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Department of Medicine, University of Washington, Seattle, Washington
ABSTRACT I. INTRODUCTION II. CELL BIOLOGY A. Structure B. Function C. Substrates D. Lipid Acceptors E. Apolipoprotein Binding F. Lipid Transport Model III. REGULATION A. Expression 1. Transcription 2. Protein stability A) APOLIPOPROTEINS. B) FREE FATTY ACIDS. C) FREE CHOLESTEROL. D) REACTIVE CARBONYLS. 3. Protein trafficking B. Activity 1. Signaling pathways A) PROTEIN KINASE A. B) PROTEIN KINASE CK2. C) JANUS KINASE 2. D) PROTEIN KINASE C. 2. Partner proteins 3. Substrate trafficking IV. IN VIVO FUNCTIONS A. HDL Production 1. Genetic variations 2. Animal models B. Reverse Cholesterol Transport C. Tissue-Specific Functions 1. Brain 2. Intestines 3. Gallbladder 4. Lung 5. Testis 6. Kidney V. ABCA1 AND CARDIOVASCULAR DISEASE A. ABCA1 Activity B. ABCA1-Interacting Apolipoproteins 1. Apolipoprotein supply 2. Dysfunctional apolipoproteins A) CHLORINATION AND NITRATION OF HDL. B) TYROSYLATION OF HDL. C) OTHER OXIDATIVE PATHWAYS. VI. ABCA1 AS A THERAPEUTIC TARGET A. Transcriptional Regulation 1. LXR agonists 2. RXR agonists 3. PPAR{gamma} agonists B. Posttranscriptional Regulation C. Apolipoprotein Supply VII. CONCLUSIONS GRANTS ACKNOWLEDGMENTS REFERENCES
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ABSTRACT |
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I. INTRODUCTION |
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Approximately two-thirds of the cholesterol in human plasma is carried in a class of lipoprotein particles called low-density lipoprotein (LDL). LDL provides a source of cholesterol for steroidogenesis and cellular membranes. This occurs through the interaction of LDL with a cell-surface receptor that mediates internalization and degradation of the lipoprotein particles (27). The hepatic LDL receptor is responsible for clearing most of the LDL cholesterol from the plasma.
Cells other than those in steroidogenic tissues and the liver cannot metabolize cholesterol. Instead, they modulate their membrane cholesterol content by a feedback system that controls the rate of cholesterol biosynthesis and uptake by the LDL receptor (26). With most cell types this system is sufficient to provide cells with enough cholesterol for membrane integrity and function without overloading them. Some cells, particularly macrophages, can ingest cholesterol by endocytotic and phagocytotic pathways that are not feedback regulated by cholesterol (213). These cells must either store this excess cholesterol as esters or secrete it.
High-density lipoprotein (HDL), which carries about one-third of the cholesterol in human plasma, is involved in the removal of excess cholesterol from cells. HDL is a multifunctional and heterogeneous class of particles that transports a variety of lipids and lipophilic molecules between tissues and other lipoproteins. One of the major functions of HDL is to transport cholesterol from peripheral tissues to the liver for elimination in the bile (69, 90, 210). This process, called reverse cholesterol transport, is widely believed to account for much of the inverse relationship between plasma HDL levels and CVD revealed by population studies.
HDL components can remove cellular cholesterol by multiple mechanisms (210, 237). HDL phospholipids absorb cholesterol that diffuses from the plasma membrane into the aqueous phase, a passive process that is facilitated by the interaction of HDL particles with scavenger receptor B1 (143). Four cell membrane transporters have been identified that mediate cholesterol efflux from cells to HDL components by metabolically active pathways. All four belong to a superfamily of ATP-binding cassette transporters (ABCs). ABCA1 mediates the transport of cellular cholesterol, phospholipids, and other metabolites to HDL proteins (apolipoproteins) that are associated with no or very little lipid (204, 287). ABCA1 is highly expressed in the liver and tissue macrophages (149, 296). ABCA7, a close homolog of ABCA1, selectively transports phospholipids to lipid-depleted apolipoproteins (1, 116, 133, 140, 164, 285). It is highly expressed in myelolymphatic tissues, lung, adrenal, and brain. ABCG1 and ABCG4 mediate cholesterol transport from cells to HDL particles and are highly expressed in tissue macrophages and brain cells, respectively (120, 188, 202, 244, 284).
ABCA1 has been more extensively characterized than the other three ABC lipid transporters. Numerous studies of cultured cells, human HDL deficiencies, and animal models have shown that ABCA1 is a major determinant of plasma HDL levels and a potent atheroprotective factor (4, 131, 204, 255, 287). This transporter has therefore become an important new therapeutic target for drug development designed for clearing cholesterol from arterial macrophages and preventing CVD. This review focuses on the biology and pathophysiology of ABCA1.
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II. CELL BIOLOGY |
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ABC transporters are the largest membrane transporter family, with members in all phyla (54). ABCs are grouped into seven subclasses labeled ABCA through ABCG. Of the 49 ABCs in humans, 13 are in the ABCA subclass (54). Mutations in ABC genes cause a variety of diseases, including cystic fibrosis, Startgardt's macular degeneration, and disturbances in lipid and lipoprotein metabolism. All ABC transporters utilize ATP to generate the energy needed to transport metabolites across membranes. Structurally, ABCs fall into two groups: 1) whole, transporters having two similar structural units joined covalently; and 2) half, transporters of single structural units that form active heterodimers or homodimers (54). ABCA1 and ABCA7 are whole transporters, whereas ABCG1 and ABCG4 are homodimeric half transporters.
ABCA1 is a 2,261-amino-acid integral membrane protein that comprises two halves of similar structure (71). Each half has a transmembrane domain containing six helices and a nucleotide binding domain (NBD) containing two conserved peptide motifs known as Walker A and Walker B, which are present in many proteins that utilize ATP, and a Walker C signature unique to ABC transporters (Fig. 1) (54). ABCA1 is predicted to have an NH2 terminus oriented into the cytosol and two large extracellular loops that are highly glycosylated and linked by one or more cysteine bonds (Fig. 1) (31, 54).
