Liver X Receptor Activation Controls Intracellular Cholesterol Trafficking and Esterification in Human Macrophages
http://www.100md.com
循环研究杂志 2005年第9期
the UR 545 Inserm (E.R., L.H., S.L., M.L., C.F., M.A.B., S.B., J.C.F., V.C., B.S., G.C.-G.), Institut Pasteur de Lille and Universitee de Lille 2, France
the Institute of Biotechnology (A.L.M., E.I.), University of Helsinki, Finland.
Abstract
Liver X receptors (LXRs) are nuclear receptors that regulate macrophage cholesterol efflux by inducing ATP-binding cassette transporter A1 (ABCA1) and ABCG1/ABCG4 gene expression. The Niemann-Pick C (NPC) proteins NPC1 and NPC2 are located in the late endosome, where they control cholesterol trafficking to the plasma membrane. The mobilization of cholesterol from intracellular pools to the plasma membrane is a determinant governing its availability for efflux to extracellular acceptors. Here we investigated the influence of LXR activation on intracellular cholesterol trafficking in primary human macrophages. Synthetic LXR activators increase the amount of free cholesterol in the plasma membrane by inducing NPC1 and NPC2 gene expression. Moreover, ABCA1-dependent cholesterol efflux induced by LXR activators was drastically decreased in the presence of progesterone, which blocks postlysosomal cholesterol trafficking, and reduced when NPC1 and NPC2 mRNA expression was depleted using small interfering RNA. The stimulation of cholesterol mobilization to the plasma membrane by LXRs led to a decrease in cholesteryl ester formation and AcyleCcoenzyme A cholesterol acyltransferase-1 activity. These data indicate that LXR activation enhances cholesterol trafficking to the plasma membrane, where it becomes available for efflux, at the expense of esterification, thus contributing to the overall effects of LXR agonists in the control of macrophage cholesterol homeostasis.
Key Words: nuclear receptor gene regulation metabolism atherosclerosis
Introduction
During the initial stages of atherogenesis, monocytes migrate into the subendothelial space of blood vessels and differentiate into macrophages. The uptake of excessive amounts of lipoprotein-derived lipids converts them into foam cells.1 After uptake, modified low-density lipoprotein (LDL)eCderived cholesteryl esters (CEs) are hydrolyzed in late endosomes/lysosomes to free cholesterol, which then traffics to and integrates into the plasma membrane.1 Excess membrane cholesterol is transported back to the endoplasmic reticulum, where it is re-esterified with fatty acids (FAs) by acyleCcoenzyme A:cholesterol acyltransferase-1 (ACAT1) and stored in lipid droplets.2 However, not all LDL-derived cholesterol traffics to the plasma membrane because a fraction is directly transported to the endoplasmic reticulum for esterification. The amount of lipid accumulation in macrophages reflects the balance between the rate of cholesterol accumulation/uptake and its removal via the reverse cholesterol transport pathway.3 Availability of cholesterol in the plasma membrane is a crucial step for cholesterol efflux.
The molecular mechanisms responsible for regulating intracellular cholesterol transport and homeostasis are subject of intense investigation, and much of the knowledge has been gained from studies on the Niemann-Pick C (NPC) disease, a fatal recessive disorder caused by mutations in either the NPC1 or NPC2 genes.4 Mutations in NPC1 cause the majority of cases of NPC disease, whereas mutations in the NPC2 gene account for <10% of the cases.5 NPC1 is a transmembrane glycoprotein localized in the late endosomal compartment with a sterol-sensing domain projected into the lumen, whereas NPC2 is a soluble cholesterol-binding protein localized in the core of late endosomes.5 NPC-deficient cells are characterized by the accumulation of LDL-derived free cholesterol within late endosomes/lysosomes attributable to a defective movement of sterols out of the lysosomal expanding pool to other locations, particularly the plasma membrane. Recently, MLN64, a transmembrane protein containing a steroidogenic acute regulatory protein (StAR) domain, was found to be localized with NPC1 in late endosomes, raising the possibility that MLN64 functions in concert with NPC1 and NPC2.6 The StAR domain binds cholesterol and stimulates its movement from donor vesicles to the acceptor membrane. However, mutations in the StAR domain of MLN64 did not markedly impair cholesterol dynamics and had a minor impact on cholesterol movement.7 These findings suggest that proteins with redundant functions can substitute for MLN64 in the cell.
The liver X receptors (LXRs) LXR and LXR are nuclear receptors activated by oxysterols involved in lipid uptake and efflux, lipogenesis, and lipoprotein metabolism.8 LXR activators promote apolipoprotein AI (apoAI)eCmediated cholesterol efflux through the induction of ATP-binding cassette transporter A1 (ABCA1), a direct LXR target gene in human and murine macrophages.9,10 Recently, ABCG1 and ABCG4, involved in lipid efflux to high-density lipoprotein (HDL), were identified as LXR target genes in macrophages.11,12
Although the effect of LXRs on cholesterol efflux from macrophages is well documented, whether LXRs also regulate intracellular metabolism and trafficking of cholesterol has not been studied yet. Here we demonstrate that LXR activation controls positively the postlysosomal mobilization of cholesterol, leading to an enrichment of cholesterol in the plasma membrane, where it redistributes in the external leaflet domains. In addition, we demonstrate that the stimulation of cholesterol mobilization to the plasma membrane by LXRs leads to a reduction of CE content and formation. Our results identify a role for LXR in the control of intracellular cholesterol homeostasis in macrophages upstream of cholesterol efflux.
Materials and Methods
Cell Culture
Human mononuclear cells were isolated from healthy donor blood.13 Mature monocyte-derived macrophages were used after 10 days of culture. Murine bone marroweCderived macrophages (BMDMs) were isolated from C57BL/6J mice.14 For experiments, medium was changed to medium without serum supplemented with 1% Nutridoma HU (Boehringer Mannheim).
RNA Extraction and Analysis
Total cellular RNA isolated from human macrophages treated or not for 24 hours with LXR ligands T0901317 or GW3965 (250 nmol/L, 500 nmol/L, and 1 eol/L) or from mouse BMDM 24-houreCtreated with T0901317 or GW3965 (2 eol/L) using Trizol (Invitrogen), was reverse transcribed using random hexameric primers and Superscript reverse transcriptase (Invitrogen). cDNAs were quantified by quantitative polymerase chain reaction (Q-PCR) on an MX4000 apparatus (Stratagene) using specific primers (see the online supplement, available at http://circres.ahajournals.org).
Filipin Labeling of Membrane Cholesterol
Human macrophages were pretreated for 24 hours and thereafter every 24 hours with T0901317 (1 eol/L) or GW3965 (1 eol/L) and cholesterol loaded by incubation with acetylated LDL (AcLDL; 50 e/mL) in the absence or presence of progesterone (33 eol/L) for 48 hours, and filipin labeling was performed (see online supplement).
Measurement of Cholesterol in the Outer Layer of the Plasma Membrane
Cholesterol in the outer layer of the plasma membrane was assessed by measuring specific oxidation of cholesterol with exogenous cholesterol oxidase from Pseudomonas fluorescens.15 Macrophages were pretreated for 24 hours and thereafter every 24 hours with T0901317 or GW3965 (1 eol/L) and cholesterol loaded by incubation with [3H]cholesterol-AcLDL (50 e/mL) for 48 hours. Cholesterol oxidase accessibility was measured as described15 (see online supplement).
Cholesterol Efflux
Human macrophages were pretreated for 24 hours and thereafter every 24 hours with T0901317 (1 eol/L) and cholesterol loaded by incubation with [3H]cholesterol-AcLDL (50 e/mL) for 48 hours. ApoAI-mediated cholesterol efflux was measured (see online supplement). Where indicated, progesterone (33 eol/L) was added during cholesterol loading and cholesterol efflux periods.
