Review
Nuclear receptor mediated mechanisms of macrophage cholesterol metabolism

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Abstract

Macrophages comprise a family of multi-faceted phagocytic effector cells that differentiate “in situ” from circulating monocytes to exert various functions including clearance of foreign pathogens as well as debris derived from host cells. Macrophages also possess the ability to engulf and metabolize lipids and this way connect lipid metabolism and inflammation. The molecular link between these processes is provided by certain members of the nuclear receptor family. For instance, peroxisome proliferator activated receptors (PPAR) and liver X receptors (LXR) are able to sense the dynamically changing lipid environment and translate it to gene expression changes in order to modulate the cellular phenotype. Atherosclerosis embodies both sides of this coin: it is a disease in which macrophages with altered cholesterol metabolism keep the arteries in a chronically inflamed state. A large body of publications has accumulated during the past few decades describing the role of nuclear receptors in the regulation of macrophage cholesterol homeostasis, their contribution to the formation of atherosclerotic plaques and their crosstalk with inflammatory pathways. This review will summarize the most recent findings from this field narrowly focusing on the contribution of various nuclear receptors to macrophage cholesterol metabolism.

Highlights

► Various nuclear receptors (NR) mediate macrophage cholesterol homeostasis. ► NR pathways form a regulatory loop in mediating macrophage cholesterol metabolism. ► Elimination of certain NRs leads to alteration of atherosclerotic lesion formation.

Introduction

In their pioneering work Brown and Goldstein described that macrophages of patients with familial hypercholesterolemia – whose cells lack the receptor for low-density lipoprotein (LDL) – are able to accumulate cholesteryl-esters suggesting that macrophage uptake of LDL involves another group of receptors distinct from the LDL receptor (LDLR) (reviewed in (Brown and Goldstein, 1983)). It was later recognized that the LDLR independent uptake of LDL by macrophages within the atherosclerotic lesions requires LDL to first undergo chemical or enzymatic alteration, which in humans is now proven to be an oxidative modification in vivo (Quinn et al., 1987). Last but not least, the notion that the uptake of oxLDL initiates a positive feedback in foam cells by inducing the expression of the CD36 scavenger receptor gave rise to the hypothesis that an oxLDL driven intracellular mechanism should be accountable for the generation of foam cells and the development of atherosclerosis (Han et al., 1997). These above results pointed to a highly relevant question: what are the molecular links that connect the derivatives of oxLDL and the enhanced lipid uptake by atherosclerotic macrophages? The groundbreaking results of the last 15 years in this field of study revealed that lipid activated nuclear receptors are not only involved in regulating macrophage lipid uptake but play critical roles in the regulation of lipid intracellular trafficking as well as efflux. The current review will focus on how nuclear receptors regulate receptor mediated cholesterol uptake, cellular storage, transport and efflux, as well as production of apolipoproteins and lipoprotein remodeling enzymes.

Macrophages play central roles in host defense in both innate and adaptive immunity. They are also linked to the pathology of many disease processes. Macrophages are professional phagocytes that reside in nearly every tissue and are responsible for clearance of pathogens, dying cells and oxidized lipid molecules. Macrophages – with the exception of microglia (Ginhoux et al., 2010) – are derived from multipotent hematopoietic stem cells (Friedman, 2002) characterized by long term repopulating capacity. Monocytes differentiate from common myeloid progenitors through monoblasts to circulating monocytes maturing to various tissue-specific macrophages. During activation macrophages become equipped with a plethora of features by which they are able to sense, engulf and eliminate foreign invaders and toxic host-derived particules. The activation of macrophages is stimulated by signaling pathways of cytokines and growth factors that lead to the activation of specific transcription factors. Macrophages also take part in tissue repair and the resolution phase of inflammation.

It is now evident that activated macrophages can be classified into classically (M1) and alternatively (M2) activated subtypes (Gordon and Martinez, 2010). M1 macrophages develop in response to Th1 cell derived cytokines such as IFNγ, or microbial lipopolysaccharide (LPS). These cells produce inflammatory cytokines, reactive oxygen species and perform cytotoxic functions to eradicate foreign invaders. However, if not kept under tight control at the resolution phase of inflammation, these cells can be very harmful to the host cells and tissues and can contribute to the pathogenesis of certain metabolic disorders such as atherosclerosis and obesity induced insulin resistance (Olefsky and Glass, 2010). M2 macrophages, on the other hand, develop upon stimulation with IL-4 or IL-13 cytokines, are linked to Th2 responses and involved in anti-inflammatory processes (Gordon and Martinez, 2010).

