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• W2023607090 abstract "The mobilization of cholesterol from intracellular pools to the plasma membrane is a determinant that governs its availability for efflux to extracellular acceptors. NPC1 and NPC2 are proteins localized in the late endosome and control cholesterol transport from the lysosome to the plasma membrane. Here, we report that NPC1 and NPC2 gene expression is induced by oxidized LDL (OxLDL) in human macrophages. Because OxLDLs contain natural activators of peroxisome proliferator-activated receptor α (PPARα), a fatty acid-activated nuclear receptor, the regulation of NPC1 and NPC2 by PPARα and the consequences on cholesterol trafficking were further studied. NPC1 and NPC2 expression is induced by synthetic PPARα ligands in human macrophages. Furthermore, PPARα activation leads to an enrichment of cholesterol in the plasma membrane. By contrast, incubation with progesterone, which blocks postlysosomal cholesterol trafficking, as well as NPC1 and NPC2 mRNA depletion using small interfering RNA, abolished ABCA1-dependent cholesterol efflux induced by PPARα activators.These observations identify a novel regulatory role for PPARα in the control of cholesterol availability for efflux that, associated with its ability to inhibit cholesterol esterification and to stimulate ABCA1 and scavenger receptor class B type I expression, may contribute to the stimulation of reverse cholesterol transport. The mobilization of cholesterol from intracellular pools to the plasma membrane is a determinant that governs its availability for efflux to extracellular acceptors. NPC1 and NPC2 are proteins localized in the late endosome and control cholesterol transport from the lysosome to the plasma membrane. Here, we report that NPC1 and NPC2 gene expression is induced by oxidized LDL (OxLDL) in human macrophages. Because OxLDLs contain natural activators of peroxisome proliferator-activated receptor α (PPARα), a fatty acid-activated nuclear receptor, the regulation of NPC1 and NPC2 by PPARα and the consequences on cholesterol trafficking were further studied. NPC1 and NPC2 expression is induced by synthetic PPARα ligands in human macrophages. Furthermore, PPARα activation leads to an enrichment of cholesterol in the plasma membrane. By contrast, incubation with progesterone, which blocks postlysosomal cholesterol trafficking, as well as NPC1 and NPC2 mRNA depletion using small interfering RNA, abolished ABCA1-dependent cholesterol efflux induced by PPARα activators. These observations identify a novel regulatory role for PPARα in the control of cholesterol availability for efflux that, associated with its ability to inhibit cholesterol esterification and to stimulate ABCA1 and scavenger receptor class B type I expression, may contribute to the stimulation of reverse cholesterol transport. Macrophages play a pivotal role in the development of atherosclerosis. After recruitment in the subendothelial space, monocytes differentiate into macrophages and accumulate lipids, thus forming foam cells. The mobilization of cellular cholesterol to the plasma membrane and its efflux to extracellular acceptors is an important mechanism in the regulation of cellular cholesterol levels. This constitutes the first step of reverse cholesterol transport (RCT), a pathway of cholesterol transport from peripheral tissues to the liver. In addition to the interaction of the HDL particle with its membrane receptors, such as scavenger receptor class B type I (SR-BI), the availability of cholesterol in the plasma membrane is an important determinant for efficient cholesterol efflux. Within the cells, modified LDL-derived cholesteryl esters (CEs) are hydrolyzed in lysosomes to free cholesterol (FC). This FC is probably initially transported to the plasma membrane, where it integrates the cell membrane (1Tabas I. Cholesterol and phospholipid metabolism in macrophages.Biochim. Biophys. Acta. 2000; 1529: 164-174Google Scholar). Excess cholesterol is transported back to the endoplasmic reticulum, where it is reesterified by ACAT1and stored as CEs in lipid droplets (2Buhman K.F. Accad M. Farese R.V. Mammalian acyl-CoA:cholesterol acyltransferases.Biochim. Biophys. Acta. 