Supplemental Material can be found at:
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 48, pp. 33357–33364, November 28, 2008
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Identification of Neutral Cholesterol Ester Hydrolase, a Key
Enzyme Removing Cholesterol from Macrophages*□
S

Received for publication, April 7, 2008, and in revised form, September 8, 2008 Published, JBC Papers in Press, September 9, 2008, DOI 10.1074/jbc.M802686200

Hiroaki Okazaki‡1, Masaki Igarashi‡1, Makiko Nishi‡, Motohiro Sekiya‡, Makiko Tajima‡, Satoru Takase‡,
Mikio Takanashi‡, Keisuke Ohta‡, Yoshiaki Tamura‡, Sachiko Okazaki‡, Naoya Yahagi‡, Ken Ohashi‡,
Michiyo Amemiya-Kudo‡§, Yoshimi Nakagawa¶, Ryozo Nagai储, Takashi Kadowaki‡, Jun-ichi Osuga‡,and Shun Ishibashi‡**2
From the ‡Departments of Metabolic Diseases and 储Cardiovascular Diseases, Graduate School of Medicine, University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, §Okinaka Memorial Institute for Medical Research and Toranomon Hospital,
Tokyo 105-0001, ¶Metabolism, Endocrinology, and Atherosclerosis, Institute of Clinical Medicine, University of Tsukuba,
Ibaraki 305-8575, and **Division of Endocrinology and Metabolism, Department of Medicine, School of Medicine, Jichi Medical
University, Tochigi 329-0498, Japan

Atherosclerotic cardiovascular diseases are the leading
causes of mortality in industrialized countries, despite advancesin the management of coronary risk factors. Heart attacks arise
from thrombotic occlusion of coronary arteries following the
rupture of plaques. Lipid-rich plaques, which are characterized
by a plethora of CE3-laden macrophage foam cells, are prone to* This work was supported by a grant-in-aid for scientific research from the
Ministry of Education, Science, and Culture and the Program for Promotionof Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation and Takeda Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1 and S2.
1
Both authors contributed equally to this work.
2
To whom correspondence should be addressed: Division of Endocrinology
and Metabolism, Dept. of Medicine, Jichi Medical University, 3311-1
Yakushiji, Shimotsuke, Tochigi 329-0498, Japan. Tel.: 81-285-58-7355; Fax:
81-285-40-6035; E-mail: ishibash@jichi.ac.jp.
3
The abbreviations used are: CE, cholesterol ester; HSL, hormone-sensitive
lipase; CEH, cholesteryl ester hydrolase; nCEH, neutral cholesteryl esterhydrolase; Ad, adenovirus; BSA, bovine serum albumin; LDL, low density

NOVEMBER 28, 2008 • VOLUME 283 • NUMBER 48

rupture (1). Thus, it is important to clarify the mechanism thateliminates CE from macrophage-derived foam cells.
Foam cells are generated by the unlimited uptake of modified
lipoproteins through scavenger receptors (2). Cholesterol in the
lipoproteins is stored in lipid droplets as CE after re-esterification by acyl-CoA:cholesterol acyltransferase 1 (ACAT1) (3).
Hydrolysis of CE is the initial step toward elimination of cholesterol from foam cells (4). Free cholesterol thus generated isre-esterified or is released from the cells primarily throughATP-binding cassette transporters (5). Thus, the balance
between synthesis and hydrolysis of CE conceivably governs thelevel of CE in macrophages.
Hydrolysis of CE in macrophages has been known for over 40
years (6). However, its molecular mechanism has yet to be fully
understood. Circumstantial evidence suggests that the hydrolysis of CE in foam cell macrophages is mediated by hormonesensitive lipase (HSL), a multifunctional enzyme that catalyzes
the hydrolysis of triacylglyerol (TG), diacylglycerol, CE, andretinyl ester in various organs such as adipose tissue, muscle,
and testis (7, 8). This belief is supported by the following facts.
First, it has been demonstrated that various lines of macrophages express HSL (9 –12). Second, HSL expression is regulated coordinately with nCEH activity in murine macrophages(13). Third, we (14) and others (15) demonstrated that overexpression of HSL is associated with increased hydrolysis of CE
stores in THP-1 and RAW264.7 macrophages.
However, recent studies by us (16) and others (17) have challenged this notion by finding that peritoneal macrophages isolated from HSL-deficient mice retained almost a normal level ofnCEH activity, suggesting that enzyme(s) other than HSL are
responsible for nCEH activity in macrophages. Moreover, HSL
is expressed at a low level (11, 12) or is undetectable (17) inhuman macrophages. Recently, Ghosh et al. (18) reported cloning of cholesteryl ester hydrolase (CEH) which mediates thehydrolysis of CE in human monocyte-derived macrophages.
They have further shown that macrophage-specific transgenic
expression of CEH significantly reduced atherosclerosis in low
lipoprotein; acLDL, acetylated LDL; DMEM, Dulbecco’s modified Eagle’s
medium; MPM, murine peritoneal macrophage; shRNA, short hairpin RNA;
PBS, phosphate-buffered saline; TGH, triacylglycerol hydrolase; GST, glutathione S-transferase; m.o.i., multiplicity of infection; TG, triacylglycerol.

