Liver X Receptor Activation Induces the Uptake of Cholesteryl Esters From High Density Lipoproteins in Primary Human Macrophages
Objective— Liver X receptors (LXRs) are oxysterol-activated nuclear receptors regulating reverse cholesterol transport, in part by modulating cholesterol efflux from macrophages to apoAI and HDL via the ABCA1 and ABCG1/ABCG4 pathways. Moreover, LXR activation increases intracellular cholesterol trafficking via the induction of NPC1 and NPC2 expression. However, implication of LXRs in the selective uptake of cholesteryl esters from lipoproteins in human macrophages has never been reported.
Methods and Results— Our results show that (1) selective CE uptake from HDL3 is highly efficient in human monocyte-derived macrophages; (2) surprisingly, HDL3-CE uptake is strongly increased by LXR activation despite antiatherogenic effects of LXRs; (3) HDL3-CE uptake increase is not linked to SR-BI expression modulation but it is dependent of proteoglycan interactions; (4) HDL3-CE uptake increase is associated with increased expression and secretion of apoE and LPL, two proteins interacting with proteoglycans; (5) HDL3-CE uptake increase depends on the integrity of raft domains and is associated with an increased caveolin-1 expression.
Conclusions— Our study identifies a new role for LXRs in the control of cholesterol homeostasis in human macrophages. LXR activation results in enhanced dynamic intracellular cholesterol fluxes through an increased CE uptake from HDL and leads to an increased cholesterol availability to efflux to apoAI and HDL.
Macrophages play a pivotal role in the development of atherosclerosis.1 During the initial stages of the disease, monocytes migrate into the subendothelial space of blood vessels and differentiate into macrophages. Macrophage scavenger receptors, CD36 and SR-A, which lack negative feedback regulation by cholesterol, mediate the uptake of oxidized lipids in the subendothelial space. Under physiological conditions, the net accumulation of lipids in macrophages is the result of a complex balance and reflects the rate of cholesterol endocytosis and its removal via the reverse cholesterol transport pathway. Caveolae are free cholesterol rich, invaginated microdomains (50 to 100 nm in diameter) at the surface of most peripheral cells. Caveolin-1, the main structural protein of caveolae, is involved in the regulation of cellular cholesterol metabolism and lipid uptake, as well as efflux.2 The entrance of excessive amounts of lipoprotein-derived lipids into macrophages leads to their conversion into foam cells.
Selective cholesteryl ester (CE) uptake is a mechanism where CE are taken up by the cells independently from the entry of the lipoparticles. It mainly occurs from HDL in liver and steroidogenic tissues and involves principally SR-BI, a transmembrane glycoprotein receptor.3,4 In macrophages, it has been mainly reported in cell lines like THP-1 and J774 but may also occur in primary human cells.5
Liver X receptors (LXRs) are oxysterol-activated transcription factors which after heterodimerization with the 9-cis-retinoic acid receptor (RXR), bind to specific LXR response elements (LXREs), thus regulating the expression of target genes involved in intra- and extracellular lipid metabolism. LXRβ is ubiquitously expressed,6 whereas the expression of LXRα is predominantly restricted to tissues known to play important roles in lipid metabolism, such as liver, adrenal glands, kidney, macrophages, intestine, and adipose tissue.7 In the last few years LXRs have emerged as key regulators of macrophage cholesterol homeostasis. LXR activators promote apoAI-mediated cholesterol efflux through the increase of cholesterol trafficking to the plasma membrane8 and the induction of ABCA1, which is a direct target gene of LXR in human and murine macrophages.9–11 Other members of the ATP binding cassette family, ABCG1 and ABCG4, which are involved in lipid efflux to HDL, were also identified as LXR target genes in macrophages.12,13 Although LXRs do not promote cholesterol accumulation via the scavenger receptor pathway,8 it would be important to know whether LXRs control other pathways to provide cells with cholesterol, namely selective CE uptake.
