THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 279, No. 30, Issue of July 23, pp. 30938 –30944, 2004
Printed in U.S.A.

Scavenger Receptor Expressed by Endothelial Cells I (SREC-I)Mediates the Uptake of Acetylated Low Density Lipoproteins by
Macrophages Stimulated with Lipopolysaccharide*
Received for publication, December 1, 2003, and in revised form, May 11, 2004
Published, JBC Papers in Press, May 15, 2004, DOI 10.1074/jbc.M313088200

Yoshiaki Tamura‡, Jun-ichi Osuga‡, Hideki Adachi§, Ryu-ichi Tozawa‡, Yasukazu Takanezawa¶,
Ken Ohashi‡, Naoya Yahagi‡, Motohiro Sekiya‡, Hiroaki Okazaki‡, Sachiko Tomita‡,
Yoko Iizuka‡, Hiroyuki Koizumi¶, Toshihiro Inaba储, Hiroaki Yagyu储, Nobuo Kamada**,Hiroshi Suzuki‡‡, Hitoshi Shimano§§, Takashi Kadowaki‡, Masafumi Tsujimoto§, Hiroyuki Arai¶,Nobuhiro Yamada§§, and Shun Ishibashi‡储¶¶
From the ‡Department of Metabolic Diseases, Faculty of Medicine, University of Tokyo 7-3-1 Hongo, Bunkyo-ku, Tokyo
113-8655, Japan, the §Laboratory of Cellular Biochemistry, Institute of Physical and Chemical Research (RIKEN),
Wako-shi, Saitama 351-0198, Japan, the ¶Department of Health Chemistry, Graduate School of Pharmaceutical Science,
University of Tokyo 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, the 储Department of Internal Medicine, Jichi Medical
School, 3311-1 Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi 329-0498, Japan, the **Chugai ResearchInstitute for Medical Science Incorporated, 1-135 Komakado, Gotenba, Shizuoka 412-8513, Japan, the ‡‡Unit for
Functional Genomics, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary
Medicine, Nishi-2, Inada, Obihiro, Hokkaido 080-8555, Japan, and §§Metabolism, Endocrinology, and Atherosclerosis,
Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan

Scavenger receptor expressed by endothelial cells I(SREC-I) is a novel endocytic receptor for acetylated low
density lipoprotein (LDL). Here we show that SREC-I isexpressed in a wide variety of tissues, including macrophages and aortas. Lipopolysaccharide (LPS) robustly
stimulated the expression of SREC-I in macrophages. In
an initial attempt to clarify the role of SREC-I in the
uptake of modified lipoproteins as well as in the development of atherosclerosis, we generated mice with atargeted disruption of the SREC-I gene by homologous
recombination in embryonic stem cells. To exclude theoverwhelming effect of the type A scavenger receptor(SR-A) on the uptake of Ac-LDL, we further generated
mice lacking both SR-A and SREC-I (SR-Aⴚ/ⴚ;SREC-Iⴚ/ⴚ)by cross-breeding and compared the uptake and degradation of Ac-LDL in the isolated macrophages. The contribution of SR-A and SREC-I to the overall degradationof Ac-LDL was 85 and 5%, respectively, in a non-stimulated condition. LPS increased the uptake and degradation of Ac-LDL by 1.8-fold. In this condition, the contribution of SR-A and SREC-I to the overall degradation of
Ac-LDL was 90 and 6%, respectively. LPS increased theabsolute contribution of SR-A and SREC-I by 1.9- and2.3-fold, respectively. On the other hand, LPS decreased
the absolute contribution of other pathways by 31%.
Consistently, LPS did not increase the expression ofother members of the scavenger receptor family such as
CD36. In conclusion, SREC-I serves as a major endocytic
receptor for Ac-LDL in LPS-stimulated macrophageslacking SR-A, suggesting that it has a key role in thedevelopment of atherosclerosis in concert with SR-A.
* This work was supported by a grant-in-aid for Scientific Researchfrom the Ministry of Education, Science, and Culture and by the Takeda
Medical Research 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.
¶¶ To whom correspondence should be addressed: Division of Endocrinology and Metabolism, Department of Internal Medicine, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-machi, Kawachi-gun,Tochigi 329-0498, Japan. Tel.: 81-285-58-7355; Fax: 81-285-40-6035;
E-mail: ishibash@jichi.ac.jp.