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Unstable lipid-rich plaques in atherosclerosis are characterized by the accumulation of macrophage foam cells loaded withcholesterol ester (CE). Although hormone-sensitive lipase andcholesteryl ester hydrolase (CEH) have been proposed to mediate the hydrolysis of CE in macrophages, circumstantial evidence suggests the presence of other enzymes with neutral cholesterol ester hydrolase (nCEH) activity. Here we show that themurine orthologue of KIAA1363, designated as neutral cholesterol ester hydrolase (NCEH), is a microsomal nCEH with highexpression in murine and human macrophages. The effect ofvarious concentrations of NaCl on its nCEH activity resembles
that on endogenous nCEH activity of macrophages. RNA silencing of NCEH decreases nCEH activity at least by 50%; conversely, its overexpression inhibits the CE formation in macrophages. Immunohistochemistry reveals that NCEH is expressedin macrophage foam cells in atherosclerotic lesions. These data
indicate that NCEH is responsible for a major part of nCEH
activity in macrophages and may be a potential therapeutic target for the prevention of atherosclerosis.

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2008/09/10/M802686200.DC1.html

Neutral Cholesterol Ester Hydrolase in Macrophages
density lipoprotein receptor knock-out mice (19). However, theCEH gene is identical to the human orthologue of TGH, which
was originally reported as a microsomal TG lipase in rat liver byLehner et al. (20). In our hands, both the mouse orthologue of
TGH, designated as TGH-1, and its paralogue with 70.4% identity to TGH-1, designated as TGH-2, have negligible nCEH
activity (21). These considerations have prompted us to employ
a bioinformatic approach to identify the enzyme responsible for
nCEH activity in macrophages.