Small Interfering RNAeCMediated Macrophage RNA Interference
Complementary RNA oligonucleotides derived from human NPC1 (small interfering RNA [siRNA] ID 8092) and NPC2 (siRNA ID 18116) sequences (Ambion) were used to downregulate NPC1 and NPC2 expression in primary human macrophages. The scrambled control RNA oligonucleotides were also from Ambion. For cholesterol efflux study, macrophages were transfected with siRNA using jetSI (Polyplus Transfection). Nine hours after transfection, macrophages were pretreated for 24 hours and thereafter every 24 hours with T0901317 (1 eol/L) and cholesterol loaded by incubation with [3H]cholesterol-AcLDL (50 e/mL) in RPMI 1640 medium supplemented with 1% Nutridoma for 48 hours, and [3H]-cholesterol efflux was measured as described above.
Protein Extraction and Western Blot Analysis
Cells were harvested in lysis buffer containing PBS, 1% Nonidet P-40 and protease inhibitors. Proteins (20 e) were separated by SDS-PAGE, transferred to Hybond-C Extra membrane (Amersham), and immunoblotted using antibodies against NPC116 or NPC2.17 Intensity of the bands was quantified using TINA 2.1 software.
Cellular Cholesterol Measurement
Human macrophages were pretreated for 24 hours and thereafter every 24 hours with T0901317 (1 eol/L) or GW3965 (1 eol/L) and cholesterol loaded by incubation with [3H]cholesterol-AcLDL (50 e/mL) for 48 hours. Intracellular lipids were extracted, and total cholesterol and cholesterol distribution were measured (see online supplement).
Measurement of CE Formation
CE formation was assessed by measuring the incorporation of [14C]-oleate into CEs. Human macrophages were cholesterol loaded by incubation with AcLDL (50 e/mL) for 48 hours. T0901317 (1 eol/L) or GW3965 (1 eol/L) was added 24 hours before cholesterol loading and thereafter every 24 hours. After the cholesterol-loading period, CE formation was measured (see online supplement).
Statistical Analysis
Statistical differences between groups were analyzed by Student t and ANOVA tests and were considered significant when P0.05.
Results
LXR Activation Induces Plasma Membrane Cholesterol Enrichment Without Affecting Cholesterol Accumulation in Primary Human Macrophages
To evaluate whether LXR plays a role in cholesterol trafficking to the plasma membrane, filipin staining was performed in primary human differentiated macrophages loaded with AcLDL and treated with two different LXR agonists. LXR activation increased the amount of free cholesterol in the plasma membrane, as demonstrated by an increase of the filipin fluorescent signal (Figure 1A through 1C). Quantitative analysis of the filipin fluorescent signals demonstrated that LXR agonists significantly increased the fluorescence at the plasma membrane level with a concomitant reduction of the signal intensity in the perinuclear region (Figure 1D; ratio of membrane/intracellular intensity: T0901317=2.0±0.4 and GW3965=1.6±0.1 compared with control untreated cells set as 1).
To determine whether LXR activation results in a change in cholesterol distribution within the plasma membrane, a cholesterol oxidase accessibility test was performed. Human macrophages were loaded with [3H]cholesterol-AcLDL, treated with T0901317 or GW3965 (1 eol/L), and then incubated with cholesterol oxidase (1 U/mL). The amount of specifically oxidized [3H]cholesterol in LXR agonist-treated macrophages was 3-fold higher relative to control untreated cells (Figure 2). Filipin staining and cholesterol oxidase assays indicate that LXR ligands increase plasma membrane cholesterol content and induce a redistribution of free cholesterol to the outer layer of the plasma membrane, where it may be more available for efflux.
To verify that the observed plasma membrane cholesterol enrichment on LXR activation is not a consequence of a global effect on lipid accumulation, intracellular cholesterol was measured in macrophages loaded with AcLDL and treated with T0901317 or GW3965 (1 eol/L). LXR activation did not modify macrophage cholesterol accumulation (cholesterol content untreated cells: 29.1±2.4 e/mg protein, T0901317: 32.4±2.8, GW3965: 27.7±2.3, AcLDL: 63.6±6.7, AcLDL+ T0901317: 73.1±2.7, AcLDL+ GW3965: 67.8±7.7), thus excluding the possibility that plasma membrane cholesterol enrichment by LXR is secondary to an effect on cholesterol uptake.
LXR Activation Increases Expression of Genes Involved in Cholesterol Trafficking to the Plasma Membrane
NPC1 and NPC2, and possibly MLN64, mediate intracellular cholesterol trafficking from the endosome/lysosome to the plasma membrane. To investigate a possible role of LXR on the expression of these genes, Q-PCR analysis was performed on primary human macrophages treated with LXR ligands. T0901317 or GW3965 induced NPC1, NPC2, and MLN64 mRNA levels (Figure 3A through 3C), an effect dependent of the ligand concentration used (Figure 3D through 3F). In the same experiments, ABCA1 gene expression was measured as positive control of LXR activation (fold induction of ABCA1 gene: T0901317=2.8±0.3 and GW3965=4.0±1.2 compared with untreated cells set as 1). In contrast, NPC1, NPC2, and MLN64 gene expression was not regulated by LXR agonists in mouse BMDM (Figure 4A through 4C), despite a positive regulation of ABCA1 expression (fold induction of ABCA1 gene: T0901317=7.0±1.7 and GW3965=5.5±0.6 compared with untreated cells set as 1). Interestingly, no induction of cholesterol oxidase accessibility was observed in BMDM on LXR activation (Figure 4D). These data suggest that cholesterol enrichment in the plasma membrane by LXR agonists is induced in a species-specific manner and is not merely a consequence of ABCA1 induction.
LXR-Induced Plasma Membrane Cholesterol Enrichment and Efflux Are Blocked by Progesterone
To analyze the mechanism of LXR-induced cholesterol trafficking from the endosome/lysosome to the plasma membrane, the effect of progesterone, which inhibits postlysosomal cholesterol transport and mimics the phenotype of NPC1-deficient cells,18 on LXR-induced cholesterol trafficking was measured.
First, filipin staining was performed on human macrophages loaded with AcLDL for 24 hours in the presence or not of progesterone (33 eol/L) and treated with T0901317 (1 eol/L). In the absence of progesterone, T0901317 induced an enrichment of cholesterol in the plasma membrane (Figure 5A). In contrast, when cells were treated with T0901317 in the presence of progesterone, filipin fluorescence was enhanced in perinuclear regions, suggesting cholesterol accumulation in this cellular compartment and abolishment of the inductive effect of the LXR ligand (Figure 5A).
Second, primary human macrophages were loaded with AcLDL in the presence or not of progesterone (33 eol/L), treated with T0901317 (1 eol/L), and subsequently exposed to apoAI to induce cholesterol efflux. In the absence of progesterone, apoAI-specific cholesterol efflux was 5-fold induced by T0901317 compared with control untreated cells. The presence of progesterone significantly reduced basal as well as T0901317-stimulated apoAI-specific efflux (Figure 5B). Thus, the control of cholesterol trafficking from the lysosomal compartment to the plasma membrane is a critical intermediate in the induction of cholesterol efflux by LXR.
Suppression of NPC1 and NPC2 Expression by siRNA Decreases LXR Induction of Cholesterol Efflux in Human Macrophages
To determine the contribution of NPC1 and NPC2 induction to cholesterol efflux stimulation by the LXR agonists, an siRNA approach was used to reduce NPC1 or NPC2 gene expression. Q-PCR analysis indicated that the specific siRNAs significantly suppressed NPC1 and NPC2 gene expression, by 80% and 70%, respectively, when compared with scrambled siRNA-transfected cells (Figure 6A and 6B). In addition, NPC1 and NPC2 protein levels were significantly reduced (NPC1 55% of reduction compared with scrambled siRNA-transfected cells, and NPC2 50% of reduction compared with scrambled siRNA-transfected cells). Moreover, no cross-interference was observed between the NPC1 and NPC2 siRNAs (Figure 6A and 6B). NPC1 and NPC2 knockdown did not affect ABCA1 gene expression, which was still positively regulated by T0901317 (data not shown). The reduced expression of NPC1 or NPC2 led to a significant decrease of basal cholesterol efflux (Figure 6C). In the absence of LXR ligand, cholesterol efflux in NPC1 or NPC2 siRNA macrophages was reduced by 40% or 30%, respectively. Scrambled siRNA did not affect cholesterol efflux when compared with untransfected cells (data not shown). In the presence of T0901317 (1 eol/L), cholesterol efflux in NPC1 or NPC2 siRNA macrophages was significantly decreased by 30% or 20%, but the magnitude of the induction by LXR was comparable to those observed in scrambled siRNA-transfected macrophages. This effect could be attributable to a residual LXR regulation of the other NPC, which is not affected by the siRNA (Figure 6C). However, when cholesterol efflux was measured in macrophages transfected with NPC1 and NPC2 siRNA, the stimulation of efflux by T0901317 was significantly decreased compared with scrambled siRNA-transfected macrophages treated with the same compound. Moreover, the fold induction of cholesterol efflux on LXR activation of NPC1/NPC2 siRNA-transfected macrophages was significantly lower than in scrambled siRNA-transfected cells (Figure 6D).