Scavenger receptors mediate the endocytosis of chemicallymodified lipoproteins such as acetylated low density lipoprotein (LDL),1 thereby contributing to the development of atherosclerosis (1). The scavenger receptor gene family comprises a
series of unlinked genes encoding membrane proteins withdiverse ligand binding activity (2). The class A type I/type II
scavenger receptor (SR-A) is the prototype receptor belonging
to this family (3) and accounts for ⬃80% of the uptake of
Ac-LDL in macrophages (4, 5).
Recently, we identified scavenger receptor expressed by endothelial cells I (SREC-I), which encodes a protein of 830 aminoacids and binds fluorescent DiI-labeled Ac-LDL when expressed in Chinese hamster ovary cells (6), and its paralogous
gene, SREC-II (7). The SREC-I protein is composed of an Nterminal extracellular ligand binding domain with seven epidermal growth factor receptor-like cysteine pattern signatures,a membrane-spanning domain, and an unusually long C-terminal cytoplasmic domain that includes a Ser/Pro-rich region
followed by a Gly-rich region. SREC-II encodes an 834-aminoacid protein with 35% homology to SREC-I. Although SREC-II
has little activity to internalize modified LDL, SREC-I-expressing fibroblasts are intensely aggregated with SREC-II-expressing fibroblasts, indicating the association of SREC-I and
SREC-II (7). However, the precise functions of these two proteins are currently unknown.
In atherosclerotic lesions, macrophages are laden with lipids
and immunologically activated (8). In line with this, the development of atherosclerosis is accelerated by LPS (9), a major
component of Gram-negative bacteria that stimulates the production of various cytokines in vivo, thereby contributing to thepathogenesis of endotoxin shock (10). Conversely, the absenceof toll-like receptor 4, a receptor for LPS, inhibits its progression (11). These considerations have prompted us to examine
the effects of LPS on the expression of SREC-I. In the present
study, we show that LPS robustly stimulated the expression of

1
The abbreviations used are: LDL, low density lipoprotein; SR-A,
class A type I/type II scavenger receptor; SR-BI, class B type I scavenger receptor; SREC, scavenger receptor expressed by endothelial
cells.

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This paper is available on line at http://www.jbc.org

SREC-I-mediated Uptake of Ac-LDL in LPS-treated Macrophages
SREC-I in macrophages. In an initial attempt to clarify the roleof SREC-I in the uptake of modified lipoproteins as well as in
the development of atherosclerosis, we generated mice withtargeted disruption of the SREC-I gene by homologous recombination in embryonic stem cells. To exclude the overwhelming
effect of SR-A on the uptake of Ac-LDL, we further generated
mice lacking both SR-A and SREC-I (SR-A⫺/⫺;SREC-I⫺/⫺). By
comparing the uptake and degradation of Ac-LDL in peritoneal
macrophages isolated from these mice, we found that SREC-I
plays a significant role in the uptake of Ac-LDL in the settingof SR-A deficiency, especially when stimulated with LPS. Fromthese results, we propose that SREC-I contributes to the development of atherosclerosis in concert with SR-A.
EXPERIMENTAL PROCEDURES

