role of acat1-positive late endosomes in macrophages...

INTRODUCTION

Macrophages are the principal cell of innate im-munity. They show active phagocytosis, producecytokines, present antigens to lymphocytes, and playan important role in host defense against various mi-croorganisms. In addition, macrophages actively me-tabolize cholesterol and contribute to atherogenesisby producing various biologically active molecules

(1). Macrophages in atheromatous plaques com-monly accumulate numerous lipid droplets in theircytoplasm. These unique macrophages are calledfoamy transformed macrophages. Macrophages inhyperlipidemic conditions actively internalize low-density lipoprotein (LDL), with or without chemicalmodification, via scavenger receptor-mediated endo-cytosis or fluid-phase pinocytosis (2, 3). Internalizedmodified or native LDL is hydrolyzed at acid lipase-positive late endosomes, which results in the gen-eration of free cholesterol in loco (4, 5). Free choles-terol generated at late endosomes is transferred toplasma membranes by Niemann-Pick disease typeC1/C2 protein (NPC1/2), and excess amounts oftransferred free cholesterol are further transported

REVIEW

Role of ACAT1-positive late endosomes inmacrophages :Cholesterol metabolism and therapeuticapplications for Niemann-Pick disease type C

Naomi Sakashita1, XiaoFeng Lei2, Masashi Kamikawa3, and Kazuchika Nishitsuji1

1Department of Human Pathology, Institute of Health Biosciences, the University of Tokushima Gradu-

ate School, Tokushima, Japan ; 2Department of Biochemistry, Showa University, Tokyo, Japan ; 3Depart-

ment of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan

Abstract : Macrophages in hyperlipidemic conditions accumulate cholesterol esters anddevelop into foamy transformed macrophages. During this transformation, macrophagesdemonstrate endoplasmic reticulum fragmentation and consequently produce acyl co-enzyme A : cholesterol acyltransferase 1 (ACAT1)-positive late endosomes (ACAT1-LE).ACAT1-LE-positive macrophages effectively esterify modified or native low-density lipo-protein-derived free cholesterol, which results in efficient cholesterol esterification aswell as atherosclerotic plaque formation. These macrophages show significant cholesterolester formation even when free cholesterol egress from late endosomes is impaired, whichindicates that free cholesterol is esterified at ACAT1-LE. Genetic blockade of cholesterolegress from late endosomes causes Niemann-Pick disease type C (NPC), an inheritedlysosomal storage disease with progressive neurodegeneration. Induction of ACAT1-LEin macrophages with the NPC phenotype led to significant recovery of cholesterol esteri-fication. In addition, in vivo ACAT1-LE induction significantly extended the lifespan ofmice with the NPC phenotype. Thus, ACAT1-LE not only regulates intracellular choles-terol metabolism but also ameliorates NPC pathophysiology. J. Med. Invest. 61 : 270-277,August, 2014

Keywords : ACAT1, late endosomes, macrophages, cholesterol metabolism, Niemann-Pick disease type C

Received for publication February 4, 2014 ; accepted June 8, 2014.

Address correspondence and reprint requests to NaomiSakashita, MD, PhD, Department of Human Pathology, Instituteof Health Biosciences, the University of Tokushima GraduateSchool, 3 -18-15 Kuramotocho, Tokushima 770-8503, Japan andFax : +81-88-633-9423.

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to the endoplasmic reticulum (ER), where acyl co-enzyme A : cholesterol acyltransferase (ACAT) re-sides. ACAT catalyzes the esterification of free cho-lesterol, so that modified or native LDL-derived freecholesterol is re-esterified at the ER, released intothe cytoplasm, and stored as lipid droplets. Theseprocesses cause foamy transformation of the macro-phages (4, 5).

During our morphological analysis of foamy trans-formed macrophages, we discovered a unique, cho-lesterol-induced, functional endosomal organelle :ACAT1-positive late endosomes (ACAT1-LE). Inthis review article, we describe our discovery ofACAT1-LE and discuss the functional significance ofthis unique cholesterol-induced organelle. In addi-tion, we describe the clinical significance of ACAT1-LE induction on the congenital endosomal storagedisease--Niemann Pick disease type C (NPC).