Similar effects on NPC1 and NPC2 mRNA and protein expression, as well as on cholesterol efflux, were observed when NPC1 or NPC2 gene expression was decreased with a second set of siRNA for NPC1 and NPC2 (data not shown).
These observations indicate that the regulation of NPC1 and NPC2, both involved in the postlysosomal cholesterol mobilization to the plasma membrane, is an important step modulating the stimulatory effect of LXR on cholesterol efflux.
LXR Activation Decreases the CE Content in Human Macrophage Foam Cells
Because LXR activation stimulates cholesterol mobilization to the plasma membrane, it was investigated whether this effect could lead to changes in intracellular cholesterol distribution.
Human macrophages were loaded with [3H]cholesterol-AcLDL for 48 hours and treated with T0901317 or GW3965 added 24 hours before cholesterol loading and thereafter every 24 hours. LXR agonists significantly decreased the amount of [H3]cholesterol present in the CEs in a dose-dependent manner (Figure 7A and 7B), suggesting a role for LXR in the control of macrophage cholesterol esterification.
LXR Activation Decreases CE Formation in Human Macrophage Foam Cells
Because LXR activation reduced the amount of CE in macrophages, the role of LXR agonists on ACAT1 activity and gene expression was investigated. Treatment of AcLDL-loaded macrophages with T0901317 or GW3965 (1 eol/L) resulted in a reduction of CE formation as measured by a decreased incorporation of [14C]oleic acid into CEs (Figure 8A). To determine whether LXR decreases CE formation through regulation of ACAT1 gene expression, Q-PCR analysis was performed in human macrophages incubated with T0901317 or GW3965 (1 eol/L) for 24 hours. LXR activation did not affect ACAT1 mRNA levels, excluding that LXR acts via changes in ACAT1 gene expression (Figure 8B). The decreased CE formation by LXR could be attributable to a reduced supply of free cholesterol as substrate for the ACAT1 enzyme.
Discussion
LXRs are oxysterol-activated nuclear receptors expressed in macrophages, where they control cholesterol metabolism and inflammation response.19eC21 In macrophages, LXR ligands enhance ABCA1 expression as well as apoAI-mediated cholesterol efflux9,10 and regulate the expression of the transporters ABCG1 and ABCG4,11 thus stimulating cholesterol efflux to HDL.12 Macrophage cholesterol homeostasis maintenance is the result of a balance between influx, endogenous synthesis, esterification/hydrolysis, and efflux. One determinant governing the rate of cholesterol efflux is the availability of cholesterol in the plasma membrane. In this report, we studied the influence of LXR activation on cholesterol homeostasis in human macrophages upstream of cholesterol efflux.
The control of macrophage cholesterol metabolism begins at the level of lipid uptake. However, LXR activation does not affect AcLDL loading. In contrast, LXR agonists enhance the mobilization of free cholesterol to the plasma membrane, resulting in enriched cholesterol content in the outer layer, thus becoming more available for efflux. The induction of NPC1, NPC2, and MLN64 genes, all involved in the cholesterol trafficking from late endosome/lysosomes to plasma membrane, contributes, at least partly, to the observed effects of the LXR ligands. This regulation occurs in a species-specific manner because no induction was observed in murine BMDM on stimulation. These observations provide evidence for the existence of species differences in response to LXR agonists that may result in distinct regulation of cholesterol homeostasis, as already reported for cholesterol 7-hydroxylase in the liver.22
The enrichment of cholesterol in the plasma membrane and induction of apoAI-specific cholesterol efflux by LXR activation were reduced drastically in the presence of progesterone, which blocks cholesterol mobilization from the late endosome/lysosome and mimics a phenotype comparable to the one observed in NPC-deficient cells.18,23 To further investigate the mechanism by which cholesterol trafficking could contribute to cholesterol efflux, an siRNA approach to knockdown NPC1 or NPC2 expression was used. The implication of MLN64 on cholesterol efflux was not studied because it has been reported that MLN64 plays only a minor role in cholesterol dynamics.7 Interestingly, simultaneous repression of NPC1 and NPC2 expression led to a drastic reduction of basal as well as LXR-induced cholesterol efflux without affecting ABCA1 gene expression. However, LXR activation still resulted in, albeit smaller, an induction of cholesterol efflux, suggesting the existence of other pathways involved in cholesterol movement regulated by LXR. Our observations indicate that stimulation of postlysosomal cholesterol mobilization to the plasma membrane by LXR activation via NPC1 and NPC2 induction is an important step upstream the stimulation of efflux through the ABCA1 pathway. In addition, we provide the first evidence that the NPC2 gene, which accounts for <10% of the NPC cases,5 is involved in the process of cholesterol efflux, similar to NPC1.
Recently, fibroblasts isolated from NPC patients were shown to display a reduced accessibility of plasma membrane cholesterol to cholesterol oxidase24 associated with an impaired cholesterol efflux to apoAI.25 In line, NPC1-deficient subjects have decreased plasma HDL cholesterol levels.25 The effects on NPC1 and NPC2 combined with the ability of LXR agonists to induce ABCA1-dependent apoAI-specific cholesterol efflux9,10 and to stimulate cellular cholesterol efflux to HDL, via the induction of ABCG1 and ABCG4,11,12 are supportive for a role of LXR in the control of HDL metabolism.
The induction of NPC1 and NPC2 gene expression by LXR associated to its ability to induce cholesterol efflux could have physiological consequences in vivo in cells other than macrophages. Of particular interest are recent studies suggesting a role of cholesterol metabolism in the development of Alzheimer’s disease.26 In neuronal cells, which express ABCA1,27 stimulation of cholesterol efflux decreased amyloid- peptide secretion.26
LXR activation also decreased cholesterol esterification rates and reduced CE levels in human macrophage foam cells. These actions of LXR are not attributable to a decreased gene expression of ACAT1, the enzyme responsible for cholesterol esterification in macrophages. ACAT1 enzyme activity and cholesterol esterification rate are also controlled by FA availability, which could depend partially on their catabolism by enzymes such as carnitine palmitoyltransferase 1 (CPT-1), an enzyme located in the mitochondrial outer membrane catalyzing the entry of long-chain FAs into the mitochondria for their -oxidation.28 However, LXR activation did not affect CPT-1 mRNA levels in macrophages (data not shown), thus rendering the possibility that reduced cholesterol esterification is attributable to lowered FA substrate availability unlikely. Moreover, LXR activators did not affect neutral CE hydrolase activity in macrophages (data not shown), thus indicating that the effects of LXR occur rather via inhibition of cholesterol esterification than by stimulation of CE hydrolysis. Thus, it is likely that the stimulation of cholesterol mobilization to the plasma membrane by LXR activators results in a reduced availability of cholesterol as substrate for ACAT1.
The observed effects on cholesterol esterification on LXR activation could also reflect the capacity of LXR to induce spontaneous cholesterol efflux from macrophages, even in the absence of any extracellular acceptor in the medium, as demonstrated by Cignarella et al in macrophages simultaneously treated with LXR and retinoid X receptor (RXR) ligands.29 Our results, obtained on incubation with specific LXR agonists in the absence of RXR agonists, indicate that LXR activation increases only very weakly the spontaneous cholesterol efflux to culture medium compared with the observed induction in the presence of apoAI (data not shown). Thus, it is unlikely that under the condition studied here, the reduction of cholesterol esterification on LXR stimulation is a consequence of an induction of passive cholesterol efflux.