General Methods—Standard molecular biology techniques were used(12). The current experiments were performed in accordance with institutional guidelines for animal experiments at the University of Tokyo and the Jichi Medical School.
SREC-I Antibody Preparation—Two milligrams of the carboxyl-end
peptide of mouse SREC-I (amino acid residues 801– 820, KEQEEPLYENVVPMSVPPQH) was conjugated with keyhole limpet hemocyanin
using the Imject sulfhydryl-reactive antibody production kit (Pierce).
The keyhole limpet hemocyanin-peptide was gel-purified and emulsified with an equal volume of complete Freund’s adjuvant (Calbiochem).
A female Wister rat was immunized with the emulsions. One week after
the boost injection, blood was collected, and the antiserum was purified
and eluted through an affinity column (Sulfolink coupling gel; Pierce) towhich the antigen peptide was coupled.
Mice—SR-A knock-out mice were generated previously (4). ApoE
knock-out mice were purchased from the Jackson Laboratory (Bar
Harbor, ME) (13). Both mice had been back-crossed to C57BL/6J genetic
background and fed a normal chow diet (MF diet from Oriental Yeast
Co., Tokyo, Japan) that contained 5.6% (w/w) fat with 0.09% (w/w)
cholesterol, and the mice were allowed access to water and foodad libitum.
Cells—Thioglycolate-elicited peritoneal macrophages (14) and mouseembryonic fibroblasts (15) were prepared as described previously. Cells
were treated with varying concentrations of LPS (Escherichia coli
O127:B8; Sigma) for 12 h before the experiments.
Northern Blot Analysis—For the SREC-I cDNA probe, two probes
were prepared, namely Probe A, a 5⬘ 2.0-kb fragment spanning theextracellular and intracellular domains, and Probe B, a 0.1-kb fragment
consisting of only the transmembrane domain. Poly (A)⫹ RNA was
purified using Oligotex-dT30TM, an oligo(dT) latex (Roche Applied Science) from 100 –150 ␮g of total RNA that was extracted by TRIzol
reagent (Invitrogen) from either cultured cells or tissues. One to three
milligrams of poly(A)⫹ RNA was subjected to 1% agarose gel electrophoresis in the presence of formalin, transferred to Hybond N (Amersham Biosciences), and hybridized to the 32P-labeled probes for SREC-I
and other scavenger receptors as described previously (16).
Western Blot Analysis—Cells were lysed with 0.1% SDS. After centrifugation, 50 ␮g of the supernatant was subjected to SDS-PAGE andtransferred to Hybond ECLTM, a nitrocellulose membrane (Amersham
Biosciences). After incubation with the anti-SREC-I antibody (1:400
dilution), the membrane was incubated with a goat anti-rat IgG conjugated with horseradish peroxidase (1:2000 dilution; Amersham Biosciences). The secondary antibody was visualized by an enhanced
chemiluminescence kit (Amersham Biosciences).
Generation of the SREC-I Knock-out Mice—The SREC-I gene wascloned from the 129/Sv mouse genomic library (Clontech) using themouse cDNA as a probe. A replacement-type targeting vector wasconstructed so that a 35-bp segment in exon 8, which encodes 3⬘ twothirds of the transmembrane domain, was replaced with a polIIneo
cassette (Fig. 5A). Long arm consists of a 10-kb NotI/KpnI fragment
spanning the 5⬘ untranslated region and exon 8; short arm consists of a
0.9-kb SacI/XbaI fragment within intron 9. These were inserted together into the vector pPolIIshort-neobpA-HSVTK, as described previously (17). After digestion with SalI, the vector was electroporated into
JH-1 embryonic stem cells (a generous gift from Dr. Herz at Universityof Texas Southwestern Medical Center at Dallas, TX). Targeted clones,
which had been selected in the presence of G418 and 1-(2-deoxy,
2-fluoro-␤-D-arabinofuranosyl)-5 iodouracil, were identified by PCR using the primers 5⬘-GATTGGGAAGACAATAGCAGGCATGC-3⬘ and 5⬘CAGAGAGTGTCACCACAACAAGAGGA-3⬘ (Fig. 5A). Homologous recombination was verified by Southern blot analysis after digestion with

