Clinica Chimica Acta 429 (2014) 69–75
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NPC1, intracellular cholesterol trafficking and atherosclerosis Xiao-Hua Yu a, Na Jiang c, Ping-Bo Yao d, Xi-Long Zheng e, Francisco S. Cayabyab f, Chao-Ke Tang a,b,⁎ a
Life Science Research Center, University of South China, Hengyang, Hunan 421001, China Institute of Cardiovascular Research, Key Laboratory for Atherosclerology of Hunan Province, University of South China, Hengyang, Hunan 421001, China c Department of Electrocardiogram, the Second Affiliated Hospital, University of South China, Hengyang, Hunan 421001, China d Department of Intensive Care, the Affiliated Nanhua Hospital, University of South China, Hengyang, Hunan 421001, China e Department of Biochemistry and Molecular Biology, The Libin Cardiovascular Institute of Alberta, The University of Calgary, Health Sciences Center, 3330 Hospital Dr NW, Calgary, Alberta, T2N 4 N1, Canada f Department of Physiology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada b
a r t i c l e
i n f o
Article history: Received 27 September 2013 Received in revised form 17 November 2013 Accepted 23 November 2013 Available online 1 December 2013 Keywords: NPC1 NPC2 Cholesterol ORP5 Atherosclerosis
a b s t r a c t Post-lysosomal cholesterol trafficking is an important, but poorly understood process that is essential to maintain lipid homeostasis. Niemann-Pick type C1 (NPC1), an integral membrane protein on the limiting membrane of late endosome/lysosome (LE/LY), is known to accept cholesterol from NPC2 and then mediate cholesterol transport from LE/LY to endoplasmic reticulum (ER) and plasma membrane in a vesicle- or oxysterolbinding protein (OSBP)related protein 5 (ORP5)-dependent manner. Mutations in the NPC1 gene can be found in the majority of NPC patients, who accumulate massive amounts of cholesterol and other lipids in the LE/LY due to a defect in intracellular lipid trafficking. Liver X receptor (LXR) is the major positive regulator of NPC1 expression. Atherosclerosis is the pathological basis of coronary heart disease, one of the major causes of death worldwide. NPC1 has been shown to play a critical role in the atherosclerotic progression. In this review, we have summarized the role of NPC1 in regulating intracellular cholesterol trafficking and atherosclerosis. © 2013 Elsevier B.V. All rights reserved.
Contents 1. 2. 3. 4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure and characterization . . . . . . . . . . . . . . . . . . . . . . . . . Cholesterol trafficking function of NPC1 and NPC disease . . . . . . . . . . . . . Mechanisms of NPC1-mediated cholesterol transport . . . . . . . . . . . . . . 4.1. Cholesterol “handoff” from NPC2 to NPC1 . . . . . . . . . . . . . . . . 4.2. NPC1 promotes post-lysosomal cholesterol trafficking in a vesicular manner 4.3. NPC1 cooperates with ORP5 to deliver cholesterol out of LE/LY . . . . . . 5. Regulation of expression . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Role in atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Conclusion and future perspectives . . . . . . . . . . . . . . . . . . . . . . Disclosure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abbreviations: NPC1, Niemann-Pick type C1; LE/LY, late endosome/lysosome; LDL, lowdensity lipoprotein; LDL-C, LDL-cholesterol; ER, endoplasmic reticulum; SSD, sterol-sensing domain; NTD, N-terminal domain; apoE, apolipoprotein E; LXR, liver X receptor; OSBP, oxysterolbinding protein; ORP5, OSBPrelated protein 5; TGN, trans-Golgi network; oxLDL, oxidized low-density lipoprotein; ERK1/2, extracellular signal-regulated kinase 1/2; COX-2, cyclooxygenase-2; PPARα, peroxisome proliferator-activated receptor α; SREBP, sterol regulatory element-binding protein; LDLR, low-density lipoprotein receptor; ABCA1, ATP-binding cassette transporter A1; DHC, dihydrocapsaicin; apoA-I, apolipoprotein A-I. ⁎ Corresponding author at: Institute of Cardiovascular Research, University of South China, Hengyang, Hunan 421001, China. Tel./fax: +86 734 8281853. E-mail address: [email protected] (C.-K. Tang). 0009-8981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2013.11.026
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1. Introduction Cholesterol is an abundant metabolite in mammalian tissues that plays important roles in normal embryonic development, cell differentiation, and nerve conduction. Membrane fluidity of all cells is tightly regulated by the ordered packing of cholesterol between phospholipid molecules. It is also the precursor of a variety of biologically active molecules such as bile acids, vitamin D and steroid hormones. Thus, an insufficient supply of cholesterol will have detrimental effects on
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cell function, tissue development as well as whole-body physiology. Too much cholesterol, however, can also result in pathological consequences. Both clinical and animal studies have established a direct link between plasma cholesterol levels and the risks of coronary heart disease, one of the major causes of death worldwide [1,2]. The control of intracellular cholesterol levels is a complicated process, involving cholesterol uptake, biosynthesis, transport, metabolism and secretion. Interestingly, cholesterol uptake and secretion have been well described in several recent reviews [3,4]. Nevertheless, intracellular cholesterol trafficking remains elusive. Growing evidence shows that Niemann-Pick type C1 (NPC1) plays a key role in the process [5]. NPC1 is a transmembrane glycoprotein located in the limiting membrane of late endosome/lysosome (LE/LY). It can transfer low-density lipoprotein (LDL)-cholesterol (LDL-C) from LE/LY to endoplasmic reticulum (ER) for esterification or to plasma membrane for efflux, a crucial process governing the balance between macrophage cholesterol import and export, with potential consequences in atherogenesis [6]. This review focuses on the molecular characterization, cholesterol transport function and involved mechanisms of NPC1, and explores its emerging pathogenic significance and therapeutic potential in atherosclerosis. 2. Structure and characterization The NPC1 gene maps to chromosome 18q11–12, spans more than 47 kb, and consists of 25 exons that range in size from 74 to 788 nucleotides, and introns that range from 0.097 to 7 kb in length [7]. The human NPC1 protein is composed of 1278 amino acid residues, with 13 putative transmembrane helices, three large loops projecting into the lumen of LE/LY, four small luminal loops, six small cytoplasmic loops and a cytoplasmic tail (Fig. 1). Five segments (residues 615–797) from the third to seventh transmembrane helix form the so-called sterol-sensing domain (SSD), which shares high sequence homology with those of several other cholesterol metabolism-related membrane proteins, such as Patched (a regulatory protein in the Hedgehog signaling pathway), sterol regulatory element binding protein cleavage activating protein (SCAP), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and NPC1-like protein 1 (a protein responsible for dietary cholesterol and biliary cholesterol absorption). SSD is an important binding site between cholesterol and NPC1. It can bind azocholestanol, a photoactivatable analog of cholesterol, in cells, and two loss-of-function mutations (P692S and Y635C) in the region severely block this effect [8]. In contrast, two gain-of-function mutations (L657F and D787N) in the SSD lead to a nearly 2-fold increase in the rates of moving LDL-C out of LE/LY [9]. NPC1 is synthesized in ER, transited through the Golgi and finally targeted to LE/LY. The dileucine motif present at the C-terminus has been reported to promote NPC1 trafficking to the endosomal compartment by direct interaction with the clathrin adaptor protein complex
1 [10]. A motif located in the SSD also contributes to this process [11]. Therefore, although the precise function of NPC1 SSD in vivo is still not fully understood, it is essential for normal cholesterol efflux from LE/LY. NPC1 has three large luminal loops. The first loop (residues 25–264) at the N-terminus is called the N-terminal domain (NTD) or NPC1 domain, which is highly conserved in vertebrate and yeast orthologs of NPC1. It is composed of 18 conserved cysteine residues involved in intra-domain disulfide links, and a leucine zipper motif. When NTD was secreted as a soluble dimer in cultured cells, high affinity binding to cholesterol and 25-hydroxycholesterol was observed, suggesting NTD as a possible sterol binding site for NPC1 [12]. Nevertheless, the full-length NPC1 protein with a Q79A mutation, which disrupts the sterol binding by NTD, can still restore normal cholesterol transport out of LE/LY in NPC1-deficient Chinese hamster ovary cells, indicating that other cholesterol binding sites such as SSD except NTD may be present in the NPC1 protein [12]. The second loop, namely the region between transmembrane helices 2 and 3, can interact with NPC2 [13]. The third loop (residues 855–1098) locates between membrane-spanning domains 8 and 9. It is rich in conserved cysteine residues and consists of a ring finger motif. To date, of the over 200 NPC disease-causing mutations identified in the NPC1 gene [14], approximately half of these mutations are localized to this region, including the most common mutation in Western Europeans, I1061T, which accounts for about 20% of the total number of NPC disease cases [15]. 3. Cholesterol trafficking function of NPC1 and NPC disease Cells can obtain cholesterol by two ways. One is de novo synthesis, a well-defined energy-consuming and feedback-regulated process which is involved in a variety of enzymatic reactions [16]. The other is exogenous uptake mainly from circulating LDL through receptor-mediated endocytosis [3]. After delivery from the early endosome to LE/LY, cholesterol esters carried by LDL are hydrolyzed by lysosomal acid lipase to release free cholesterol. Then, NPC1 mediates the trafficking of free cholesterol from LE/LY to other cellular compartments, including the plasma membrane and ER. In NPC1-absent cells, although the endocytic uptake of LDL-C and subsequent hydrolysis of cholesterol esters are normal, the transfer of unesterified cholesterol from LE/LY is impaired. Consequently, normal amounts of cholesterol fail to reach the plasma membrane and ER to modulate cholesterol balance [17]. In agreement, overexpression of human NPC1 protein greatly promotes LDL-C influx into plasma membrane and ER in Chinese hamster ovary cell lines [18]. NPC disease is a rare, fatal autosomal recessive neurovisceral condition characterized by progressive neurological deterioration and premature death, with an estimated incidence of 1:150,000 live births. Approximately 95% of cases are caused by mutations in the NPC1 gene, while the other 5% is related to mutations in the NPC2 gene [19]. Loss of function of either of these proteins leads to impaired trafficking
Fig. 1. Schematic of NPC1 protein structure. NPC1 protein has 13 transmembrane domains, three large luminal loops and a cytoplasmic tail. Loop 1 or NTD containing a leucine zipper motif can bind cholesterol. SSD is also a binding site for cholesterol and serves as an LE/LY targeting motif. A dileucine motif at the C-terminus is important for NPC1 localization in LE/LY. Loop 2 is able to directly bind NPC2. Loop 3 is composed of a ring finger motif.