ACAT

The presence of a cholesterol-esterifying enzymein mammalian cells has been known since the 1960s.Its enzymatic activity was usually detected in micro-somal fractions, so ACAT is said to reside in the ER.Chang et al. first cloned ACAT in 1993 (6), and ex-tensive genetic investigations then produced thecloned ACAT isozyme ACAT2 (after which the firstcloned ACAT was renamed ACAT1) (7). Most hu-man somatic cells express ACAT1 ; in particular,macrophages, adrenal cortex cells, and sebaceouscells express a significant amount of ACAT1 (8). Incontrast, ACAT2 expression is limited to the apicalregion of enterocytes, fetal hepatocytes, and well-differentiated macrophages (9-11). Under physi-ological conditions, ACAT functions in intracellularcholesterol homeostasis in mammalian cells, espe-cially in macrophages and steroid hormone-produc-ing cells ; in addition, it regulates lipoprotein syn-thesis in hepatocytes and enterocytes (5). Artificialinhibition of ACAT enzymatic activity thereforecauses impaired lipoprotein synthesis as well asER stress-mediated apoptosis via free cholesterol-induced cell toxicity (12).

In pathological situations, ACAT, by esterifyingcholesterol, serves a critical function as a protectoragainst free cholesterol-induced cell toxicity. Thepresence of foamy transformed macrophages inatherosclerotic plaques therefore indicates ACAT-de-pendent macrophage protection against massive cho-lesterol loading. Foamy transformed macrophages

in atheromatous plaques, i.e. macrophages withmassive cholesterol loading, continuously internalizemodified LDL via scavenger receptors. The key is-sue for foamy transformed macrophages in athero-matous plaques relates to sufficient ACAT1 expres-sion (mature macrophages express both ACAT1and ACAT2, and 90% of enzymatic activity derivesfrom ACAT1) (11). In fact, a high-fat diet fed toatherogenic mice with ACAT1-null macrophagesled to a reduced number of foamy transformedmacrophages and consequently severe inflamma-tion, which resulted in increased atheromatousplaque formation (13). These data prompted us toquestion how macrophages protect themselves fromcholesterol-induced cell toxicity. The answer to thisbasic question is ACAT1-LE.

ACAT1 VESICLES AND ACAT1-LE

As noted above, ACAT is an ER-resident enzyme.We confirmed the location of ACAT1 to be the tu-bular ER membrane in normal human macrophages(Figure 1A, intact M ) (14). Next, we tried to detectthe ACAT1 signal in macrophages under chole-sterol-rich conditions. Treatment with a cholesteroldonor (acetylated LDL) caused the ACAT1 signalin human macrophages to appear on small vesiclesapproximately 100 nm in diameter (Figure 1A, cho-lesterol-rich M ). The ER marker protein GRP78also appeared on small vesicles after cholesteroldonor treatment (Figure 1B, cholesterol-rich M ).Furthermore, both ACAT1 and GRP78 signals cor-related with each other before and after foamy trans-formation (Figure 1C), and the expression level ofACAT1 did not change before and after cholesteroldonor treatment (data not shown). These data thusindicate that cholesterol-rich, foamy transformedmacrophages produce ACAT1-positive vesicles viaER fragmentation.

ACAT is a representative allosteric enzyme whoseenzymatic activity increases after cholesterol binds toits allosteric site. We thus believed that formation ofACAT1 vesicles would be related to allosteric regu-lation. A series of investigations of the significanceof ACAT1 vesicle formation led to the discovery ofACAT1-LE. As Figure 2 shows, separate ACAT1 sig-nals and signals of the late endosomal marker proteinlysosomal-associated membrane protein 2 (LAMP2)were detected in macrophages with no lipid accumu-lation (Figure 2A, intact M ). In contrast, approxi-mately 20% of the ACAT1 signal corresponded to the

The Journal of Medical Investigation Vol. 61 August 2014 271

intact M cholesterol rich MA

B

C

Figure 1 ACAT1 vesicle formation in cholesterol -rich macrophagesHuman macrophages derived from peripheral blood monocytes with or without acetylated LDL treatment were subjected to immu-noelectron microscopy (post-embedding method) and confocal laser scanning microscopy. (A) Immunoelectron microscopy afterusing ACAT1-specific antibody. (B) Immunoelectron microscopy after using GRP78 (ER marker protein)-specific antibody. The insetin A is an enlarged image. Arrows indicate specific signals. (C) Confocal laser scanning microscopy after using ACAT1-specific anti-body (green) and GRP78-specific antibody (red). Yellow in the merged image indicates a colocalization signal. Details are provided inthe text. Adapted from references 9 and 14.