The macrophage population in chronic disorders such as atherosclerosis (foam cells) is very likely of a mixed M1/M2 phenotype (Olefsky and Glass, 2010). Nuclear receptors are widely expressed in macrophages and are intimately implicated in regulating their metabolic and immune homeostasis (reviewed in (Rigamonti et al., 2008)).

Various cell types are involved in the development of atherosclerosis including endothelial cells, monocytes/macrophages, T cells and smooth muscle cells (Ross, 1999). At the initial stage of atherosclerosis, cholesterol, cholesteryl-ester and phospholipid containing LDL is accumulated and oxidized in the subendothelial layer of arterial walls (Tabas et al., 2007). oxLDL causes endothelial cell activation which leads to the recruitment of blood derived monocytes to the subendothelial layer (Mestas and Ley, 2008). These monocytes then differentiate into macrophages and take up oxidized and native LDL via scavenger receptors such as CD36 and SR-A, as well as LDLR (Suzuki et al., 1997, Nozaki et al., 1995, Fogelman et al., 1988). Further uptake of modified LDL by macrophages leads to foam cell formation and early atherosclerotic lesions (Moore and Tabas, 2011). Activated T cell and macrophage derived cytokines and growth factors induce smooth muscle cell migration and proliferation as well as extracellular matrix production leading the development of fibrous plaques (Lusis, 2000). Later, the fibrous cap thinning and necrotic core formation lead to the formation of vulnerable plaques. The previous process is induced by massively dying smooth muscle cells and the decreased capacity of smooth muscle cells to synthesize collagen. In addition, macrophage produced matrix metalloproteinases can degrade the different components of the extracellular matrix resulting in fibrous cap thinning. The other critical component of vulnerable plaque development is the necrotic core formation, which is dictated by macrophage apoptosis as well as defective phagocytosis of apoptotic macrophages in the plaques (Moore and Tabas, 2011). The final catastrophic step might occur when the vulnerable plaque ruptures the contacts of the necrotic core causing thrombosis with the potential for an occlusive thrombus (Dickson and Gotlieb, 2003).

The in vivo examination of atherosclerosis development requires well characterized animal models. Interestingly, mice – with the exception of the C57BL/6 strain – are resistant to atherosclerosis, and even in these animals only a relatively harmful and toxic high-fat diet could induce atherosclerotic lesions limited to early fatty-streak stage that were quite different from the human condition. Consequently, it became necessary to use genetically modified animal models to study atherosclerosis in vivo, of which the most wildly used are the apolipoprotein (Apo) E (Plump et al., 1992, Zhang et al., 1992) and LDLR deficient (Ishibashi et al., 1993) mice. Apo E deficient animals develop atherosclerotic lesions that convert into fibrous caps on chow diet. On the other hand, LDLR(−/−) mice robustly develop atherosclerotic lesions on Western type high fat but not on a normal diet (Jawien et al., 2004).

Within macrophages the accumulated cholesterol is stored in the form of cholesteryl-esters in cytoplasmic lipid droplets. The balance between cholesteryl-esters and free cholesterol is regulated by the cholesteryl-ester cycle, in which LDL derived cholesteryl-esters are hydrolyzed in the late endosomes/lysosomes by acid cholesteryl-ester hydrolase while cellular lipid droplet derived cholesteryl-esters are hydrolyzed in the cytoplasm by neutral cholesteryl-ester hydrolase. Liberated free cholesterol is then transported to the plasma membrane where it either becomes integrated or released by cholesterol efflux. Additionally, the fate of excess free cholesterol may be an acyl-CoA:cholesterol acyltransferase-1 (ACAT1) catalyzed re-esterification and storage in lipid droplets (Ghosh et al., 2010). Cholesterol transport from the late endosomes to the plasma membrane is regulated by the Niemann Pick type C (NPC) 1 and 2 proteins (Ory, 2004). Cholesterol efflux is the most important cholesterol disposing process in macrophages, during which free cholesterol is removed by ABCA1 and ABCG1 mediated transport to Apo A-1 and HDL (Yvan-Charvet et al., 2010). The accumulation of LDL derived cholesterol and lipids cause foam cell formation of macrophages leading to further LDL modification and amplification of inflammation as well as lesion formation in the early stages of atherosclerosis (Moore and Tabas, 2011). Therefore, the understanding of the regulation of macrophage cholesterol homeostasis is critical for the therapeutic intervention of atherosclerosis.