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of endocytosed cholesterol and intracellular accumulation of lipids including unesterified cholesterol, glycosphingolipids, sphingomyelin and sphingosine in multiple tissues [20]. In the brain, this causes neuronal degeneration. Typical neurological manifestations include vertical supranuclear gaze palsy, saccadic eye movement abnormalities, cerebellar ataxia, dystonia, dysmetria, dysarthria and dysphagia. These neurological signs arise at different ages, but invariably progress over time. In general, patients with neurological onset early in life show aggravated symptoms faster and have a shorter lifespan than those with adult onset [21]. There is currently no effective treatment for the disease. Zampieri and colleagues demonstrated that increased degradation of mutant NPC1 protein due to protein misfolding is implicated in the pathogenesis of NPC1 disease [22]. They also showed that treatment of fibroblasts carrying NPC1 missense mutations in homo or hemizygosity with 10% glycerol, a chemical chaperone known to stabilize misfolded proteins, results in a significant increase of NPC1 protein levels and an improvement of the intracellular trafficking of cholesterol, suggesting that at least some NPC1 mutations may be potentially rescued by small molecule-based chaperone therapy [22]. More recently, cyclodextrin has been reported to re-establish the normal movement of cholesterol out of LE/LY in NPC1-deficient cells [23,24], and significantly ameliorates symptoms and extends lifespan in a murine NPC disease model [25]. Administration of hydroxypropyl-β-cyclodextrin was effective in improving hepatosplenomegaly and central nervous system dysfunction in two patients with NPC disease, although it did not improve the neurological deficits in either patient [26]. However, the safety and long-term effect of this drug needs to be more fully characterized and better defined. 4. Mechanisms of NPC1-mediated cholesterol transport 4.1. Cholesterol “handoff” from NPC2 to NPC1 Homozygous mutations in either the NPC1 or NPC2 gene cause virtually indistinguishable cellular and clinical phenotypes despite the large discrepancy in their molecular structure. Similarly, when NPC1-absent mice are crossed with NPC2 hypomorphic mice, which retain 0%–4% residual NPC2 protein, the phenotypes of the progeny are identical with those of NPC1- or NPC2-absent mice [27]. NPC1's NTD consists of a deep pocket that can surround cholesterol with the 3β-hydroxyl group and tetracyclic ring buried inside, leaving the isooctyl side chain of cholesterol partially exposed on the surface [28]. NPC2 is a ubiquitously expressed 151-amino acid, soluble protein in the lumen of LE/LY. It contains a deep hydrophobic pocket that can bind cholesterol with its isooctyl side chain buried and 3β-hydroxyl group exposed, in an orientation opposite to the NTD of NPC1 [29]. Thus, it is possible that both proteins operate sequentially in the same pathway to promote cholesterol transport. More recently, the concerted action of both proteins has been investigated in two alanine-scanning mutagenesis experiments. A patch of three amino acids around the sterol-binding site of NPC2 was identified as being essential for cholesterol transfer but not for cholesterol binding [30]. Likewise, a patch of 11 amino acids surrounding the entrance to the sterol-binding pocket of NPC1 NTD was shown to be required for cholesterol binding but not for cholesterol transfer [28]. According to these data, the authors have proposed a working model for “hydrophobic handoff” of cholesterol from NPC1 to NPC2. In the model, the two surface patches on NPC2 and NPC1 interact, leading to the opening of an entry pore on the NTD of NPC1 and allowing cholesterol delivery to NPC1 for export (Fig. 2) [30]. Based on this, NPC2 can transfer its bound cholesterol to NPC1 directly, thus avoiding the necessity for insoluble cholesterol to transit the water phase and preventing cholesterol from crystallizing in the lysosomal lumen. In support of the model, bidirectional transfer of cholesterol between NPC1 and liposomes is accelerated by more than 100 fold in the presence of NPC2, and a naturally occurring human mutant of
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NPC2 (Pro120Ser) abolishes the effect [31]. Additionally, Deffieu and co-workers showed that the second large luminal loop of NPC1 is a binding site for NPC2 protein [13]. Moreover, this binding only occurs in acidic conditions that mimic the lysosomal lumen environment and are optimal for cholesterol binding to NPC2 and delivery to NPC1; whereas, human disease-causing mutations in the region inhibit NPC2 binding, providing direct evidence that NPC1 loop 2 holds NPC2 in position to promote directional cholesterol transfer from NPC2 to NPC1 [13]. Therefore, this handoff may be essential for cholesterol export from LE/LY. Of note, the reverse direction may also exist. It has been reported that NPC1 first accepts free cholesterol in the interior of LE/LY and then transfers it to NPC2 [32]. Further studies are required to clarify a bona fide cholesterol transport mechanism by NPC1 and NPC2. 4.2. NPC1 promotes post-lysosomal cholesterol trafficking in a vesicular manner Although the NPC1/NPC2 handoff model describes cholesterol transport inside LE/LY lumen, how cholesterol is exported from LE/LY has not yet been defined. One of the possible mechanisms is vesicular transport, a pathway involving vesicles that bud off from LE/LY and fuse with ER or plasma membrane (Fig. 2). This concept is supported by an earlier study, which demonstrated that organelles containing functional NPC1-fluorescent protein fusions undergo dynamic movements associated with extending strands of ER [33], although whether such movements can directly transport cholesterol is still questionable. On the other hand, the normal rapid tubulovesicular trafficking of LE is retarded in NPC1-mutated cells [34]. Also, the release of lysosomal cargo-containing membrane vesicles is significantly reduced in NPC1 mutant fibroblasts as compared to wild type cells [35]. The trans-Golgi network (TGN) is involved in NPC1-mediated cholesterol transport downstream. The SNARE complex is composed of VAMP4, syntaxin 6, syntaxin 16 and Vti1a, and plays an important role in mediating the transport of various proteins between LE/LY and TGN by vesicles [36]. Urano et al. showed that Chinese hamster ovary cells lacking NPC1 display a significant reduction of LDL-C within TGN, and knockdown by RNAi of three TGN-specific SNAREs (VAMP4, syntaxin 6, and syntaxin 16) decreases LDL-derived cholesterol transport by over 50% in intact cells, suggesting that vesicular trafficking transports a significant portion of LDL-C from NPC1-containing endosomal compartment to TGN before its arrival at ER or plasma membrane [37]. Anaplasma phagocytophilum, an obligatory intracellular and cholesterol-robbing bacterium, disrupts the NPC1 pathway of intracellular cholesterol trafficking by inhibiting VAMP4 and syntaxin 16 [38]. Thus, the SNARE complex may be associated with vesicular transport of cholesterol out of LE/LY. The Ras-superfamily of small G proteins is a family of GTP hydrolases that is regulated by GTP/GDP binding states. Rab, one member of the Ras-superfamily, plays a crucial role in the vesicular trafficking. It has been reported that overexpression of Rab8 markedly diminishes cholesterol accumulation and restores LDL-C egress from LE/LY in NPC1-deficient fibroblasts, yet depletion of Rab8 from wild-type fibroblasts causes cholesterol deposition within late endosomal compartments [39]. Another Rab protein, Rab9, is a small GTPase enriched in the LE/LY. In NPC1-deficient fibroblasts, the level of Rab9 is elevated by 1.8-fold because of reduced degradation of the protein, and it is sequestered on endosome membranes, leading to impairment of mannose 6-phosphate receptor trafficking [40]. Ectopic expression of human telomerase reverse transcriptase induces upregulation of Rab9 in NPC1-deficient cells so that the transport of cholesterol from LE/LY to plasma membrane is restored [41]. Likewise, overexpression of Rab9 by protein transduction significantly decreases cellular free cholesterol levels in NPC1-deficient human fibroblasts and mouse neurons [42]. These results suggest that Rab9 may be able to bypass the requirement for NPC1, and that Rab9 overexpression may also have therapeutic potential for NPC disease in vivo. To test this possibility, NPC1−/− mice