2000; 1529: 142-154Google Scholar). The plasma membrane contains the highest percentage of cellular cholesterol (3Lange Y. Ramos B.V. Analysis of the distribution of cholesterol in the intact cell.J. Biol. Chem. 1983; 258: 15130-15134Google Scholar). It is likely that maintenance of this cellular cholesterol equilibrium depends on specific intracellular transport processes.Trafficking of cholesterol from the late endosome/lysosome to the plasma membrane is a process controlled by a network of proteins that includes at least two components, namely NPC1 and NPC2 (4Strauss III J.F. Liu P. Christenson L.K. Watari H. Sterols and intracellular vesicular trafficking: lessons from the study of NPC1.Steroids. 2002; 67: 947-951Google Scholar). NPC1 is a transmembrane protein containing a sterol-sensing domain localized in the late endosomal compartment, and NPC2 is a soluble endosomal/lysosomal cholesterol binding protein (5Carstea E.D. Morris J.A. Coleman K.G. Loftus S.K. Zhang D. Cummings C. Gu J. Rosenfeld M.A. Pavan W.J. Krizman D.B. et al.Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis.Science. 1997; 277: 228-231Google Scholar, 6Naureckiene S. Sleat D.E. Lackland H. Fensom A. Vanier M.T. Wattiaux R. Jadot M. Lobel P. Identification of HE1 as the second gene of Niemann-Pick C disease.Science. 2000; 290: 2298-2301Google Scholar). Mutations in the NPC1 and NPC2 genes cause Niemann Pick type C (NPC) disease, a fatal recessive disorder characterized by the accumulation of LDL-derived cholesterol in lysosomes. In the brain, this leads to neuronal degeneration due to a defective movement of sterols out of the expanding pools of lysosomal cholesterol to other locations, in particular, the plasma membrane (7Blanchette-Mackie E.J. Dwyer N.K. Amende L.M. Kruth H.S. Butler J.D. Sokol J. Comly M.E. Vanier M.T. August J.T. Brady R.O. et al.Type-C Niemann-Pick disease: low density lipoprotein uptake is associated with premature cholesterol accumulation in the Golgi complex and excessive cholesterol storage in lysosomes.Proc. Natl. Acad. Sci. USA. 1988; 85: 8022-8026Google Scholar). Mutations in NPC1 cause the majority of cases of NPC disease, whereas mutations in the NPC2 gene account for less than 10% of the cases (4Strauss III J.F. Liu P. Christenson L.K. Watari H. Sterols and intracellular vesicular trafficking: lessons from the study of NPC1.Steroids. 2002; 67: 947-951Google Scholar).Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that regulate the expression of genes controlling lipid and glucose metabolism. PPARα, which is activated by fibrates, fatty acids, and eicosanoids, is highly expressed in liver, heart, muscle, and kidney, and is also present in cells of the arterial wall, including monocytes and macrophages (8Chinetti G. Fruchart J.C. Staels B. Peroxisome proliferator-activated receptors: new targets for the pharmacological modulation of macrophage gene expression and function.Curr. Opin. Lipidol. 2003; 14: 459-468Google Scholar). PPARα controls macrophage lipid homeostasis and cholesterol efflux, the first step of the RCT pathway. PPARα activators enhance the expression of the HDL receptor CLA-1/SR-BI (9Chinetti G. Gbaguidi G.F. Griglio S. Mallat Z. Antonucci M. Poulain P. Chapman J. Fruchart J.C. Tedgui A. Najib-Fruchart J. et al.CLA-1/SR-BI is expressed in atherosclerotic lesion macrophages and regulated by activators of peroxisome proliferator-activated receptors.Circulation. 2000; 101: 2411-2417Google Scholar) and the ABC transporter ABCA1 (10Chinetti G. Lestavel S. Bocher V. Remaley A.T. Neve B. Torra I. Pineda Teissier E. Minnich A. Jaye M. Duverger N. et al.PPARα and PPARγ activators induce cholesterol removal from human macrophage foam cells through stimulation of the ABCA1 pathway.Nat. Med. 2001; 7: 53-58Google Scholar). In addition, PPARα activation inhibits cellular CE formation activity, thus limiting CE accumulation in vascular macrophages and preventing foam cell formation (11Chinetti G. Lestavel S. Fruchart J.C. Clavey V. Staels B. Peroxisome proliferator-activated receptor alpha reduces cholesterol esterification in macrophages.Circ. Res. 2003; 92: 212-217Google Scholar). Along with the induction of ABCA1 expression, a decrease in the CE:FC ratio may