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EXPERIMENTAL PROCEDURES
Materials—Phorbol 12-myristate 13-acetate, triolein, lecithin, bovine serum albumin fraction V (BSA), p-nitrophenyl
butyrate, and leupeptin were purchased from Sigma. Dexamethasone, 3-isobutyl-2-methylxanthine, fatty acid-free BSA,
cholesterol esterase from Pseudomonas sp., cholesterol oxidase,
horseradish peroxidase, p-hydroxyphenylacetic acid, andsodium taurocholate were purchased from Wako Pure Chemicals (Osaka, Japan). Pioglitazone was provided by Takeda
Pharmaceutical (Osaka, Japan). Tri[3H]oleoylglycerol, cholesterol [1-14C]oleate, and [1-14C]oleic acid were purchased fromGE Healthcare. LDL was isolated by ultracentrifugation andacetylated to prepare acetylated LDL (acLDL) as described previously (14).
Cells—HEK293 or RAW264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) high glucose containing 10% (v/v) fetal bovine serum without or with 1 mM sodium
pyruvate, respectively. THP-1 cells were cultured in RPMI 1640
medium containing 10% (v/v) fetal bovine serum and differentiated to THP-1 macrophages by treatment with 100 nM phorbol 12-myristate 13-acetate for 48 h. Murine peritoneal macrophages (MPM) were harvested as described previously (16). In
brief, 1 ml of 5% thioglycolate broth was injected into the peritoneal cavities of mice. After 4 days, the peritoneal cavities werelavaged with 16 ml of ice-cold saline. The cells were washedthree times with PBS and resuspended in DMEM to give a concentration of 106 cells per ml, and 10 ml per dish was plated in
10-cm dishes. After incubation at 37 °C for 2 h, the nonadherent cells were removed by washing three times with warmed
PBS. Cells were incubated in DMEM containing 10% fetal calf
serum for 24 h and harvested for the experiments. 3T3-L1 cells
were cultured in medium A (DMEM containing 10% calf
serum, supplemented with calcium pantothenate and biotin).
To induce differentiation of 3T3-L1 cells into adipocytes, 2 day
postconfluent preadipocytes (day 0) were incubated with 1 ␮M
dexamethasone and 0.5 mM isobutylmethylxanthine, 5 ␮g/ml
insulin, 1 ␮M pioglitazone for 48 h, following treatment with 5
␮g/ml insulin and 1 ␮M pioglitazone for 48 h. After the incubation period, cells were switched to medium A, and the medium
was renewed every other day. Mononuclear cells were isolated
from a normal volunteer using Lymphoprep (NYCOMED,
Roskilde, Denmark), and cells that attached to plastic dishes
were cultured in RPMI 1640 medium containing 10% fetal calf
serum and used as human monocyte-derived macrophages.
Mice—C57BL/6J and apoE knock-out mice were purchasedfrom Clea (Tokyo, Japan) and The Jackson Laboratories (Bar
Harbor, ME), respectively. Mice were maintained and cared foraccording to the regulations of the Animal Care Committee ofthe University of Tokyo. For the preparation of atherosclerotic
aortas, mice were caged separately with 12-h light/dark cycles
and given free access to standard chow diet containing 0.075%
cholesterol (MF; Oriental Yeast Co., Ltd.; Osaka, Japan).
Data Base Search and Amino Acid Sequence Analysis—The
search for a novel lipase containing lipase consensus motifs and␣/␤-hydrolase folds was performed in the GENES protein database of Kyoto Encyclopedia of Genes and Genomes by using the
MOTIF search program (22), followed by prediction of secondary structure by PSIPRED and the PREDICT PROTEIN PHDsec program (23). The search for orthologues or paralogues of
NCEH was performed by Sequence Similarity Database in the
Kyoto Encyclopedia of Genes and Genomes Database (22).
Alignment of the deduced protein sequence with other lipases,
calculation of the percent identity, and generation of a phylogenetic tree were performed using the ClustalW program. Prediction of the transmembrane domain was performed using
SOSUI and the PREDICT PROTEIN PHDhtm program (23).
cDNA Cloning for the Expression of Recombinant Proteins—
Total RNA isolated from murine peritoneal macrophages wasreverse-transcribed into cDNA using reverse transcriptase following the manufacturer’s protocol (Thermoscript RNaseHreverse transcriptase (Invitrogen)). The coding sequences ofmurine NCEH were amplified by PCR from macrophage cDNA
using cDNA polymerase (AmpliTaq DNA polymerase (Roche
Applied Science)). The primers used for PCR were as follows:
murine NCEH forward 5⬘-CCGCCTGGGGACAATGAG-3⬘
and murine NCEH reverse 5⬘-TCTGAGGAGTTTCCTCCGTCA-3⬘.
The construction of cDNA for HSL was described previously. The products containing the complete open reading
frame were ligated into compatible sites of pGEM T easy
vector (Promega).
Recombinant Adenovirus for NCEH and HSL Expression (AdNCEH, Ad-HSL)—Murine NCEH or HSL cDNA fragment was
amplified by PCR from mouse cDNA containing plasmids(described above) using forward and reverse primers. LacZ
cDNA was amplified by PCR from SV40 ␤-galactosidase vector(Promega). The PCR products were ligated to pENTER4 vector(Invitrogen). Subsequently, murine NCEH or HSL cDNA fragment was subcloned into pAd/CMV/V5-DEST vector by Gateway Technology (Invitrogen). The adenoviral vectors were linearized using restriction enzyme and transfected into HEK293
using Superfect reagent (Promega). Large scale production ofhigh titer recombinant Ad-NCEH, Ad-HSL, or Ad-LacZ was
performed as described elsewhere (14). The purified viruses
were stored in 10% (v/v) glycerol/phosphate-buffered saline(PBS) at ⫺80 °C.
Recombinant Adenovirus Expressing Short Hairpin (sh) RNA
Directed against Murine NCEH—Recombinant adenovirus
expressing shRNA for NCEH (Ad-shNCEH) was produced by
the Gateway Technology (Invitrogen). The sense and antisense
templates of NCEH shRNA were produced from nucleotides
136 to 154 of the NCEH coding sequence.
The following oligonucleotides were used: shRNA for NCEH
sense template, 5⬘-GT GAG TAA TCT GAT ACG TTA CGTGTG CTG TCC GTA ATG TAT CAG GTT ACT CAC-3⬘;
shRNA for NCEH antisense template, 5⬘-GT GAG TAA CCTSupplemental Material can be found at:
http://www.jbc.org/content/suppl/2008/09/10/M802686200.DC1.html