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Fig. 2. A working model of NPC1-mediated intracellular cholesterol trafficking. Cholesterol is transferred from NPC2 within the lumen to the NTD of NPC1 in the limiting membrane of LE/LY in a reverse orientation. After delivery to the LE/LY limiting membrane, cholesterol is transferred to ER and plasma membrane in a vesicular manner. Alternatively, with the help of VPS4/SKD1, ORP5 can form a transient protein complex with NPC1 at the membrane contact sites connecting LE/LY and ER to export cholesterol into ER. Upon completion of cholesterol transfer, VPS4/SKD1 breaks down the complex and recycles the ORP5.
were crossed with Rab9 transgenic mice, resulting in reduced storage of lipids and prolonged lifespan [43]. 4.3. NPC1 cooperates with ORP5 to deliver cholesterol out of LE/LY In addition to membrane vesicles, cholesterol transport can be performed by carrier proteins in a nonvesicular manner. Oxysterolbinding protein (OSBP) and its relatives (OSBPrelated proteins, ORPs), which constitute a large conserved protein family in eukaryotes, have recently been identified as putative sterol carriers [44]. OSBP has been shown to act as a highaffinity cytosolic receptor for oxysterols, such as 25hydroxycholesterol. ORPs consist of 12 members in human and 7 members in budding yeast. All ORPs share a conserved ~ 400 amino acid OSBP-related domain (ORD) that is found at the C-terminus of OSBP, and is the binding site of cholesterol, oxysterols and phosphatidylinositol 4-phosphate [45]. ORD can also simultaneously bind two different organelle membranes, thus allowing a rapid but regulated sterol transfer between the contact sites [46]. Additionally, OSBP and ORPs have membrane targeting domains such as the pleckstrin homology domain. Osh4p, a yeast ORP, has been reported to transport sterols from ER to LE/LY and, in turn, transport phosphatidylinositol 4-phosphate backward [47]. In contrast, the sterol transport from plasma membrane to ER in yeast is severely impaired when all ORPs are knocked out [48]. These observations provide further support to an in vivo role for ORPs as sterol carriers. ORP5 is an ERlocalized, tailanchored protein that possesses a highly conserved pleckstrin homology domain near the N-terminus and an ORD near the C-terminus. ORP5 is able to bind cholesterol and 25hydroxycholesterol in COS7 cells [49]. It is thus possible that ORP5 may accept LDLC from NPC1 in a nonvesicular sterol transfer manner. In support of this, a recent study showed that ORP5 knockdown
markedly inhibits LDL-C transport to ER and causes LE/LY cholesterol accumulation in HeLa cells [50]. Moreover, cholesterol appears to accumulate in the limiting membrane of LE/LY in ORP5-depleted cells, whereas depletion of NPC1 or both ORP5 and NPC1 leads to luminal accumulation of cholesterol, suggesting that ORP5 may be localized at the downstream of NPC1 [50]. Thus, in response to an increased cholesterol level in the limiting membrane, ORP5 may form a transient protein complex with NPC1 at the membrane contact sites connecting LE/LY and ER, thereby leading to transportation of cholesterol from LE/LY directly to ER (Fig. 2). Of note, the formation of the NPC1/ORP5 junction may also require assistance from other proteins such as VPS4/SKD1, which is implicated in endosomal cholesterol transport and also interacts with both ORPs and NPC1 [51,52]. Importantly, the interaction between VPS4/SKD1 and ORPs/NPC1 is more efficient upon sterol depletion [51,52]. It is likely that VPS4/SKD1 splits the NPC1/ORP5 complex to recycle ORP5 when cholesterol transport is completed. However, such an effect remains to be further investigated. 5. Regulation of expression NPC1 is ubiquitously expressed in all tissues, with the most abundant expression in the liver [53]. Since NPC1 is so critical to intracellular cholesterol homeostasis, its expression is highly controlled at the transcriptional and posttranscriptional levels (Table 1). The sterol-sensing nuclear transcription factor liver X receptor (LXR) is regarded as the major regulator of NPC1 expression. In support of this concept, previous studies from our laboratory showed that administration of LXR agonist T0901317 markedly increases NPC1 mRNA and protein in the small intestine, liver and aorta of apolipoprotein E (apoE)−/− mice [54]. Our group has also reported that NO-1886, a potent agonist of lipoprotein lipase, can upregulate NPC1 expression at both the mRNA and protein
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Table 1 Regulation of NPC1 expression. Factors
Cells/Tissues
Pathway
NPC1 expression
References
T0901317 NO-1886 Clozapine, haloperidol OxLDL Alzheimer's disease Weight loss TCDD, BP Dietary fatty acids MiR-33a/b
Small intestine, liver and aorta THP-1 macrophage-derived foam cells Human glioblastoma cells Macrophages Hippocampus, frontal cortex White adipose tissues Macrophages Peritoneal fibroblasts, liver Human hepatic cells
LXR LXRα LXR ERK1/2/COX-2/PPARα ? ? AHR SREBP SREBP-2,-1
↑mRNA, protein ↑mRNA, protein ↑ mRNA ↑mRNA, protein ↑mRNA, protein ↓mRNA ↓mRNA, protein ↓mRNA, protein ↓mRNA, protein
[54] [55] [56] [57] [58] [59] [60] [61] [63]
levels by activating the LXRα signaling pathway in THP-1 macrophagederived foam cells [55]. In another study, treatment of human glioblastoma cells with psychotropic drugs such as clozapine and haloperidol significantly induces NPC1 transcription through the activation of LXR [56]. However, whether LXR response elements exist in the NPC1 gene remains unclear. Our recent investigation has demonstrated that oxidized low-density lipoprotein (oxLDL) can enhance the expression of NPC1 mRNA and protein in macrophages, and the mechanisms are associated with the extracellular signal-regulated kinase 1/2 (ERK1/2)/ cyclooxygenase-2 (COX-2)/peroxisome proliferator-activated receptor α (PPARα) pathway [57]. It is suggested that NPC1 expression is upregulated at both the mRNA and protein levels in the hippocampus and frontal cortex of Alzheimer's disease patients as compared to control individuals [58]. Bambace and co-workers observed that NPC1 mRNA is significantly elevated in the subcutaneous and omental white adipose tissues of obese individuals, and is reduced by weight loss [59]. On the other hand, contaminants such as 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) and benzo(a)pyrene (BP) inhibit NPC1 mRNA and protein expression in macrophages in an aryl hydrocarbon receptor (AHR)-dependent manner [60]. The NPC1 gene and protein are also downregulated by dietary fatty acids, but not dietary cholesterol, in peritoneal fibroblasts and liver of C57BL/6J mice through feedback inhibition of the sterol regulatory element-binding protein (SREBP) pathway [61]. MicroRNA (miR)-33a/b are intronic miRNAs located within the SREB-2 and -1 genes, respectively [62]. It has been reported that miR-33a/b can markedly decrease the levels of NPC1 mRNA and protein in human hepatic cells, indicating a novel regulatory role of miR-33a/b in lipid metabolism [63]. Thus, miR-33 a/b inhibitors can be potentially used to treat atherosclerosis. Taken together, many other factors regulating NPC1 expression may be discovered through future research. 