The goal of this study was thus to investigate the role of LXR agonists in selective CE uptake from HDL3 (HDL3-CE uptake) in differentiated human macrophages. We demonstrate that HDL3-CE uptake is highly efficient in these cells and strongly increased by LXR activators in an LXR-dependent manner. Modulation of SR-BI protein levels does not explain this increase. In the presence of heparin, which inhibits protein interactions with proteoglycans14 or of β-d-xyloside, a general inhibitor of proteoglycan synthesis,15 basal and LXR-activated HDL3-CE uptake are strongly decreased. HDL3-CE uptake increase is associated with the increase of gene expression, synthesis, and secretion of apoE and LPL, two proteins interacting with proteoglycans. Moreover, methyl-β-cyclodextrin (MBCD), a reagent which destabilizes raft domains, reduces HDL3-CE uptake and its induction by LXRs. Finally, caveolin-1 and apoE are both strongly induced in raft fractions on LXR activation which could suggest a potential role of caveolin-1 in apoE effects on HDL3-CE uptake.
In conclusion, these results suggest that LXR activation increases HDL3-CE uptake through upregulation of secreted proteins interacting with proteoglycans, namely apoE and LPL, and this regulation requires raft domain integrity and may involve caveolae.
Materials and Methods
An expanded Materials and Methods section is available online (please see http://atvb.ahajournals.org).
Isolation, Culture, and Activation of Human Monocyte-Derived Macrophages
Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient centrifugation of healthy normolipemic donor blood.16 On day 12 of the primary culture, human monocyte-derived macrophages (HMDMs) were incubated in serum-free RPMI medium supplemented with 1% Nutridoma HU (Roche). HMDMs were activated for 48 hours with synthetic LXR ligands T0901317 (1 μmol/L) or GW3965 (1 μmol/L) or with the natural ligand 22-R-Hydroxycholesterol (1 μmol/L) or DMSO (control cells).
Lipoprotein Interaction With Cells
Total lipids were extracted by chloroform/methanol (2/1, vol/vol). Free cholesterol (FC) and CE of each sample were separated by thin layer chromatography (TLC). Corresponding spots were scrapped and radioactivity counted.
RNA were extracted by Trizol and SR-BI, apoE, LPL, and caveolin-1 mRNA expression levels were analyzed by quantitative polymerase chain reaction (PCR).
Raft Domain Isolation
Raft domain isolation was performed by discontinuous sucrose gradient method and GM1 was used as raft domain marker.19,20
On postnuclear supernatants, on chloroform/methanol precipitated cell proteins from each fraction of the sucrose gradients and on trichloroacetic acid (TCA) precipitated proteins from culture supernatants, immunoblot was performed followed by SR-BI, apoE, LPL, and caveolin-1 protein analysis.
Primary human monocytes were grown on BD Falcon CultureSlides (1.106 cells per well) in presence of M-colony stimulating factor (CSF) to promote their differentiation to macrophages. Caveolin-1 and apoE were immunolocalized by confocal microscopy.
Statistically differences between control and activated conditions were analyzed by Student t test and were considered significant when P≤0.05 (*P<0.05; **P<0.01).
LXR Activation Increases Selective CE Uptake From HDL3 but Does Not Induce CE Accumulation
To determine whether human macrophages display a selective CE uptake from HDL3, HMDMs were incubated with [3H]-CE-HDL3 or [125I]-HDL3 for 4 hours at 37°C. As previously described,5 HDL3-CE uptake level is much more important than HDL3 cell-association and degradation (supplemental Figure IA, available online at http://atvb.ahajournals.org), indicating that basal HDL3-CE uptake occurs in HMDM and does not involve significant endocytosis and degradation of the HDL3 particle (less than 2%).