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ABCA1 mediates the transport of cholesterol, phospholipids, and other lipophilic molecules across cellular membranes, where they are removed from cells by lipid-poor HDL apolipoproteins (204). Its homology with other better-characterized ABC transporters suggests that ABCA1 forms a channel in the membrane that promotes flipping of lipids from the inner to outer membrane leaflet by an ATPase-dependent process (203, 204). ABCA1 localizes to the plasma membrane and intracellular compartments (193, 194), where it could potentially facilitate transport of lipids to either cell surface-bound or internalized apolipoproteins.
ABCA1 appears to target specific membrane domains for lipid secretion. These are likely to be regions that are sensitive to accumulation of cholesterol and other lipophilic compounds. This same source of cholesterol feeds into intracellular compartments that are the preferred substrate for the esterifying enzyme acyl CoA:cholesterol acyltransferase (ACAT) (176, 304). Thus ABCA1 removes cholesterol that would otherwise accumulate as cytosolic cholesteryl ester lipid droplets. One possibility for the link between ABCA1 and ACAT is that both proteins function to protect cells from incorporating too much free cholesterol into the endoplasmic reticulum where it may disrupt the peptide biosynthetic machinery. When cholesterol esterification is blocked in sterol-laden macrophages, the potentially cytotoxic free cholesterol that accumulates is a preferred substrate for the ABCA1 pathway (136).
These observations imply that ABCA1 associates with cholesterol-rich membrane lipid domains. The properties of these domains are unknown, but they appear to be separate from sphingolipid-rich rafts and caveolae (177). A fraction of ABCA1, however, may associate with membrane rafts that are selectively solubilized by the detergent Lubrol and are relatively enriched with cholesterol and phosphatidylcholine (60). Immunogold electron microscopy showed that apolipoproteins interact with diffuse structures protruding from the plasma membrane (163). It is likely that these structures are formed by the lipid transport activity of ABCA1, as has been shown for another ABC phospholipid transporter (52).
Two models have been proposed to account for the ability of ABCA1 to target specific lipid domains (Fig. 2). The exocytosis model implies that excess intracellular cholesterol is packaged into transport vesicles or rafts, perhaps in the Golgi apparatus, which translocate to domains in the plasma membrane containing ABCA1 (205). In support of this model are results showing that induction of ABCA1 in the absence of apolipoproteins increases the appearance of cholesterol on the cell surface (276). The retroendocytosis model suggests that ABCA1- and apolipoprotein-containing vesicles endocytose to intracellular lipid deposits, where ABCA1 pumps lipids into the vesicle lumen for release by exocytosis (242, 261). Consistent with this mechanism are studies showing that ABCA1 recycles rapidly between the plasma membrane and late endosomal/lysosomal compartments, that these compartments accumulate cholesterol in cells with dysfunctional ABCA1, and that ABCA1-containing intracellular vesicles also contain apolipoproteins (193, 194). It is still unclear, however, which model represents the dominant pathway for ABCA1-dependent lipid efflux.
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ABCA1 appears to mediate the transport of diverse types of molecules. In addition to cholesterol and phospholipids, ABCA1 has been reported to promote secretion of 
-tocopherol (208), apoE (279), and interleukin-1
(101, 313). ABCA1-mediated secretion of -tocopherol mimics that of cholesterol (208), suggesting similar transport mechanisms for these substrates.
ABCA1 can promote phospholipid efflux from cells even when membranes are depleted of cholesterol (276, 286), consistent with phospholipids being the primary substrate for ABCA1. Analysis of lipids removed by apolipoproteins implicates phosphatidylcholine as the major phospholipid substrate (70, 166). There is evidence that ABCA1 translocates phosphatidylserine to the exofacial side of the plasma membrane (34, 233, 309), which was proposed to account for ABCA1-mediated cellular apolipoprotein binding and cholesterol efflux (34). Another study, however, suggests that the increased phosphatidylserine translocation associated with ABCA1 expression is too small to account for the enhanced apolipoprotein binding and cholesterol efflux (257). Moreover, there is no selective enrichment of phosphatidylserine in the phospholipids removed by apolipoproteins (70, 166), implicating little interaction with phosphatidylserine-rich domains. Nevertheless, there is evidence that ABCA1 promotes engulfment of apoptotic cells by macrophages (100) and generates microparticles that bleb from plasma membranes (49, 166), two processes that may require phosphatidylserine surfacing.
The broad substrate specificity of ABCA1 is consistent with targeting specific lipid domains for removal from the cell. It is possible that ABCA1 can simultaneously transport several molecules, provided they are associated with phosphatidylcholine. These could include cholesterol and other lipophiles (e.g., -tocopherol or peptides) that accumulate in membrane domains accessible to ABCA1. Although ABCA1 substrates may be diverse, the most physiologically relevant of these are likely to be cholesterol and phospholipids, because overloading cells with cholesterol induces ABCA1 expression (see below) and phospholipids are required cofactors for cholesterol transport.
HDL apolipoproteins selectively interact with plasma membrane lipid domains that are formed by the action of ABCA1 (86, 87, 166). This interaction is specific for apolipoproteins that contain no or very little lipids. This was evident from studies showing that purified HDL apolipoproteins promote cholesterol and phospholipid efflux from cells exclusively by this pathway (75, 226). In contrast, lipidated apolipoproteins, such as mature HDL particles, promote cholesterol efflux by multiple mechanisms involving passive diffusion, interaction with SR-B1, and the transport activities of ABCG1, ABCG4, and ABCA1. Whether or not an HDL particle has activity for the ABCA1 pathway probably depends on its ability to act as a donor of lipid-free apolipoproteins that dissociate from the particles (178, 201).
The ABCA1 pathway has broad specificity for multiple HDL apolipoproteins, including apolipoproteins AI, AII, E, CI, CII, CIII, and AIV (226). These apolipoproteins contain 11–22 amino acid repeats of amphipathic -helices (249). In this type of helix, the charged amino acids align along one face of the long axis while the hydrophobic residues align along the other face.
