Oxysterols: Sources, cellular storage and metabolism, and new insights into their roles in cholesterol homeostasis

Molecular Aspects of Medicine 30 (2009) 111–122

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Molecular Aspects of Medicine journal homepage: www.elsevier.com/locate/mam

Review

Oxysterols: Sources, cellular storage and metabolism, and new insights into their roles in cholesterol homeostasis Andrew J. Brown a, Wendy Jessup b,* a b

School of Biotechnology and Biomolecular Sciences, Sydney, Australia Centre for Vascular Research, School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia

a r t i c l e

i n f o

Article history: Received 19 December 2008 Accepted 10 February 2009

Keywords: 7-Ketocholesterol 24(S),25-Epoxycholesterol Efflux LXR Oxidised LDL SREBP

a b s t r a c t Oxysterols are structurally identical to cholesterol, but with one or more additional oxygen containing functional groups (such as alcohol, carbonyl or epoxide groups). The wide array of oxysterols encountered in human health and disease vary in their origin (either enzymic or non-enzymic), and their putative effects and/or function(s). Some are thought to be damaging, whereas others may play important physiological roles, including in the regulation of cholesterol homeostasis. In this review, we will concentrate on the major cellular oxysterols. We summarise their location, generation, metabolism and elimination, as well as providing insights into the latest research into their regulatory roles in cholesterol homeostasis. Ó 2009 Elsevier Ltd. All rights reserved.

Contents 1. 2.

3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological sources of oxysterols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Non-enzymic oxidation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Enzymic oxidation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Dietary versus endogenous sources of oxysterols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxysterol cellular levels, metabolism and elimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Cellular concentrations and locations of oxysterols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Oxysterol metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Elimination of oxysterols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. Sterol export to apolipoprotein AI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2. Sterol export to HDL. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

112 112 112 113 113 114 114 115 116 116 116

* Corresponding author. Tel.: +61 2 9385 1219; fax: +61 2 9385 1389. E-mail address: [email protected] (W. Jessup). Abbreviations: 7OOHC, 7-hydroperoxycholesterol; 7aHC, 7a-hydroxycholesterol; 7bHC, 7b-hydroxycholesterol; 7KC, 7-ketocholesterol; 11b-HSD1, 11b-hydroxysteroid dehydrogenase type 1; 24,25EC, 24(S),25-epoxycholesterol; 24HC, 24(S)-hydroxycholesterol; 25HC, 25-hydroxycholesterol; 27HC, 27hydroxycholesterol; ABCA1, ATP-binding cassette, subfamily A, member 1; ABCG1, ATP-binding cassette, subfamily G, member 1; ACAT, acyl CoA cholesterol acyl transferase; AcLDL, acetylated low density lipoprotein; apoAI, apolipoprotein AI; DOS, 2,3(S); 22(S), 23-dioxidosqualene; ER, endoplasmic reticulum; HDL, high density lipoprotein; HMG-CoA reductase, 3-hydroxy-3-methyl-glutaryl-CoA reductase; Ld, liquid disordered domain; Insig, insulininduced gene; Lo, liquid ordered domain; LCAT, lecithin cholesteryl acyl transferase; LDL, low density lipoprotein; LXR, liver X receptor; MOS, 2,3(S)monooxidosqualene; OSC, 2,3-oxidosqualene cyclase; OxLDL, oxidised LDL; ROS, reactive oxygen species; Scap, SREBP cleavage activating protein; SM, squalene monooxygenase; SR, scavenger receptor; SREBP, sterol regulatory element binding protein; SULT, sulfotransferase. 0098-2997/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.mam.2009.02.005