N. Sakashita, et al. M with ACAT1-LE in metabolic disease272

LAMP2 signal in foamy transformed macrophages(Figure 2A, cholesterol-rich M , Figure 2B). Thepurified ACAT1-positive organelles from cholesterol-rich macrophages contained LAMP2, but those fromintact macrophages did not (Figure 2C), and im-munoelectron microscopy confirmed the presence

of the ACAT1 signal on beads containing phago-somal membranes from only foamy transformedmacrophages (Figure 2D), not intact macrophages.These data thus indicate that foamy transformedmacrophages form ACAT1-LE via ER fragmenta-tion (14, 15).

A

B

C D

Figure 2 ACAT1-LE in intact and cholesterol -rich macrophages(A, B) Human macrophages derived from blood-borne monocytes with or without aggregated LDL treatment were subjected to im-munofluorescence staining and quantitative colocalization analysis. The green signal indicates ACAT1, the magenta signal indicatesLAMP2, and the white signal indicates colocalization. DIC indicates differential interference contrast microscopy. (C) Macrophageswith or without aggregated LDL treatment were homogenized and subjected to immunoadsorption with beads conjugated to ACAT1-specific antibody. Immunoadsorbed ACAT1-positive particles were analyzed by means of immunoblotting. Sup., supernatant. (D) Macro-phages with or without methyl -β -cyclodextrin-cholesterol complex (mβCD-cho) treatment (cholesterol donor) were incubated withfluorescent beads to investigate phagocytosis. Macrophages after phagocytosis were homogenized and subjected to ultracentrifugationto collect beads containing phagosomes, and the phagosomal fraction was further subjected to immunoelectron microscopy (pre-embedding technique). Arrows indicate ACAT1-positive colloidal gold signals. Details are provided in the text. Adapted from refer-ences 14 and 15.


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ACAT1-LE AND CHOLESTEROL METABO-LISM

As described above, the late endosome is an or-ganelle that contains free cholesterol derived frominternalized LDL, and ACAT1 is an enzyme that es-terifies free cholesterol. ACAT1-LE would thereforebe an organelle that would re-esterify LDL-derivedfree cholesterol in loco . To demonstrate efficientcholesterol esterification at endosomes, we investi-gated cholesterol esterification kinetics in macro-phages with ACAT1-LE. Figure 3A illustrates thatintact macrophages required at least 2 hours to re-esterify LDL-derived free cholesterol because ofthe complex intracellular cholesterol trafficking

system described here, in the Introduction. In con-trast, foamy transformed macrophages with ACAT1-LE esterified a significant amount of LDL-derivedcholesterol within 30 minutes. This finding suggestsefficient cholesterol esterification in ACAT1-LE-positive macrophages. To demonstrate directly thatACAT1-LE is the site of cholesterol esterification,we performed an experiment in which cholesterolegress was inhibited. U18666A is an amphiphilicamino-steroid that blocks free cholesterol egressfrom late endosomes by interfering with the choles-terol trafficking protein NPC1 (5). Macrophagestreated with U18666A showed negligible cholesterolesterification because no cholesterol reached theER, where ACAT1 resides. In contrast, foamy trans-formed ACAT1-LE-positive macrophages esterifieda significant amount of free cholesterol even in thepresence of U18666A (Figure 3B). These data aredirect evidence of cholesterol esterification at theACAT1-LE, i.e. this unique organelle effectively es-terified LDL-derived free cholesterol just after LDLhydrolysis at late endosomes. This finding stimu-lated us to pursue the therapeutic application ofACAT1-LE to NPC.