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EcoRI using a 0.5-kb SpeI/SmaI fragment, which was downstream ofthe short arm, as a probe (Fig. 5A). Targeted embryonic stem clones
were injected into C57BL/6J blastocysts, yielding one line of chimericmice that transmitted the disrupted allele through the germline.
Generation of the SR-A/SREC-I Double Knock-out Mice—The SRA⫺/⫺ mice were crossed with the SREC-I⫺/⫺ mice, which were a
C57BL/6J ⫻ 129/Sv hybrid, to obtain SR-A⫹/⫺;SREC-I⫹/⫺ mice, whichwere interbred to obtain four types of mice, namely wild-type, SR-A⫹/⫹;
SREC-I⫺/⫺, SR-A⫺/⫺;SREC-I⫹/⫹, and SR-A⫺/⫺;SREC-I⫺/⫺ mice.
Thus, the genetic background of these mice was 75% C57BL/6J and 25%
129/Sv. Littermates were used for the experiments.
Biochemical Analyses—Blood was collected from the retro-orbital
venous plexus after a 12-h fast. Plasma glucose (ANTSENSE II, Bayer
Medical, Tokyo, Japan), cholesterol (Determiner TC, Kyowa Medex,
Tokyo), and triglycerides (TGLH; Wako Chemicals, Tokyo, Japan)
were measured.
Histology—Mice were sacrificed by decapitation. Tissues were excised, fixed in 10% neutral buffered formalin, embedded in paraffin, andstained with hematoxylin-eosin.
Preparation of Lipoproteins—LDL (d 1.019 –1.063 g/ml) and lipoprotein-deficient serum (d ⬎1.21 g/ml) were prepared by stepwise ultracentrifugation from plasma obtained from healthy volunteers. The lipoproteins and lipoprotein-deficient serum were dialyzed against 10
mM sodium phosphate, pH 7.4, 150 mM NaCl, 0.01%(w/v) EDTA, and
0.01% (w/v) NaN3. LDL was acetylated with acetic anhydrate andradioiodinated by the iodine monochloride method as described (18).
Protein concentrations were determined by the BCA protein assay
reagent kit (Pierce).
Cellular Uptake and Degradation of 125I-Ac-LDL—Peritoneal macrophages were plated in 12-well plates at a density of 1 ⫻ 106/well andtreated with or without 100 ng/ml of LPS for 12 h. After stringent
washing with PBS, the cells were incubated with a medium containing
varying concentrations of 125I-Ac-LDL and 5 mg/ml lipoprotein-deficient serum, with or without a 50-fold excess of unlabeled Ac-LDL, for
5 h at 37 °C. The amounts of 125I-Ac-LDL either degraded by or associated with the cells were measured according to a modified method (19)of Goldstein et al. (18).
Statistics—The differences of the means were compared by Student’s
t test.
RESULTS

Tissue Distribution of mRNA Expression of SREC-I and -II—We performed Northern blot analyses to examine the expression of SREC-I and II in various organs of a mouse (Fig. 1).
SREC-I was expressed in a wide variety of organs, most predominantly in liver, lung, kidney, and heart. On the other
hand, the expression of SREC-II was restricted to lungand kidney.
LPS Stimulates the Expression of SREC-I in Peritoneal
Macrophages—LPS robustly increased the mRNA expressionof both SREC-I and SR-A in macrophages (Fig. 2). The peak of
the stimulation was reached by the 12-h time point of thestimulation (Fig. 2A), and the maximal responses were obtained at the concentration of 10 ng/ml (Fig. 2B). The relative
increase in the expression of SREC-I was 4-fold, which wasmore prominent than that of SR-A (1.8-fold) (Fig. 3). It is ofnote that the treatment with LPS did not significantly change
the expression of MARCO (macrophage receptor with collagenous structure) (20) and SR-BI (21) and that it even decreased
the expression of CD36 (22) and FEEL-1 (fasciclin, EGF-like,laminin-type EGF-like, and link domain-containing scavenger
receptor-1) (23).
Expression of SREC-I in Aortas—We compared the mRNA
expression levels of SREC-I in the atherosclerotic aortas, whichwere taken from 12-month-old apoE knock-out mice, with normal aortas, mouse embryonic fibroblasts, peritoneal macrophages, kidney, or lung from wild-type mice (Fig. 4). Normal
and atherosclerotic aortas expressed 1.7- and 2.1-fold higher
levels of SREC-I mRNA than the non-stimulated macrophages,
respectively. The expression levels were comparable with thosein the kidney, but much lower than those of the LPS-treatedmacrophages.

