PPARs (peroxisome-proliferator-activated receptors) and LXRs (liver X receptors) are ligand-activated transcription factors that control lipid and glucose metabolism, as well as the inflammatory response. Since the macrophage plays an important role in host defence and immuno-inflammatory pathologies, particular attention has been paid to the role of PPARs and LXRs in the control of macrophage gene expression and function. Altered macrophage functions contribute to the pathogenesis of many infectious, immunological and inflammatory disease processes, including atherosclerosis. Research over the last few years has revealed important roles for PPARs and LXRs in macrophage inflammation and cholesterol homoeostasis with consequences in atherosclerosis development. This review will discuss the role of these transcription factors in the control of cholesterol trafficking in macrophages.

Introduction

Altered macrophage functions contribute to the pathogenesis of many infectious, immunological and inflammatory disease processes. Pharmacological modulation of macrophage gene expression therefore represents an important strategy for the prevention and treatment of inflammation-related diseases, such as atherosclerosis.

Atherosclerosis is a chronic inflammatory disease of the large arteries that can lead to an acute clinical event due to plaque erosion or rupture and ensuing thrombosis. The atherosclerosis process is initiated by the recruitment and retention of monocytes and lymphocytes in the arterial wall. This event is triggered by elevated LDL (low-density lipoprotein) cholesterol levels leading to the retention and accumulation of oxidized LDL in the subendothelial space which induce a pro-inflammatory reaction by vascular cells. Lipid-loaded macrophage-derived foam cells, generated at the earliest stages of atherogenesis, initiate and promote atherosclerotic lesion formation. Intermediate and advanced lesions consist of foam cells, and migrating and proliferating smooth muscle cells and are characterized by a local inflammatory response [1].

Epidemiological studies have revealed several genetic and environmental risk factors predisposing to atherosclerosis, including dyslipidaemia and hypertension, as well as insulin resistance and Type 2 diabetes. PPARs (peroxisome-proliferator-activated receptors) and LXRs (liver X receptors) are transcription factors that regulate the expression of genes that control lipid and lipoprotein metabolism and glucose homoeostasis. Thus PPARs and LXRs modulate the major metabolic disorders predisposing to atherosclerosis and constitute interesting molecular targets for its treatment [2].

The PPAR subfamily contains three genes, PPARα, PPARβ/δ and PPARγ, which, upon ligand activation, act as a heterodimer with the RXR (retinoid X receptor) and bind to specific PPREs (PPAR-response elements) in the promoter region of target genes, thus regulating their expression. Moreover, PPARs inhibit the activation of a number of inflammatory response genes through interference with pro-inflammatory transcription factor pathways [3,4]. All PPARs are activated by naturally occurring FA (fatty acid)-derived molecules. Several isotype-specific pharmacological compounds, including fibrates (PPARα ligands), have been and continue to be developed.

The LXRs (LXRα and LXRβ) are nuclear receptors activated by oxysterols that play a central role in the transcriptional control of lipid metabolism [5]. LXRα is expressed highly in the liver and at lower levels in the adrenal glands, intestine, adipose tissue, macrophages, lung and kidney, whereas LXRβ is ubiquitously expressed. As with PPARs, LXRs also form heterodimers with RXR and bind to LXREs (LXR-responsive elements) located in the promoter of target genes.

Over the last 5 years, important progress has been made in understanding the role of PPARs and LXRs in the control of macrophage functions. We will discuss below the role of these transcription factors in the control of cholesterol homoeostasis in human macrophages.

Macrophage cholesterol metabolism: lipid accumulation and cholesterol efflux

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 RCT (reverse cholesterol transport), a pathway of cholesterol transport from peripheral tissues to the liver. In addition to the interaction of the HDL (high-density lipoprotein) particle with its membrane receptors, such as SR-B1 (scavenger receptor B1), the availability of cholesterol in the plasma membrane is an important determinant for efficient cholesterol efflux. Within the cells, modified LDL-derived CEs (cholesteryl esters) are hydrolysed in lysosomes to FC [free (unbound) cholesterol]. This FC is probably initially transported to the plasma membrane where it integrates the cell membrane [6]. Excess cholesterol is transported back to the endoplasmic reticulum where it is re-esterified by ACAT1 (acyl-CoA:cholesterol acyltransferase 1) and stored as (CE) lipid droplets [7]. The plasma membrane contains the highest percentage of cellular cholesterol [8]. 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 (Niemann–Pick type C1) and NPC2 [9]. NPC1 is a transmembrane protein containing a sterol-sensing domain localized in the late endosomal compartment, whereas NPC2 is a soluble endosomal/lysosomal cholesterol-binding protein [10,11]. Mutations in the NPC1 and NPC2 genes cause NPC disease, a fatal recessive disorder characterized by the accumulation of LDL-derived cholesterol in lysosomes. In the brain, this leads to neuronal degeneration, owing to a defective movement of sterols out of the expanding pools of lysosomal cholesterol to other locations, in particular the plasma membrane [12]. Mutations in NPC1 cause the majority of cases of NPC disease, whereas mutations in NPC2 account for less than 10% of the cases [9].