NPC AND ACAT1-LE INDUCTION

Niemann-Pick disease is a congenital lysosomalstorage disease characterized by massive sphingo-myelin accumulation. This disease was traditionallyclassified as type A to type E according to its clinicalmanifestations, but molecular analysis of the diseaseled to reclassification of types according to two cri-teria : Niemann-Pick disease type A, with an acidsphingomyelinase deficiency, and NPC, with anNPC1 protein deficiency (16). Deletion of the NPC1protein causes free cholesterol accumulation in lateendosomes, which results in inhibition of acid sphin-gomyelinase. As a result, NPC accumulates freecholesterol as well as sphingomyelin, with conse-quent cell injury. One possible therapeutic strategyfor NPC is using methyl-β -cyclodextrin (mβCD),which is a cyclic oligosaccharide, to incorporatehydrophobic molecules such as cholesterol. mβCDis internalized via pinocytosis, remains at late en-dosomes, and removes accumulated free cholesterolfrom late endosomes and plasma membranes ofcells with the NPC phenotype (npc-/-) (17). Admini-stration of mβCD to npc-/- mice ameliorated tissueinjury and prolonged survival, and this beneficial ef-fect was also observed in patients with NPC (18, 19).

A

B

Figure 3 Functional analysis of ACAT1-LE(A) Human macrophages derived from blood-borne monocyteswith or without mβCD-cho (cholesterol donor) treatment wereincubated with [3H]cholesterol - labeled acetylated LDL. Macro-phages after incubation with radiolabeled acetylated LDL weresubjected to the [3H]cholesteryl ester formation assay. (B) Hu-man macrophages with or without treatment with mβCD-choand the amphiphilic amino-steroid U18666A were incubated with[3H]cholesterol - labeled acetylated LDL. They were then sub-jected to the [3H]cholesteryl ester formation assay. Details areprovided in the text. Adapted from references 14 and 15.


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In contrast to the approach of removing choles-terol by using mβCD, we believed that inducingACAT1-LE in npc-/- macrophages would also effec-tively reduce the intracellular cholesterol level bymeans of cholesterol esterification. Indeed, treat-ment with mβCD-cholesterol complex (mβCD-cho),a cholesterol donor, effectively produced ACAT1-LE-positive npc-/- macrophages. These ACAT1-LE-positive npc-/- macrophages showed significant

cholesterol esterification and effective reduction ofthe intracellular cholesterol level (Figure 4A). Inaddition, administration of mβCD-cho to npc-/- neo-nate mice effectively extended their survival to thesame level as did conventional cholesterol removertreatment (Figure 4B) (20). These data thus showthat induction of ACAT1-LE in patients with NPCmay be an alternative therapeutic approach (Figure5).

A B

Figure 4 Therapeutic application of ACAT1-LE to NPC(A) Bone marrow-derived macrophages with the NPC phenotype (npc1-/-) with or without mβCD-cho treatment were then incubatedwith [3H]cholesterol - labeled acetylated LDL, after which they were subjected to the [3H]cholesteryl ester formation assay. (B) At 7 daysafter birth, npc1-/- neonates were treated with mβCD-cho (cholesterol donor) or mβCD (cholesterol remover). Survival of npc1-/-mice after treatment was determined. Details are provided in the text. Adapted from reference 20.

Figure 5 Schematic explanation of ACAT1-LE formation in NPCIn NPC, deficiency of the NPC1 protein causes massive accumulation of exogenous LDL-derived free cholesterol in late endosomes/lysosomes (LE/LS) in macrophages. This process results in significant cell injury and cell death. In contrast, pretreatment of macro-phages with mβCD-cho induces ACAT1-LE and esterification of free cholesterol in loco , which results in reduced cell injury and greatercell survival. Adapted from reference 20.


CLOSING REMARKS

The question of whether intracellular ACAT1 lo-calization would change under cholesterol-rich con-ditions (i.e. substrate-rich conditions for ACAT1)in human macrophages led us to discover ACAT1-LE and an alternative treatment strategy for NPC.Although in this review we did not mention theACAT1-LE degradative pathway, ACAT1-LE hasan impaired degradative pathway that depends onACAT enzymatic activity. The late endosome is notonly the principal organelle in the intracellular degra-dative pathway but is also the sorting compartmentfor protein synthesis. Therefore, ACAT1-LE-positivefoamy transformed macrophages may manifest im-paired protein sorting, which in hyperlipidemic con-ditions would result in disturbed protein synthesissuch as cytokine production. Further research mustclarify the details of ACAT1-LE function and itsmedical significance.

CONFLICT OF INTEREST

none

ACKNOWLEDGEMENT

This work was supported by Grants-in-Aid forScientific Research C-23590448, C-20590384, andB-17390115 to N. S. from the Japan Society for thePromotion of Science (JSPS).

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