30940

SREC-I-mediated Uptake of Ac-LDL in LPS-treated Macrophages

FIG. 1. Northern blot analysis of SREC-I (upper panel) and SREC-II (lower panel) in various organs of a mouse. Two micrograms ofpoly(A)⫹ RNA was subjected to Northern blot analysis.

FIG. 2. Northern blot analysis of SREC-I and SR-A in macrophages treated with LPS. A, thioglycolate-elicited macrophages from
C57BL/6 wild-type mice were treated with 100 ng/ml LPS for theindicated times. B, cells were treated with the indicated concentrationsof LPS for 12 h. Two micrograms of poly(A)⫹ RNA was subjected to
Northern blot analysis. 36B4 was used as a loading control.

Generation of Mice Lacking the SREC-I and/or SR-A Gene—
The intercross of the progeny (SREC-I⫹/⫺) resulted in offspring of both sexes with all three genotypes at the SREC locus
with the expected Mendelian ratios (105:191:85 ␹2 ⫽ 1.06, p ⬎0.05) (Fig. 5B). Fig. 5C shows the results of Northern blot
analyses of SREC-I in peritoneal macrophages. When hybridized with Probe A, which contains nearly the whole coding
region of the SREC-I cDNA, Northern blot revealed a bandwith mRNA size of 2.9 kb in wild-type macrophages and a bandof 2.4 kb in SREC-I⫺/⫺ macrophages. When hybridized with
Probe B, which contains the transmembrane domain, no band
was detectable in SREC-I⫺/⫺ macrophages. These results indicate that SREC-I⫺/⫺ mice express a truncated transcript

FIG. 3. Northern blot analysis of scavenger receptors for AcLDL in macrophages treated with or without LPS. Thioglycolateelicited macrophages from C57BL/6 wild-type mice were treated with(⫹) or without (⫺) 100 ng/ml LPS for 12 h. Two micrograms of poly(A)⫹RNA was subjected to Northern blot analysis. Probes used were
SREC-I, SR-A, MARCO, CD36, SR-BI, and FEEL-1. Signal intensity
was corrected against the intensity of 36B4, and the relative signal
increase ratio was calculated.

that lacks the transmembrane domain. SREC-I⫺/⫺ mice werefertile and apparently normal. There were no significant differences in the growth curves of wild-type and SREC-I⫺/⫺mice. As shown in Table I, plasma levels of glucose, cholesterol,and triglycerides were not different between wild-type and
SREC-I⫺/⫺ mice. We failed to detect any pathological findingsin the brain, lung, heart, liver, kidney, and testis of SREC-I⫺/⫺
mice.
Northern blot analysis of four types of mice confirmed theabsence of the expression of SR-A in both SR-A⫺/⫺;SREC-I⫹/⫹
and SR-A⫺/⫺;SREC-I⫺/⫺ mice as well as the expression of the
disrupted allele of SREC-I in both SR-A⫹/⫹;SREC-I⫺/⫺ and SRA⫺/⫺;SREC-I⫺/⫺ mice (Fig. 6A). Western blot analysis confirmed the absence of SREC-I protein (⬃140 and160 kDa) in both
SR-A⫹/⫹; SREC-I⫺/⫺ and SR-A⫺/⫺;SREC-I⫺/⫺ mice (Fig. 6B).