The amphipathic -helices in HDL apolipoproteins fall into two major subclasses based on the distribution of charged amino acids (Fig. 3). Class A helices, the most common subclass, tend to cluster their positively charged amino acids along the polar-nonpolar boundaries and the negatively charged amino acids along the center of the polar region (249). Type Y amphipathic -helices have a similar distribution pattern, except they have a positive charge disrupting the cluster of negative charges on the polar face (Fig. 3). Type Y helices have a higher lipidaffinity than type A helices (86). Because of these novel distributions of charged residues, apolipoproteins associate with lipid surfaces but can freely exchange between surfaces through the aqueous environment. These properties also allow lipid free apolipoproteins to assemble phospholipids and free cholesterol to generate nascent HDL particles.
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70% of the total HDL protein content. ApoA-I contains eight 22-amino acid and two 11-amino acid tandem amphipathic -helical domains (Fig. 3). Studies of synthetic peptides corresponding to each of these helices showed that helices 1 and 10 have the greatest affinity for phospholipids (86, 277). These peptides, however, failed to mediate ABCA1-dependent cholesterol efflux unless they were covalently linked to the 11-mer helix 9 (189). This linkage produced a linear array of acidic amino acids along the polar face of the helices, suggesting that this may be an important apoA-I structural determinant for lipid removal by the ABCA1 pathway (189). The importance of the COOH-terminal helices in this process is supported by studies showing that truncation mutants of apoA-I lacking the 10th helix were unable to remove cholesterol from cells by the ABCA1 pathway (215).
A synthetic 18-amino acid peptide that is an analog of class A amphipathic -helices (Fig. 3, top left) can mimic apoA-I in removing cholesterol and phospholipids by the ABCA1 pathway. A dimer of this peptide is more efficacious (176, 306), suggesting cooperativity between tandem helices. These studies imply that the amphipathic -helix is the major structural motif required for removing ABCA1-transported lipids. Interestingly, the D-isomer of the 18-mer -helix is just as active as the L-isomer (9, 227), suggesting that there are no stereoselective requirements for these peptides to interact with ABCA1 lipid transport.
The broad specificity for amphipathic -helices implies that proteins other than apolipoproteins containing this structural motif could remove cellular lipids by the ABCA1 pathway. Indeed, it was shown that phospholipid transfer protein (PLTP), which contains amphipathic -helices, can interact with ABCA1 and remove cellular cholesterol and phospholipids (209). Because of its low lipid binding capacity, PLTP tends to transfer these lipids to HDL rather than generate new lipoprotein particles. PLTP plays important and diverse roles in lipoprotein metabolism, including transferring phospholipids between lipoproteins, remodeling HDL to generate lipid-poor particles, and facilitating the production of triglyceride-rich apoB-containing particles in the liver (5, 123, 125, 126, 271). At physiological levels, PLTP may help overcome some of its atherogenic effects by transferring excess cholesterol from macrophages to HDL. Because of its low lipid binding, however, high concentrations of PLTP may actually block lipid removal from cells by interfering with apolipoprotein interactions. These observations may partially explain why both deleting and hyperexpressing PLTP in mice increases atherosclerosis (126, 273).
Serum amyloid A (SAA) also removes cellular lipids by the ABCA1 pathway (258). SAA is an acute-phase protein that is induced over 1,000-fold during inflammation (110). SAA mostly associates with HDL in the plasma. It contains two tandem amphipathic -helices that differ in amino acid charge distribution from those in apolipoproteins. Although the function of SAA is unknown, one activity may be to remove excess lipids through its interactions with ABCA1. This may become necessary at sites of inflammation where macrophages ingest cholesterol-rich membranes from apoptotic and necrotic cells.
Covalent cross-linking studies revealed that apolipoproteins bind directly to ABCA1 with saturability and high affinity (Kd <10–7 M) (45, 72). This binding is temperature sensitive and readily reversible. The apolipoprotein binding sites on ABCA1 have a broad specificity for multiple HDL apolipoproteins, including apos A-I, A-II, C-I, C-II, and C-III (72). These sites also recognize the 18-mer synthetic amphipathic -helix (72) and PLTP (209). Thus the broad specificity of the ABCA1 binding sites closely mirrors the ABCA1-dependent lipid transport activity of the acceptors. These binding sites have not yet been identified, but their loose specificity for amphipathic -helices suggests that they may be rich in hydrophobic amino acids.
Mutational analyses have uncovered a dissociation between the lipid transport activity of ABCA1 and its apolipoprotein binding activity. Nearly all missense mutations in ABCA1 that impair lipid efflux to apolipoproteins also impair apolipoprotein binding to ABCA1 (73). One substitution mutation (W590S), however, severely reduces apolipoprotein-mediated lipid efflux without having much effect on apolipoprotein binding (73). These results imply that apolipoprotein binding to ABCA1 is essential but not sufficient for removing lipids. As discussed below, the intrinsic lipid transport activity of ABCA1 and its apolipoprotein binding activity can also be regulated independently. It is therefore likely that these two activities depend on different properties of ABCA1.
Although the structure of ABCA1 has not been characterized, electron microscopy and X-ray crystallography of other ABC transporters have generated molecular models that may apply to ABCA1. The two most studied ABC structures belong to mammalian P-glycoprotein (234, 235), which extrudes a variety of lipophilic compounds from cells, and bacterial/pathogenic MsbA (35, 36), which translocates lipids from the inner to outer membrane leaflets. Despite some disagreement about mechanisms involved, the consensus of these analyses suggests that the two symmetrical transmembrane bundles come together to form a chamber that scans the inner leaflet of the membrane for substrates, incorporates them into the chamber, and flips them to the outer leaflet for extrusion from the cell. This involves a series of conformational changes in the ABC protein that is probably driven by the NBD domains (Fig. 4) (35, 235).