In conclusion, our results demonstrate a novel role for LXR in the control of cholesterol mobilization and distribution, an effect which, associated with the induction of ABCA1 and ABCG1/ABCG4, 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 the Fondation Leducq and the Nouvelle Societee Franaise d’Atheeroscleerose (L.H.). We thank C. Duhem and D. Deslee for their technical assistance, and K. Bertrand (Genfit SA, France) for providing the T0901317 and GW3965 compounds.
References
Tabas I. Cholesterol and phospholipid metabolism in macrophages. Biochim Biophys Acta. 2000; 1529: 164eC174.
Buhman KF, Accad M, Farese RV. Mammalian acyl-CoA:cholesterol acyltransferases. Biochim Biophys Acta. 2000; 1529: 142eC154.
von Eckardstein A, Nofer JR, Assmann G. High density lipoproteins and arteriosclerosis. Role of cholesterol efflux and reverse cholesterol transport. Arterioscler Thromb Vasc Biol. 2001; 21: 13eC27.
Carstea ED, Morris JA, Coleman KG, Loftus SK, Zhang D, Cummings C, Gu J, Rosenfeld MA, Pavan WJ, Krizman DB, Nagle J, Polymeropoulos MH, Sturley SL, Ioannou YA, Higgins ME, Comly M, Cooney A, Brown A, Kaneski CR, Blanchette-Mackie EJ, Dwyer NK, Neufeld EB, Chang TY, Liscum L, Strauss JF III, Ohno K, Zeigler M, Carmi R, Sokol J, Markie D, O’Neill RR, van Diggelen OP, Elleder M, Patterson MC, Brady RO, Vanier MT, Pentchev PG, Tagle DA. Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis. Science. 1997; 277: 228eC231.
Naureckiene S, Sleat DE, Lackland H, Fensom A, Vanier MT, Wattiaux R, Jadot M, Lobel P. Identification of HE1 as the second gene of Niemann-Pick C disease. Science. 2000; 290: 2298eC2301.
Watari H, Arakane F, Moog-Lutz C, Kallen CB, Tomasetto C, Gerton GL, Rio MC, Baker ME, Strauss JF III. MLN64 contains a domain with homology to the steroidogenic acute regulatory protein (StAR) that stimulates steroidogenesis. Proc Natl Acad Sci U S A. 1997; 94: 8462eC8427.
Kishida T, Kostetskii I, Zhang Z, Martinez F, Liu P, Walkley SU, Dwyer NK, Blanchette-Mackie EJ, Radice GL, Strauss JF III. Targeted mutation of the MLN64 START domain causes only modest alterations in cellular sterol metabolism. J Biol Chem. 2004; 279: 19276eC19285.
Janowski BA, Willy PJ, Devi TR, Falck JR, Mangelsdorf DJ. An oxysterol signalling pathway mediated by the nuclear receptor LXR alpha. Nature (London). 1996; 383: 728eC731.
Costet P, Luo Y, Wang N, Tall AR. Sterol-dependent transactivation of the human ABC1 promoter by LXR/RXR. J Biol Chem. 2000; 275: 28240eC28245.
Venkateswaran A, Laffitte BA, Joseph SB, Mak PA, Wilpitz DC, Edwards PA, Tontonoz P. Control of cellular cholesterol efflux by the nuclear oxysterol receptor LXRalpha. Proc Natl Acad Sci U S A. 2000; 97: 12097eC12102.
Engel T, Lorkowski S, Lueken A, Rust S, Schluter B, Berger G, Cullen P, Assmann G. The human ABCG4 gene is regulated by oxysterols and retinoids in monocyte-derived macrophages. Biochem Biophys Res Comm. 2001; 288: 483eC488.
Wang N, Lan D, Chen W, Matsuura F, Tall AR ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipoproteins. Proc Natl Acad Sci U S A. 2004: 9774eC9779.
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: 25573eC25580.
Van Eck M, De Winther MP, Herijgers N, Havekes LM, Hofker MH, Groot PH, Van Berkel TJ. Effect of human scavenger receptor class A overexpression in bone marrow-derived cells on cholesterol levels and atherosclerosis in apoE-deficient mice. Arterioscler Thromb Vasc Biol. 2000; 20: 2600eC2606.
Underwood KW, Andemariam B, McWilliams GL, Liscum L. Quantitative analysis of hydrophobic amine inhibition of intracellular cholesterol transport. J Lipid Res. 1996; 37: 1556eC1568.
Lusa S, Blom TS, Eskelinen EL, Kuismanen E, Mansson JE, 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: 1893eC1900.
Blom TS, Linder MD, Snow K, Pihko H, Hess MW, Jokitalo E, Veckman V, Syvanen AC, Ikonen E. Defective endocytic trafficking of NPC1 and NPC2 underlying infantile Niemann-Pick type C disease. Hum Mol Genet. 2003; 12: 257eC272.
Butler JD, Blanchette-Mackie J, Goldin E, O’Neill RR, Carstea G, Roff CF, Patterson MC, Patel S, Comly ME, Cooney A, et al. Progesterone blocks cholesterol translocation from lysosomes. J Biol Chem. 1992; 267: 23797eC23805.
Tontonoz P, Mangelsdorf DJ. Liver X receptor signaling pathways in cardiovascular disease. Mol Endocrinol. 2003; 17: 985eC993.
Joseph SB, Castrillo A, Laffitte BA, Mangelsdorf DJ, Tontonoz P. Reciprocal regulation of inflammation and lipid metabolism by liver X receptors. Nat Med. 2003; 9: 213eC219.
Terasaka N, Hiroshima A, Ariga A, Honzumi S, Koieyama T, Inaba T, Fujiwara T. Liver X receptor agonists inhibit tissue factor expression in macrophages. FEBS J. 2005; 272: 1546eC1556.
Menke JG, Macnaul KL, Hayes NS, Baffic J, Chao YS, Elbrecht A, Kelly LJ, Lam MH, Schmidt A, Sahoo S, Wang J, Wright SD, Xin P, Zhou G, Moller DE, Sparrow CP. A novel liver X receptor agonist establishes species differences in the regulation of cholesterol 7alpha-hydroxylase (CYP7a). Endocrinology. 2002; 143: 2548eC2558.
Sato M, Akaboshi S, Katsumoto T, Taniguchi M, Higaki K, Tai T, Sakuraba H, Ohno K. Accumulation of cholesterol and GM2 ganglioside in cells cultured in the presence of progesterone: an implication for the basic defect in Niemann-Pick disease type C. Brain Dev. 1998; 20: 50eC52.
Garver WS, Krishnan K, Gallagos JR, Michikawa M, Francis GA, Heidenreich RA. Niemann-Pick C1 protein regulates cholesterol transport to the trans-Golgi network and plasma membrane caveolae. J Lipid Res. 2002; 43: 579eC589.
Choi HY, Karten B, Chan T, Vance JE, Greer WL, Heidenreich RA, Garver WS, Francis GA. Impaired ABCA1-dependent lipid efflux and hypoalphalipoproteinemia in human Niemann-Pick type C disease. J Biol Chem. 2003; 278: 32569eC32577.
Sun Y, Yao J, Kim TW, Tall AR. Expression of liver X receptor target genes decreases cellular amyloid beta peptide secretion. J Biol Chem. 2003; 278: 27688eC27694.
Panzenboeck U, Balazs Z, Sovic A, Hrzenjak A, Levak-Frank S, Wintersperger A, Malle E, Sattler W. ABCA1 and scavenger receptor class B, type I, are modulators of reverse sterol transport at an in vitro blood-brain barrier constituted of porcine brain capillary endothelial cells. J Biol Chem. 2002; 277: 42781eC42789.
McGarry JD, Brown NF. The mitochondrial carnitine palmitoyltransferase system. From concept to molecular analysis. Eur J Biochem. 1997; 244: 1eC14.