6. Role in atherosclerosis In atherosclerosis, cellular cholesterol accumulates in lipid-engorged macrophage foam cells and this drives lipid deposition in atherosclerotic plaque. The control of macrophage cholesterol homeostasis is of great importance in the prevention of atherosclerosis. Dysregulation of the balance of cholesterol influx and efflux will lead to excessive accumulation of cholesterol in macrophages and their transformation into foam cells [3]. Because NPC1 is a key participant in promoting intracellular cholesterol trafficking, it is possible that NPC1 plays an important role in the progression of atherosclerosis. Indeed, NPC1 variants have been shown to increase the occurrence of coronary heart disease in Chinese population [64]. High-fat-diet-fed chimeric NPC1−/− mice reconstituted with LDL receptor (LDLR) −/− NPC1−/− macrophages display accelerated aortic atherosclerosis despite lower serum cholesterol levels when compared with mice reconstituted with wild-type macrophages [65]. Mechanistically, macrophages derived from chimeric NPC1−/− mice exhibit reduced synthesis of 27-hydroxycholesterol, an endogenous LXR ligand, leading to decreased expression of LXR-regulated cholesterol transporters such as ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1), impaired cholesterol efflux and increased
cholesterol-induced oxidative stress [65]. Similarly, the introduction of a NPC1−/− mutation into apoE−/− mice significantly facilitates atherosclerotic lesion formation and atherothrombosis [66]. It has been reported that NPC1 promoter methylation reduces NPC1 expression, impairs movement of cholesterol out of LE/LY and contributes to atherosclerosis progression [67]. Conversely, we found that the synthetic LXR agonist T0901317 strongly increases NPC1 expression in apoE−/− mice, resulting in reduced cholesterol accumulation in foam cells and decreased atherosclerotic lesions in the artery wall [68]. Dihydrocapsaicin (DHC), one of the major components of capsaicinoids, has been shown to attenuate atherosclerotic plaque formation in apoE−/− mice fed a high-fat/high-cholesterol diet through PPARγ/LXRα-signalingdependent upregulation of NPC1 expression [69]. Since the cholesterol in LE/LY is a priority as ABCA1-mediated cholesterol efflux [70] and NPC1 inactivation can decrease the expression and activity of ABCA1 [71], it is likely that the atheroprotective effects of NPC1 are dependent on ABCA1 which can mediate the rate-limiting step in high density lipoprotein (HDL) particle formation. Plasma HDL levels show a strong inverse association with the risks of atherosclerotic cardiovascular disease. To test the hypothesis, the synthetic sphingosine analog FTY720 significantly reduces the delivery of scavenged lipoprotein cholesterol to ER and promotes its release to apolipoprotein A-I (apoA-I) in human primary monocyte-derived macrophages, thus producing profound protection against atherosclerosis [72]. This is accompanied by enhanced levels of NPC1 and ABCA1 through the activation of LXR, and FTY720-induced upregulation of ABCA1 is also NPC1-dependent [72]. Thus, NPC1 may be antiatherogenic by enhancing cholesterol availability to efflux to apoA-I and HDL (Fig. 3). Macrophage apoptosis in advanced atherosclerotic lesions promotes plaque instability, a precursor of acute vascular events [73]. Macrophages in advanced lesions accumulate large amounts of unesterified cholesterol, which is a potent inducer of macrophage apoptosis. Induction of apoptosis in cultured macrophages is entirely dependent on cholesterol delivery to ER [74]. Feng and colleagues have demonstrated that although advanced lesions in NPC1+/+ or apoE−/− mice have extensive acellular areas rich in unesterified cholesterol and macrophage debris, the lesions of NPC1+/− apoE−/− mice exhibit substantially more cells and less necrosis [75]. Moreover, in comparison with NPC1+/+ or apoE−/− lesions, NPC1+/− apoE−/− lesions have reduced TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling)-positive areas surrounding necrotic areas, indicative of macrophage apoptosis [75]. These findings suggest that intact intracellular cholesterol transport is important for macrophage apoptosis in advanced atherosclerotic lesions, and that NPC1 heterozygosity confers resistance to lesional necrosis and macrophage apoptosis in murine atherosclerosis, thereby providing a therapeutic strategy to stabilize atherosclerotic plaque (Fig. 3). This is paradoxical with the above observations which regard NPC1 as an antiatherogenic agent [65–69]. Perhaps the discrepancy might be due to different atherosclerotic stages and experimental protocols. To date, the interaction between NPC1 and patients with atherosclerosis remains unclear, and heterozygous family members do not seem at a massively increased or decreased risks for vascular phenotypes.
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Fig. 3. The association between NPC1 and atherosclerosis. T0901317, DHC and FTY720 induce NPC1 expression in an LXR-dependent manner, which leads to increased transport of cholesterol from LE/LY to plasma membrane and promotes ABCA1upregulation. Subsequently, ABCA1 mediates cholesterol efflux to apoA-I to form nascent HDL particles, thereby protecting against atherosclerosis. On the other hand, NPC1 transfers cholesterol from LE/LY to ER and then stimulates macrophage apoptosis in advanced stage of atherosclerosis, resulting in plaque rupture.
Therefore, further translational studies on the vascular functions of NPC1 will ultimately benefit patients with NPC1-related atherosclerosis.
Disclosure The authors have declared no conflict of interest.
7. Conclusion and future perspectives In summary, NPC1 first accepts cholesterol from NPC2 in the LE/LY, and then mediates the transfer of cholesterol to ER and plasma membrane in a vesicular or nonvesicular manner. Defective NPC1 is linked to NPC disease. Upregulation of NPC1 expression is able to promote intracellular cholesterol transport and increases its availability to efflux to apoA-I. Hence, NPC1 activators may be effective in facilitating sterol elimination and inhibiting foam cell formation. Most current attempts at regulating NPC1-dependent sterol excretion involve the use of synthetic LXR agonists, which can substantially induce NPC1 transcription in the liver [76]. LXR activation thus elicits the unwanted side effect of increased endogenous lipogenesis, leading to pronounced hepatic steatosis [77], ruling out synthetic LXR agonists as a safe way to facilitate NPC1 function. To avoid this side effect, alternative strategies for NPC1 modulation need to be pursued. It remains likely that NPC1 may be more directly targeted by small molecule activators. With in vitro and cell-based functional assays for NPC1-dependent cholesterol transport in place, it may now be possible to screen for these compounds. Notably, the link between NPC1 and atherosclerosis is complex, and thus the contribution of this protein to disease protection in humans remains to be further investigated. In addition, there are still many problems left to be solved. What is the function of the four small luminal loops and six small cytoplasmic loops in NPC1? Do other ORPs besides ORP5 cooperate with NPC1 to transfer cholesterol out of LE/LY? Is NPC1-mediated cholesterol transport from LE/LY to plasma membrane required for ORP5? Also, it will be of interest to determine whether full length NPC1 would provide evidence for a more direct interaction with NPC2. Understanding the answers to these questions will provide insightful knowledge regarding the underlying mechanisms of intracellular cholesterol trafficking and accelerate the future design of novel therapeutic reagents.
Acknowledgments The authors gratefully acknowledge the financial support from the National Natural Sciences Foundation of China (81070220 and 81170278), and Aid Program for Science and Technology Innovative Research Team in Higher Educational Institutions (2008-244) of Human Province, China, and the construct program of the key discipline in Hunan Province.
