherogenesis: Regulators of Gene Expression in Vascular Cells Circ. Res., May 14, 2004; 94(9): 1168 - 1178. [Abstract] [Full Text] [PDF]

				J. L. Hargrove, P. Greenspan, and D. K. Hartle Nutritional Significance and Metabolism of Very Long Chain Fatty Alcohols and Acids from Dietary Waxes Experimental Biology and Medicine, March 1, 2004; 229(3): 215 - 226. [Abstract] [Full Text] [PDF]

				M. Ricote, A. F. Valledor, and C. K. Glass Decoding Transcriptional Programs Regulated by PPARs and LXRs in the Macrophage: Effects on Lipid Homeostasis, Inflammation, and Atherosclerosis Arterioscler Thromb Vasc Biol, February 1, 2004; 24(2): 230 - 239. [Abstract] [Full Text]

This Article Abstract Full Text (PDF) All Versions of this Article: 92/2/212    most recent 01.RES.0000053386.46813.E9v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chinetti, G. Articles by Staels, B. Search for Related Content PubMed PubMed Citation Articles by Chinetti, G. Articles by Staels, B. Related Collections Cell signalling/signal transduction Gene regulation Lipid and lipoprotein metabolism