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Oxysterols and cholesterol homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Accelerated degradation of HMG-CoA reductase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Suppression of activation of the sterol regulatory element binding protein (SREBP) pathway . . . . . . . . . . . . . . . . . . . . . 4.3. Increased cholesterol efflux by activating liver X receptors (LXR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Increasing cholesterol storage as esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Contribution of oxysterol–membrane interactions in regulating cellular cholesterol homeostasis . . . . . . . . . . . . . . . . . . 4.6. 24(S),25-Epoxycholesterol: a monitor and modulator of cholesterol synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7. In vivo evidence for oxysterols playing a role in cholesterol homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction This volume reflects a recent renewed interest in oxysterols, a century after Lifshultz first identified ‘oxycholesterol’ (Lifschutz, 1913), and three decades after Kandutsch and colleagues proposed what has become known as the ‘Oxysterol Hypothesis of Cholesterol Homeostasis’ (Kandutsch et al., 1978). The revised interest in oxysterols probably arises from recent advances in our understanding of the importance of cholesterol in controlling eukaryotic membrane structure and function. Specific oxysterols have the potential to either control cholesterol biosynthesis and/or to directly interfere with normal cellular functions of cholesterol. The following chapters detail specific roles of oxysterols in lipid homeostasis and signalling, as well as potentially deleterious effects on cell function and their pathobiology. In this review, we will focus on cellular oxysterols, their location, generation, metabolism and elimination. For details of plasma oxysterols, the reader is directed to several earlier detailed reviews (Brown and Jessup, 1999; Schroepfer, 2000). 2. Biological sources of oxysterols Oxysterols are largely derived from cholesterol by a variety of routes. Several previous reviews have comprehensively discussed the major biological oxysterols and the routes for their formation (Brown and Jessup, 1999; Schroepfer, 2000; Gill et al., 2008; Smith and Murphy, 2008). Fig. 1 shows the structures of several major biological oxysterols and their relationship to the structure of cholesterol. In general, biological oxysterols fall into two main categories; those oxygenated on the sterol ring, mainly at the 7-position (e.g., 7a/b-hydroperoxycholesterol (7OOHC), 7-ketocholesterol (7KC) and 7a/b-hydroyxcholesterol (7HC)) and those oxygenated on the side-chain (e.g., 24S-hydroxycholesterol (24HC), 25-hydroxycholesterol (25HC) and 27-hydroxycholesterol (27HC)). Generally, ring-oxygenated sterols tend to be formed non-enzymically, whereas side-chain oxygenated sterols usually have an enzymic origin. However, there are exceptions to this rule; for example 25HC and 7aHC can be produced by both enzymic and non-enzymic routes (see Gill et al., 2008 for further discussion). 2.1. Non-enzymic oxidation Direct radical attack on cholesterol by reactive oxygen species (ROS), such as the hydroxyl radical, leads to abstraction of an allylic hydrogen atom at C-7. The carbon-centred radical generated at C-7 is relatively long-lived and can react further with molecular oxygen to form a cholesterol peroxyl radical (COO). Further hydrogen abstraction from another lipid generates the relatively stable cholesterol hydroperoxides (7a- and 7b-OOHC) (Fig. 1). Cholesterol hydroperoxides have been detected at low levels in some biological samples, including human atherosclerotic plaque (Chisolm et al., 1994; Brown et al., 1997; Adachi et al., 2000). 7OOHC is the major oxysterol formed at the early stages of non-enzymic oxidation of cholesterol (Brown et al., 1997). However, tissue levels of 7OOHC are usually quite low relative to downstream 7-oxygenated products, most probably due to the further processing of the hydroperoxides both by further non-enzymic lipid oxidation as well as by enzymic reduction (Brown et al., 1997). In the presence of trace levels of transition metals, cholesterol hydroperoxides are further decomposed non-enzymically to 7a/b-alkoxy radicals (CO), which in turn can undergo further reactions to generate 7a/b-hydroxycholesterols and 7-ketocholesterol (Fig. 1). These are the major non-enzymically generated oxysterols that are present in most tissues (Brown and Jessup, 1999). The major locations of cholesterol are in plasma lipoproteins and in cell membranes, where cholesterol is invariably present together with other lipids, predominantly phospholipids. Cholesterol is chemically less susceptible to oxidative attack than the polyunsaturated fatty acyl moieties present in phospholipids. This is seen in in vitro oxidation of cholesterol in plasma and in isolated low density lipoprotein (LDL), where oxidation of fatty acyl groups occurs earlier and more extensively than that of cholesterol (Noguchi et al., 1998). This means that, in these situations, oxysterols occur in a generally oxidised milieu, and will be accompanied by many other, potentially toxic, oxidised compounds. This is an important consideration when the biological activities of complex species such as oxidised LDL (OxLDL) are measured. Interestingly, a recent study of the kinetic of lipid oxidation in cultured cells exposed to oxidative stress reported that cholesterol was relatively more oxidised than polyunsaturated fatty acids (Saito et al., 2007). On the face of it, this suggests that cholesterol in cell membranes is

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NON-ENZYMIC

ENZYMIC

Cholesterol C

ROS

B

Ch 25 h

7α/β-Hydroperoxy D ec -cholesterol om p

os itio n

24S-Hydroxy -cholesterol

25-Hydroxy -cholesterol

CYP7A

7-Ketocholesterol

CY P4 6

27 YP C

A

D

27-Hydroxy -cholesterol

7β-Hydroxycholesterol 7α-Hydroxycholesterol Fig. 1. Sources and structures of selected oxysterols. The complete structure of cholesterol is shown with rings lettered (A–D) and carbons numbered (1–27). Truncated structures of the oxysterols are shown, emphasising the additional oxygenation. Ch25h, cholesterol 25-hydroxylase, a non-cytochrome P450 enzyme; ROS, reactive oxygen species. Please refer to the text for further details.

more susceptible to oxidation than surrounding polyunsaturated fatty acids. However, as the authors point out, the levels of lipid oxidation products in cells reflect a balance between their rates of formation, metabolism and export. As cholesterol hydroperoxides are cleared less efficiently by cellular peroxidases than fatty acyl peroxides, their higher levels in stressed cells may be more a reflection of their less efficient clearance by cellular glutathione peroxide than their rates of formation. 2.2. Enzymic oxidation Enzymic side-chain hydroxylation of cholesterol can generate the 24-, 25- and 27-hydroxycholesterols; each oxysterol is generated by separate enzymes (Russell, 2000) (Fig. 1). Sterol 27-hydroxylase (CYP27A1) is a mitochondrial P450 enzyme expressed in many tissues, particularly highly in liver and macrophages. The enzyme catalyses the first step in the ‘alternative’ pathway for bile acid synthesis. CYP27A1 catalyses the addition of a hydroxyl group to the side-chain of cholesterol and also to a range of related sterols, forming 27-hydroylated sterols. It is unusual in that it also further oxidises the same methyl group to generate the carboxylic acid derivative. Genetic deficiency of CYP27A1 leads to the disease cerebrotendinous xanthomatosis. Cholesterol 24-hydroxylase (CYP46A1) is also a P450 enzyme which is located in the endoplasmic reticulum (ER) (Lund et al., 1999) and most highly expressed in neural cells of the brain and retina (Björkhem et al., 1998; Bretillon et al., 2007). The brain is the major source of circulating 24HC (Björkhem et al., 1998). Cholesterol 25-hydroxylase is a non-haeme iron-containing protein that is expressed at very low levels in most tissues. Despite this, there is interest in the enzyme because of the activity of its product (25HC) as a regulator of the sterol regulatory element binding protein (SREBP) pathway for cholesterol-dependent transcriptional regulation (Russell, 2000). Overexpression of cholesterol 25-hydroxylase in a cell line increased generation of 25HC, and suppressed SREBP processing and cholesterol synthesis (Lund et al., 1998). The enzyme is located in the membranes of the ER and Golgi, where SREBP, SREBP cleavage activating protein (Scap) and insulin-induced gene (Insig) protein are also found, consistent with a functional association with this system in vivo. 2.3. Dietary versus endogenous sources of oxysterols Relatively high levels of oxysterols are found in some foods, particularly cholesterol-rich foods such as meat, eggs and dairy products. These are most probably generated non-enzymically during cooking, processing and storage. Therefore, there is an interest in which dietary sources contribute to circulating and tissue levels of oxysterols. Unfortunately, there is no