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Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
NPC1, intracellular cholesterol trafficking and atherosclerosis Xiao-Hua Yu a, Na Jiang c, Ping-Bo Yao d, Xi-Long Zheng e, Francisco S. Cayabyab f, Chao-Ke Tang a,b,⁎ a
Life Science Research Center, University of South China, Hengyang, Hunan 421001, China Institute of Cardiovascular Research, Key Laboratory for Atherosclerology of Hunan Province, University of South China, Hengyang, Hunan 421001, China c Department of Electrocardiogram, the Second Affiliated Hospital, University of South China, Hengyang, Hunan 421001, China d Department of Intensive Care, the Affiliated Nanhua Hospital, University of South China, Hengyang, Hunan 421001, China e Department of Biochemistry and Molecular Biology, The Libin Cardiovascular Institute of Alberta, The University of Calgary, Health Sciences Center, 3330 Hospital Dr NW, Calgary, Alberta, T2N 4 N1, Canada f Department of Physiology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada b
a r t i c l e
i n f o
Article history: Received 27 September 2013 Received in revised form 17 November 2013 Accepted 23 November 2013 Available online 1 December 2013 Keywords: NPC1 NPC2 Cholesterol ORP5 Atherosclerosis
a b s t r a c t Post-lysosomal cholesterol trafficking is an important, but poorly understood process that is essential to maintain lipid homeostasis. Niemann-Pick type C1 (NPC1), an integral membrane protein on the limiting membrane of late endosome/lysosome (LE/LY), is known to accept cholesterol from NPC2 and then mediate cholesterol transport from LE/LY to endoplasmic reticulum (ER) and plasma membrane in a vesicle- or oxysterolbinding protein (OSBP)related protein 5 (ORP5)-dependent manner. Mutations in the NPC1 gene can be found in the majority of NPC patients, who accumulate massive amounts of cholesterol and other lipids in the LE/LY due to a defect in intracellular lipid trafficking. Liver X receptor (LXR) is the major positive regulator of NPC1 expression. Atherosclerosis is the pathological basis of coronary heart disease, one of the major causes of death worldwide. NPC1 has been shown to play a critical role in the atherosclerotic progression. In this review, we have summarized the role of NPC1 in regulating intracellular cholesterol trafficking and atherosclerosis. © 2013 Elsevier B.V. All rights reserved.
Contents 1. 2. 3. 4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure and characterization . . . . . . . . . . . . . . . . . . . . . . . . . Cholesterol trafficking function of NPC1 and NPC disease . . . . . . . . . . . . . Mechanisms of NPC1-mediated cholesterol transport . . . . . . . . . . . . . . 4.1. Cholesterol “handoff” from NPC2 to NPC1 . . . . . . . . . . . . . . . . 4.2. NPC1 promotes post-lysosomal cholesterol trafficking in a vesicular manner 4.3. NPC1 cooperates with ORP5 to deliver cholesterol out of LE/LY . . . . . . 5. Regulation of expression . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Role in atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Conclusion and future perspectives . . . . . . . . . . . . . . . . . . . . . . Disclosure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abbreviations: NPC1, Niemann-Pick type C1; LE/LY, late endosome/lysosome; LDL, lowdensity lipoprotein; LDL-C, LDL-cholesterol; ER, endoplasmic reticulum; SSD, sterol-sensing domain; NTD, N-terminal domain; apoE, apolipoprotein E; LXR, liver X receptor; OSBP, oxysterolbinding protein; ORP5, OSBPrelated protein 5; TGN, trans-Golgi network; oxLDL, oxidized low-density lipoprotein; ERK1/2, extracellular signal-regulated kinase 1/2; COX-2, cyclooxygenase-2; PPARα, peroxisome proliferator-activated receptor α; SREBP, sterol regulatory element-binding protein; LDLR, low-density lipoprotein receptor; ABCA1, ATP-binding cassette transporter A1; DHC, dihydrocapsaicin; apoA-I, apolipoprotein A-I. ⁎ Corresponding author at: Institute of Cardiovascular Research, University of South China, Hengyang, Hunan 421001, China. Tel./fax: +86 734 8281853. E-mail address: [email protected] (C.-K. Tang). 0009-8981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2013.11.026
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1. Introduction Cholesterol is an abundant metabolite in mammalian tissues that plays important roles in normal embryonic development, cell differentiation, and nerve conduction. Membrane fluidity of all cells is tightly regulated by the ordered packing of cholesterol between phospholipid molecules. It is also the precursor of a variety of biologically active molecules such as bile acids, vitamin D and steroid hormones. Thus, an insufficient supply of cholesterol will have detrimental effects on
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cell function, tissue development as well as whole-body physiology. Too much cholesterol, however, can also result in pathological consequences. Both clinical and animal studies have established a direct link between plasma cholesterol levels and the risks of coronary heart disease, one of the major causes of death worldwide [1,2]. The control of intracellular cholesterol levels is a complicated process, involving cholesterol uptake, biosynthesis, transport, metabolism and secretion. Interestingly, cholesterol uptake and secretion have been well described in several recent reviews [3,4]. Nevertheless, intracellular cholesterol trafficking remains elusive. Growing evidence shows that Niemann-Pick type C1 (NPC1) plays a key role in the process [5]. NPC1 is a transmembrane glycoprotein located in the limiting membrane of late endosome/lysosome (LE/LY). It can transfer low-density lipoprotein (LDL)-cholesterol (LDL-C) from LE/LY to endoplasmic reticulum (ER) for esterification or to plasma membrane for efflux, a crucial process governing the balance between macrophage cholesterol import and export, with potential consequences in atherogenesis [6]. This review focuses on the molecular characterization, cholesterol transport function and involved mechanisms of NPC1, and explores its emerging pathogenic significance and therapeutic potential in atherosclerosis. 2. Structure and characterization The NPC1 gene maps to chromosome 18q11–12, spans more than 47 kb, and consists of 25 exons that range in size from 74 to 788 nucleotides, and introns that range from 0.097 to 7 kb in length [7]. The human NPC1 protein is composed of 1278 amino acid residues, with 13 putative transmembrane helices, three large loops projecting into the lumen of LE/LY, four small luminal loops, six small cytoplasmic loops and a cytoplasmic tail (Fig. 1). Five segments (residues 615–797) from the third to seventh transmembrane helix form the so-called sterol-sensing domain (SSD), which shares high sequence homology with those of several other cholesterol metabolism-related membrane proteins, such as Patched (a regulatory protein in the Hedgehog signaling pathway), sterol regulatory element binding protein cleavage activating protein (SCAP), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and NPC1-like protein 1 (a protein responsible for dietary cholesterol and biliary cholesterol absorption). SSD is an important binding site between cholesterol and NPC1. It can bind azocholestanol, a photoactivatable analog of cholesterol, in cells, and two loss-of-function mutations (P692S and Y635C) in the region severely block this effect [8]. In contrast, two gain-of-function mutations (L657F and D787N) in the SSD lead to a nearly 2-fold increase in the rates of moving LDL-C out of LE/LY [9]. NPC1 is synthesized in ER, transited through the Golgi and finally targeted to LE/LY. The dileucine motif present at the C-terminus has been reported to promote NPC1 trafficking to the endosomal compartment by direct interaction with the clathrin adaptor protein complex
1 [10]. A motif located in the SSD also contributes to this process [11]. Therefore, although the precise function of NPC1 SSD in vivo is still not fully understood, it is essential for normal cholesterol efflux from LE/LY. NPC1 has three large luminal loops. The first loop (residues 25–264) at the N-terminus is called the N-terminal domain (NTD) or NPC1 domain, which is highly conserved in vertebrate and yeast orthologs of NPC1. It is composed of 18 conserved cysteine residues involved in intra-domain disulfide links, and a leucine zipper motif. When NTD was secreted as a soluble dimer in cultured cells, high affinity binding to cholesterol and 25-hydroxycholesterol was observed, suggesting NTD as a possible sterol binding site for NPC1 [12]. Nevertheless, the full-length NPC1 protein with a Q79A mutation, which disrupts the sterol binding by NTD, can still restore normal cholesterol transport out of LE/LY in NPC1-deficient Chinese hamster ovary cells, indicating that other cholesterol binding sites such as SSD except NTD may be present in the NPC1 protein [12]. The second loop, namely the region between transmembrane helices 2 and 3, can interact with NPC2 [13]. The third loop (residues 855–1098) locates between membrane-spanning domains 8 and 9. It is rich in conserved cysteine residues and consists of a ring finger motif. To date, of the over 200 NPC disease-causing mutations identified in the NPC1 gene [14], approximately half of these mutations are localized to this region, including the most common mutation in Western Europeans, I1061T, which accounts for about 20% of the total number of NPC disease cases [15]. 3. Cholesterol trafficking function of NPC1 and NPC disease Cells can obtain cholesterol by two ways. One is de novo synthesis, a well-defined energy-consuming and feedback-regulated process which is involved in a variety of enzymatic reactions [16]. The other is exogenous uptake mainly from circulating LDL through receptor-mediated endocytosis [3]. After delivery from the early endosome to LE/LY, cholesterol esters carried by LDL are hydrolyzed by lysosomal acid lipase to release free cholesterol. Then, NPC1 mediates the trafficking of free cholesterol from LE/LY to other cellular compartments, including the plasma membrane and ER. In NPC1-absent cells, although the endocytic uptake of LDL-C and subsequent hydrolysis of cholesterol esters are normal, the transfer of unesterified cholesterol from LE/LY is impaired. Consequently, normal amounts of cholesterol fail to reach the plasma membrane and ER to modulate cholesterol balance [17]. In agreement, overexpression of human NPC1 protein greatly promotes LDL-C influx into plasma membrane and ER in Chinese hamster ovary cell lines [18]. NPC disease is a rare, fatal autosomal recessive neurovisceral condition characterized by progressive neurological deterioration and premature death, with an estimated incidence of 1:150,000 live births. Approximately 95% of cases are caused by mutations in the NPC1 gene, while the other 5% is related to mutations in the NPC2 gene [19]. Loss of function of either of these proteins leads to impaired trafficking
Fig. 1. Schematic of NPC1 protein structure. NPC1 protein has 13 transmembrane domains, three large luminal loops and a cytoplasmic tail. Loop 1 or NTD containing a leucine zipper motif can bind cholesterol. SSD is also a binding site for cholesterol and serves as an LE/LY targeting motif. A dileucine motif at the C-terminus is important for NPC1 localization in LE/LY. Loop 2 is able to directly bind NPC2. Loop 3 is composed of a ring finger motif.