Next we studied whether [3H]-CE-HDL3 uptake in macrophages is regulated by LXRs. Cells treated during 48 hours with different LXR agonists displayed an enhanced HDL3-CE uptake compared to control cells (Figure 1A). Under the same condition of LXR activation, no significant difference was found in HDL3 cell-association or degradation (supplemental Figure IB and IC). The role of LXRs in this upregulation is unequivocally established because LXR agonist treatment did not induce HDL3-CE uptake after LXRα/β gene silencing (Figure 1B). Incubation with [3H]-CE-HDL for 4 hours led to the intracellular appearance of labeled free cholesterol (FC) indicating that approximately 30% of internalized CE was hydrolyzed during this time period (Figure 1C). Both labeled FC and CE increased after LXR activation. However, intracellular CE/TC (TC: total cholesterol) and FC/TC ratios were not modified indicating that the increase of HDL3-CE uptake induced by LXR activation does not promote cellular CE accumulation.
Induction of HDL3-CE Uptake After LXR Activation Is Not Linked to SR-BI Modulation but Is Dependent on Proteoglycan-Protein Interactions
First, we verified whether SR-BI protein modulation could explain HDL3-CE uptake regulation by LXRs. HMDMs were incubated with LXR agonists for 48 hours and SR-BI expression measured. Although SR-BI mRNA level was slightly increased, SR-BI protein expression in cellular post nuclear extracts was not modified (supplemental Figure II). Although a role of SR-BI in basal selective uptake in HMDMs cannot be excluded, HDL3-CE uptake induction by LXRs is not attributable to change in SR-BI protein expression.
It was reported that selective CE uptake mechanisms could be independent on SR-BI.21,22 Two complementary approaches were used to assign a role of proteoglycans: (1) HDL3-CE uptake was measured with or without heparin which removes proteins interacting with proteoglycans,14 (2) HDL3-CE uptake was measured in the presence or the absence of β-d-xyloside which inhibits proteoglycan synthesis.15 CE uptake from HDL3 in control conditions and its upregulation by T0901317 or GW3965 were completely abolished on heparin incubation (Figure 2A). In the presence of β-d-xyloside, basal HDL3-CE uptake was strongly decreased and no longer induced after LXR activation with the natural ligand 22-R-Hydroxycholesterol (Figure 2B). LXR activation by T0901317 still enhanced HDL3-CE uptake but to a lesser extent than in the absence of β-d-xyloside.
Hence, HDL3-CE uptake appears to be dependent not only on proteoglycans but also on interacting proteins both in basal and LXR activated conditions. Thus, induction of expression of proteins which interact with proteoglycans likely plays a role in the upregulation of HDL3-CE uptake after LXR activation.
LXR Activation Increases Intracellular Pools and Secretion of ApoE and LPL
ApoE and LPL are proteins that interact with proteoglycans and are involved in the mechanism of CE uptake.17,22 Because LPL and apoE are also LXR target genes in certain cell lines,23,24 the regulation of expression of apoE and LPL by LXR was studied in HMDMs. Incubation of HMDMs with synthetic LXR ligands for 48 hours resulted in a strong increase of apoE and LPL gene expression and a tendency with the natural ligand (Figure 3A and Figure 3B). A 48-hour LXR activation with T0901317 led to a strong induction of LPL and apoE secretion in the culture medium, which was paralleled with an induction of intracellular apoE.
To determine the relevance of a potential role of secreted apoE and LPL in the induction of HDL3-CE uptake, HMDMs were incubated with [3H]-CE-HDL3 for 4 hours at 37°C with or without exogenous free human apoE (10 μg/mL) or exogenous bovine LPL (100 ng/mL). Our results show that free apoE and LPL induce an increase of HDL3-CE uptake (Figure 3C), moreover heparin treatment decreases this induction.