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The MsbA model predicts that the transmembrane chamber of ABCA1 is initially open at the bottom (Fig. 4, top left) (35, 36). Lipids in the inner membrane leaflet are laterally transported into the chamber by a process that is facilitated by high-affinity phospholipid binding sites. This phospholipid recognition induces ATP binding to the NBDs, which promotes their dimerization and thus closes the chamber (Fig. 4, step A). The interactions of phospholipid polar head groups with charged amino acids in the chamber flips the trapped lipids to the outer leaflet. The hydrolysis of ATP by the NBDs forms an ADP-bound intermediate that changes the conformation of the transmembrane domains, opening the chamber at the mem-brane outer leaflet (Fig. 4, step B). Because of a decreased affinity for phospholipid binding, lipids are extruded from the chamber into cholesterol-rich domains on the cell surface, where they are removed by apolipoproteins to form nascent HDL particles (Fig. 4, step C). The structure of the ABCA1 chamber reverts back to its substrate uptake conformation after ADP dissociates from the NBDs (Fig. 4, step D). Although this is a plausible model for ABCA1 function, more direct evidence is needed to confirm that it actually applies to this transporter.
The removal of lipids by apolipoproteins appears to involve a two-step process, whereby apolipoproteins first bind to ABCA1 and then solubilize the ABCA1-transported lipids (Fig. 4, step C) (45). Apolipoprotein binding to ABCA1 is not required for lipid flippase activity, as ABCA1 translocates cholesterol to the cell surface in the absence of apolipoproteins (276). This binding may serve to target apolipoproteins to the lipid domains formed by ABCA1. It might also promote extrusion of lipids from the open chamber by dissociating phospholipids from their binding sites.
There is evidence that ABCA1 forms oligomers in both intracellular membranes and the plasma membrane and that the homotetramer is the major functional unit (55). It was postulated that the interaction of apolipoproteins with each of these units may be responsible for generation of nascent HDL particles containing four or more apolipoprotein molecules per particle. Binding of apolipoproteins to ABCA1 does not appear to be required for its oligomerization. The oligomeric structure of functional ABCA1 is consistent with what has been observed for other ABC transporters (225, 236).
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III. REGULATION |
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As expected for a transporter that mediates secretion of excess cellular cholesterol, transcription of ABCA1 is markedly induced by overloading cells with cholesterol. This induction occurs exclusively through activation of the nuclear receptors liver X receptor (LXR and/or LXR) and retinoid X receptor (RXR) (51, 247). LXR and RXR form obligate heterodimers that preferentially bind to response elements within the ABCA1 gene promoter and the first intron. LXRs and RXRs bind to and are activated by oxysterols and retinoic acid, respectively (230). Binding of either one or both ligands can activate transcription. Treatment of cells with either an oxysterol or 9-cis-retinoic acid induces ABCA1, but their combined treatment has marked synergistic effects (51, 247). The LXR gene promoter in human macrophages contains a LXR response element (146, 297), indicating that LXR can autoregulate its own expression. This would serve to amplify the effects of oxysterols on the ABCA1 lipid efflux pathway.
Because uptake of nonoxidized cholesterol by cells increases ABCA1 expression, cholesterol must be converted to oxysterols before inducing ABCA1. The most potent LXR ligands contain a single stereoselective oxygen on the side chain that functions as a hydrogen bond acceptor (121). It is believed that 22-hydroxycholesterol, 24-hydroxycholesterol, and 24,25-epoxycholesterol are the major naturally occurring liver LXR ligands (121). Most oxysterols are generated by cytochrome P-450 enzymes that are particularly prevalent in the liver and play a role in bile acid metabolism. One of these enzymes, sterol 27-hydroxylase (Cyp27), is broadly distributed in various tissues and cell types, including macrophages, suggesting that 27-hydroxycholesterol is the major LXR ligand in macrophages and other peripheral cells (80). Consistent with this idea is the accelerated atherosclerosis that is associated with cerebrotendinous xanthomatosis, a rare inherited disease characterized by a lack of Cyp27 (124). Recent studies with mouse models lacking or overexpressing Cyp27, however, suggest that 27-hydroxycholesterol has little impact on whole body cholesterol homeostasis (175).
Lipid metabolites other than sterols can modulate ABCA1 expression by the LXR system (Table 1). Polyunsaturated fatty acids act as antagonists to oxysterol binding to response elements in the LXR gene (214), potentially interfering with induction of ABCA1 by sterols (272). Geranylgeranyl pyrophosphate (GGPP), a product of the mevalonate pathway that isoprenylates proteins, was shown to suppress LXR-induced ABCA1 synthesis by two mechanisms: as an antagonist of the interaction of LXR with its nuclear coactivator SRC-1 and as an activator of Rho GTP-binding proteins (82). This second mechanism might alter sterol trafficking in cells, reducing their availability as ligands for LXR. Activators of peroxisome proliferator activating nuclear receptors (PPARs) also enhance ABCA1 transcription in some cells. PPAR
activators stimulate cholesterol efflux from cells by activating transcription of the LXR gene, which in turn induces ABCA1 transcription (37, 41). Thyroid hormone receptor was reported to suppress ABCA1 transcription by forming a complex with RXR that competes for LXR/RXR binding to its response elements (115).
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(216), transforming growth factor 1 (10), and oncostatin M (148) have been reported to modulate ABCA1 transcription in cultured macrophages and hepatoma cells. Sterol regulatory element binding protein 2, a transcription factor that regulates sterol synthesis, appears to interact with the ABCA1 promoter in vascular endothelial cells and to suppress ABCA1 transcription (308), thus providing an additional mechanism for suppressing cholesterol efflux when cells are sterol depleted. The only posttranscriptional processes so far identified that affect ABCA1 expression involve modulation of ABCA1 protein stability (Table 1). When ABCA1 inducers are removed in the absence of apolipoproteins, ABCA1 mRNA and protein are degraded at a fast rate (half-life of 1–2 h) (290). The rapid protein turnover is largely due to a sequence at amino acids 1283–1306 in the first intracellular loop that is enriched in proline-glutamate-serine-threonine (PEST motif) (283). Phosphorylation of T1286 and T1305 in this motif (Fig. 5) promotes ABCA1 proteolysis by an unknown member of the calpain protease family (173, 283). There are several metabolic factors that modulate the rate of ABCA1 protein degradation by either this calpain system or other processes.