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of endocytosed cholesterol and intracellular accumulation of lipids including unesterified cholesterol, glycosphingolipids, sphingomyelin and sphingosine in multiple tissues [20]. In the brain, this causes neuronal degeneration. Typical neurological manifestations include vertical supranuclear gaze palsy, saccadic eye movement abnormalities, cerebellar ataxia, dystonia, dysmetria, dysarthria and dysphagia. These neurological signs arise at different ages, but invariably progress over time. In general, patients with neurological onset early in life show aggravated symptoms faster and have a shorter lifespan than those with adult onset [21]. There is currently no effective treatment for the disease. Zampieri and colleagues demonstrated that increased degradation of mutant NPC1 protein due to protein misfolding is implicated in the pathogenesis of NPC1 disease [22]. They also showed that treatment of fibroblasts carrying NPC1 missense mutations in homo or hemizygosity with 10% glycerol, a chemical chaperone known to stabilize misfolded proteins, results in a significant increase of NPC1 protein levels and an improvement of the intracellular trafficking of cholesterol, suggesting that at least some NPC1 mutations may be potentially rescued by small molecule-based chaperone therapy [22]. More recently, cyclodextrin has been reported to re-establish the normal movement of cholesterol out of LE/LY in NPC1-deficient cells [23,24], and significantly ameliorates symptoms and extends lifespan in a murine NPC disease model [25]. Administration of hydroxypropyl-β-cyclodextrin was effective in improving hepatosplenomegaly and central nervous system dysfunction in two patients with NPC disease, although it did not improve the neurological deficits in either patient [26]. However, the safety and long-term effect of this drug needs to be more fully characterized and better defined. 4. Mechanisms of NPC1-mediated cholesterol transport 4.1. Cholesterol “handoff” from NPC2 to NPC1 Homozygous mutations in either the NPC1 or NPC2 gene cause virtually indistinguishable cellular and clinical phenotypes despite the large discrepancy in their molecular structure. Similarly, when NPC1-absent mice are crossed with NPC2 hypomorphic mice, which retain 0%–4% residual NPC2 protein, the phenotypes of the progeny are identical with those of NPC1- or NPC2-absent mice [27]. NPC1's NTD consists of a deep pocket that can surround cholesterol with the 3β-hydroxyl group and tetracyclic ring buried inside, leaving the isooctyl side chain of cholesterol partially exposed on the surface [28]. NPC2 is a ubiquitously expressed 151-amino acid, soluble protein in the lumen of LE/LY. It contains a deep hydrophobic pocket that can bind cholesterol with its isooctyl side chain buried and 3β-hydroxyl group exposed, in an orientation opposite to the NTD of NPC1 [29]. Thus, it is possible that both proteins operate sequentially in the same pathway to promote cholesterol transport. More recently, the concerted action of both proteins has been investigated in two alanine-scanning mutagenesis experiments. A patch of three amino acids around the sterol-binding site of NPC2 was identified as being essential for cholesterol transfer but not for cholesterol binding [30]. Likewise, a patch of 11 amino acids surrounding the entrance to the sterol-binding pocket of NPC1 NTD was shown to be required for cholesterol binding but not for cholesterol transfer [28]. According to these data, the authors have proposed a working model for “hydrophobic handoff” of cholesterol from NPC1 to NPC2. In the model, the two surface patches on NPC2 and NPC1 interact, leading to the opening of an entry pore on the NTD of NPC1 and allowing cholesterol delivery to NPC1 for export (Fig. 2) [30]. Based on this, NPC2 can transfer its bound cholesterol to NPC1 directly, thus avoiding the necessity for insoluble cholesterol to transit the water phase and preventing cholesterol from crystallizing in the lysosomal lumen. In support of the model, bidirectional transfer of cholesterol between NPC1 and liposomes is accelerated by more than 100 fold in the presence of NPC2, and a naturally occurring human mutant of
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NPC2 (Pro120Ser) abolishes the effect [31]. Additionally, Deffieu and co-workers showed that the second large luminal loop of NPC1 is a binding site for NPC2 protein [13]. Moreover, this binding only occurs in acidic conditions that mimic the lysosomal lumen environment and are optimal for cholesterol binding to NPC2 and delivery to NPC1; whereas, human disease-causing mutations in the region inhibit NPC2 binding, providing direct evidence that NPC1 loop 2 holds NPC2 in position to promote directional cholesterol transfer from NPC2 to NPC1 [13]. Therefore, this handoff may be essential for cholesterol export from LE/LY. Of note, the reverse direction may also exist. It has been reported that NPC1 first accepts free cholesterol in the interior of LE/LY and then transfers it to NPC2 [32]. Further studies are required to clarify a bona fide cholesterol transport mechanism by NPC1 and NPC2. 4.2. NPC1 promotes post-lysosomal cholesterol trafficking in a vesicular manner Although the NPC1/NPC2 handoff model describes cholesterol transport inside LE/LY lumen, how cholesterol is exported from LE/LY has not yet been defined. One of the possible mechanisms is vesicular transport, a pathway involving vesicles that bud off from LE/LY and fuse with ER or plasma membrane (Fig. 2). This concept is supported by an earlier study, which demonstrated that organelles containing functional NPC1-fluorescent protein fusions undergo dynamic movements associated with extending strands of ER [33], although whether such movements can directly transport cholesterol is still questionable. On the other hand, the normal rapid tubulovesicular trafficking of LE is retarded in NPC1-mutated cells [34]. Also, the release of lysosomal cargo-containing membrane vesicles is significantly reduced in NPC1 mutant fibroblasts as compared to wild type cells [35]. The trans-Golgi network (TGN) is involved in NPC1-mediated cholesterol transport downstream. The SNARE complex is composed of VAMP4, syntaxin 6, syntaxin 16 and Vti1a, and plays an important role in mediating the transport of various proteins between LE/LY and TGN by vesicles [36]. Urano et al. showed that Chinese hamster ovary cells lacking NPC1 display a significant reduction of LDL-C within TGN, and knockdown by RNAi of three TGN-specific SNAREs (VAMP4, syntaxin 6, and syntaxin 16) decreases LDL-derived cholesterol transport by over 50% in intact cells, suggesting that vesicular trafficking transports a significant portion of LDL-C from NPC1-containing endosomal compartment to TGN before its arrival at ER or plasma membrane [37]. Anaplasma phagocytophilum, an obligatory intracellular and cholesterol-robbing bacterium, disrupts the NPC1 pathway of intracellular cholesterol trafficking by inhibiting VAMP4 and syntaxin 16 [38]. Thus, the SNARE complex may be associated with vesicular transport of cholesterol out of LE/LY. The Ras-superfamily of small G proteins is a family of GTP hydrolases that is regulated by GTP/GDP binding states. Rab, one member of the Ras-superfamily, plays a crucial role in the vesicular trafficking. It has been reported that overexpression of Rab8 markedly diminishes cholesterol accumulation and restores LDL-C egress from LE/LY in NPC1-deficient fibroblasts, yet depletion of Rab8 from wild-type fibroblasts causes cholesterol deposition within late endosomal compartments [39]. Another Rab protein, Rab9, is a small GTPase enriched in the LE/LY. In NPC1-deficient fibroblasts, the level of Rab9 is elevated by 1.8-fold because of reduced degradation of the protein, and it is sequestered on endosome membranes, leading to impairment of mannose 6-phosphate receptor trafficking [40]. Ectopic expression of human telomerase reverse transcriptase induces upregulation of Rab9 in NPC1-deficient cells so that the transport of cholesterol from LE/LY to plasma membrane is restored [41]. Likewise, overexpression of Rab9 by protein transduction significantly decreases cellular free cholesterol levels in NPC1-deficient human fibroblasts and mouse neurons [42]. These results suggest that Rab9 may be able to bypass the requirement for NPC1, and that Rab9 overexpression may also have therapeutic potential for NPC disease in vivo. To test this possibility, NPC1−/− mice