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clear quantitative answer to this question. However, several studies in animals and humans have shown that oxysterols can be absorbed in the gut and transported into the circulation within chylomicrons (reviewed in Brown and Jessup, 1999; Carpenter, 2002). Some of the absorbed oxysterols are taken up from the circulation into tissues, but the relative abundance of individual oxysterols is different in diet, serum and tissues, indicating dietary oxysterols are modified in vivo by differential uptake, metabolism and elimination. The extent to which dietary sources contribute to tissue oxysterol levels is expected to depend heavily on the specific types as well as the levels of their intake.

3. Oxysterol cellular levels, metabolism and elimination 3.1. Cellular concentrations and locations of oxysterols The effects of oxysterols on cells are many and varied (Smith and Johnson, 1989). The physiological roles of specific oxysterols (covered in other parts of this issue) are as intermediates in bile acid synthesis, in sterol transport between tissues and in the regulation of gene expression. Under normal conditions, oxysterols involved in these processes are maintained at very low and controlled levels, usually in the presence of a large excess (1000-fold) of cholesterol. In studies that aim to explore the biological effects of cholesterol oxidation products, it is important that the experimental systems reproduce as closely as possible the conditions under which oxysterols are present in vivo. The major biological role of cholesterol is to define the physical structure of cell membranes. Cholesterol has a condensing effect on the phospholipid bilayer, the basic structural feature of all cell membranes. It restricts phospholipid acyl chain motion and decreases membrane permeability. The distribution of cholesterol and its associated phospholipids within membranes is not uniform; cholesterol forms liquid ordered (Lo) domains or ‘lipid rafts’ by associating tightly with phospholipids containing saturated acyl chains. These Lo domains are surrounded by more fluid, liquid disordered (Ld) domains. The heterogeneity in membrane lipid distribution induced by cholesterol is believed to control the association and activity of some proteins with membranes, regulating signal transduction events across the membrane. There is also variation in the amount of cholesterol present in different cell membranes, from relatively high levels at the plasma membrane (30–50% total lipid) to very low (a few percent) levels in the ER and mitochondria. Like cholesterol, many oxysterols are very hydrophobic and confined within cell membranes. However, small differences in the structures of oxysterols relative to cholesterol can affect how they interact with other membrane lipids, and this in turn can have significant effects on membrane structure (Wang et al., 2004; Massey and Pownall, 2005). For cells in vivo, usually the very low oxysterol:cholesterol ratio means that oxysterols have little impact on cell membrane structure and function. However, in some experimental (and possibly pathophysiological) conditions, where oxysterols represent a much larger proportion (>20%) of total cell sterols, the effects of oxysterols on membrane structure can become significant. There are very few reliable measures of cellular oxysterol concentrations and subcellular locations. This effort has been hampered by the difficulties that are associated with making accurate measurement of the very low concentrations of oxysterols that are present in biological samples, often in the presence of a vast excess of native cholesterol (Brown and Jessup, 1999; Van Reyk and Jessup, 1999). Particularly for non-enzymically generated oxysterols, even a small degree of cholesterol autoxidation during sample storage, extraction and analysis has the potential to make a significant contribution to the measured levels of oxysterols (Breuer and Björkhem, 1995). At present, there are only a very few reports of the in vivo cellular levels of oxysterols (Hultén et al., 1996; Van Reyk et al., 2006) and further research is certainly warranted. More extensive and accurate analysis should provide a better idea of the range of levels exhibited in various pathological conditions/tissues. This need is presently being addressed, with recent development of mass spectrometric-based lipidomic analyses and the publication of several new methods for analysing a range of sterols, including traditionally hard-to-measure oxysterols like 24(S),25-epoxycholesterol (24,25EC) (Watson, 2006; McDonald et al., 2007). A further issue that is frequently neglected in considering in vivo levels of oxysterols is the proportion that is esterified. Esterification of excess cholesterol in cells, mediated by acyl CoA cholesterol acyl transferase (ACAT), is a normal cellular mechanism for limiting the levels of free (unesterified) cholesterol in cell membranes within tight limits, in order to maintain normal membrane structure. The esterified cholesterol generated by ACAT is stored as a metabolically inert store of excess cholesterol in cytoplasmic lipid droplets. ACAT also esterifies many oxidised forms of cholesterol (Zhang et al., 2003), and in fact oxysterols are predominantly present as esters in vivo. For example, in human atherosclerotic lesions and in human macrophage foam cells isolated from lesions, 80–95% of all oxysterols measured are esterified (Brown et al., 1997; Brown and Jessup, 1999; Van Reyk et al., 2006). Similarly the majority of circulating sterols in the circulation are also esterified (Dzeletovic et al., 1995), in this case mediated by lecithin cholesteryl acyl transferase (LCAT). If we assume that esterified oxysterols are, like cholesteryl esters, metabolically inert, then the effective in vivo cellular levels of oxysterols will be 10–20-fold lower than the total oxysterol levels that are usually measured. This is an important consideration when designing experiments intended to deliver oxysterols at ‘physiological levels’ to cells in vitro. It is advisable to measure the extent to which oxysterols are esterified under the specific experimental conditions used, as this can vary depending on experimental conditions. For example, when free 7KC is supplied to macrophages incorporated into acetylated LDL (AcLDL), >90% of the 7KC accumulated in the cells esterified; in contrast, when 7KC is incorporated into native LDL only 30–50% becomes esterified (Gelissen et al., 1996). This probably reflects the differences in the amounts of free fatty acids that are available as co-substrates in the different loading conditions. 7KC-enriched AcLDL particles are endocytosed