Cignarella A, Engel T, von Eckardstein A, Kratz M, Lorkowski S, Lueken A, Assmann G, Cullen P. Pharmacological regulation of cholesterol efflux in human monocyte-derived macrophages in the absence of exogenous cholesterol acceptors. Atherosclerosis. 2005; 179: 229eC236.(E. Rigamonti, L. Helin, S)
the Institute of Biotechnology (A.L.M., E.I.), University of Helsinki, Finland.
Abstract
Liver X receptors (LXRs) are nuclear receptors that regulate macrophage cholesterol efflux by inducing ATP-binding cassette transporter A1 (ABCA1) and ABCG1/ABCG4 gene expression. The Niemann-Pick C (NPC) proteins NPC1 and NPC2 are located in the late endosome, where they control cholesterol trafficking to the plasma membrane. The mobilization of cholesterol from intracellular pools to the plasma membrane is a determinant governing its availability for efflux to extracellular acceptors. Here we investigated the influence of LXR activation on intracellular cholesterol trafficking in primary human macrophages. Synthetic LXR activators increase the amount of free cholesterol in the plasma membrane by inducing NPC1 and NPC2 gene expression. Moreover, ABCA1-dependent cholesterol efflux induced by LXR activators was drastically decreased in the presence of progesterone, which blocks postlysosomal cholesterol trafficking, and reduced when NPC1 and NPC2 mRNA expression was depleted using small interfering RNA. The stimulation of cholesterol mobilization to the plasma membrane by LXRs led to a decrease in cholesteryl ester formation and AcyleCcoenzyme A cholesterol acyltransferase-1 activity. These data indicate that LXR activation enhances cholesterol trafficking to the plasma membrane, where it becomes available for efflux, at the expense of esterification, thus contributing to the overall effects of LXR agonists in the control of macrophage cholesterol homeostasis.
Key Words: nuclear receptor gene regulation metabolism atherosclerosis
Introduction
During the initial stages of atherogenesis, monocytes migrate into the subendothelial space of blood vessels and differentiate into macrophages. The uptake of excessive amounts of lipoprotein-derived lipids converts them into foam cells.1 After uptake, modified low-density lipoprotein (LDL)eCderived cholesteryl esters (CEs) are hydrolyzed in late endosomes/lysosomes to free cholesterol, which then traffics to and integrates into the plasma membrane.1 Excess membrane cholesterol is transported back to the endoplasmic reticulum, where it is re-esterified with fatty acids (FAs) by acyleCcoenzyme A:cholesterol acyltransferase-1 (ACAT1) and stored in lipid droplets.2 However, not all LDL-derived cholesterol traffics to the plasma membrane because a fraction is directly transported to the endoplasmic reticulum for esterification. The amount of lipid accumulation in macrophages reflects the balance between the rate of cholesterol accumulation/uptake and its removal via the reverse cholesterol transport pathway.3 Availability of cholesterol in the plasma membrane is a crucial step for cholesterol efflux.
The molecular mechanisms responsible for regulating intracellular cholesterol transport and homeostasis are subject of intense investigation, and much of the knowledge has been gained from studies on the Niemann-Pick C (NPC) disease, a fatal recessive disorder caused by mutations in either the NPC1 or NPC2 genes.4 Mutations in NPC1 cause the majority of cases of NPC disease, whereas mutations in the NPC2 gene account for <10% of the cases.5 NPC1 is a transmembrane glycoprotein localized in the late endosomal compartment with a sterol-sensing domain projected into the lumen, whereas NPC2 is a soluble cholesterol-binding protein localized in the core of late endosomes.5 NPC-deficient cells are characterized by the accumulation of LDL-derived free cholesterol within late endosomes/lysosomes attributable to a defective movement of sterols out of the lysosomal expanding pool to other locations, particularly the plasma membrane. Recently, MLN64, a transmembrane protein containing a steroidogenic acute regulatory protein (StAR) domain, was found to be localized with NPC1 in late endosomes, raising the possibility that MLN64 functions in concert with NPC1 and NPC2.6 The StAR domain binds cholesterol and stimulates its movement from donor vesicles to the acceptor membrane. However, mutations in the StAR domain of MLN64 did not markedly impair cholesterol dynamics and had a minor impact on cholesterol movement.7 These findings suggest that proteins with redundant functions can substitute for MLN64 in the cell.
The liver X receptors (LXRs) LXR and LXR are nuclear receptors activated by oxysterols involved in lipid uptake and efflux, lipogenesis, and lipoprotein metabolism.8 LXR activators promote apolipoprotein AI (apoAI)eCmediated cholesterol efflux through the induction of ATP-binding cassette transporter A1 (ABCA1), a direct LXR target gene in human and murine macrophages.9,10 Recently, ABCG1 and ABCG4, involved in lipid efflux to high-density lipoprotein (HDL), were identified as LXR target genes in macrophages.11,12
Although the effect of LXRs on cholesterol efflux from macrophages is well documented, whether LXRs also regulate intracellular metabolism and trafficking of cholesterol has not been studied yet. Here we demonstrate that LXR activation controls positively the postlysosomal mobilization of cholesterol, leading to an enrichment of cholesterol in the plasma membrane, where it redistributes in the external leaflet domains. In addition, we demonstrate that the stimulation of cholesterol mobilization to the plasma membrane by LXRs leads to a reduction of CE content and formation. Our results identify a role for LXR in the control of intracellular cholesterol homeostasis in macrophages upstream of cholesterol efflux.
Materials and Methods
Cell Culture
Human mononuclear cells were isolated from healthy donor blood.13 Mature monocyte-derived macrophages were used after 10 days of culture. Murine bone marroweCderived macrophages (BMDMs) were isolated from C57BL/6J mice.14 For experiments, medium was changed to medium without serum supplemented with 1% Nutridoma HU (Boehringer Mannheim).
RNA Extraction and Analysis
Total cellular RNA isolated from human macrophages treated or not for 24 hours with LXR ligands T0901317 or GW3965 (250 nmol/L, 500 nmol/L, and 1 eol/L) or from mouse BMDM 24-houreCtreated with T0901317 or GW3965 (2 eol/L) using Trizol (Invitrogen), was reverse transcribed using random hexameric primers and Superscript reverse transcriptase (Invitrogen). cDNAs were quantified by quantitative polymerase chain reaction (Q-PCR) on an MX4000 apparatus (Stratagene) using specific primers (see the online supplement, available at http://circres.ahajournals.org).
Filipin Labeling of Membrane Cholesterol
Human macrophages were pretreated for 24 hours and thereafter every 24 hours with T0901317 (1 eol/L) or GW3965 (1 eol/L) and cholesterol loaded by incubation with acetylated LDL (AcLDL; 50 e/mL) in the absence or presence of progesterone (33 eol/L) for 48 hours, and filipin labeling was performed (see online supplement).
Measurement of Cholesterol in the Outer Layer of the Plasma Membrane
Cholesterol in the outer layer of the plasma membrane was assessed by measuring specific oxidation of cholesterol with exogenous cholesterol oxidase from Pseudomonas fluorescens.15 Macrophages were pretreated for 24 hours and thereafter every 24 hours with T0901317 or GW3965 (1 eol/L) and cholesterol loaded by incubation with [3H]cholesterol-AcLDL (50 e/mL) for 48 hours. Cholesterol oxidase accessibility was measured as described15 (see online supplement).
Cholesterol Efflux
Human macrophages were pretreated for 24 hours and thereafter every 24 hours with T0901317 (1 eol/L) and cholesterol loaded by incubation with [3H]cholesterol-AcLDL (50 e/mL) for 48 hours. ApoAI-mediated cholesterol efflux was measured (see online supplement). Where indicated, progesterone (33 eol/L) was added during cholesterol loading and cholesterol efflux periods.
Small Interfering RNAeCMediated Macrophage RNA Interference
Complementary RNA oligonucleotides derived from human NPC1 (small interfering RNA [siRNA] ID 8092) and NPC2 (siRNA ID 18116) sequences (Ambion) were used to downregulate NPC1 and NPC2 expression in primary human macrophages. The scrambled control RNA oligonucleotides were also from Ambion. For cholesterol efflux study, macrophages were transfected with siRNA using jetSI (Polyplus Transfection). Nine hours after transfection, macrophages were pretreated for 24 hours and thereafter every 24 hours with T0901317 (1 eol/L) and cholesterol loaded by incubation with [3H]cholesterol-AcLDL (50 e/mL) in RPMI 1640 medium supplemented with 1% Nutridoma for 48 hours, and [3H]-cholesterol efflux was measured as described above.