In the human adrenal cell line NCI-H295R which does not secrete apoE, the LXR ligand T0901317 did not influence HDL3-CE uptake (1.542±0.113 μg TC/mg cellular proteins in control cell versus 1.608±0.122 μg TC/mg cellular proteins in T0901317 activated cells). Additionally, we have previously shown that exogenous apoE strongly increases HDL3-CE uptake in this cell line.17 Taken together these data support the hypothesis that apoE is important in the upregulation of the CE uptake through LXR activation in HMDM.
Therefore, the increase in HDL3-CE uptake by LXR ligands could be mediated through upregulation of apoE and LPL which interact with proteoglycans.
Basal and LXR-Induced CE Uptake Involve Raft Domains and Correlate With Caveolin-1 Expression
Lipid rafts are specialized membrane domains involved in selective CE uptake in certain cell types.25 Methyl-β-cyclodextrin (MBCD) is a cholesterol-binding reagent used to deplete plasma membranes from cholesterol, thus disrupting structural integrity of raft domains. Incubation of cells with MBCD led to a decrease of HDL3-CE uptake in control and LXR-activated cells (Figure 4A).
Because regulation of HDL3-CE uptake by LXR was sensible to disruption of raft domains by methyl-β-cyclodextrin, it was analyzed whether LXR modulates expression of caveolin-1, the major protein involved in caveolae formation.
Caveolin-1 mRNA and protein were detected in HMDM cells in basal condition (Figure 4B and 4C). In LXR activated cells caveolin-1 mRNA (Figure 4B) and protein (Figure 4C) were strongly increased compared to control cells suggesting that LXR activation could participate in lipid homeostasis via the modulation of caveolin-1 expression.
SR-BI, ApoE, LPL, and Caveolin-1 Are Located in Raft Domains and LXR Induces Caveolin-1 and ApoE in These Domains
SR-BI–mediated HDL3-CE uptake may be related to its localization in raft domains. Analysis of GM1 allows locating raft domains among different fractions isolated by a discontinuous sucrose gradient (see Materials and Methods; Figure 5A). Presence of SR-BI in raft (fraction 3 represents 57±3% of total SR-BI) and in nonraft fractions (fractions from 4 to 11 represent 43±4% of total SR-BI) was not significantly different after LXR activation. A significant amount of apoE or LPL was found in rafts (apoE and LPL respectively represent approximately 25% and 37% of total protein). On LXR activation, apoE increased to the same extent both in raft and in nonraft fractions whereas LPL was induced in both fractions but to a higher extent in the nonraft fractions. LXR activation increased caveolin-1 protein expression in the raft fraction.
Immunofluorescence staining in Figure 5B, in total concordance with panel A, revealed that both apoE and caveolin-1 are induced by T0901317. More, the colocalization of the two proteins already observed in control cells is more important in LXR activated cells as quantified according to Van Steensel and Costes.26,27
LXRs are nuclear receptors involved in lipogenesis and lipoprotein metabolism.28 LXRs have emerged as key regulators of macrophage cholesterol homeostasis, and particularly in the process of lipid efflux to extracellular acceptors.10,11,29,30 Although LXRs have no effect on the cholesterol entry via the scavenger receptor pathway,8 LXR regulation of selective CE uptake from lipoproteins has not yet been reported. In vitro and in vivo experiments demonstrated that SR-BI is a physiologically relevant HDL3 receptor that can mediate selective CE uptake in the liver and in steroidogenic tissues4,31,32 as well as in primary human macrophages.5 In this study we confirm that, in HMDMs, selective CE uptake from HDL3 is highly efficient but with high interindividual variations. This uptake is independent of HDL3 internalization and degradation. CE uptake from HDL3 by LXR ligands is highly stimulated. This increase is found for each macrophage preparation with a variable factor from 1.7 to 7 (22 donors) and is LXR-dependent as it is lost after LXRα/β mRNA silencing. Although SR-BI contributes to basal HDL3-CE uptake, it appears not to account for the stimulation of CE uptake on LXR activation.