Neutral Cholesterol Ester Hydrolase in Macrophages

NOVEMBER 28, 2008 • VOLUME 283 • NUMBER 48

pended and used as the microsomal fraction (14). nCEH activity
was measured essentially as described by Hajjar et al. (26), using
a reaction mixture containing 6.14 ␮M cholesterol [1-14C]oleate(48.8 ␮Ci/␮mol; 1 ␮Ci ⫽ 37 kBq). Triacylglycerol lipase activity
was measured according to a modification of the method of
Hajjar et al. (26). In brief, the samples were incubated at 37 °C
for 30 min in a final volume of 200 ␮l of a reaction mixture
containing 105 ␮M tri[3H]oleoylglycerol (99.4 ␮Ci/␮mol), 23.7
␮M lecithin, 12.5 ␮M sodium taurocholate, 1 M NaCl, and 85 mM
potassium phosphate, pH 7.0. The high concentration of NaCl
was included to inactivate lipoprotein lipase.
In Vitro Adenovirus Experiments—Two days after THP-1
cells were treated with phorbol 12-myristate 13-acetate, THP-1
macrophages were incubated in RPMI 1640 medium containing 5 mg/ml BSA and transduced with recombinant adenovirus
carrying NCEH, HSL, or LacZ as a control. After 24 h, acLDL
was added to the medium at a final concentration of 100 ␮g/ml
without or with 1% [14C]oleate-BSA complex (14), and cells
were incubated for 48 h at 37 °C. Cellular lipids were extractedusing hexane/isopropyl alcohol (3:2), and cholesterol content
was determined by enzymatic fluorometric microassay according to the method of Heider and Boyett (27), with minor modifications (25). The radioactivity of intracellular CE formed
from [14C]oleate was determined as described (14).
Immunohistochemical Localization—Cells were fixed with
3% paraformaldehyde after washing with PBS. Tissue samples
were fixed in 10% formalin, pH 7.0, embedded in paraffin, cut
into sections, and placed on glass slides. Tissue samples weredeparaffinized and rehydrated before use. The samples wereincubated in 3% H2O2 in PBS to quench endogenous peroxidase
activity and washed with PBS, and nonspecific binding sites
were blocked by incubation with 1% goat serum. The sections
were then incubated with affinity-purified anti-NCEH polyclonal antibody. After washing with PBS, the sections wereincubated with a biotinylated goat anti-rabbit antibody (1:200,
Vector Laboratories, Burlingame, CA). The antibody-specificstaining was visualized with a Vecstatin ABC reagent (Vector
Laboratories) and diamine benzidine, resulting in a brown precipitate. Cell nuclei were counterstained with methyl green.
Combined immunohistochemical staining was performed to
identify the cell types expressing NCEH as described previouslywith minor modifications (28). NCEH was first localized byusing the protocol above. Subsequently, the sections weretreated with 0.1% pepsin (Roche Applied Science) in 0.1 N HCl
for 30 min and incubated with rat monoclonal anti-murine
F4/80 (1:10, serotec) for 30 min. The respective sections wereincubated with a biotinylated rabbit anti-rat antibody (1:200,
Vector Laboratories) for 30 min, followed by a 30-min incubation with an alkaline phosphatase-coupled ABC reagent (1:200,
Vector Laboratories). Alkaline phosphatase activity was visualized by using the Alkaline Phosphatase Kit III (Vector Laboratories), resulting in a blue precipitate.
Statistical Analyses—Results are presented as means ⫾ S.E.
Student’s t test was employed to compare the means. All calculations were performed with STAT view version 5.0 for Macintosh (SAS Institute Inc.).
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GAT ACA TTA CGG ACA GCA CAC GTA ACG TAT CAG
ATT ACT CAC-3⬘. An adenovirus containing shRNA for ␤-galactosidase (Ad-shLacZ) was used as a control.
Murine peritoneal macrophages were seeded in 6-well plates
at a cell density of 1.7 ⫻ 106 cells/well. After transduction with
Ad-shNCEH or Ad-shLacZ for 48 h at 37 °C, cells were harvested, and whole cell lysates were assayed for enzymatic
activities.
Generation of Rabbit Polyclonal Antibodies against Murine
NCEH—To prepare polyclonal anti-murine NCEH antiserum,
amino acid residues containing the catalytic domain of murine
NCEH (amino acids 99 –250) were expressed in bacteria as a
glutathione S-transferase (GST) fusion protein, which was
purified by glutathione affinity chromatography, and used for
immunization of rabbits according to a standard protocol as
described previously (24). Serum of rabbits before immunization was used as a control. From serum samples with high antibody titers, IgG fractions were isolated using a protein G column (GE Healthcare).
Northern Blot Analyses—Total RNA was isolated from various tissues or cultured cells using TRIzol reagent according to
the manufacturer’s protocol (Invitrogen). RNA (10 ␮g) was
subjected to formaldehyde/agarose gel electrophoresis andblotted to a Hybond-N membrane (GE Healthcare). Aftercross-linking, mouse NCEH, HSL, and TGH-1 mRNA weredetected using 32P-labeled probes. Probes for murine NCEH
were originally prepared by reverse transcription-PCR frommouse peritoneal macrophage mRNA. Probes for HSL (exon 8
probe), TGH-1 were constructed from cDNA fragments amplified by reverse transcription-PCR using cDNA obtained frommouse adipose tissue as a template (14). After hybridization andwashing, signals were visualized by exposure to a PhosphorImager Screen (FUJIFILM) and analyzed using BASTATION(FUJIFILM) software.
Western Blot Analysis—Cells were sonicated in buffer A andcentrifuged at 40,000 or 100,000 ⫻ g for 45 min at 4 °C. The
supernatant was used as S-40 or S-100 cytosolic fraction, andthe precipitates were resuspended and used as the microsomal
fraction (14). Ten micrograms of proteins of various cellular
fractions were separated on a 10% SDS-PAGE and transferred
to a nitrocellulose membrane. For detection of the proteins, themembranes were incubated with either anti-murine NCEH,
anti-murine HSL antiserum, monoclonal anti-mouse ACAT1
antibody (25), or anti-F4/80 antibody (AbD serotec) at a dilution of 1:100 –1,000. Specifically bound immunoglobulins weredetected in a second reaction with a horseradish peroxidaselabeled IgG conjugate and visualized by enhanced chemiluminescence detection (ECL Plus, GE Healthcare) using a Kodak
image system, and the intensity of immunoreactive bands were
quantified by NIH-image software.
Assays for CE Hydrolase and TG Lipase Activities—Wholecell lysates were prepared from transfected HEK293, murine
peritoneal macrophages, or THP1 cells and used for the enzyme
activity assays. These cells were sonicated in buffer A (50 mmol/
liter Tris-HCl, pH 7.0, 250 mmol/liter sucrose, 1 mmol/liter
EDTA, 2 ␮g/ml leupeptin) and centrifuged at 100,000 ⫻ g for 45
min at 4 °C. The supernatant was used as S-100 cytosolic fraction, respectively. The precipitates at 100,000 ⫻ g were resus-Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2008/09/10/M802686200.DC1.html