Certain members of the nuclear receptor superfamily are lipid-responsive transcription factors that are able to translate signals from the constantly changing lipid environment to gene expression changes. The human and the mouse genome encode 48 or 49 different nuclear receptors, respectively. The divergent members of the superfamily are traditionally categorized into three groups: “classic” members with known endogenous ligands are typically endocrine receptors such as the thyroid or the estrogen receptors, “orphan” receptors are those whose endogenous ligands remained unknown, and “adopted orphan” receptors’ ligands were originally unknown but later became identified (Kliewer et al., 1999). However, the constant identification of new ligands – that are often end products or intermediates of metabolic pathways (Blumberg and Evans, 1998) – is a major weakness to the above grouping; hence a phylogenetic classification model based on sequence similarity was also introduced (Nuclear Receptors Nomenclature Committee, 1999).

Nuclear receptors share a common domain structure consisting of an N-terminal activation function (AF-1), a DNA binding (DBD), a ligand binding (LBD), and a second C-terminal activation (AF-2) domain (Evans, 1988). While DBDs containing two zinc-binding motifs are highly conserved and responsible for nuclear localization, the LBDs are more diverse with specialized ligand binding pockets for each receptor. The AF-2 domain mediates the displacement of co-repressors and the recruitment of co-activators. The chemically non-polar natural or synthetic agonists and antagonists of the receptors pass freely through the lipid bilayer of the cell membrane and the specific ligand–receptor interaction leads to allosteric changes. In a simplified way, co-repressor complexes bound to unliganded, inactive nuclear receptors are replaced with co-activator complexes in the presence of activating ligands to turn on the signaling cascade (Nagy et al., 1999). Another form of nuclear receptor mediated gene expression regulation is transrepression, where the ligand-bound receptor sequesters other transcription factors (Glass and Saijo, 2010).

Section snippets

Basics of PPAR biology

There are three subtypes of the vertebrate PPAR family: PPARα, PPARβ/δ and PPARγ (based on their position in the phylogenetic tree also called NR1C1, NR1C2 and NR1C3, respectively). PPARα is an activator of mitochondrial and peroxisomal fatty acid β-oxidation and is expressed where lipid metabolism is highly dynamic, such as the kidney, heart, liver and skeletal muscle. PPARγ is an activator of fatty acid synthesis and storage, and is present in white and brown adipose tissue. There are at

Basics of LXR biology

LXRα (NR1H3) and LXRβ (NR1H2) are ligand activated nuclear receptors that were discovered over 15 years ago by multiple groups (Apfel et al., 1994, Willy et al., 1995, Song et al., 1994, Shinar et al., 1994, Teboul et al., 1995, Seol et al., 1995) and act as key regulators of lipid metabolism and inflammation (Bensinger and Tontonoz, 2008, N and Castrillo, 2011). They formulate obligate heterodimers with RXRs to bind LXR response elements AGGTCA separated by four nucleotides (DR-4) (Willy et

Other nuclear receptors

Although the best known nuclear receptors in the regulation of macrophage cholesterol homeostasis and the development of atherosclerosis are PPARs and LXRs, recent studies showed the importance of other nuclear receptors such as the pregnane X receptor (PXR), the farnesoid X receptor (FXR), the glucocorticoid receptor (GR), the retinoic acid receptors (RARs) and the NR4A nuclear receptor family members.

Conclusions and future perspectives

It appears that nuclear receptors, as one would have predicted, have a major role in regulating macrophage cholesterol metabolism. It is particularly striking that several of the receptors not only respond to ligand and regulate a set of target genes but also form an interrelated network which includes molecules involved in lipid uptake, processing and efflux. Moreover, induction of ligand production and the expression of other receptors create a complete regulatory loop. A particularly good

Acknowledgments

The authors would like to thank Dr. István Szatmári for carefully reading the manuscript and providing helpful suggestions and Ms. Erika Sári for the skillful preparation of the illustrations. L.N. is supported by Grants from the Hungarian Scienctific Research Fund (OTKA NK72730 and K100196), EU FP7 (MOLMEDREX FP7-REGPOT-2008-1 #229920), TAMOP-4.2.2/08/1, and TÁMOP-4.2.1/B-09/1/KONV-2010-0007 implemented through the New Hungary Development Plan co-financed by the European Social Fund and the

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