Protein Extraction and Western Blot Analysis
Cells were harvested in lysis buffer containing PBS, 1% Nonidet P-40 and protease inhibitors. Proteins (20 e) were separated by SDS-PAGE, transferred to Hybond-C Extra membrane (Amersham), and immunoblotted using antibodies against NPC116 or NPC2.17 Intensity of the bands was quantified using TINA 2.1 software.
Cellular Cholesterol Measurement
Human macrophages were pretreated for 24 hours and thereafter every 24 hours with T0901317 (1 eol/L) or GW3965 (1 eol/L) and cholesterol loaded by incubation with [3H]cholesterol-AcLDL (50 e/mL) for 48 hours. Intracellular lipids were extracted, and total cholesterol and cholesterol distribution were measured (see online supplement).
Measurement of CE Formation
CE formation was assessed by measuring the incorporation of [14C]-oleate into CEs. Human macrophages were cholesterol loaded by incubation with AcLDL (50 e/mL) for 48 hours. T0901317 (1 eol/L) or GW3965 (1 eol/L) was added 24 hours before cholesterol loading and thereafter every 24 hours. After the cholesterol-loading period, CE formation was measured (see online supplement).
Statistical Analysis
Statistical differences between groups were analyzed by Student t and ANOVA tests and were considered significant when P0.05.
Results
LXR Activation Induces Plasma Membrane Cholesterol Enrichment Without Affecting Cholesterol Accumulation in Primary Human Macrophages
To evaluate whether LXR plays a role in cholesterol trafficking to the plasma membrane, filipin staining was performed in primary human differentiated macrophages loaded with AcLDL and treated with two different LXR agonists. LXR activation increased the amount of free cholesterol in the plasma membrane, as demonstrated by an increase of the filipin fluorescent signal (Figure 1A through 1C). Quantitative analysis of the filipin fluorescent signals demonstrated that LXR agonists significantly increased the fluorescence at the plasma membrane level with a concomitant reduction of the signal intensity in the perinuclear region (Figure 1D; ratio of membrane/intracellular intensity: T0901317=2.0±0.4 and GW3965=1.6±0.1 compared with control untreated cells set as 1).
To determine whether LXR activation results in a change in cholesterol distribution within the plasma membrane, a cholesterol oxidase accessibility test was performed. Human macrophages were loaded with [3H]cholesterol-AcLDL, treated with T0901317 or GW3965 (1 eol/L), and then incubated with cholesterol oxidase (1 U/mL). The amount of specifically oxidized [3H]cholesterol in LXR agonist-treated macrophages was 3-fold higher relative to control untreated cells (Figure 2). Filipin staining and cholesterol oxidase assays indicate that LXR ligands increase plasma membrane cholesterol content and induce a redistribution of free cholesterol to the outer layer of the plasma membrane, where it may be more available for efflux.
To verify that the observed plasma membrane cholesterol enrichment on LXR activation is not a consequence of a global effect on lipid accumulation, intracellular cholesterol was measured in macrophages loaded with AcLDL and treated with T0901317 or GW3965 (1 eol/L). LXR activation did not modify macrophage cholesterol accumulation (cholesterol content untreated cells: 29.1±2.4 e/mg protein, T0901317: 32.4±2.8, GW3965: 27.7±2.3, AcLDL: 63.6±6.7, AcLDL+ T0901317: 73.1±2.7, AcLDL+ GW3965: 67.8±7.7), thus excluding the possibility that plasma membrane cholesterol enrichment by LXR is secondary to an effect on cholesterol uptake.
LXR Activation Increases Expression of Genes Involved in Cholesterol Trafficking to the Plasma Membrane
NPC1 and NPC2, and possibly MLN64, mediate intracellular cholesterol trafficking from the endosome/lysosome to the plasma membrane. To investigate a possible role of LXR on the expression of these genes, Q-PCR analysis was performed on primary human macrophages treated with LXR ligands. T0901317 or GW3965 induced NPC1, NPC2, and MLN64 mRNA levels (Figure 3A through 3C), an effect dependent of the ligand concentration used (Figure 3D through 3F). In the same experiments, ABCA1 gene expression was measured as positive control of LXR activation (fold induction of ABCA1 gene: T0901317=2.8±0.3 and GW3965=4.0±1.2 compared with untreated cells set as 1). In contrast, NPC1, NPC2, and MLN64 gene expression was not regulated by LXR agonists in mouse BMDM (Figure 4A through 4C), despite a positive regulation of ABCA1 expression (fold induction of ABCA1 gene: T0901317=7.0±1.7 and GW3965=5.5±0.6 compared with untreated cells set as 1). Interestingly, no induction of cholesterol oxidase accessibility was observed in BMDM on LXR activation (Figure 4D). These data suggest that cholesterol enrichment in the plasma membrane by LXR agonists is induced in a species-specific manner and is not merely a consequence of ABCA1 induction.
LXR-Induced Plasma Membrane Cholesterol Enrichment and Efflux Are Blocked by Progesterone
To analyze the mechanism of LXR-induced cholesterol trafficking from the endosome/lysosome to the plasma membrane, the effect of progesterone, which inhibits postlysosomal cholesterol transport and mimics the phenotype of NPC1-deficient cells,18 on LXR-induced cholesterol trafficking was measured.
First, filipin staining was performed on human macrophages loaded with AcLDL for 24 hours in the presence or not of progesterone (33 eol/L) and treated with T0901317 (1 eol/L). In the absence of progesterone, T0901317 induced an enrichment of cholesterol in the plasma membrane (Figure 5A). In contrast, when cells were treated with T0901317 in the presence of progesterone, filipin fluorescence was enhanced in perinuclear regions, suggesting cholesterol accumulation in this cellular compartment and abolishment of the inductive effect of the LXR ligand (Figure 5A).
Second, primary human macrophages were loaded with AcLDL in the presence or not of progesterone (33 eol/L), treated with T0901317 (1 eol/L), and subsequently exposed to apoAI to induce cholesterol efflux. In the absence of progesterone, apoAI-specific cholesterol efflux was 5-fold induced by T0901317 compared with control untreated cells. The presence of progesterone significantly reduced basal as well as T0901317-stimulated apoAI-specific efflux (Figure 5B). Thus, the control of cholesterol trafficking from the lysosomal compartment to the plasma membrane is a critical intermediate in the induction of cholesterol efflux by LXR.
Suppression of NPC1 and NPC2 Expression by siRNA Decreases LXR Induction of Cholesterol Efflux in Human Macrophages
To determine the contribution of NPC1 and NPC2 induction to cholesterol efflux stimulation by the LXR agonists, an siRNA approach was used to reduce NPC1 or NPC2 gene expression. Q-PCR analysis indicated that the specific siRNAs significantly suppressed NPC1 and NPC2 gene expression, by 80% and 70%, respectively, when compared with scrambled siRNA-transfected cells (Figure 6A and 6B). In addition, NPC1 and NPC2 protein levels were significantly reduced (NPC1 55% of reduction compared with scrambled siRNA-transfected cells, and NPC2 50% of reduction compared with scrambled siRNA-transfected cells). Moreover, no cross-interference was observed between the NPC1 and NPC2 siRNAs (Figure 6A and 6B). NPC1 and NPC2 knockdown did not affect ABCA1 gene expression, which was still positively regulated by T0901317 (data not shown). The reduced expression of NPC1 or NPC2 led to a significant decrease of basal cholesterol efflux (Figure 6C). In the absence of LXR ligand, cholesterol efflux in NPC1 or NPC2 siRNA macrophages was reduced by 40% or 30%, respectively. Scrambled siRNA did not affect cholesterol efflux when compared with untransfected cells (data not shown). In the presence of T0901317 (1 eol/L), cholesterol efflux in NPC1 or NPC2 siRNA macrophages was significantly decreased by 30% or 20%, but the magnitude of the induction by LXR was comparable to those observed in scrambled siRNA-transfected macrophages. This effect could be attributable to a residual LXR regulation of the other NPC, which is not affected by the siRNA (Figure 6C). However, when cholesterol efflux was measured in macrophages transfected with NPC1 and NPC2 siRNA, the stimulation of efflux by T0901317 was significantly decreased compared with scrambled siRNA-transfected macrophages treated with the same compound. Moreover, the fold induction of cholesterol efflux on LXR activation of NPC1/NPC2 siRNA-transfected macrophages was significantly lower than in scrambled siRNA-transfected cells (Figure 6D).