Neutral Cholesterol Ester Hydrolase in Macrophages
RESULTS AND DISCUSSION

33360 JOURNAL OF BIOLOGICAL CHEMISTRY

FIGURE 1. Structure of NCEH. Sequence alignment of murine NCEH withhuman, rat, and mouse HSL. The deduced amino acid sequence of murine NCEH
protein (NCEH) is aligned and compared with murine, rat, and human HSL(murHSL, ratHSL, and humHSL). The first Met residue is numbered as 1. HSL has
four functional domains as follows: 1) the N-terminal 300 residues domain, containing the ALBP-binding site (boldface); 2) catalytic core domain (line), containing the HG dipeptide oxyanion motif (shaded box) and GXSXG active serine motif(shaded box); 3) regulatory module domain, containing five serine residues, Ser557,Ser-559,Ser-591,Ser-650,andSer-65,whichcanbephosphorylatedbycAMPdependent protein kinase (solid circle) or other kinases such as AMP-activatedprotein kinase or extracellular signal-regulated kinase (ERK) (open circle); 4) lipidbinding domain (dashed line), containing a putative lipid-binding site and two of
the catalytic triad, i.e. Asp-694 and His-724 (shaded box). The predicted secondary
structure was shown as follows: ␣-helix is indicated by a line; ␤-sheet is indicatedby an arrow; secondary structure of HSL and NCEH is shown in gray and black,respectively. The mature NCEH protein is highly conserved during evolution andhas homology to HSL in these motifs and secondary structures, except for the
regulatory domain and the N-terminal 300 residue domain. NCEH has an N-terminal transmembrane region (outlined letters). Identical amino acids are shownby asterisks. Amino acids considered as strongly conserved are as follows: STA,
NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, and FYW, and are indicated by colons.
Amino acids considered as weakly conserved are as follows: CSA, ATV, SAG, STNK,
STPA, SGND, SNDEQK, NDEQHK, NEQHRK, FVLIM, and HFY, and are indicated by
dots.