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B) FREE FATTY ACIDS. Unsaturated free fatty acids directly destabilize ABCA1 protein in cultured cells (289, 290). When ABCA1 is induced by LXR-independent mechanisms, mono-, di-, and polyunsaturated fatty acids increase the rate of ABCA1 protein degradation (290). In contrast, saturated fatty acids have no effect on ABCA1 turnover in these cells. However, if ABCA1 is induced by LXR ligands, the saturated fatty acids palmitate and stearate also destabilize ABCA1 (289). This is because one of the other genes induced by LXR encodes an enzyme called stearoyl-CoA desaturase, which converts these two saturated fatty acids to their monounsaturated derivatives, palmitoleate and oleate (289). Thus, in most cholesterol-loaded cells in vivo, where ABCA1 is induced by LXR-activating sterols, it is likely that exposure to both saturated and unsaturated fatty acids impair the ABCA1 cholesterol secretion pathway by reducing ABCA1 levels.
The effects of fatty acids on ABCA1 stability appear to be mediated by a signaling pathway involving activation of phospholipase D2 and phosphorylation of ABCA1 (291). The biologic reason for this cross-regulation by different lipid classes is unclear, but it is possible that suppressing ABCA1-mediated lipid secretion by unsaturated fatty acids plays a physiological role in cellular lipid homeostasis, perhaps to retain excess cholesterol as a reservoir for membrane synthesis. This regulation, however, may have pathological consequences. Type 2 diabetes and the metabolic syndrome are characterized by elevated fatty acids, low plasma HDL levels, and prevalent CVD (19, 88). Palmitate and oleate, the two most common fatty acids, destabilize ABCA1 over a fatty acid-to-albumin molar ratio within the range observed for subjects with these disorders (289, 290). It is possible that impaired ABCA1-mediated cholesterol secretion from cells contributes to the low plasma HDL levels and enhanced atherosclerosis in these subjects.
C) FREE CHOLESTEROL. Loading cultured macrophages with cholesterol in the presence of a drug that inhibits cholesterol esterification induces apoptosis, which is triggered by the accumulation of free cholesterol in the endoplasmic reticulum (67). This condition also destabilizes ABCA1 protein, possibly by enhancing degradation by proteosomes (66). Thus an impaired ability of ABCA1 to remove lipids from macrophages may potentiate the damaging effects of free cholesterol. Such a mechanism could contribute to progression of atherosclerosis, as macrophages progressively accumulate large amounts of free cholesterol in advanced lesions.
D) REACTIVE CARBONYLS. Treatment of cultured macrophages with the reactive carbonyl species glyoxal and glycoaldehyde acutely and severely impair the ABCA1 pathway, presumably by directly damaging ABCA1 protein (220). These carbonyls are generated during glucose metabolism and protein glycoxidation and form advanced glycated end products (AGEs) on tissue proteins (15, 28, 89, 267, 300). AGEs accumulate during aging, and their formation is enhanced by diabetes. In addition to glycoxidation, activated phagocytes can generate glycoaldehyde from amino acids using myeloperoxidase (7). Thus the inflammation that underlies multiple disorders including diabetes is likely to produce carbonyl stress in tissues and impair the ABCA1 pathway. This could be another mechanism that contributes to the increased CVD associated with these metabolic disorders.
Modulation of ABCA1 trafficking could have important influences on ABCA1 protein levels and function (Table 1). ABCA1 recycles rapidly between the plasma membrane and late endosomal/lysosomal compartments (193, 194), which may play a role in both lipid transport and protein processing. ABCA1 is selectively expressed on the basolateral membranes of cultured intestinal cells (185, 200), gallbladder epithelial cells (151), brain capillary endothelial cells (218), alveolar type II cells (312), and hepatocytes (192), indicating the presence of factors that target ABCA1 to specific membranes in polarized cells. There is emerging evidence that the interaction of proteins with the COOH-terminal domain may be involved in directing intracellular trafficking of ABCA1.
The COOH terminus of ABCA1 contains a ESTV motif that conforms to the consensus sequence for binding to PDZ domain-containing proteins (74), a diverse group of proteins that form multimeric complexes that often mediate cytoskeleton interactions and protein trafficking (139). Yeast two-hybrid screens identified three PDZ proteins (1- and 2-syntrophin and Lin7) that interact with the COOH-terminal domain of ABCA1 (29, 184). Overexpressing -syntrophin in cells stabilized ABCA1 and enhanced cholesterol efflux (184), suggesting that its interaction with ABCA1 is important for optimum function. When a mutant for ABCA1 lacking the PDZ binding domain was modestly overexpressed in cells, cholesterol efflux was less than half that seen with cells expressing a similar amount of wild-type ABCA1 (74). However, when cells were forced to express high levels of this mutant, there was no impairment of cholesterol efflux. Thus it appears that interactions of PDZ proteins with ABCA1 have modest effects on its function but are not essential for activity. The role of these interactions in trafficking of ABCA1 to membranes in polarized cells has not been explored in detail.
Intracellular protein trafficking often involves the fusion of transport vesicles, which is mediated by a group of vesicle membrane receptors called SNAREs, including the syntaxin family of proteins. Screening macrophages for syntaxins that are increased in response to cholesterol loading led to the identity of syntaxin 13 as an ABCA1 binding protein (14). Silencing of syntaxin 13 with siRNA reduced ABCA1 protein levels and lipid efflux to HDL and apoA-I without affecting ABCA1 mRNA levels (14). Thus syntaxin 13 appears to stabilize ABCA1 protein, probably by modulating its vesicular trafficking. In addition to endosomes, macrophage phagosomes also appear to contain syntaxin 13, raising the possibility that phagocytotic vesicles play a role in processing ABCA1.
Several compounds have been described that appear to affect ABCA1 levels or activity through their effects on ABCA1 trafficking. Ceramide was shown to enhance cholesterol efflux to apolipoproteins by increasing the cell surface content of ABCA1 (298). In contrast, cyclosporin A was reported to inhibit ABCA1-dependent lipid efflux by trapping ABCA1 on the cell surface where it lost activity (156). These studies show that agents that alter the trafficking patterns of ABCA1 can have significant effects on its cellular content and activity.