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Fig. 2. A working model of NPC1-mediated intracellular cholesterol trafficking. Cholesterol is transferred from NPC2 within the lumen to the NTD of NPC1 in the limiting membrane of LE/LY in a reverse orientation. After delivery to the LE/LY limiting membrane, cholesterol is transferred to ER and plasma membrane in a vesicular manner. Alternatively, with the help of VPS4/SKD1, ORP5 can form a transient protein complex with NPC1 at the membrane contact sites connecting LE/LY and ER to export cholesterol into ER. Upon completion of cholesterol transfer, VPS4/SKD1 breaks down the complex and recycles the ORP5.
were crossed with Rab9 transgenic mice, resulting in reduced storage of lipids and prolonged lifespan [43]. 4.3. NPC1 cooperates with ORP5 to deliver cholesterol out of LE/LY In addition to membrane vesicles, cholesterol transport can be performed by carrier proteins in a nonvesicular manner. Oxysterolbinding protein (OSBP) and its relatives (OSBPrelated proteins, ORPs), which constitute a large conserved protein family in eukaryotes, have recently been identified as putative sterol carriers [44]. OSBP has been shown to act as a highaffinity cytosolic receptor for oxysterols, such as 25hydroxycholesterol. ORPs consist of 12 members in human and 7 members in budding yeast. All ORPs share a conserved ~ 400 amino acid OSBP-related domain (ORD) that is found at the C-terminus of OSBP, and is the binding site of cholesterol, oxysterols and phosphatidylinositol 4-phosphate [45]. ORD can also simultaneously bind two different organelle membranes, thus allowing a rapid but regulated sterol transfer between the contact sites [46]. Additionally, OSBP and ORPs have membrane targeting domains such as the pleckstrin homology domain. Osh4p, a yeast ORP, has been reported to transport sterols from ER to LE/LY and, in turn, transport phosphatidylinositol 4-phosphate backward [47]. In contrast, the sterol transport from plasma membrane to ER in yeast is severely impaired when all ORPs are knocked out [48]. These observations provide further support to an in vivo role for ORPs as sterol carriers. ORP5 is an ERlocalized, tailanchored protein that possesses a highly conserved pleckstrin homology domain near the N-terminus and an ORD near the C-terminus. ORP5 is able to bind cholesterol and 25hydroxycholesterol in COS7 cells [49]. It is thus possible that ORP5 may accept LDLC from NPC1 in a nonvesicular sterol transfer manner. In support of this, a recent study showed that ORP5 knockdown
markedly inhibits LDL-C transport to ER and causes LE/LY cholesterol accumulation in HeLa cells [50]. Moreover, cholesterol appears to accumulate in the limiting membrane of LE/LY in ORP5-depleted cells, whereas depletion of NPC1 or both ORP5 and NPC1 leads to luminal accumulation of cholesterol, suggesting that ORP5 may be localized at the downstream of NPC1 [50]. Thus, in response to an increased cholesterol level in the limiting membrane, ORP5 may form a transient protein complex with NPC1 at the membrane contact sites connecting LE/LY and ER, thereby leading to transportation of cholesterol from LE/LY directly to ER (Fig. 2). Of note, the formation of the NPC1/ORP5 junction may also require assistance from other proteins such as VPS4/SKD1, which is implicated in endosomal cholesterol transport and also interacts with both ORPs and NPC1 [51,52]. Importantly, the interaction between VPS4/SKD1 and ORPs/NPC1 is more efficient upon sterol depletion [51,52]. It is likely that VPS4/SKD1 splits the NPC1/ORP5 complex to recycle ORP5 when cholesterol transport is completed. However, such an effect remains to be further investigated. 5. Regulation of expression NPC1 is ubiquitously expressed in all tissues, with the most abundant expression in the liver [53]. Since NPC1 is so critical to intracellular cholesterol homeostasis, its expression is highly controlled at the transcriptional and posttranscriptional levels (Table 1). The sterol-sensing nuclear transcription factor liver X receptor (LXR) is regarded as the major regulator of NPC1 expression. In support of this concept, previous studies from our laboratory showed that administration of LXR agonist T0901317 markedly increases NPC1 mRNA and protein in the small intestine, liver and aorta of apolipoprotein E (apoE)−/− mice [54]. Our group has also reported that NO-1886, a potent agonist of lipoprotein lipase, can upregulate NPC1 expression at both the mRNA and protein
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Table 1 Regulation of NPC1 expression. Factors
Cells/Tissues
Pathway
NPC1 expression
References
T0901317 NO-1886 Clozapine, haloperidol OxLDL Alzheimer's disease Weight loss TCDD, BP Dietary fatty acids MiR-33a/b
Small intestine, liver and aorta THP-1 macrophage-derived foam cells Human glioblastoma cells Macrophages Hippocampus, frontal cortex White adipose tissues Macrophages Peritoneal fibroblasts, liver Human hepatic cells
LXR LXRα LXR ERK1/2/COX-2/PPARα ? ? AHR SREBP SREBP-2,-1
↑mRNA, protein ↑mRNA, protein ↑ mRNA ↑mRNA, protein ↑mRNA, protein ↓mRNA ↓mRNA, protein ↓mRNA, protein ↓mRNA, protein
[54] [55] [56] [57] [58] [59] [60] [61] [63]
levels by activating the LXRα signaling pathway in THP-1 macrophagederived foam cells [55]. In another study, treatment of human glioblastoma cells with psychotropic drugs such as clozapine and haloperidol significantly induces NPC1 transcription through the activation of LXR [56]. However, whether LXR response elements exist in the NPC1 gene remains unclear. Our recent investigation has demonstrated that oxidized low-density lipoprotein (oxLDL) can enhance the expression of NPC1 mRNA and protein in macrophages, and the mechanisms are associated with the extracellular signal-regulated kinase 1/2 (ERK1/2)/ cyclooxygenase-2 (COX-2)/peroxisome proliferator-activated receptor α (PPARα) pathway [57]. It is suggested that NPC1 expression is upregulated at both the mRNA and protein levels in the hippocampus and frontal cortex of Alzheimer's disease patients as compared to control individuals [58]. Bambace and co-workers observed that NPC1 mRNA is significantly elevated in the subcutaneous and omental white adipose tissues of obese individuals, and is reduced by weight loss [59]. On the other hand, contaminants such as 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) and benzo(a)pyrene (BP) inhibit NPC1 mRNA and protein expression in macrophages in an aryl hydrocarbon receptor (AHR)-dependent manner [60]. The NPC1 gene and protein are also downregulated by dietary fatty acids, but not dietary cholesterol, in peritoneal fibroblasts and liver of C57BL/6J mice through feedback inhibition of the sterol regulatory element-binding protein (SREBP) pathway [61]. MicroRNA (miR)-33a/b are intronic miRNAs located within the SREB-2 and -1 genes, respectively [62]. It has been reported that miR-33a/b can markedly decrease the levels of NPC1 mRNA and protein in human hepatic cells, indicating a novel regulatory role of miR-33a/b in lipid metabolism [63]. Thus, miR-33 a/b inhibitors can be potentially used to treat atherosclerosis. Taken together, many other factors regulating NPC1 expression may be discovered through future research. 6. Role in atherosclerosis In atherosclerosis, cellular cholesterol accumulates in lipid-engorged macrophage foam cells and this drives lipid deposition in atherosclerotic plaque. The control of macrophage cholesterol homeostasis is of great importance in the prevention of atherosclerosis. Dysregulation of the balance of cholesterol influx and efflux will lead to excessive accumulation of cholesterol in macrophages and their transformation into foam cells [3]. Because NPC1 is a key participant in promoting intracellular cholesterol trafficking, it is possible that NPC1 plays an important role in the progression of atherosclerosis. Indeed, NPC1 variants have been shown to increase the occurrence of coronary heart disease in Chinese population [64]. High-fat-diet-fed chimeric NPC1−/− mice reconstituted with LDL receptor (LDLR) −/− NPC1−/− macrophages display accelerated aortic atherosclerosis despite lower serum cholesterol levels when compared with mice reconstituted with wild-type macrophages [65]. Mechanistically, macrophages derived from chimeric NPC1−/− mice exhibit reduced synthesis of 27-hydroxycholesterol, an endogenous LXR ligand, leading to decreased expression of LXR-regulated cholesterol transporters such as ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1), impaired cholesterol efflux and increased