SREC-I-mediated Uptake of Ac-LDL in LPS-treated Macrophages

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TABLE I
Plasma levels of glucose, total cholesterol, and triglycerides
After a 12-h fast, blood was collected from the retro-orbital venous
plexus of mice aged 8 weeks. Plasma glucose, total cholesterol, andtriglycerides were measured. All values are expressed as means ⫾ S.E.
No significant deference between wild-type and SREC-I ⫺/⫺ mice.
SREC-I ⫺/⫺Wild-type (⫹/⫹)

FIG. 4. Northern blot analysis of SREC-I in normal and atherosclerotic aortas. RNA were extracted from the following cells or organs: mouse embryonic fibroblasts (MEF) treated with (⫹) or without(⫺) 100 ng/ml LPS for 12 h; mouse peritoneal macrophages (MPM)
treated with or without 100 ng/ml LPS for 12 h; kidney; lung; normal
aortas from wild-type mice (Aorta ⫹/⫹); and atherosclerotic aortas
from apoE⫺/⫺ mice (Aorta ⫺/⫺). Aortic arches and the thoracic part ofdescending aortas with rampant visible plaques were excised from five
12-month-old mice. After adipose tissues surrounding the aortas were
removed as much as possible, the aortas were used for the preparationof RNA. Three micrograms of poly(A)⫹ RNA was subjected to Northernblot analysis of SREC-I. 36B4 was used as a loading control.

FIG. 5. Targeted disruption of SREC-I gene. A, map of the
SREC-I gene and targeting construct. Long boxes represent exons. The
exon coding transmembrane domain was replaced with the neomycin
resistance gene (Neor) of targeting vector, which has a herpes simplex
virus thymidine kinase (HSV-TK) cassette for a negative selection
downstream of its short arm. A 0.5-kb fragment was used as a probe forSouthern blot analysis (shaded box). B, Southern blot analysis. After
digestion with EcoRI, tail DNA was used for Southern blot analysis.
The size of the disrupted allele, 4 kb, was smaller than that of wild-typeallele (6.4 kb). C, mRNA expression of SREC-I in peritoneal macrophage. Two micrograms of poly(A)⫹ RNA from wild-type (⫹/⫹) and
SREC-I⫺/⫺ mice (⫺/⫺) macrophage was hybridized with two cDNA
probes, namely Probe A, a 5⬘ 2-kb fragment spanning the extracellularand intracellular domains, and Probe B, a 0.1-kb fragment consisting of
only the transmembrane domain.

LPS Stimulates Protein Expression of SREC-I—LPS significantly increased the SREC-I protein by ⬃2-fold in wild-type
and SR-A⫺/⫺;SREC-I⫹/⫹ macrophages (Fig. 6B).
LPS Increases the Contribution of SREC-I to the Cellular
Uptake and Degradation of the 125I-Ac-LDL—In non-stimulated conditions (Fig. 7A) there was no significant difference inthe specific uptake and degradation of 125I-Ac-LDL between
wild-type and SR-A⫹/⫹;SREC-I⫺/⫺ macrophages. Based on the

mg/dl

GlucoseTotal cholesterol
Triglycerides

61.1 ⫾ 3.2 (n ⫽ 31)
89.7 ⫾ 5.1 (n ⫽ 22)
89.6 ⫾ 9.4 (n ⫽ 22)

63.2 ⫾ 3.2 (n ⫽ 31)
82.0 ⫾ 3.3 (n ⫽ 27)
97.4 ⫾ 10.3 (n ⫽ 27)