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and hydrolysed completely in lysosomes, providing a rich supply of fatty acids for ACAT. In contrast, 7KC-enriched native LDL is not rapidly endocytosed and transfers 7KC through direct exchange between the LDL particle and cell membrane. Thus, the LDL particles provide little acyl substrates for ACAT. In some studies, oxysterols are supplied to cells by incubation with OxLDL. In vitro oxidation of LDL (usually by Cu2+ ± Fe2+) transforms the particle into a ligand for macrophage scavenger receptors (scavenger receptor (SR) A and CD36), allowing its rapid uptake. LDL requires quite extensive oxidation to become a ligand for these receptors, and there is no compelling evidence to support the widely-held view that material equivalent to this in vitro OxLDL is ever generated in vivo. Despite this, OxLDL is frequently used as an experimental tool to stimulate cholesterol and/or oxysterol loading to mimic atherosclerotic ‘foam cells’. It is therefore important to understand the lipid composition of OxLDL, and its impact on cell sterol content following uptake. During the course of LDL oxidation, we found that >50% of its cholesterol was converted to oxysterols, predominantly 7KC (Brown et al., 1996). In addition, the fatty acyl groups esterified to the majority (60–70%) of both cholesterol and the newlyformed oxysterols were also extensively oxidised (Brown et al., 1996). The extensive oxidation of both cholesterol and fatty acyl groups has a significant impact on the nature of the lipid-loaded ‘foam cells’ that are generated by uptake of OxLDL. In these cells, oxysterols represent 30% of total cell sterol (compared with <5% in human lesion foam cells) (Brown et al., 1996, 2000a). Further, a large proportion of the sterols esterified to oxidised acyl esters are resistant to lysosomal hydrolysis and remained trapped within the lysosomal compartment of the cells (Brown et al., 2000a). We have no comparable measurements of these species in human lesion foam cells. But overall, we recommend extreme caution in interpreting data generated using cells loaded with OxLDL and in extrapolating this to in vivo conditions. As a simpler, more reductionist approach, in some studies pure individual oxysterols are added directly to the cell culture medium. It is particularly important in these systems to directly measure the oxysterol incorporation in both absolute terms and as a percentage relative to cell cholesterol, and also as the proportion esterified. Oxysterols are never present in isolation in vivo, but exist often in combination with other oxysterols and always in the presence of a large excess of native cholesterol. Some deleterious effects of bolus addition of oxysterols in vitro are diminished when they are added together with cholesterol or even other oxysterols (Clare et al., 1995; Leonarduzzi et al., 2004). This further emphasises the care with which experimental studies of oxysterol biology must be conducted and the pressing need to accurately define the amounts and physical location of the oxysterols that accumulate within cells. 3.2. Oxysterol metabolism Many of the enzymic pathways which metabolise cholesterol are also able to act on oxysterols, as recently reviewed (Gill et al., 2008). The major routes for oxysterol metabolism are shown in Fig. 2. Both ACAT and LCAT efficiently esterify oxysterols in cells and plasma, respectively. ACAT has two sterol-binding sites, one catalytic and the other an allosteric activator domain. The structural requirement of the allosteric site is quite stringent for cholesterol (Zhang et al., 2003) and oxysterols are generally poor allosteric activators of ACAT. However, in the presence of cholesterol (as is always the case in vivo) to occupy the allosteric site, ACAT becomes quite promiscuous in its catalytic

27-hydroxylated 7-ketocholesterol

7-Ketocholesteryl ester

Esterification

Further oxidation (e.g. CYP27)

(ACAT or LCAT)

Sulfation

7-Ketocholesterol

(e.g. SULT2B1b)

7-Ketocholesterol 3-sulfate

Reduction (CYP11)

7β-Hydroxycholesterol

Fig. 2. Modes of metabolism of oxysterols. Some of the ways 7-ketocholesterol can be metabolised is given as an example. See Fig. 1 for complete sterol structure. Please refer to the text for further details.