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To search for enzymes with CE hydrolase activity, we
screened a gene data base for murine and human proteins withhomology to structures in known lipases, i.e. ␣/␤-hydrolase
folds (29), the GXSXG active serine motif for serine esterases,
and the HG dipeptide motif that is present 70 –100 amino acids
N-terminal of the catalytic site serine in many lipases (29),yielding 53 candidates that included HSL, lipoprotein lipase,hepatic lipase, endothelial lipase, pancreatic lipase, carboxyl
ester lipase, gastric lipase, lysosomal acid lipase, monoacylglycerol lipase, TGH-1, and TGH-2 (21). Thirty three genes had notbeen previously annotated. We expressed the candidate genes
in HEK293 cells for measurement of nCEH activity. Northernblot analysis was used to verify the expression in murine peritoneal macrophages. Besides HSL, only one fulfilled theserequirements.
The murine gene for NCEH (NCBI nucleotide entry
AK045363) encodes a 408-amino acid protein (GenBankTM
accession number NP_848887) with a calculated molecularmass of 45.7 kDa. KIAA1363, which is a human orthologue ofmurine brain chlorpyrifos oxon-binding protein (30) that wasrecently identified during our study, is the human orthologue of
NCEH and encodes a 440-amino acid protein (GenBankTM
accession number NP_065843) with 87.5% identity to themurine NCEH in the C-terminal 408 amino acids. The aminoacid sequences of murine NCEH and its closely related proteins, murine, rat, and human HSL, are shown in Fig. 1. The
structures of the predicted ␣-helix and ␤-sheet are well conserved between NCEH and HSL. NCEH contains domains thatare present in HSL such as the catalytic core domain and lipidbinding domain (31, 32), and is thus apparently homologous to
HSL in terms of domain structure. Based on the homology with
HSL, it can be predicted that Ser-191, Asp-348, and His-378 of
the murine NCEH protein form a putative catalytic triad, andHis-113 and Gly-114 form an HG oxyanion motif. Despite theremarkably high structural homology between NCEH and HSL,
NCEH possesses only 22.1% sequence identity to HSL. In addition to the absence of the regulatory domain of HSL, NCEH
lacks the N-terminal region of HSL. The N-terminal region of
NCEH instead contains a stretch of 23 hydrophobic aminoacids, a putative transmembrane domain, which is absent in
HSL, suggesting distinct subcellular localization of these two
proteins.
Analysis using the ClustalW program supported the phylogenetic kinship between NCEH and HSL (supplemental Fig. S1).
Neutral lipid hydrolases are clustered essentially into three large
groups as follows: 1) an extracellular lipase superfamily comprising
lipoprotein lipase, endothelial lipase, hepatic lipase, and pancreatic
lipase; 2) a carboxylesterase family comprising carboxyl ester
lipase, TGH-1, and TGH-2; and 3) a distinct “intracellular lipase”
gene family comprising HSL and NCEH. Lysosomal acid lipase
and adipose triglyceride lipase do not belong to any of these families. Arylacetamide deacetylase shows weak similarity (44.0%) tomurine NCEH, and there is no other mouse protein with muchsequence similarity to murine NCEH.
Northern blot analysis revealed that NCEH mRNA wasexpressed at high levels in peritoneal macrophages and kidney,