After a 4-hour incubation with [3H]-CE-HDL3, analysis of the radioactivity in cells revealed a paralleled increase in intracellular CE and FC in LXR activated HMDMs without any modification of the CE/TC ratio. Thus, despite the increase of CE uptake by LXR activation, there is no CE accumulation and, hence, no stimulation of foam cell formation. LXR ligands activate efflux to apoAI and to HDL by inducing ABC transporters A1, G1, and G4.11–13 We thus hypothesize that LXR activation leads to a dynamic recycling of cholesterol which was evidenced by the fact that LXR activation simultaneously upregulates CE uptake and free cholesterol efflux to HDL under the studied experimental conditions (supplemental Figure III).
Our results demonstrated that proteins interacting with proteoglycans are probably involved in the HDL3-CE uptake regulation by LXR activation because preincubation of HMDMs with β-d-xyloside or heparin led to a significant decrease of basal selective CE uptake and attenuated induction after LXR activation. Assessment of the relationship between these functional results and the modulation of expression of two LXR targets known to play a role in CE uptake and to interact with proteoglycans, ie, apoE and LPL,17,22,23,24 showed that LXR activation increased apoE and LPL expression and secretion. Moreover, in HMDMs, free exogenous apoE and LPL were able to induce HDL3-CE uptake, an effect that was inhibited by heparin. The effect of exogenous apoE and LPL on HDL3-CE uptake thus appears to be dependent on proteoglycans.
Raft domains have been shown to be involved in CE uptake.25 Both basal CE uptake and its upregulation by LXR ligands are highly dependent on raft integrity because methyl-β-cyclodextrin decreases HDL3-CE uptake in both conditions. It has been demonstrated that caveolin-1 protein expression is sufficient for caveolae formation at the cell surface.33–35 Caveolin-1 protein is expressed at low levels in primary human macrophages but LXR activation strongly enhances its expression, which could result in a modification of cell surface microdomain composition. Such changes could influence lipid uptake and efflux properties.36 Moreover, we observed that, on LXR activation, apoE expression increased in the different intracellular pools, in particular in the raft fractions, and is colocalized with caveolin-1. Thus the increase of apoE in raft domains and subsequent colocalization with other proteins (especially caveolin-1 and SR-BI) could be involved in the induction of CE uptake by LXR ligands.
In conclusion, selective CE uptake from HDL3 in HMDM is a specific phenomenon which is upregulated by natural and synthetic LXR ligands in an LXR-dependent manner. This regulation may implicate several partner proteins, ie, apoE, LPL, and caveolin-1. Although the function of each protein is not clearly elucidated, this regulation is highly dependent on proteoglycans and specialized membrane domains, namely raft domains and caveolae. Interestingly LXR activation induces a dynamic intracellular phenomenon because the increase of HDL3-CE uptake leads to cellular cholesterol mobilization associated with an increase in cholesterol efflux, avoiding a cholesterol accumulation in macrophages. We previously showed that LXR activation in HMDMs does not affect neutral CE hydrolase activity, nor acyl-coenzyme A (CoA):cholesterol acyltranferase 1 (ACAT) expression, 2 enzymes involved in cellular cholesterol homeostasis, but rather upregulates NPC proteins implicated in transport of cholesterol from intracellular pools to the plasma membrane.8 Our results suggest that LXR activation could accelerate intracellular dynamics of cholesterol resulting, despite the increase in CE uptake, in an increase of free cholesterol available for efflux to apoAI or HDL. Subsequently, the remodeled HDL with less cholesterol could have improved activity in reverse cholesterol transport as previously suggested.37
We thank Genfit SA, France for providing the T0901317 and GW3965 compounds.
Sources of Funding
This work was supported by grants from the European Vascular Genomics Network (EVGN) and from the Nouvelle Société Française d’Athérosclérose (NSFA).
S.B and L.H. contributed equally to this study.
Original received July 24, 2008; final version accepted September 4, 2008.
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