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activity towards oxysterols, which is reflected by their efficient esterification in vivo (Brown et al., 1997; Brown and Jessup, 1999; Van Reyk et al., 2006). Sterol 27-hydroxylase (CYP27A1) can act on both cholesterol and some ring-oxygenated sterols. For example, CYP27A1 can metabolise 7KC to 27-hydroxylated 7KC, which in turn is metabolised further to more water-soluble metabolites that can be excreted from the cell (Brown et al., 2000b; Lyons and Brown, 2001; Lee et al., 2006). Metabolism of 7KC by CYP27A1 was associated with reduced cellular toxicity of this oxysterol (Lee et al., 2006), suggesting that the metabolites of 7KC are less toxic and/or more easily eliminated than the parent oxysterol. 11b-Hydroxysteroid dehydrogenase type 1 (11b-HSD1 or CYP11b), another P450 enzyme, is best known for its role in glucocorticoid metabolism, where it controls the reversible interconversion between cortisol and cortisone. Recently, CYP11b was also shown to reduce 7KC to 7bHC (reviewed in Jessup and Brown, 2005). Although the enzyme can reversibly interconvert these oxysterols, it appears to act predominantly as a reductase in vivo. The importance of this pathway for oxysterol metabolism in vivo remains to be established. Interestingly, the activity and reaction direction of 11b-HSD1 in adipocytes is differentially affected by 7KC or 7bHC treatment, with potential implications for obesity and its complications (Wamil et al., 2008). Sulfotransferases (SULTs) are a superfamily of enzymes, within which the SULT2 subgroup specifically sulfate the 3b-hydroxyl group of steroids and sterols. Their best-known role is in the elimination of steroid hormones. However, many oxysterols are also substrates for the cholesterol sulfotransferase enzyme (SULT2B1b). These include both ring-oxygenated (Fuda et al., 2007) and side-chain (Chen et al., 2007) oxysterols. This appears to be a significant route for the elimination of oxysterols, as expression of SULT2B1b appears to protect cells against the toxicity of some non-enzymically generated oxysterols (Fuda et al., 2007) and also to suppress liver X receptor (LXR) signalling in cells by endogenous side-chain oxysterols (Chen et al., 2007; Ma et al., 2008). 3.3. Elimination of oxysterols The hydrophobic nature of most oxysterols normally confines them within cells in non-polar locations such as membranes and lipid droplets. Like cholesterol, they can only be eliminated directly from cells to lipophilic acceptors, a process mediated by specific membrane lipid transporters, such as ATP-binding cassette, subfamily A, member 1 (ABCA1), ATP-binding cassette, subfamily G, member 1(ABCG1) and scavenger receptor BI (SRBI) (Jessup et al., 2006). Exceptions to this rule are some of the more hydrophilic side-chain hydroxylated sterols such as 24HC and 27HC (Björkhem, 2006). Most studies of lipoprotein-mediated oxysterol elimination have measured the export of 7KC and other non-enzymically oxidised forms of cholesterol. 3.3.1. Sterol export to apolipoprotein AI ABCA1 transports phospholipids and cholesterol to lipid-free apolipoproteins such as apolipoprotein AI (apoAI), the major protein component of high density lipoprotein (HDL). These apolipoproteins contain multiple amphipathic a-helical domains which bind directly to the surface of membrane bilayers through their hydrophobic faces. It has been suggested that ABCA1 is primarily a phospholipid flippase and that its activity changes the lipid packing of the plasma membrane, increasing its binding affinity for apolipoproteins such as apoAI. The bound apoAI then desorbs from the membrane surface together with some of the membrane phospholipids. Cholesterol may be abstracted simultaneously with phospholipids, or transfer into apoAI/phospholipid particles following their formation. In vitro studies have indicated that apoAI insertion into membranes is favoured where Lo and Ld domains coexist, probably through lattice defects at the boundaries between these phases (Pownall et al., 1978, 1979), and cholesterol plays an important role in generating these domains. Incorporation of 7KC into cells has several interesting effects on ABCA1-mediated sterol export to apoAI. Firstly, the presence of 7KC inhibits export of cholesterol to apoAI (Gelissen et al., 1996; Gaus et al., 2001, 2004). This probably reflects the direct effects of 7KC on membrane structure, particularly the ability of 7KC to form ordered lipid domains and boundary bilayer defects. In model membranes, 7KC is less effective than cholesterol in generating Lo phases and in promoting apoAI binding and membrane solubilisation (Massey and Pownall, 2005). In intact cells, 7KC locates predominantly in Lo domains (Myers and Stanley, 1999; Berthier et al., 2004; Gaus et al., 2004), and this is associated with lower binding of apoAI to Lo domains and reduced cholesterol export, while apoAI binding to Ld domains was unaffected (Gaus et al., 2004). Interestingly, the relative export of membrane 7KC to apoAI is much lower than that of cholesterol. This may reflect its asymmetric distribution predominantly into Lo domains, since apoAI appears to preferentially extract sterols from the Ld phase (Fukuda et al., 2007). 3.3.2. Sterol export to HDL Cholesterol exchange between cells and HDL or other phospholipid-containing extracellular acceptors can occur spontaneously by a passive diffusion process in which the direction of net transfer depends on the relative concentrations of sterol in cells and lipoprotein. This process can be accelerated and/or modified when certain membrane proteins are expressed. For example, SRBI can accelerate bi-directional cholesterol exchange between cells and HDL, while ABCG1 is thought to mediate uni-directional export of cholesterol from cells to HDL. Some oxysterols are readily exported from cells to HDL (Gelissen et al., 1999; Jessup et al., 2002). For example, 7KC and 7bHC effluxed from OxLDL-loaded mouse macrophages to HDL at rates similar to that of cholesterol (Gelissen et al., 1999).