The lipid transport and apolipoprotein binding activities of ABCA1 are regulated by several signaling pathways that have no or little effect on ABCA1 expression (Table 2). Most of these pathways affect ABCA1 by direct phosphorylation. Several protein kinases have been described that modulate ABCA1 lipid transport and/or apolipoprotein binding activity. These signaling pathways can either increase or decrease different aspects of ABCA1 activity and thus coordinate its overall function.
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Although PKA-mediated phosphorylation of ABCA1 is required for activity, the importance of this signaling pathway as a physiological modulator of ABCA1 is unclear. Some studies have shown that incubating cells with cAMP analogs had no stimulatory effect on ABCA1 activity or phosphorylation, even though PKA inhibitors could reduce these processes, consistent with the idea that the intracellular cAMP content is already saturating for its effects on ABCA1 (207, 264). Other studies, however, have shown that increasing the cellular cAMP content enhances ABCA1 activity in association with increased ABCA1 phosphorylation (97, 98). Moreover, the interaction of apoA-I with ABCA1-expressing cells was reported to increase the cellular cAMP content and ABCA1 phosphorylation (98). The reason for these discrepancies is unknown, but it may reflect difference in cell lines and culture conditions. In support of this idea is a study showing that immortalizing primary fibroblasts increased the responsiveness of the ABCA1 pathway to a cAMP analog (207).
B) PROTEIN KINASE CK2.
Protein kinase 2 or CK2 is a serine/threonine kinase that plays a role in multiple processes related to cell survival and transformation (165, 243). Studies using recombinant peptides spanning the intracellular domains of ABCA1 showed that CK2 phosphorylates ABCA1 at threonines 1242 and 1243 and serine 1255, which are downstream from NBD-1 (Fig. 5) (233). Site-directed mutagenesis of these domains prevented CK2-mediated ABCA1 phosphorylation and enhanced lipid flipping, apolipoprotein binding to cells, and apolipoprotein-mediated lipid efflux (233). Thus CK2 acts as a downregulator of ABCA1 lipid transport and apolipoprotein binding activities. Interestingly, CK2-mediated phosphorylation of a chicken homeoprotein called Engrailed 2 inhibits its secretion from cells (170). Although transported in vesicles and secreted, Engrailed 2 lacks a classic signal sequence. A similar secretion mechanism has been described for interleukin-1, a reported substrate for ABCA1 (101, 313). These observations are consistent with an attenuating role of CK2 in ABCA1-dependent secretion of substrates that are important for normal cell function and viability.
C) JANUS KINASE 2. Janus kinase 2 (JAK2) is a tyrosine kinase that is activated by over half of the cytokine/hematopoietin superfamily of receptors (137). Activation of JAK2 is the initiating step in downstream signaling for most of these receptors. JAK2 also plays a role in modulating ABCA1 activity. A JAK2-specific inhibitor reduces apoA-I-mediated lipid transport and apoA-I binding to ABCA1, and mutant cells lacking JAK2 have a severely impaired ABCA1 pathway (264). In contrast to PKA, which regulates the lipid transport but not apolipoprotein binding activity of ABCA1, JAK2 is required for apolipoprotein binding to ABCA1 but has little effect on the intrinsic cholesterol flippase activity (264). These studies show that the lipid transport and apolipoprotein binding activities of ABCA1 can be regulated independently by different signaling processes.
The interaction of apoA-I with ABCA1-expressing cells acutely stimulates autophosphorylation of JAK2 (264), generating the active JAK2 that phosphorylates its target proteins. This suggests that apolipoproteins potentiate their own interactions with ABCA1 by activating JAK2, which in turn increases the apolipoprotein binding to ABCA1 required for lipid removal. It is unknown if the initiating step in this process is mediated by binding of apolipoproteins to ABCA1 or to other proteins, or whether the JAK2-targeted protein is ABCA1 or a partner protein.
D) PROTEIN KINASE C. In addition to the possibility that phosphorylation of ABCA1 by protein kinase C (PKC) helps stabilize the protein, there is circumstantial evidence that PKC may play a role in modulating the lipid transport activity of ABCA1. Before the discovery of ABCA1, several studies showed that inhibition or activation of PKCs respectively decreased or increased cholesterol efflux from cells to HDL or purified apoA-I (160, 179, 266, 282), implicating PKCs as modulators of ABCA1 activity. More recent evidence suggested that activation of phospholipase C and D may be critical for apoA-I-mediated cellular cholesterol efflux (99, 281). However, this also appeared to be the case in cells with defective ABCA1 (99), suggesting that it may not directly involve this transporter. Moreover, short-term treatment of ABCA1-transfected cells with a broad-spectrum PKC inhibitor was shown to have no effect on apoA-I-mediated cholesterol or phospholipid efflux (264). It is possible that PKCs do not directly activate ABCA1 but modulate trafficking of lipid substrates to ABCA1 (see below).
There is growing evidence that ABCA1 activity is modulated by the interaction of a diverse group of proteins (Table 2). ABCA1 was coimmunoprecipitated with Cdc42 (270), a member of the RhoGTPase family that forms complexes with proteins that control the cytoskeletal architecture and vesicular transport (262). This interaction may cause the changes in cell morphology associated with under- or overexpressing ABCA1 (57, 270). Moreover, forced expression of Cdc42 was shown to enhance apoA-I-mediated cholesterol efflux, whereas expression of dominant negative Cdc42 impaired this efflux, consistent with a role of Cdc42 in modulating ABCA1 function (108, 197).
Immunoprecipitation studies also identified Fas-associated death domain protein (FADD) as an ABCA1 interacting protein (30). Blocking this interaction impaired apolipoprotein-mediated phospholipid efflux. The biological significance of the FADD/ABCA1 association is unclear, as FADD is mainly involved in death receptor-induced apoptosis. These findings raise the possibility that ABCA1 has an antiapoptotic function, perhaps related to its ability to extrude lipophilic molecules from cells.