contribute to an enhanced liberation and efflux of FC to extracellular acceptors, as observed upon PPARα activation (10Chinetti G. Lestavel S. Bocher V. Remaley A.T. Neve B. Torra I. Pineda Teissier E. Minnich A. Jaye M. Duverger N. et al.PPARα and PPARγ activators induce cholesterol removal from human macrophage foam cells through stimulation of the ABCA1 pathway.Nat. Med. 2001; 7: 53-58Google Scholar, 11Chinetti G. Lestavel S. Fruchart J.C. Clavey V. Staels B. Peroxisome proliferator-activated receptor alpha reduces cholesterol esterification in macrophages.Circ. Res. 2003; 92: 212-217Google Scholar).Here, we report that the expression of NPC1 and NPC2 is strongly induced by oxidized LDL (OxLDL) and to a lesser extent by acetylated LDL (AcLDL) in cultured human macrophages in vitro. Because OxLDLs contain natural ligands for PPARα (12Delerive P. Furman C. Teissier E. Fruchart J-C. Duriez P. Staels B. Oxidized phospholipids activate PPARα in a phospholipase A2-dependent manner.FEBS Lett. 2000; 471: 34-38Google Scholar), these observations prompted us to study whether PPARα also regulates processes controlling cholesterol mobilization upstream of the efflux step. Our results demonstrate that PPARα activation stimulates the postlysosomal mobilization of cholesterol and induces NPC1 and NPC2 gene and protein expression. This results in an enrichment of cholesterol in the plasma membrane and a redistribution of cholesterol in the external cell surface domains. These results identify a new role for PPARα in the control of macrophage cholesterol trafficking, resulting in an enhanced availability of cholesterol at the cell membrane, where it is presented for efflux.MATERIALS AND METHODSTissue and cell cultureMononuclear cells isolated by Ficoll gradient centrifugation from blood of healthy normolipidemic donors were suspended in RPMI 1640 medium containing gentamycin (40 mg/ml), glutamine (0.05%), and 10% pooled human serum (13Chinetti G. Griglio S. Antonucci M. Torra I. Pineda Delerive P. Majd Z. Fruchart J.C. Chapman J. Najib J. Staels B. Activation of peroxisome proliferator-activated receptors α and γ induces apoptosis of human monocyte-derived macrophages.J. Biol. Chem. 1998; 273: 25573-25580Google Scholar). Differentiation of monocytes into macrophages occurs spontaneously by adhesion of 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 analysisTotal cellular RNA was extracted from 10-day-old primary human macrophages treated or not with OxLDL or AcLDL (50 μg/ml) or the PPARα ligands Wy14643 (10, 25, and 50 μmol/l), fenofibric acid (50 μmol/l), ciprofibrate (50 μmol/l), bezafibrate (50 μmol/l), and GW7647 (600 nmol/l) for 24 h using Trizol (Life Technologies, France). For quantitative PCR, total RNA was reverse transcribed using random hexameric primers and Superscript reverse transcriptase (Life Technologies). cDNA was quantified by real-time PCR on a MX 4000 apparatus (Stratagene), using specific primers for: NPC1, 5′-CTT AGT GCA GGA ACT CTG TCC AGG-3′ and 5′-TCC ACA TCA CGG CAG GCA TTG TAC-3′; NPC2, 5′-GGT TTG TCT TGT GAC CGC-3′ and 5′-AGG AAT GTA GCT GCC AGG-3′; ABCA1, 5′-GTG AAC AGC TCC AGC TCC TCC AC-3′ and 5′-AAG GTC TTG TTC ACC TCA GCC ATG AC-3′; and cyclophilin, 5′-GCA TAC GGG TCC TGG CAT CTT GTC C-3′ and 5′-ATG GTG ATC TTC TTG CTG GTC TTG C-3′. PCR amplification was performed in a volume of 25 μl containing 100 nmol/l of each primer, 4 mmol/l MgCl2, the Brillant Quantitative PCR Core Reagent Kit mix as recommended by the manufacturer (Stratagene), and SYBR Green 0.33× (Sigma-Aldrich). The conditions were 95°C for 10 min, followed by 40 cycles of 30 s at 95°C, 30 s at 55°C, and 30 s at 72°C. NPC1, NPC2, and ABCA1 mRNA levels were subsequently normalized to 28S or cyclophilin mRNA.Protein extraction and Western blot analysisCells were harvested in ice-cold lysis buffer containing PBS, 1% NP-40, and protease inhibitor mixture. Equal amounts of proteins (20 μg) were separated by SDS-PAGE (6% gels for NPC1 detection and 12.5% for NPC2), and transferred to Hybond-C Extra membrane (Amersham). Equal protein loading of the gels was verified by Ponceau's red staining. Membranes were blocked with 5% milk (for NPC1 blots) or 3% fatty acid-free BSA (for NPC2 blots) in Tris-buffered saline and 0.1% Tween 20, and immunoblotted using antibodies against NPC1 (14Lusa S. Blom T.S. Eskelinen E.L. Kuismanen E. Mansson J.E. Simons K. Ikonen E. Depletion of rafts in late endocytic membranes is controlled by NPC1-dependent recycling of cholesterol to the plasma membrane.J. Cell Sci. 2001; 114: 1893-1900Google Scholar), NPC2 (15Blom T.S. Linder M.D. Snow K. Pihko H. Hess M.W. Jokitalo E. Veckman V. Syvanen A.C. Ikonen E. Defective endocytic trafficking of NPC1 and NPC2 underlying infantile Niemann-Pick type C disease.Hum. Mol. Genet. 