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Acetyl CoA

ER

Synthesis

HMG-CoA Reductase

e.g. HMG-CoA reductase

1

Cholesterol

Proteasome

CE 4 Oxysterol

Uptake

SREBP

Efflux

2

e.g. LDL receptor

e.g. ABCA1 Scap

Insig

3

SREBP target genes

Nucleus

e.g. HMG-CoA reductase LDL-receptor

LXR RXR

LXR target genes e.g. ABCA1, ABCG1

Fig. 3. Certain oxysterols produced enzymically from cholesterol (e.g., 24HC) or in the cholesterol biosynthetic pathway (24,25EC) reduce cholesterol status in the cell by several molecular mechanisms: 1. accelerated degradation of HMG-CoA reductase; 2. suppression of SREBP activation; 3. increased cholesterol efflux by activating LXR-mediated gene transcription; 4. increased intracellular storage of cholesterol as esters (CE). Please refer to the text for further details. This scheme was modified from Brown (2009).

When mouse macrophages were selectively enriched simultaneously with 7KC and cholesterol, net mass transfer of both cholesterol and 7KC occurred to a wide range of phospholipid-containing acceptors, including HDL, reconstituted apoAI/ phosphatidylcholine discs and small unilamellar vesicles (Gelissen et al., 1999). Recently, ABCG1 has been suggested to promote the export of 7KC and 7bHC to HDL, and to protect cells against 7KC and 7bHC induced apoptosis and impaired endothelial cell function (Engel et al., 2007; Terasaka et al., 2007, 2008). However, it is important to note that mass transfer of 7KC can also occur in the opposite direction – from HDL to cells – if the oxysterol content of HDL is enriched (Jessup et al., 2002). Thus it is possible, for example in the case of cholesterol-loaded human macrophages exposed to 7KC-containing HDL, for net directions of cholesterol and 7KC transport to be in opposite directions. In this system, the sterol traffic is more consistent with passive or SRBI-mediated sterol transport, and less compatible with the uni-directional (outward) transport mechanism of ABCG1. 4. Oxysterols and cholesterol homeostasis Because cholesterol is essential and yet too much is deleterious, cells have multiple strategies in place to keep their cholesterol levels under exquisite control. Mechanisms operate to limit cholesterol accumulation, including reducing cholesterol synthesis and uptake, stimulating cholesterol elimination from the cell, and increasing storage as inert cholesteryl esters. Certain oxysterols seem to serve as sensors and regulators of cholesterol excess. Control of cellular cholesterol homeostasis is multifaceted. The involvement of oxysterols in various aspects of cholesterol homeostasis is summarised in Fig. 3 and briefly reviewed below. 4.1. Accelerated degradation of HMG-CoA reductase 3-Hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase is a key flux-controlling enzyme in cholesterol biosynthesis, and is the molecular target of the statin class of hypocholesterolaemic drugs. Post-translational degradation of the HMG-CoA reductase by the ubiquitin-proteasomal system is an important aspect in the control of cholesterol synthesis. Certain oxysterols can accelerate HMG-CoA reductase degradation, although the more physiologically relevant regulators for this process may be intermediates in the cholesterol biosynthetic pathway (DeBose-Boyd, 2008). 4.2. Suppression of activation of the sterol regulatory element binding protein (SREBP) pathway Many genes encoding for enzymes in cholesterol biosynthesis are governed by the SREBP family of transcription factors. There are three isoforms: SREBP-1a has overlapping gene targets between SREBP-1c and SREBP-2; SREBP-1c generally controls genes involved in the synthesis of fatty acids, phospholipids and triglycerides, whereas SREBP-2 is primarily concerned with regulating genes involved in cholesterol synthesis and uptake (including HMG-CoA reductase and the LDL receptor). The activation of SREBP entails some elegant cell biology elucidated by the laboratory of Goldstein and Brown over two