It is likely that additional proteins will be discovered that interact with ABCA1 and modulate activity. The COOH-terminal region of ABCA1 contains a highly conserved VFVNFA motif that is required for activity (74). Mutations in this motif have no effect on ABCA1 trafficking but severely impair its apoA-I interactions and lipid efflux. Peptide competition experiments suggest that this motif promotes the interaction of ABCA1 with an unknown cellular protein that is a component of the active complex (74).
The activity of the ABCA1 pathway is influenced by factors that control intracellular trafficking of lipids (Table 2). Cholesterol, phospholipids, and other lipophiles are likely to be transported between cellular compartments in vesicular membranes. ADP-ribosylation factors (ARFs) and ARF-like proteins (ARLs) are Ras-related small GTPases that control the vesicle budding involved in vesicular transport pathways. One of these ARLs (ARL7) was shown to be induced by cholesterol loading of macrophages and by LXR/RXR agonists (65). Immunofluorescent studies suggested that ARL plays a role in vesicular transport between a perinuclear compartment and the plasma membrane. Expression of native and dominant-active ARL7 stimulated cholesterol efflux from cells by the ABCA1 pathway (65). These observations suggest that ARL7 is involved in trafficking of cholesterol from intracellular compartments to plasma membrane domains containing ABCA1, although they do not exclude the possibility that these ARL7-containing vesicles target ABCA1 to lipid domains.
Trafficking of lipid substrates to ABCA1 may also be mediated by signaling processes elicited by the interaction of apolipoproteins with ABCA1-expressing cells. The ability of apolipoproteins to remove cholesterol selectively from sites of cholesterol esterification has been reported to involve activation of PKC by apolipoproteins (304).
Disorders that affect intracellular cholesterol trafficking have an impact on the ABCA1 pathway. Niemann-Pick type C (NPC) disease is a neurodegenerative disorder characterized by impaired intracellular lipid trafficking and accumulation of free cholesterol in late endosomes/lysosomes (84, 144, 195, 222). This is due to mutations in the gene for NPC1, which mediates transport of lipoprotein-derived cholesterol from these intracellular compartments to the Golgi apparatus, endoplasmic reticulum, and plasma membrane. Cells lacking NPC1 have below normal levels of ABCA1 expression and activity (42), consistent with an impaired transport of intracellular cholesterol to sites that regulate ABCA1 transcription and to membrane domains targeted for efflux. This impaired ABCA1 pathway may explain why patients with NPC have low plasma HDL levels and an overaccumulation of cellular cholesterol (42).
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IV. IN VIVO FUNCTIONS |
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Tissue culture studies predict that macrophage-rich tissues would have relatively high levels of ABCA1. As scavenger cells, macrophages ingest modified lipoproteins and damaged cell membranes and thus can accumulate large amounts of cholesterol, an inducer of ABCA1. Indeed, ABCA1 mRNA levels were shown to be higher in cholesterol-loaded cultured human macrophages than in any human tissues (138). ABCA1 is also highly expressed in the liver, where it is upregulated when mice are fed a high-cholesterol diet (296). It is therefore predictable that hepatic ABCA1 would make an important contribution to whole body lipoprotein metabolism. Current knowledge about the physiological role of ABCA1 in vivo is based largely on studies of human genetic disorders, mouse genetic models, and cultured tissue-specific cells.
Mutations in ABCA1 can cause an autosomal recessive genetic disorder called Tangier disease, which is characterized by very low levels of plasma HDL and deposition of sterols in tissue macrophages (11). Tangier disease was first identified in the 1960s as an HDL deficiency syndrome affecting families from Tangier Island in the Chesapeake Bay, United States of America (77). It was discovered in the mid 1990s that the ability of purified apoA-I to remove cholesterol and phospholipids from these cells was severely impaired (75). In 1999, four laboratories independently identified the defective Tangier disease gene as ABCA1 (21, 24, 150, 239).
Over 70 mutations in ABCA1 have been identified in subjects with low plasma HDL levels, nearly one-third of which are missense mutations (48, 78, 255). Although these mutations occur throughout the gene, they tend to cluster in the extracellular loops, the NBD domains, and the COOH-terminal region.
The functional impact of a small subset of the missense mutations has been studied in cell culture. When expressed in cells, most of these mutants appear in the plasma membrane but have severely impaired lipid transport and apolipoprotein binding activities (73). Some studies have suggested that ABCA1 proteins with substitution mutations Q597R and R587W in the first extracellular loop do not localize to the plasma membrane (232, 263), but other studies have contradicted these findings (73, 150). Missense mutations in the second extracellular loop and in the ninth membrane-spanning domain were reported to prevent ABCA1 trafficking to the plasma membrane (6). Only one mutant (W590S) has been described that has near-normal apolipoprotein binding activity but defective lipid transport (73).
The initial loss-of-function mutations in ABCA1 were discovered in case reports and family studies. More recently, rare mutations were identified by screening ABCA1 from subjects with low plasma HDL levels for single nucleotide polymorphisms (SNPs) (48, 78, 255). Most of the identified rare alleles (mutations) were absent in populations of subjects with high HDL levels (48, 78), implying that they play a causative role in lowering HDL levels. Some mutations, however, were found to be more frequent in subjects with the highest HDL levels, consistent with an enhancement of the ABCA1 pathway. On the basis of mutation frequency in those with the lowest 1% of HDL cholesterol, it was estimated that as many as 10% of the HDL-deficient individuals in the general population are heterozygous for ABCA1 mutations (78). This is likely to be an underestimate, as these studies may have missed critical intronic or regulatory mutations. Moreover, these reports did not examine allelic frequencies in subjects with moderately reduced HDL levels, which is more typical of Tangier disease heterozygotes (255). Taken together, these observations are consistent with ABCA1 being a major determinant of plasma HDL levels in humans.