Supplemental Material can be found at:
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protein.4 The ratio of NCEH protein
to HSL protein was calculated to be
11 in murine peritoneal macrophages and 1 in RAW264.7 cells,
respectively.
The preferential localization of
HSL and NCEH in the cytosol andmicrosomal fractions, respectively,
was also confirmed by measurement of the enzymatic activity ofeach fraction in cells transiently
transfected with plasmids expressing HSL or NCEH. Although mostof the nCEH activity of HSL-transfected cells was recovered in the
S-40 fraction, nCEH activity wasbarely detectable in the S-40 fraction of NCEH-transfected cells aswell as murine peritoneal macrophages (data not shown). This subFIGURE 2. Tissue and subcellular distribution of NCEH expression. Total RNA (10 ␮g) from adipose tissues cellular localization pattern isand 3T3-L1 adipocytes at various stages of differentiation and murine peritoneal macrophages (A), various compatible with the microsomal
murine tissues (B), and human monocytes/monocyte-derived macrophages (C) were subjected to Northernblot analysis. Specific mRNAs were detected with radiolabeled murine cDNAs for NCEH, HSL, and TGH-1. distribution of NCEH and its preEthidium bromide staining of the gels is shown. Abbreviations used are as follows: WAT, white adipose tissue; ponderance in murine peritoneal
BAT, brown adipose tissue; MPM, murine peritoneal macrophages; ed, epididymal; sc, subcutaneous. Subcellular distribution of nCEH activity and NCEH and HSL proteins. MPM were sonicated and centrifuged at macrophages. These results are
100,000 ⫻ g and the supernatant (S-100) or microsomal (Ms) fractions along with whole cell lysate (whole) were apparently contrary to those resubjected to the measurements of nCEH activity (D) and Western blot analysis (E) using anti-HSL and anti-NCEH ported by Khoo et al. (33), who
antisera. F, quantification of NCEH or HSL proteins in macrophages. Ten micrograms of proteins of whole celllysates from RAW264.7 and MPM were separated by SDS-PAGE on the same gel as the indicated amounts of showed that 60% of the CE hydroGST fusion proteins (supplemental Fig. S2). Immunoblotting was performed, and the densities of bands from lase activity in the homogenate of
HSL or NCEH of RAW264.7 or MPM were quantified using NIH Image. Blots for ACAT1 and F4/80 are shown as
J774 cells was recovered in the S-40
controls. The moles of HSL or NCEH in 10 ␮g of protein from RAW264.7 or MPM were calculated from equations
fraction; this difference could be
relating moles and densities of GST fusion proteins.
attributed to the high level ofexpression of HSL in J774 cells (34)and to a lesser degree in heart and adrenal tissue (Fig. 2, A and the high level of expression of NCEH in murine peritoneal
and B). Compared with other enzymes with nCEH activity macrophages.
expressed in macrophages, such as HSL and TGH-1, a murine
To further determine enzymological characteristics oforthologue of CEH reported by Ghosh (18), the expression of NCEH, we overexpressed NCEH in HEK293 cells (Fig. 3A) and
NCEH was the most abundant and specific for macrophages measured enzymatic activities of their whole cell lysate at vari(Fig. 2A). Although NCEH expression was barely detectable in ous concentrations of NaCl (Fig. 3B), pH (Fig. 3D), and concenfreshly isolated human monocytes, it was robustly induced dur- trations of cholesterol oleate (Fig. 3E) or triolein (Fig. 3F) as
ing the differentiation to mature macrophages (Fig. 2C).
substrates.
To determine the subcellular localization of endogenous
The nCEH activity in the whole cell lysates from NCEHnCEH activity and NCEH protein, we isolated S-100 and micro- transfected cells was slightly stimulated by low concentrations
somal fractions from murine peritoneal macrophages, meas- of sodium chloride (6.5% at 0.05 M), and inhibited only by 31% atured the nCEH activity (Fig. 2D), and performed Western blot higher concentrations (1 M) (Fig. 3B). This pattern closely
analysis for either NCEH or HSL (Fig. 2E). The nCEH activity resembled that of murine peritoneal macrophages (Fig. 3C).
was distributed preferentially in the microsomal fraction. The CE hydrolase activity of NCEH-transfected cells was optiNCEH proteins, duplets with molecular mass of 45 and 50 kDa, mal at neutral pH (7.2) (Fig. 3D), which is close to the optimal
were also distributed preferentially in the microsomal fraction. pH for HSL, suggesting that both enzymes are not active or at
HSL was more equally distributed between the two fractions. least not fully functional in lysosomes.
Thus, the subcellular distribution pattern of nCEH activity is
The whole cell lysate of HSL-transfected cells exhibitedcloser to that of NCEH protein than that of the HSL protein.
increased hydrolase activities for both TG and CE, with apparWe compared the amounts of NCEH protein with those of ent Km of 11.7 and 22.8 ␮M, respectively. On the other hand,
HSL protein in murine peritoneal macrophages and RAW264.7 whole cell lysate of NCEH-transfected cells hydrolyzed TG andcells using recombinant proteins as standards for calculation CE with apparent Km of 30.3 and 324 ␮M, respectively.
(Fig. 2F and see supplemental Fig. S2). Here again, two bands
were reacted with anti-NCEH antibody. Subsequent study
showed that both bands are products of glycation of the 40-kDa 4 M. Igarashi, manuscript in preparation.

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2008/09/10/M802686200.DC1.html

Neutral Cholesterol Ester Hydrolase in Macrophages

33362 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 283 • NUMBER 48 • NOVEMBER 28, 2008