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decades (Goldstein et al., 2006). Briefly, SREBP is produced in the ER as an inactive precursor bound to a cholesterol sensing protein, Scap, which measures the amount of cholesterol in the ER membranes. When this falls below a critical threshold (5% of total ER lipid) (Radhakrishnan et al., 2008), Scap accompanies SREBP to the Golgi apparatus where it is clipped sequentially by two proteases. The active transcription factor produced is imported into the nucleus where it upregulates lipogenic gene transcription. If cholesterol levels are above the critical threshold, the SREBP–Scap complex is held back in the ER by a protein restraint, Insig. The current paradigm is that cholesterol binds to Scap and oxysterols bind to Insig (Radhakrishnan et al., 2004, 2007). But in response to either signal of sterol excess, the net result is the same: Insig retains the SREBP–Scap complex, preventing the proteolytic activation of SREBP, and hence arresting lipogenic gene expression. 4.3. Increased cholesterol efflux by activating liver X receptors (LXR) Certain oxysterols are natural ligands for the LXR nuclear receptors. These control the expression of many genes encoding proteins involved in cholesterol export such as ABCA1, ABCG1, and apolipoprotein E. It should be noted that many genes involved in fatty acid and triglyceride synthesis, e.g., fatty acid synthase and SREBP-1, are also upregulated by LXR. However, oxysterol ligands for LXR have the advantage over many synthetic agonists, in that any transcriptional upregulation of SREBP-1c tends to be offset by suppression of SREBP-1c activation. Two enzymes in cholesterol biosynthesis have been demonstrated to be negatively regulated via LXR, indicating yet another way that oxysterols may inhibit cholesterol synthesis (Wang et al., 2008). 4.4. Increasing cholesterol storage as esters Above a certain threshold, excess cellular cholesterol is stored away as cholesteryl esters by the action of ACAT in the ER (Xu and Tabas, 1991). Oxysterols like 25HC stimulate cholesterol esterification. The mechanism has been thought to involve the oxysterol intercalating into the plasma membrane, displacing cholesterol which then traffics to the ER where it stimulates esterification and suppresses SREBP activation. Hence, 25HC acutely stimulates esterification of radiolabelled cholesterol delivered as a pulse to the plasma membrane of cells (Du et al., 2004). However, with the binding of oxysterols to Insig now providing a plausible explanation of how oxysterols suppress SREBP activation, doubt has been cast on whether or not oxysterols indeed induce cholesterol transfer from the plasma membrane to the ER. Moreover, when cellular cholesterol contents were manipulated and the cholesterol contents of highly purified ER membranes were related to SREBP processing, no effect of 25HC treatment was found on ER cholesterol levels, despite maximal suppression of SREBP processing (Radhakrishnan et al., 2008). It may be that ACAT is allosterically regulated by certain oxysterols, although this is not a general property of oxysterols (Cheng et al., 1995). More work will be required to define the molecular mechanism(s) by which certain oxysterols stimulate cholesterol esterification. 4.5. Contribution of oxysterol–membrane interactions in regulating cellular cholesterol homeostasis Sterol enantiomers are a clever approach to deconvolute the membrane effects of sterols from the effects of sterol–protein interactions. Gale et al. (2008) employed the enantiomer (structural mirror image) of 25HC to investigate the molecular mechanism of the action of side-chain oxysterols in cholesterol homeostasis. Somewhat surprisingly, they reported that the enantiomeric form of 25HC suppressed SREBP-mediated transcriptional activity and increased HMG-CoA reductase degradation to a similar extent as the naturally occurring isomer. This behaviour was abrogated by increasing the saturation of the phospholipid acyl chain constituents. This study raises the possibility that oxysterols may contribute to the regulation of cellular cholesterol homeostasis through modulation of the properties of the lipid environment in which proteins like Insig and Scap are embedded. Further investigations will be required to determine the relative importance of oxysterol–membrane interactions versus specific oxysterol–protein interactions in controlling cholesterol homeostasis at physiologically relevant oxysterol concentrations. 4.6. 24(S),25-Epoxycholesterol: a monitor and modulator of cholesterol synthesis Recent work on 24,25EC has opened up a new vista on the role of endogenously produced oxysterols in cholesterol homeostasis. We first became interested in 24,25EC when we found that the cholesterol-lowering drugs, the statins, decreased cholesterol efflux from macrophages and ABCA1 expression by an LXR-mediated mechanism (Wong et al., 2004). As inhibitors of HMG-CoA reductase, a key early step in the mevalonate pathway, we reasoned that statins may be inhibiting the formation of a sterol ligand for LXR. The existence of 24,25EC had been known since the early 1980s with the work of Spencer and coworkers (Nelson et al., 1981). In addition, Rowe et al. (2003) had recently published that mouse macrophages can synthesise 24,25EC. We extended this work by demonstrating 24,25EC production in human macrophages which was inhibited by statin treatment (Wong et al., 2004). 24,25EC is produced in a shunt branching from the cholesterol synthesis pathway. The enzyme squalene monooxygenase (SM) performs the first oxygenation step in this pathway, converting the hydrocarbon, squalene, to monooxidosqualene (MOS). At the beginning of the shunt pathway, SM introduces a second epoxide group to form dioxido (or diepoxy) squalene (DOS). This is preferentially cyclised by the next enzyme in the pathway, 2,3-oxidosqualene cyclase (OSC) or lanosterol syn-

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HMG-CoA reductase Acetyl-CoA

HMG-CoA

Mevalonate isoprenoids etc

Statins Squalene

Shunt Pathway DOS

SM MOS

Oxidosqualene cyclase 24(S),25-Epoxylanosterol

Lanosterol

24(S),25-Epoxy cholesterol

Cholesterol

Fig. 4. Structure and synthesis of 24(S),25-epoxycholesterol. This oxysterol is made in a shunt in the mevalonate pathway, parallel to cholesterol synthesis. Please refer to the text for further details. DOS, 2,3(S);22(S),23-dioxidosqualene; MOS, 2,3(S)-monooxidosqualene; OSC, 2,3-oxidosqualene cyclase; SM, squalene monooxygenase. This scheme was modified from Brown (2009).