The role of ABCA1 as a modulator of HDL levels and size was supported by two independent reports that measured cholesterol efflux from cells cultured from Tangier disease heterozygotes. Although these patients as a group have approximately half normal plasma HDL levels, there is a wide variation among patients (255). The type of ABCA1 mutation may have some influence on the relative activity of the ABCA1 pathway, since truncation mutants may form dominant-negative interactions with the wild-type allele (255). This range in activity allowed for comparisons of apoA-I-mediated cholesterol efflux from skin fibroblasts cultured from these patients with their plasma HDL levels and particle size. Both studies showed a significant correlation between ABCA1 activity in their cultured skin fibroblasts and the level and size of plasma HDL particles (25, 47). Thus, at least among this subject group, the intrinsic activity of the ABCA1 pathway is a major determinant of plasma HDL cholesterol levels.
SNP analyses have also identified over 20 common polymorphisms (>1% allelic frequency) in the coding, promoter, and 5'-UTR regions of ABCA1 (48, 78, 255). Several of these are associated with either low or high plasma HDL levels. The most studied of these SNPs is the R219K variant, with the K allele being associated with higher levels of HDL (255). V771M and V825I SNPs have also been reported to be associated with increased HDL levels, whereas R1587K is associated with low levels of HDL (78, 255). Although the frequencies vary between groups, all of these more common SNPs were present in individuals having both high and low HDL levels, suggesting that they do not have a strong phenotypic effect.
Studies of animal models have provided additional evidence that ABCA1 is a major determinant of plasma HDL levels. The only naturally occurring animal model of Tangier disease is the Wisconsin Hypoalpha Mutant (WHAM) chicken. ABCA1 in these chickens has a missense mutation near the NH2 terminus that produces a defective protein (13). Like Tangier patients, the WHAM chicken hypercatabolizes apoA-I and accumulates cholesteryl esters in tissues, particularly in hepatic parenchymal and intestinal epithelial cells. Targeted disruption of the Abca1 gene in mice produces a phenotype similar to that of human Tangier disease (4, 174, 211), including an HDL deficiency and accumulation of sterols in some tissues. Conversely, overexpressing ABCA1 in mice elevates plasma HDL levels (131, 256, 294).
Several lines of evidence suggest that the liver ABCA1 pathway in mice is responsible for generating most of the plasma HDL. Selective hyperexpression of ABCA1 in the liver using adenovirus transgenes markedly increases plasma HDL levels (294). Bone marrow transplantation showed that the ABCA1 pathway in peripheral macrophages in mice makes only a minor contribution to HDL formation (96). Another study showed that targeted disruption of liver ABCA1 in chow-fed mice reduced plasma HDL levels by 80% (153). These observations imply that the major cause of the HDL deficiency in Tangier patients and ABCA1 knockout mice is an impaired liver ABCA1 pathway.
B. Reverse Cholesterol Transport
It is widely believed that a major function of HDL is to transport cholesterol from peripheral cells to the liver for elimination in the bile (90). The studies described above suggest that ABCA1 plays a major role in this reverse cholesterol transport pathway. The presumed major precursor for this pathway is lipid-poor apoA-I (69, 210), which is initially synthesized and secreted by the liver (Fig. 6). Most of this apoA-I may immediately interact with liver ABCA1, but some may circulate to the periphery where it interacts with ABCA1 on cholesterol-loaded cells, particularly macrophages. It is more likely, however, that most of the lipid-poor apoA-I in peripheral tissues is generated from the surface of mature HDL particles that are transported there (Fig. 6, dotted arrow) (240). The ABCA1-bound apoA-I rapidly acquires free cholesterol and phospholipids, becoming partially lipidated. Most of these nascent particles then mature into spherical HDL particles (Fig. 6), which deliver their cholesteryl esters to the liver for secretion in the bile after binding to SR-B1, the HDL receptor that selectively transfers cholesteryl esters into hepatocytes. These cholesteryl esters can also be delivered to the liver after transfer to other lipoproteins, such as LDL (Fig. 6).
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In support of this concept are studies showing that HDL, not LDL, is the major source of cholesterol used for bile acid production (246) and that overexpressing SR-B1 in mice increases transport of lipoprotein cholesterol into the bile (254). It has been reported, however, that biliary cholesterol secretion was unimpaired in mice lacking ABCA1 (94), suggesting that either ABCA1 is unnecessary for this process or that other hepatic mechanisms can compensate for the absence of ABCA1 and low HDL levels in these animals. Additional studies are needed to define the role of hepatic ABCA1 in lipoprotein metabolism.
The brain is the most sterol-rich organ in the body, containing 25% of the total body cholesterol (58). Most of the brain cholesterol is synthesized locally, with only a small amount transported from the circulation across the blood-brain barrier (58). Flux of cholesterol out of the brain occurs largely after conversion to the more polar 24S-hydroxycholesterol, which passively traverses the blood-brain barrier (20). ABCA1 is highly expressed in glial and neuronal cells of the central nervous system, particularly in Purkinje cells and pyramidal cortical neurons (142, 149, 296). There is emerging evidence that ABCA1 plays an important role in maintaining brain cholesterol homeostasis and protecting against disease.
The major ABCA1-interacting apolipoprotein in the brain is apoE, which is synthesized locally by glial cells (169). ABCA1 is highly induced in cultured astrocytes, neuronal cells, and microglial cells by LXR/RXR ligands, where it promotes cholesterol and phospholipid efflux to lipid-free apolipoproteins, including apoE (81, 142, 259). Interestingly, glial expression of ABCA1 appears to modulate synthesis and secretion of apoE. Glial cells cultured from mice lacking ABCA1 secreted smaller lipoprotein particles with a markedly reduced cholesterol and apoE content compared with cells from ABCA1-expressing animals (109, 280). The cortex and cerebral spinal fluid of ABCA1-deficient mice have a much lower level of apoE than those expressing ABCA1 (280). These findings suggest that glial ABCA1 has the dual function of promoting lipid efflux and generating apolipoprotein lipid acceptors for lipoprotein particle formation.
ABCA1 is also expressed in cultured brain capillary endothelial cells (218), where it may play a role in sterol transport across the blood-brain barrier. These cells synthesize and secrete apoA-I, which can serve as the acceptor for the lipids extruded by ABCA1 (218). With these cells, however, ABCA1 is localized to the basolateral membrane and apoA-I is secreted in this direction, consistent with generation of