2003; 12: 257-272Google Scholar), or α-calnexin (kindly provided by Dr. Ari Helenius). Western blots were scanned, and the intensity of the bands was quantified using TINA 2.1 software.Labeling of membrane FC with filipinHuman macrophages were pretreated for 24 h and thereafter every 24 h with the PPARα activators bezafibrate (50 μmol/l) or GW7647 (600 nmol/l), and cholesterol-loaded by incubation for 48 h with AcLDL (50 μg/ml) in the presence or absence of progesterone (33 μmol/l) in RPMI 1640 medium supplemented with 1% Nutridoma. After incubation, cells were rinsed twice with ice-cold PBS and fixed with paraformaldehyde (4% in PBS) for 30 min at 4°C before incubation with filipin (50 μg/ml in PBS) for 30 min in the dark at room temperature. Labeling was then visualized by a fluorescence microscope (Axiovert 135 TV Zeiss), and fluorescent signals were quantified using Jimage 1.31v software.Measurement of cholesterol in the outer plasma membrane layerCholesterol content in the outer layer of the plasma membrane was assessed by measuring specific oxidation using exogenous cholesterol oxidase from Pseudomonas fluorescens (Sigma, France) (16Underwood K.W. Andemariam B. McWilliams G.L. Liscum L. Quantitative analysis of hydrophobic amine inhibition of intracellular cholesterol transport.J. Lipid Res. 1996; 37: 1556-1568Google Scholar). Macrophages were pretreated for 24 h and thereafter every 24 h with the PPARα activators Wy14643 (50 μmol/l), bezafibrate (50 μmol/l), ciprofibrate (50 μmol/l), fenofibric acid (50 μmol/l), or GW7647 (600 nmol/l), and cholesterol-loaded by incubation for 48 h with [3H]cholesterol-AcLDL (50 μg/ml) in RPMI 1640 medium supplemented with 1% Nutridoma. Macrophage cultures were then rinsed twice with ice-cold PBS and fixed with paraformaldehyde (4% in PBS) for 30 min at 4°C before incubation for 1 h at 37°C in PBS with or without cholesterol oxidase (1 U/ml). Thereafter, cells were rinsed twice with PBS, and intracellular lipids were extracted by hexane-isopropanol (3:2; v/v) and separated by TLC in petroleum ether-diethyl ether-acetic acid (160:40:2; v/v/v). Spots corresponding to FC and 4-cholestenone were scraped, and radioactivity was measured by scintillation counting. Accessibility of the membrane cholesterol pool to cholesterol oxidase is expressed as the 4-cholestenone:FC ratio.Cholesterol effluxHuman macrophages were pretreated for 24 h and thereafter every 24 h with the PPARα activator GW7647 (600 nmol/l), and cholesterol-loaded by incubation for 48 h with [3H]cholesterol-AcLDL (50 μg/ml) in RPMI 1640 medium supplemented with 1% Nutridoma. After this incubation period, cells were washed twice in PBS, and cholesterol efflux studies mediated by apolipoprotein A-I (apoA-I) were immediately performed by adding fresh RPMI medium without Nutridoma, containing or not 10 μg/ml of apoA-I for 24 h. [3H]cholesterol radioactivity was measured by scintillation counting in centrifuged medium and in cells after extraction of lipids with hexane-isopropanol. Where indicated, progesterone (33 μmol/l) was added during cholesterol-loading and cholesterol efflux periods.Small interfering RNA (siRNA)-mediated knock-down of NPC1 and NPC2Complementary mRNA oligonucleotides derived from the human NPC1 (siRNA ID #8092) and NPC2 (siRNA ID #18116) sequences (Ambion Europe) were used to downregulate NPC1 and NPC2 expression in primary human macrophages. GAPDH siRNA oligonucleotides used as control were also obtained from Ambion. For the cholesterol efflux assays, 10-day-old macrophages were transfected with siRNA using jetSI (Polyplus Transfection, France) according to the manufacturer's instructions and cholesterol-loaded