thase, to channel into the shunt pathway producing 24,25EC (Fig. 4). This oxysterol was originally identified in human liver (Spencer et al., 1985), but should theoretically be made in all cell-types that make cholesterol. For example, we have shown that primary human astrocytes synthesise and secrete 24,25EC (Wong et al., 2007a). We speculate that astrocytes, as support cells to neurons, may use this oxysterol packaged together with cholesterol to signal to neurons to turn down cholesterol synthesis, thus conserving the neuron’s energy for their primary function of neurotransmission. Further studies are required to test this extension of the emerging idea that neurons outsource various metabolic functions to their support cells. Oxysterols derived from cholesterol are thought to be sensors of cholesterol accumulation. However, due to feedback regulation of the mevalonate pathway, cholesterol accumulation inhibits synthesis of both 24,25EC and cholesterol synthesis. This indicated to us that 24,25EC must have a distinct role from other oxysterols derived from cholesterol. We observed that 24,25EC tended to be produced in a constant ratio to cholesterol across a number of conditions in which cholesterol biosynthetic rates were altered, suggesting that 24,25EC serves as a measure of cholesterol synthesis (Wong et al., 2007b). 24,25EC is only a minor sterol in cells, representing less than 1% of cholesterol levels or synthesis (Wong et al., 2004, 2007a; Yang et al., 2006). Many studies have investigated the effects of added oxysterols, including 24,25EC. Some studies have used pharmacological means, namely partial inhibition of OSC, to artificially increase 24,25EC synthesis (Rowe et al., 2003; Wong et al., 2007a). But what is the role of the normally low levels of endogenous 24,25EC? To investigate this question, we employed a novel approach to selectively inhibit 24,25EC synthesis, by overexpressing the enzyme which follows the initiation of the shunt pathway, OSC (Wong et al., 2008). Overexpression of OSC prevents accumulation of MOS and hence diversion into the shunt pathway, inhibiting 24,25EC synthesis without significantly increasing the flux through to cholesterol. We found that selective inhibition of 24,25EC predictably decreased LXR-mediated transcription, confirming previous in vitro studies (Lehmann et al., 1997; Janowski et al., 1999) that this oxysterol is a potent bona fida ligand for LXR. Perhaps most surprisingly, selective inhibition of 24,25EC resulted in a burst of acute cholesterol synthesis that was normalised by 24 h. Indeed, in the absence of 24,25EC, cholesterol homeostatic responses were wild and inflated. These observations led us to propose that this oxysterol fine-tunes cholesterol homeostatic responses, ensuring that they change smoothly over time (Wong et al., 2008). These studies support our contention that 24,25EC has a special role in monitoring and modulating cholesterol synthesis, and help in the formulation of a revised oxysterol hypothesis. Rather than placing oxysterols at the centre of cholesterol homeostasis as was the case with the original oxysterol hypothesis (Kandutsch et al., 1978), this revised hypothesis provides oxysterols (and other sterol regulators) with a key supporting role to cholesterol, and proposes that these oxysterols will exert more or less influence in different tissues under different physiological conditions (Gill et al., 2008). For example, 24,25EC will be particularly important when cholesterol synthesis is active, whereas when cholesterol accumulates in tissues, 24,25EC production is inhibited and other oxysterols derived from cholesterol will come to the fore. These oxysterols include 27HC produced in macrophages or 24HC made in neurons (Björkhem et al., 2009).

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4.7. In vivo evidence for oxysterols playing a role in cholesterol homeostasis In support of 24,25EC’s special role as a measure and modulator of cholesterol synthesis, Zhang et al. (2001) found that hepatic concentrations of 24,25EC increased in rats in response to administration of the sterol precursor mevalonate. Moreover, they found that 24,25EC (but not 27HC) preferentially accumulated in the nucleus in accord with this serving as an activator of the nuclear receptor, LXR. In an important study, Chen et al. (2007) employed two approaches in mice models to demonstrate that oxysterols are LXR activators. Firstly, they utilised adenovirus-mediated expression of the enzyme, SULT2B1b sulfotransferase, capable of catabolising oxysterols. They reported that expression of this enzyme prevents dietary induction of hepatic LXR target genes by cholesterol but not by the nonsterol agonist, T0901317. As has been pointed out (Björkhem, 2008), SULT2B1b is also capable of catabolising cholesterol which could possibly confound the effects seen. The second approach was a mouse model in which three oxysterol-producing enzymes were knocked out, eliminating production of three proposed in vivo ligands for LXR: 24HC, 25HC, 27HC. Again, the mice showed impaired responses to dietary cholesterol with respect to LXR target gene induction. However, it should be noted that although dietary T0901317 induced LXR target gene expression, this response was also blunted in the triple knockout mice compared to the wild-type animals, suggesting that LXR responses in general may be compromised to some extent in these knockout mice. It has been pointed out that the lack of an alternative bile acid pathway due to CYP27 deletion may have differentially affected cholesterol absorption between the control mice and the triple knockout animals, potentially confounding the results (Björkhem, 2008). Chen et al.’s pioneering study highlights the difficulty of investigating the role of oxysterols in vivo: effects are often subtle in keeping with the auxiliary role proposed for oxysterols above, and perhaps also suggesting a certain degree of redundancy. Moreover, a challenge, such as dietary cholesterol as used by Chen et al. (2007), may be required to tease out effects. In addition, knocking out an enzyme like CYP27 may produce confounding effects since it will inhibit the production of molecules apart from 27HC. More studies are needed to delineate the roles of endogenous oxysterols in vivo on cholesterol homeostasis. OSC overexpression has been shown to selectively inhibit 24,25EC in proof-of principle experiments in cultured cells (Wong et al., 2008), and presents an excellent opportunity to investigate the effect of inhibiting 24,25EC production in vivo. 5. Overview and conclusions Oxysterols are a large and diverse group of compounds, with many sources and a multiplicity of biological activities. They have a long history of study, from initial chemical characterisation in the early 1900s, through later recognition of their roles in cholesterol homeostasis, based on the seminal studies of Goldstein and Brown on the SREBP–Scap pathway, to more recent identification of oxysterols as ligands for the LXR transcription factors. To date, most of the experimental studies of the biological activities of oxysterols have been based on exposure of cells or organisms to exogenously added oxysterols, in the hope that this faithfully replicates conditions encountered in vivo. The study of oxysterol biology is now on the brink of a new era, in which lipidomics will provide a more detailed and complete understanding of the cellular and subcellular lipid environment in which oxysterols function. 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