Macrophage cholesterol homoeostasis: role of PPARα and LXRs

Cholesterol accumulation and cholesterol efflux

Net lipid accumulation in macrophages is influenced by the rates of both lipid uptake and cholesterol efflux. Neither PPARα nor LXR activation induces lipid accumulation of acetylated LDL in human macrophages, indicating that PPARα and LXR are not involved in foam cell formation [13]. In contrast, PPARα reduces macrophage uptake of glycated LDL [14], a subtype of pro-atherogenic particles characterized by apoB (apolipoprotein B) glycation and taken up by macrophages through an LPL (lipoprotein lipase)-dependent mechanism [15]. Interestingly, PPARα induces LPL gene expression [14,16], but decreases secretion and enzyme activity [14], corroborating observations in hepatocytes that PPARs also act at the post-transcriptional level [17]. This action on macrophage LPL may contribute to the beneficial effects of PPARα agonists on macrovascular disease in diabetes. In addition, PPARα activators reduce triacylglycerol-rich lipoprotein accumulation in human macrophages, as a consequence of the suppression of apoB48 receptor expression [18].

PPARα and LXR control the first steps of RCT by acting on macrophages in different ways. PPARα activators enhance the expression of the HDL receptor CLA-1 [CD36 and LIMPII (lysosomal integral membrane protein II) analogous-1]/SR-B1 [19] and the ABC (ATP-binding cassette) transporter ABCA1; the former by a post-translational mechanism, the latter by an indirect mechanism involving the stimulation of LXRα expression [13,20]. The increase in ABCA1 expression promotes apoAI-mediated cholesterol efflux [13], thus generating nascent HDL and facilitating cholesterol removal from peripheral tissue macrophages and its transport back to the liver, where cholesterol is excreted directly or after conversion into bile acids.

LXR activators promote apoAI-mediated cholesterol efflux through the induction of ABCA1, a direct LXR target gene in human and murine macrophages [21,22]. ABCG1 and ABCG4, involved in lipid efflux to HDL, have been identified as LXR target genes in macrophages [23,24].

The dynamic balance between lipid import and export in macrophages, and its control by nuclear receptors, is undoubtedly an important determinant governing the progression of atherosclerosis.

Intracellular cholesterol trafficking

PPARα and LXR activation increase the expression of NPC1 and NPC2, leading to an enrichment of cholesterol in the plasma membrane accompanied by a redistribution of cholesterol within the plasma membrane to the outer layer (Figure 1) [25,26]. The enrichment of plasma membrane cholesterol as well as the induction of apoAI-specific cholesterol efflux by PPARα or LXR ligands is abolished in the presence of progesterone used at concentrations known to block cholesterol mobilization from the late endosome/lysosome and to mimic a phenotype comparable with the one observed in NPC-deficient cells [27,28]. To investigate further the mechanism by which cholesterol trafficking could contribute to cholesterol efflux, an siRNA (small interfering RNA) approach to knock-down NPC1 or NPC2 expression was used. Interestingly, repression of NPC1 and NPC2 expression leads to a drastic reduction of basal as well as to an abolishment of PPARα- or LXR-induced cholesterol efflux. These observations indicate that stimulation of post-lysosomal cholesterol mobilization to the plasma membrane by PPARα and LXR activation via NPC1 and NPC2 induction is a crucial step upstream of the stimulation of its efflux through the ABCA1 pathway.

Role of PPARα and LXRs in macrophage cholesterol trafficking

Figure 1
Role of PPARα and LXRs in macrophage cholesterol trafficking

PPARα and LXR activation stimulates the post-lysosomal mobilization of cholesterol by regulating NPC1 and NPC2 expression. This leads to an enrichment of cholesterol in the plasma membrane and a redistribution of cholesterol in the outer cell-surface domains, where it is more available for efflux through the ABCA1 pathway. As a consequence, PPARα and LXR activation decreases the availability of cholesterol for ACAT1 and thus the cellular amount of CEs.