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41%, respectively, compared with
Ad-shLacZ. In parallel with the
inhibition of NCEH, nCEH activities were reduced by 49 and 42% by
infection with 300 and 600 m.o.i.
of Ad-shNCEH, respectively (Fig.
4B). These results indicate that atleast half of the nCEH activity is
mediated by NCEH in murine
peritoneal macrophages.
To further examine whether
NCEH inhibits accumulation of
CE in macrophages, we infected
THP-1 cells, which had been
induced to differentiate to mature
macrophages by incubation withphorbol ester, with Ad-NCEH,
Ad-HSL, or Ad-LacZ, and incubated the cells with acetyl-LDL,
and compared the amounts of CE in
the cells (Fig. 5C) and CE formationfrom [14C]oleate (Fig. 5D). The cells
infected
with Ad-HSL or Ad-NCEH
FIGURE 3. Enzymological characteristics of NCEH. HEK293 cells were infected with Ad-LacZ, Ad-HSL, or
Ad-NCEH and used for the experiments. A, expression of NCEH or HSL was confirmed by Western blot analysis. expressed HSL or NCEH proteins,
Effects of various concentrations of NaCl on the nCEH activity of whole cell lysate from cells infected with
Ad-NCEH (B) or of MPM (C). D, effects of various pH on the nCEH activity of whole cell lysate from cells infected respectively, in a dose-dependent
with Ad-NCEH using 50 mM acetate buffer (pH ⬉ 5), 50 mM phosphate buffer (5 ⬍ pH ⬍ 7.6), or 50 mM Tris-HCl manner (Fig. 5A) and showed dosebuffer (pH ⭌ 7.6). Shown are the effects of various concentrations of cholesterol oleate (E) or triolein (F) on the dependent increases of nCEH activnCEH or triglyceride lipase activities, respectively, of whole cell lysate from cells infected with Ad-NCEH or
Ad-HSL. Values for Ad-LacZ were subtracted from those for Ad-NCEH or Ad-HSL and plotted against the con- ity in whole cell lysates (Fig. 5B). At
centrations of substrates. Km values were calculated by fitting lines in Lineweaver-Burk plots.
300 m.o.i., the nCEH activity in AdNCEH-infected cells was 14% thatin Ad-HSL-infected cells (Fig. 5B). This increase in nCEH activity in cells overexpressing HSL or NCEH was associated withdecreases in the intracellular CE content (Fig. 5C) as well as in
the rate of CE formation from [14C]oleate (Fig. 5D). More pronounced inhibition of CE accumulation by Ad-HSL than by
Ad-NCEH may be explained by the higher nCEH activity
attained by Ad-HSL.
To determine whether NCEH protein is expressed in atherosclerotic lesions, we performed immunohistochemistry(Fig. 6). Cultured peritoneal macrophages were positively
stained with anti-NCEH antibody (brown, Fig. 6B) but notwith nonimmune IgG (Fig. 6A). Next, we stained tissue sections of aorta from apoE⫺/⫺ mice (Fig. 6C), which contained
FIGURE 4. Effects of RNA interference of NCEH to inhibit protein expression and nCEH activity in murine peritoneal macrophages. Recombinant advanced atherosclerotic lesions filled with cholesterol cleft.
adenoviruses coding for shRNA against LacZ (Ad-shLacZ) or NCEH (Ad- Double staining with F4/80, a macrophage-specific antibody
shNCEH) were used to infect murine peritoneal macrophages at 300 and
600 m.o.i. Two days after infection, whole cell lysates were subjected to West- (blue, Fig. 6E), showed prominent expression of NCEH proern blotting (A) and the measurement of nCEH activity (B). This is a represent- tein (brown, Fig. 6, D and E) in macrophages surrounding theative result of two independent experiments.
cholesterol cleft in the subintimal space of apoE⫺/⫺ aorta.
In summary, NCEH is substantially expressed in macroTo determine more directly how much of the nCEH activ- phages in atherosclerotic plaques and significantly contribity is accounted for by NCEH in murine peritoneal macro- utes to nCEH activity of murine macrophages. This study isphages, we used RNA silencing technology (Fig. 4). Infection the first demonstration of the molecular identity of the domwith increasing doses of Ad-shLacZ nonspecifically reduced inant nCEH in macrophages, a long unidentified enzyme catathe protein expression of both HSL and NCEH (Fig. 4A) as lyzing the counter-reaction of acyl-CoA:cholesterol acyltranswell as nCEH activities in the infected cells (Fig. 4B). ferase. Although our data do not exclude the possibility that
Whereas infection with 300 and 600 m.o.i. of Ad-shNCEH HSL or possibly other enzymes account at least in part for the
did not specifically reduce the protein expression of HSL, it nCEH activity in macrophages, it is tempting to speculate that
reduced the protein expression of NCEH by 51 and HSL evolves to have a more specialized role in adipocyte lipol-Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2008/09/10/M802686200.DC1.html

Neutral Cholesterol Ester Hydrolase in Macrophages

Acknowledgments—We thank Drs. Joseph L. Goldstein, Michael S.
Brown, David W. Russell, Hitoshi Shimano, and Nobuhiro Yamada
for helpful discussions.

REFERENCESFIGURE 6. Expression of NCEH in murine peritoneal macrophages andin foamy macrophages in atherosclerotic plaques. Expression of NCEH
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Peroxidase activity was visualized using 3,3⬘-diaminobenzidine (brown) or
Vector blue (blue), and sections were counterstained with 3% methyl
green. As a control experiment, staining of MPM (A) and the aorta (C) withpreimmune rabbit IgG as a primary antibody did not produce any horseradish peroxidase cross-reactivity. Objective magnifications are ⫻400 (Aand B) and ⫻100 (C–E).

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Based on the negligible nCEH activity of overexpressed TGH-1(21) and its marginal expression in murine peritoneal macrophages (Fig. 2A), it is unlikely that CEH plays a more significant
role in CE hydrolysis than HSL or NCEH at least in mice. FurNOVEMBER 28, 2008 • VOLUME 283 • NUMBER 48

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Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2008/09/10/M802686200.DC1.html

Neutral Cholesterol Ester Hydrolase in Macrophages

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