cholesterol-induced oxidative stress [65]. Similarly, the introduction of a NPC1−/− mutation into apoE−/− mice significantly facilitates atherosclerotic lesion formation and atherothrombosis [66]. It has been reported that NPC1 promoter methylation reduces NPC1 expression, impairs movement of cholesterol out of LE/LY and contributes to atherosclerosis progression [67]. Conversely, we found that the synthetic LXR agonist T0901317 strongly increases NPC1 expression in apoE−/− mice, resulting in reduced cholesterol accumulation in foam cells and decreased atherosclerotic lesions in the artery wall [68]. Dihydrocapsaicin (DHC), one of the major components of capsaicinoids, has been shown to attenuate atherosclerotic plaque formation in apoE−/− mice fed a high-fat/high-cholesterol diet through PPARγ/LXRα-signalingdependent upregulation of NPC1 expression [69]. Since the cholesterol in LE/LY is a priority as ABCA1-mediated cholesterol efflux [70] and NPC1 inactivation can decrease the expression and activity of ABCA1 [71], it is likely that the atheroprotective effects of NPC1 are dependent on ABCA1 which can mediate the rate-limiting step in high density lipoprotein (HDL) particle formation. Plasma HDL levels show a strong inverse association with the risks of atherosclerotic cardiovascular disease. To test the hypothesis, the synthetic sphingosine analog FTY720 significantly reduces the delivery of scavenged lipoprotein cholesterol to ER and promotes its release to apolipoprotein A-I (apoA-I) in human primary monocyte-derived macrophages, thus producing profound protection against atherosclerosis [72]. This is accompanied by enhanced levels of NPC1 and ABCA1 through the activation of LXR, and FTY720-induced upregulation of ABCA1 is also NPC1-dependent [72]. Thus, NPC1 may be antiatherogenic by enhancing cholesterol availability to efflux to apoA-I and HDL (Fig. 3). Macrophage apoptosis in advanced atherosclerotic lesions promotes plaque instability, a precursor of acute vascular events [73]. Macrophages in advanced lesions accumulate large amounts of unesterified cholesterol, which is a potent inducer of macrophage apoptosis. Induction of apoptosis in cultured macrophages is entirely dependent on cholesterol delivery to ER [74]. Feng and colleagues have demonstrated that although advanced lesions in NPC1+/+ or apoE−/− mice have extensive acellular areas rich in unesterified cholesterol and macrophage debris, the lesions of NPC1+/− apoE−/− mice exhibit substantially more cells and less necrosis [75]. Moreover, in comparison with NPC1+/+ or apoE−/− lesions, NPC1+/− apoE−/− lesions have reduced TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling)-positive areas surrounding necrotic areas, indicative of macrophage apoptosis [75]. These findings suggest that intact intracellular cholesterol transport is important for macrophage apoptosis in advanced atherosclerotic lesions, and that NPC1 heterozygosity confers resistance to lesional necrosis and macrophage apoptosis in murine atherosclerosis, thereby providing a therapeutic strategy to stabilize atherosclerotic plaque (Fig. 3). This is paradoxical with the above observations which regard NPC1 as an antiatherogenic agent [65–69]. Perhaps the discrepancy might be due to different atherosclerotic stages and experimental protocols. To date, the interaction between NPC1 and patients with atherosclerosis remains unclear, and heterozygous family members do not seem at a massively increased or decreased risks for vascular phenotypes.
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Fig. 3. The association between NPC1 and atherosclerosis. T0901317, DHC and FTY720 induce NPC1 expression in an LXR-dependent manner, which leads to increased transport of cholesterol from LE/LY to plasma membrane and promotes ABCA1upregulation. Subsequently, ABCA1 mediates cholesterol efflux to apoA-I to form nascent HDL particles, thereby protecting against atherosclerosis. On the other hand, NPC1 transfers cholesterol from LE/LY to ER and then stimulates macrophage apoptosis in advanced stage of atherosclerosis, resulting in plaque rupture.
Therefore, further translational studies on the vascular functions of NPC1 will ultimately benefit patients with NPC1-related atherosclerosis.
Disclosure The authors have declared no conflict of interest.
7. Conclusion and future perspectives In summary, NPC1 first accepts cholesterol from NPC2 in the LE/LY, and then mediates the transfer of cholesterol to ER and plasma membrane in a vesicular or nonvesicular manner. Defective NPC1 is linked to NPC disease. Upregulation of NPC1 expression is able to promote intracellular cholesterol transport and increases its availability to efflux to apoA-I. Hence, NPC1 activators may be effective in facilitating sterol elimination and inhibiting foam cell formation. Most current attempts at regulating NPC1-dependent sterol excretion involve the use of synthetic LXR agonists, which can substantially induce NPC1 transcription in the liver [76]. LXR activation thus elicits the unwanted side effect of increased endogenous lipogenesis, leading to pronounced hepatic steatosis [77], ruling out synthetic LXR agonists as a safe way to facilitate NPC1 function. To avoid this side effect, alternative strategies for NPC1 modulation need to be pursued. It remains likely that NPC1 may be more directly targeted by small molecule activators. With in vitro and cell-based functional assays for NPC1-dependent cholesterol transport in place, it may now be possible to screen for these compounds. Notably, the link between NPC1 and atherosclerosis is complex, and thus the contribution of this protein to disease protection in humans remains to be further investigated. In addition, there are still many problems left to be solved. What is the function of the four small luminal loops and six small cytoplasmic loops in NPC1? Do other ORPs besides ORP5 cooperate with NPC1 to transfer cholesterol out of LE/LY? Is NPC1-mediated cholesterol transport from LE/LY to plasma membrane required for ORP5? Also, it will be of interest to determine whether full length NPC1 would provide evidence for a more direct interaction with NPC2. Understanding the answers to these questions will provide insightful knowledge regarding the underlying mechanisms of intracellular cholesterol trafficking and accelerate the future design of novel therapeutic reagents.
Acknowledgments The authors gratefully acknowledge the financial support from the National Natural Sciences Foundation of China (81070220 and 81170278), and Aid Program for Science and Technology Innovative Research Team in Higher Educational Institutions (2008-244) of Human Province, China, and the construct program of the key discipline in Hunan Province.
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