Similar effects on NPC1 and NPC2 mRNA and protein expression, as well as on cholesterol efflux, were observed when NPC1 or NPC2 gene expression was decreased with a second set of siRNA for NPC1 and NPC2 (data not shown).
These observations indicate that the regulation of NPC1 and NPC2, both involved in the postlysosomal cholesterol mobilization to the plasma membrane, is an important step modulating the stimulatory effect of LXR on cholesterol efflux.
LXR Activation Decreases the CE Content in Human Macrophage Foam Cells
Because LXR activation stimulates cholesterol mobilization to the plasma membrane, it was investigated whether this effect could lead to changes in intracellular cholesterol distribution.
Human macrophages were loaded with [3H]cholesterol-AcLDL for 48 hours and treated with T0901317 or GW3965 added 24 hours before cholesterol loading and thereafter every 24 hours. LXR agonists significantly decreased the amount of [H3]cholesterol present in the CEs in a dose-dependent manner (Figure 7A and 7B), suggesting a role for LXR in the control of macrophage cholesterol esterification.
LXR Activation Decreases CE Formation in Human Macrophage Foam Cells
Because LXR activation reduced the amount of CE in macrophages, the role of LXR agonists on ACAT1 activity and gene expression was investigated. Treatment of AcLDL-loaded macrophages with T0901317 or GW3965 (1 eol/L) resulted in a reduction of CE formation as measured by a decreased incorporation of [14C]oleic acid into CEs (Figure 8A). To determine whether LXR decreases CE formation through regulation of ACAT1 gene expression, Q-PCR analysis was performed in human macrophages incubated with T0901317 or GW3965 (1 eol/L) for 24 hours. LXR activation did not affect ACAT1 mRNA levels, excluding that LXR acts via changes in ACAT1 gene expression (Figure 8B). The decreased CE formation by LXR could be attributable to a reduced supply of free cholesterol as substrate for the ACAT1 enzyme.
Discussion
LXRs are oxysterol-activated nuclear receptors expressed in macrophages, where they control cholesterol metabolism and inflammation response.19eC21 In macrophages, LXR ligands enhance ABCA1 expression as well as apoAI-mediated cholesterol efflux9,10 and regulate the expression of the transporters ABCG1 and ABCG4,11 thus stimulating cholesterol efflux to HDL.12 Macrophage cholesterol homeostasis maintenance is the result of a balance between influx, endogenous synthesis, esterification/hydrolysis, and efflux. One determinant governing the rate of cholesterol efflux is the availability of cholesterol in the plasma membrane. In this report, we studied the influence of LXR activation on cholesterol homeostasis in human macrophages upstream of cholesterol efflux.
The control of macrophage cholesterol metabolism begins at the level of lipid uptake. However, LXR activation does not affect AcLDL loading. In contrast, LXR agonists enhance the mobilization of free cholesterol to the plasma membrane, resulting in enriched cholesterol content in the outer layer, thus becoming more available for efflux. The induction of NPC1, NPC2, and MLN64 genes, all involved in the cholesterol trafficking from late endosome/lysosomes to plasma membrane, contributes, at least partly, to the observed effects of the LXR ligands. This regulation occurs in a species-specific manner because no induction was observed in murine BMDM on stimulation. These observations provide evidence for the existence of species differences in response to LXR agonists that may result in distinct regulation of cholesterol homeostasis, as already reported for cholesterol 7-hydroxylase in the liver.22
The enrichment of cholesterol in the plasma membrane and induction of apoAI-specific cholesterol efflux by LXR activation were reduced drastically in the presence of progesterone, which blocks cholesterol mobilization from the late endosome/lysosome and mimics a phenotype comparable to the one observed in NPC-deficient cells.18,23 To further investigate the mechanism by which cholesterol trafficking could contribute to cholesterol efflux, an siRNA approach to knockdown NPC1 or NPC2 expression was used. The implication of MLN64 on cholesterol efflux was not studied because it has been reported that MLN64 plays only a minor role in cholesterol dynamics.7 Interestingly, simultaneous repression of NPC1 and NPC2 expression led to a drastic reduction of basal as well as LXR-induced cholesterol efflux without affecting ABCA1 gene expression. However, LXR activation still resulted in, albeit smaller, an induction of cholesterol efflux, suggesting the existence of other pathways involved in cholesterol movement regulated by LXR. Our observations indicate that stimulation of postlysosomal cholesterol mobilization to the plasma membrane by LXR activation via NPC1 and NPC2 induction is an important step upstream the stimulation of efflux through the ABCA1 pathway. In addition, we provide the first evidence that the NPC2 gene, which accounts for <10% of the NPC cases,5 is involved in the process of cholesterol efflux, similar to NPC1.
Recently, fibroblasts isolated from NPC patients were shown to display a reduced accessibility of plasma membrane cholesterol to cholesterol oxidase24 associated with an impaired cholesterol efflux to apoAI.25 In line, NPC1-deficient subjects have decreased plasma HDL cholesterol levels.25 The effects on NPC1 and NPC2 combined with the ability of LXR agonists to induce ABCA1-dependent apoAI-specific cholesterol efflux9,10 and to stimulate cellular cholesterol efflux to HDL, via the induction of ABCG1 and ABCG4,11,12 are supportive for a role of LXR in the control of HDL metabolism.
The induction of NPC1 and NPC2 gene expression by LXR associated to its ability to induce cholesterol efflux could have physiological consequences in vivo in cells other than macrophages. Of particular interest are recent studies suggesting a role of cholesterol metabolism in the development of Alzheimer’s disease.26 In neuronal cells, which express ABCA1,27 stimulation of cholesterol efflux decreased amyloid- peptide secretion.26
LXR activation also decreased cholesterol esterification rates and reduced CE levels in human macrophage foam cells. These actions of LXR are not attributable to a decreased gene expression of ACAT1, the enzyme responsible for cholesterol esterification in macrophages. ACAT1 enzyme activity and cholesterol esterification rate are also controlled by FA availability, which could depend partially on their catabolism by enzymes such as carnitine palmitoyltransferase 1 (CPT-1), an enzyme located in the mitochondrial outer membrane catalyzing the entry of long-chain FAs into the mitochondria for their -oxidation.28 However, LXR activation did not affect CPT-1 mRNA levels in macrophages (data not shown), thus rendering the possibility that reduced cholesterol esterification is attributable to lowered FA substrate availability unlikely. Moreover, LXR activators did not affect neutral CE hydrolase activity in macrophages (data not shown), thus indicating that the effects of LXR occur rather via inhibition of cholesterol esterification than by stimulation of CE hydrolysis. Thus, it is likely that the stimulation of cholesterol mobilization to the plasma membrane by LXR activators results in a reduced availability of cholesterol as substrate for ACAT1.
The observed effects on cholesterol esterification on LXR activation could also reflect the capacity of LXR to induce spontaneous cholesterol efflux from macrophages, even in the absence of any extracellular acceptor in the medium, as demonstrated by Cignarella et al in macrophages simultaneously treated with LXR and retinoid X receptor (RXR) ligands.29 Our results, obtained on incubation with specific LXR agonists in the absence of RXR agonists, indicate that LXR activation increases only very weakly the spontaneous cholesterol efflux to culture medium compared with the observed induction in the presence of apoAI (data not shown). Thus, it is unlikely that under the condition studied here, the reduction of cholesterol esterification on LXR stimulation is a consequence of an induction of passive cholesterol efflux.