by incubation for 48 h with AcLDL (50 μg/ml, containing [3H]cholesterol) in RPMI 1640 supplemented with 1% Nutridoma in the presence or absence of GW7647 (600 nmol/l). [3H]cholesterol efflux was measured as described above. NPC1 and NPC2 mRNA levels were determined by real-time PCR and normalized to cyclophilin mRNA.Statistical analysisStatistically significant differences between groups were analyzed by Student's t-test and considered significant at P ≤ 0.05.RESULTSNPC1 and NPC2 mRNA levels are induced by OxLDL in differentiated human monocyte-derived macrophagesNPC1 and NPC2 belong to a network of proteins mediating postlysosomal cholesterol trafficking to the plasma membrane, a crucial process governing the balance between macrophage cholesterol import and export, with potential consequences in atherogenesis. To determine whether foam cell formation is accompanied by induction of NPC1 and NPC2 gene expression, differentiated human macrophages were incubated with OxLDL or AcLDL (50 μg/ml) and gene expression was analyzed by quantitative PCR. Our results demonstrate that OxLDL, but not AcLDL, strongly induces NPC1 and NPC2 mRNA levels (Fig. 1B, C, E, F). As a control, ABCA1 gene expression, whose mRNA levels are positively regulated by OxLDL and AcLDL, was measured (Fig. 1A, D).PPARα activation increases NPC1 and NPC2 gene and protein expression in primary human macrophagesBecause OxLDLs, and the oxidized phospholipids contained therein, are natural ligands for PPARα (12Delerive P. Furman C. Teissier E. Fruchart J-C. Duriez P. Staels B. Oxidized phospholipids activate PPARα in a phospholipase A2-dependent manner.FEBS Lett. 2000; 471: 34-38Google Scholar), a nuclear receptor expressed in macrophages controlling cholesterol metabolism (8Chinetti G. Fruchart J.C. Staels B. Peroxisome proliferator-activated receptors: new targets for the pharmacological modulation of macrophage gene expression and function.Curr. Opin. Lipidol. 2003; 14: 459-468Google Scholar), we investigated whether the expression of the NPC1 and NPC2 genes is also controlled by PPARα. Treatment of primary human macrophages with different PPARα activators [Wy14643 (50 μmol/l), GW7647 (600 nmol/l), fenofibric acid (50 μmol/l), bezafibrate (50 μmol/l), and ciprofibrate (50 μmol/l)] resulted in the induction of NPC1 and NPC2 gene expression, an effect that was concentration dependent (Fig. 2).Fig. 2NPC1 and NPC2 gene expression is regulated by peroxisome proliferator-activated receptor α (PPARα) agonists in human macrophages in a dose-dependent manner. Quantitative PCR analysis of NPC1 (A) and NPC2 (B) was performed on RNA isolated from primary human macrophages treated or not with Wy14643 (50 μM), GW7647 (600 nmol/l), fenofibric acid (50 μmol/l), ciprofibrate (50 μM), or bezafibrate (50 μM) for 24 h. Induction of NPC1 (C) and NPC2 (D) expression in human macrophages treated for 24 h with increasing concentrations of Wy14643 (10, 25, and 50 μmol/l). NPC1 and NPC2 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. Statistically significant differences between treatments are indicated (ANOVA followed by Student's t-test; *P < 0.05, **P < 0.01).View Large Image Figure ViewerDownload (PPT)In addition, PPARα activation using fenofibric acid (50 μmol/l) led to a significant increase in NPC1 and NPC2 protein levels in primary macrophages (Fig. 3).Fig. 3PPARα activation induces NPC1 and NPC2 protein expression. Cell proteins were isolated from differentiated human macrophages treated or not with fenofibric acid (50 μmol/l) for 24 h, and NPC1 (A) and NPC2 (B) protein levels were measured by Western blot analysis, as described in Materials and Methods. Specific signals were quantified by densitometry. NPC1 and NPC2 protein levels in fenofibric acid-treated macrophages were normalized to those in untreated control cells set as 1. Results are expressed as mean ± SD of results obtained from three independent cell preparations. Statistically significant differences between treatments are indicated (Student's t-test; *P < 0.05).View Large Image Figure ViewerDownload (PPT)PPARα activation leads to an enrichment of cholesterol in the plasma membrane in primary human macrophagesTo determine whether the regulation of genes involved