Figure 1
Role of PPARα and LXRs in macrophage cholesterol trafficking

PPARα and LXR activation stimulates the post-lysosomal mobilization of cholesterol by regulating NPC1 and NPC2 expression. This leads to an enrichment of cholesterol in the plasma membrane and a redistribution of cholesterol in the outer cell-surface domains, where it is more available for efflux through the ABCA1 pathway. As a consequence, PPARα and LXR activation decreases the availability of cholesterol for ACAT1 and thus the cellular amount of CEs.

One determinant governing the efflux rate of cholesterol is the availability of cholesterol in the plasma membrane. Results from the cholesterol oxidase accessibility test demonstrate that PPARα and LXR activation also leads to a redistribution of cholesterol within the plasma membrane, with an enrichment of cholesterol in the outer layer leading to more availability for the interaction with an extracellular acceptor, such as apoAI, an effect that would accelerate its efflux [25,26]. It has been demonstrated that fibroblasts isolated from subjects with NPC are characterized by a reduced accessibility to cholesterol oxidase, resulting in an impaired cholesterol efflux to apoAI [29,30]. In line with this observation, it has been reported that NPC1-deficient subjects have decreased plasma HDL cholesterol levels [30]. Thus regulation of NPC1 and proteins associated in its pathway appears to be important in the control of plasma HDL levels. The stimulatory role of PPARα activators on hepatic HDL production and apoAI expression is well documented [31].

Intracellular cholesterol distribution

Foam cells are characterized by cytoplasmic accumulation of CE and triacylglycerol-rich droplets, which confer the foamy aspect. Activation of PPARα and LXR in human macrophages and foam cells leads to a decrease of CE levels (Figure 1) [32,33]. These actions are not due to a decreased expression of ACAT1, the enzyme responsible for cholesterol esterification in macrophages. ACAT1 enzyme activity and cholesterol esterification rates are also controlled by FA availability which could depend partially on their catabolism by enzymes such as CPT-1 (carnitine palmitoyltransferase 1), an enzyme located in the mitochondrial outer membrane catalysing the entry of long-chain FAs into the mitochondria for β-oxidation [34]. PPARα increases the expression of CPT-1, which may result in a reduced availability of FAs as substrate for ACAT1 [32]. However, LXR activation does not affect CPT-1 mRNA levels in macrophages, thus rendering the possibility that reduced cholesterol esterification is due to lowered FA substrate availability unlikely. Moreover, PPARα and LXR activators do not affect NCEH (neutral cholesteryl ester hydrolase) activity in macrophages, thus indicating that these effects occur rather via inhibition of cholesterol esterification than by stimulation of CE hydrolysis. It is therefore likely that the stimulation of cholesterol mobilization to the plasma membrane by PPARα and LXR activators results in a reduced availability of cholesterol as substrate for ACAT1, thus leading to a reduction in the CE content.

Our results demonstrating that PPARα activators induce cholesterol trafficking in macrophages resulting in an enhanced availability of plasma membrane cholesterol for cholesterol efflux, provide a mechanism which may contribute to the observed clinical effects of PPARα activators on RCT and HDL metabolism [35]. On the basis of these data, it appears evident that PPARα and LXR are key controllers of macrophage cholesterol homoeostasis.

Conclusion

Over the last 5 years, important progress has been made in understanding how PPAR and LXRs control macrophage functions. At this stage, a growing body of evidence from in vitro and in vivo studies in animals and humans indicates that these transcription factors have beneficial effects in inflammatory disorders, including atherosclerosis. On the basis of these findings, the development of new molecules targeting these nuclear receptors provides exciting opportunities to reduce atherosclerosis and its complications.

Nuclear Receptors: Structure, Mechanisms and Therapeutic Targets: A Focus Topic at BioScience 2006, held at SECC Glasgow, U.K., 23–27 July 2006. Edited by C. Bevan (Imperial College London, U.K.), D. Black (Organon, U.K.) and I. McEwan (Aberdeen, U.K.).

Abbreviations

     
  • ABC

    ATP-binding cassette

  •  
  • ACAT1

    acyl-CoA:cholesterol acyltransferase 1

  •  
  • apoB (etc.)

    apolipoprotein B (etc.)

  •  
  • CE

    cholesteryl ester

  •  
  • CPT-1

    carnitine palmitoyltransferase 1

  •  
  • FA

    fatty acid

  •  
  • FC

    free cholesterol

  •  
  • HDL

    high-density lipoprotein

  •  
  • LDL

    low-density lipoprotein

  •  
  • LXR

    liver X receptor

  •  
  • NPC

    Niemann–Pick type C

  •  
  • PPAR

    peroxisome-proliferator-activated receptor

  •  
  • RCT

    reverse cholesterol transport

  •  
  • RXR

    retinoid X receptor

  •  
  • SR-B1

    scavenger receptor B1

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