In conclusion, our results demonstrate a novel role for LXR in the control of cholesterol mobilization and distribution, an effect which, associated with the induction of ABCA1 and ABCG1/ABCG4, 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 the Fondation Leducq and the Nouvelle Societee Franaise d’Atheeroscleerose (L.H.). We thank C. Duhem and D. Deslee for their technical assistance, and K. Bertrand (Genfit SA, France) for providing the T0901317 and GW3965 compounds.
References
Tabas I. Cholesterol and phospholipid metabolism in macrophages. Biochim Biophys Acta. 2000; 1529: 164eC174.
Buhman KF, Accad M, Farese RV. Mammalian acyl-CoA:cholesterol acyltransferases. Biochim Biophys Acta. 2000; 1529: 142eC154.
von Eckardstein A, Nofer JR, Assmann G. High density lipoproteins and arteriosclerosis. Role of cholesterol efflux and reverse cholesterol transport. Arterioscler Thromb Vasc Biol. 2001; 21: 13eC27.
Carstea ED, Morris JA, Coleman KG, Loftus SK, Zhang D, Cummings C, Gu J, Rosenfeld MA, Pavan WJ, Krizman DB, Nagle J, Polymeropoulos MH, Sturley SL, Ioannou YA, Higgins ME, Comly M, Cooney A, Brown A, Kaneski CR, Blanchette-Mackie EJ, Dwyer NK, Neufeld EB, Chang TY, Liscum L, Strauss JF III, Ohno K, Zeigler M, Carmi R, Sokol J, Markie D, O’Neill RR, van Diggelen OP, Elleder M, Patterson MC, Brady RO, Vanier MT, Pentchev PG, Tagle DA. Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis. Science. 1997; 277: 228eC231.
Naureckiene S, Sleat DE, Lackland H, Fensom A, Vanier MT, Wattiaux R, Jadot M, Lobel P. Identification of HE1 as the second gene of Niemann-Pick C disease. Science. 2000; 290: 2298eC2301.
Watari H, Arakane F, Moog-Lutz C, Kallen CB, Tomasetto C, Gerton GL, Rio MC, Baker ME, Strauss JF III. MLN64 contains a domain with homology to the steroidogenic acute regulatory protein (StAR) that stimulates steroidogenesis. Proc Natl Acad Sci U S A. 1997; 94: 8462eC8427.
Kishida T, Kostetskii I, Zhang Z, Martinez F, Liu P, Walkley SU, Dwyer NK, Blanchette-Mackie EJ, Radice GL, Strauss JF III. Targeted mutation of the MLN64 START domain causes only modest alterations in cellular sterol metabolism. J Biol Chem. 2004; 279: 19276eC19285.
Janowski BA, Willy PJ, Devi TR, Falck JR, Mangelsdorf DJ. An oxysterol signalling pathway mediated by the nuclear receptor LXR alpha. Nature (London). 1996; 383: 728eC731.
Costet P, Luo Y, Wang N, Tall AR. Sterol-dependent transactivation of the human ABC1 promoter by LXR/RXR. J Biol Chem. 2000; 275: 28240eC28245.
Venkateswaran A, Laffitte BA, Joseph SB, Mak PA, Wilpitz DC, Edwards PA, Tontonoz P. Control of cellular cholesterol efflux by the nuclear oxysterol receptor LXRalpha. Proc Natl Acad Sci U S A. 2000; 97: 12097eC12102.
Engel T, Lorkowski S, Lueken A, Rust S, Schluter B, Berger G, Cullen P, Assmann G. The human ABCG4 gene is regulated by oxysterols and retinoids in monocyte-derived macrophages. Biochem Biophys Res Comm. 2001; 288: 483eC488.
Wang N, Lan D, Chen W, Matsuura F, Tall AR ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipoproteins. Proc Natl Acad Sci U S A. 2004: 9774eC9779.
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: 25573eC25580.
Van Eck M, De Winther MP, Herijgers N, Havekes LM, Hofker MH, Groot PH, Van Berkel TJ. Effect of human scavenger receptor class A overexpression in bone marrow-derived cells on cholesterol levels and atherosclerosis in apoE-deficient mice. Arterioscler Thromb Vasc Biol. 2000; 20: 2600eC2606.
Underwood KW, Andemariam B, McWilliams GL, Liscum L. Quantitative analysis of hydrophobic amine inhibition of intracellular cholesterol transport. J Lipid Res. 1996; 37: 1556eC1568.
Lusa S, Blom TS, Eskelinen EL, Kuismanen E, Mansson JE, 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: 1893eC1900.
Blom TS, Linder MD, Snow K, Pihko H, Hess MW, Jokitalo E, Veckman V, Syvanen AC, Ikonen E. Defective endocytic trafficking of NPC1 and NPC2 underlying infantile Niemann-Pick type C disease. Hum Mol Genet. 2003; 12: 257eC272.
Butler JD, Blanchette-Mackie J, Goldin E, O’Neill RR, Carstea G, Roff CF, Patterson MC, Patel S, Comly ME, Cooney A, et al. Progesterone blocks cholesterol translocation from lysosomes. J Biol Chem. 1992; 267: 23797eC23805.
Tontonoz P, Mangelsdorf DJ. Liver X receptor signaling pathways in cardiovascular disease. Mol Endocrinol. 2003; 17: 985eC993.
Joseph SB, Castrillo A, Laffitte BA, Mangelsdorf DJ, Tontonoz P. Reciprocal regulation of inflammation and lipid metabolism by liver X receptors. Nat Med. 2003; 9: 213eC219.
Terasaka N, Hiroshima A, Ariga A, Honzumi S, Koieyama T, Inaba T, Fujiwara T. Liver X receptor agonists inhibit tissue factor expression in macrophages. FEBS J. 2005; 272: 1546eC1556.
Menke JG, Macnaul KL, Hayes NS, Baffic J, Chao YS, Elbrecht A, Kelly LJ, Lam MH, Schmidt A, Sahoo S, Wang J, Wright SD, Xin P, Zhou G, Moller DE, Sparrow CP. A novel liver X receptor agonist establishes species differences in the regulation of cholesterol 7alpha-hydroxylase (CYP7a). Endocrinology. 2002; 143: 2548eC2558.
Sato M, Akaboshi S, Katsumoto T, Taniguchi M, Higaki K, Tai T, Sakuraba H, Ohno K. Accumulation of cholesterol and GM2 ganglioside in cells cultured in the presence of progesterone: an implication for the basic defect in Niemann-Pick disease type C. Brain Dev. 1998; 20: 50eC52.
Garver WS, Krishnan K, Gallagos JR, Michikawa M, Francis GA, Heidenreich RA. Niemann-Pick C1 protein regulates cholesterol transport to the trans-Golgi network and plasma membrane caveolae. J Lipid Res. 2002; 43: 579eC589.
Choi HY, Karten B, Chan T, Vance JE, Greer WL, Heidenreich RA, Garver WS, Francis GA. Impaired ABCA1-dependent lipid efflux and hypoalphalipoproteinemia in human Niemann-Pick type C disease. J Biol Chem. 2003; 278: 32569eC32577.
Sun Y, Yao J, Kim TW, Tall AR. Expression of liver X receptor target genes decreases cellular amyloid beta peptide secretion. J Biol Chem. 2003; 278: 27688eC27694.
Panzenboeck U, Balazs Z, Sovic A, Hrzenjak A, Levak-Frank S, Wintersperger A, Malle E, Sattler W. ABCA1 and scavenger receptor class B, type I, are modulators of reverse sterol transport at an in vitro blood-brain barrier constituted of porcine brain capillary endothelial cells. J Biol Chem. 2002; 277: 42781eC42789.
McGarry JD, Brown NF. The mitochondrial carnitine palmitoyltransferase system. From concept to molecular analysis. Eur J Biochem. 1997; 244: 1eC14.
Cignarella A, Engel T, von Eckardstein A, Kratz M, Lorkowski S, Lueken A, Assmann G, Cullen P. Pharmacological regulation of cholesterol efflux in human monocyte-derived macrophages in the absence of exogenous cholesterol acceptors. Atherosclerosis. 2005; 179: 229eC236.(E. Rigamonti, L. Helin, S)