in cholesterol trafficking from the lysosome by PPARα is accompanied by an accumulation of cholesterol in the plasma membrane, filipin staining experiments were performed on differentiated macrophages loaded with AcLDL (50 μg/ml) for 48 h and treated with PPARα ligands. PPARα activation by bezafibrate increased the amount of FC present in the plasma membrane, as demonstrated by an increase in filipin staining (Fig. 4A). Quantitative determination of the relative filipin fluorescent signal between the perinuclear and plasma membrane subcellular domains was performed using computer-assisted analysis (Fig. 4B). PPARα activation significantly increased the fluorescence at the plasma membrane level, with a concomitant reduction of the signal intensity in the perinuclear region.Fig. 4PPARα activation induces enrichment of cholesterol in the plasma membrane. A: Primary human macrophages were cholesterol-loaded by incubation with 50 μg/ml AcLDL for 24 h. Bezafibrate (50 μmol/l) was added 24 h before and during cholesterol loading. Cells were fixed with paraformaldehyde (4% in PBS for 30 min at 4°C) and stained with filipin (50 μg/ml) for 30 min at room temperature. The figure is representative of two independent experiments. Original magnification, ×40. Arrows indicate cholesterol membrane enrichment in treated cells. B: Fluorescence intensity of filipin signals was quantified using Jimage 1.31v in the perinuclear regions and the plasma membrane. Results are expressed as mean ± SD. Statistically significant differences between treatments are indicated (Student's t-test; ***P < 0.001). AU, arbitrary units.View Large Image Figure ViewerDownload (PPT)To investigate whether PPARα activation results in a change in cholesterol distribution within the plasma membrane, a cholesterol accessibility test using cholesterol oxidase was performed on [3H]cholesterol-AcLDL-loaded macrophages treated with different synthetic PPARα ligands. Treatment with all PPARα ligands tested increased the amount of FC in the outer layer of the plasma membrane, as indicated by an enhanced accessibility of this cholesterol pool to cholesterol oxidase (Fig. 5).Fig. 5PPARα agonists increase outer-layer plasma membrane cholesterol content. Human macrophages were pretreated for 24 h and thereafter every 24 h with Wy14643 (50 μmol/l), GW7647 (600 nmol/l), fenofibric acid (50 μmol/l), ciprofibrate (50 μmol/l), and bezafibrate (50 μmol/l) and cholesterol-loaded by incubation with [3H]cholesterol-AcLDL (50 μg/ml) for 48 h. Cells were then fixed with paraformaldehyde before incubation for 1 h at 37°C in PBS with or without cholesterol oxidase from Pseudomonas fluorescens (1 U/ml). Cellular lipids were then extracted by hexane-isopropanol and separated by TLC. Spots corresponding to free cholesterol and 4-cholestenone were scraped, and radioactivity was measured by scintillation counting. Results are expressed relative to untreated cells set as 1. Statistically significant differences between treatments are indicated (Student's t-test; **P < 0.01, ***P < 0.001). Error bars indicate mean and SD.View Large Image Figure ViewerDownload (PPT)Progesterone blocks the enrichment of plasma membrane cholesterol and cholesterol efflux by PPARα activatorsTo determine the role of the NPC1 and NPC2 pathway on the induction of lysosomal cholesterol mobilization to the plasma membrane by PPARα, the influence of progesterone (33 μmol/l), an inhibitor of postlysosomal cholesterol transport (17Butler J.D. Blanchette-Mackie J. Goldin E. O'Neill R.R. Carstea G. Roff C.F. Patterson M.C. Patel S. Comly M.E. Cooney A. et al.Progesterone blocks cholesterol translocation from lysosomes.J. Biol. Chem. 1992; 267: 23797-23805Google Scholar), on PPARα activator-induced cholesterol trafficking was assessed in AcLDL-loaded macrophages. As previously shown with bezafibrate (Fig. 4A), treatment with the PPARα activator GW7647 induced an enrichment of cholesterol in the plasma membrane. By contrast, when cells were treated with GW7647 in the presence of progesterone, the inductive effect of the PPARα ligand was lost and filipin staining localized mostly in the perinuclear region, suggesting cholesterol accumulation in this cellular compartment (Fig. 6A).Fig. 6Induction of plasma membrane cholesterol enrichment and efflux by PPARα activation is blocked by progesterone. A: Primary human macrophages were cholesterol-loaded by incubation with 50 μg/ml AcLDL for 24 h in the presence (Prog) or absence (−) of progesterone (33 μmol/l). GW7647 (600 nmol/l) was added 24 h before and during cholesterol loading. Cells were fixed with paraformaldehyde and stained with filipin (50 μg/ml for 30 min at room temperature). The figure is representative of two independent experiments. Original magnification, ×40. Arrows indicate cholesterol membrane enrichment in treated cells. B: [3H]cholesterol-loaded macrophages were treated or not with GW7647 (600 nmol/l) in the presence or absence of progesterone (33 μmol/l) and subsequently incubated with RPMI 1640 medium with or without apolipoprotein A-I (apoA-I) (10 μg/ml). ApoA-I-induced [3H]cholesterol efflux was measured as described. Values are expressed relative to the untreated controls set as 1. Statistically significant differences between treatments are indicated (Student's t-test; **P < 0.01, ***P < 0.001). Prog, progesterone. Error bars indicate mean and SD.View Large Image Figure ViewerDownload (PPT)To further investigate the impact of the regulation of cholesterol trafficking by PPARα on the induction of cholesterol efflux, primary human macrophages were loaded with AcLDL (50 μg/ml) for 24 h in the presence or absence of progesterone (33 μmol/l), treated with GW7647 (600 nmol/l), and subsequently exposed to apoA-I for 24 h to induce cholesterol efflux. In the absence of progesterone, apoA-I-specific cholesterol efflux was induced approximately 2-fold in macrophages treated with GW7647 compared with control untreated cells. The presence of progesterone significantly reduced basal efflux and blocked GW7647-stimulated apoA-I-specific efflux (Fig. 6B). These data suggest that the control of cellular cholesterol transport at the lysosomal level by PPARα is a critical intermediate in the induction of cholesterol efflux by its ligands.Suppression of NPC1 and NPC2 expression by siRNA blocks PPARα-induced cholesterol efflux in human macrophagesTo firmly establish the contribution of NPC1 and NPC2 in the regulation of cholesterol efflux by PPARα, an siRNA approach was used to reduce NPC1 or NPC2 gene expression. Quantitative PCR analysis indicated that the specific siRNAs significantly suppressed NPC1 and NPC2 gene expression, by 90% and 70%, respectively, whereas control GAPDH siRNA did not influence their expression (Fig. 7A, B). Reduction of NPC1 or NPC2 expression led to a significant decrease of basal cholesterol efflux to apoA-I (Fig. 7C). In the absence of PPARα ligand, cholesterol efflux in NPC1 or NPC2 siRNA-transfected macrophages was reduced by 40% and 30%, respectively. Transfection with GAPDH siRNA did not affect cholesterol efflux when compared with untransfected cells (data not shown). However, induction of cholesterol efflux upon stimulation with GW7647 (600 nmol/l) was completely abolished in macrophages transfected with NPC1 or NPC2 siRNA, compared with GAPDH siRNA-transfected macrophages. These observations indicate that the regulation of the NPC1 and NPC2 genes, both involved in the postlysosomal cholesterol mobilization to the plasma membrane, participates in the stimulation of cholesterol efflux by PPARα.Fig. 7Suppression of NPC1 and NPC2 expression by small interfering RNA (siRNA) blocks PPARα induction of cholesterol efflux in human macrophages. mRNA levels of NPC1 (A) and NPC2 (B) were determined by quantitative PCR analysis on RNA from siRNA-transfected primary human macrophages treated or not with GW7647 (600 nmol/l) for 24 h. Results are normalized to cyclophilin mRNA and expressed relative to the levels in GAPDH" @default.
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• W2023607090 date "2005-12-01" @default.
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• W2023607090 title "Peroxisome proliferator-activated receptor α controls cellular cholesterol trafficking in macrophages" @default.
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• W2023607090 doi "https://doi.org/10.1194/jlr.m500326-jlr200" @default.
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