FIG. 6. Expression of SR-A and SREC-I in macrophages isolated from wild-type, SR-Aⴙ/ⴙ;SREC-Iⴚ/ⴚ, SR-Aⴚ/ⴚ;SREC-Iⴙ/ⴙ, and
SR-Aⴚ/ⴚ;SREC-Iⴚ/ⴚ mice. Thioglycolate-elicited peritoneal macrophages were prepared from wild-type, SR-A⫹/⫹;SREC-I⫺/⫺, SR-A⫺/⫺;
SREC-I⫹/⫹, and SR-A⫺/⫺;SREC-I⫺/⫺ mice. A, Northern blot analysis
of SR-A and SREC-I. One microgram of poly(A)⫹ RNA from macrophages was hybridized with SR-A and SREC-I probes (Probe A). B,
Western blot analysis of SREC-I in macrophages treated with or without LPS. After stimulation with (⫹) or without (⫺) 100 ng/ml LPS for
12 h, the cells were lysed with 0.1% SDS, and 50 ␮g of protein was
subjected to SDS-PAGE. Immunoblotting was performed using the rat
anti-mouse SREC-I antibody and an enhanced chemiluminescence kit.

values for 20 ␮g/ml degraded 125I-Ac-LDL, SR-A⫺/⫺;SRECI⫹/⫹ macrophages degraded significantly smaller amounts of125
I-Ac-LDL (15%) than did wild-type macrophages, supporting
the dominant role of SR-A in the uptake and degradation of
125
I-Ac-LDL in macrophages. Compared with SR-A⫺/⫺;SREC⫹/⫹I
macrophages, SR-A⫺/⫺;SREC-I⫺/⫺ macrophages showeda further reduction in the specific uptake (21%) and degradation of 125I-Ac-LDL (31%). Based on the values for 20 ␮g/ml
degraded 125I-Ac-LDL, the contribution of SR-A and SREC-I to
the overall degradation of Ac-LDL was calculated to be 85 and
5%, respectively, in the non-stimulated condition.
LPS increased the uptake and degradation of Ac-LDL by
1.8-fold (Fig. 7B). In this condition, there was no significant
difference in the specific uptake and degradation of 125IAc-LDL between wild-type and SR-A⫹/⫹;SREC-I⫺/⫺ macrophages. Based on the values for 20 ␮g/ml degraded 125I-Ac-LDL,

30942

SREC-I-mediated Uptake of Ac-LDL in LPS-treated Macrophages

FIGⴚ/ⴚ
. 7. Cell association and degradation of 125I-Ac-LDL by peritoneal macrophages isolated from wild-type, SR-Aⴙ/ⴙ;SREC-Iⴚ/ⴚ,SR-A ;SREC-I ⴙ/ⴙ, and SR-Aⴚ/ⴚ;SREC-Iⴚ/ⴚ mice. Thioglycolate-elicited peritoneal macrophages were prepared from four types of mice (n ⫽ 5),
namely wild-type (solid circle), SR-A⫹/⫹;SREC-I⫺ (open circle), SR-A⫺/⫺;SREC-I⫹/⫹ (solid square), and SR-A⫺/⫺;SREC-I⫺/⫺ (open square). After
treatment with (panel B) or without (panel A) 100 ng/ml LPS for 12 h, the cells were incubated with the indicated concentrations of 125I-Ac-LDL
with or without a 50-fold excess of unlabeled Ac-LDL at 37 °C. After 5 h, the amounts of 125I-Ac-LDL associated or degraded were determined.
Specific values were calculated by subtracting the nonspecific values from the total values. All values are expressed as means ⫾ S.E. of five mice.
*, p ⬍ 0.05 versus SR-A⫺/⫺;SREC-I⫹/⫹ mice; **, p ⬍ 0.01 versus SR-A⫺/⫺; SREC-I⫹/⫹ mice.

SR-A⫺/⫺;SREC-I⫹/⫹ macrophages degraded significantly
smaller amounts of 125I-Ac-LDL (10%) than did wild-type macrophages. Compared with SR-A⫺/⫺;SREC-I⫹/⫹ macrophages, SRA⫺/⫺;SREC-I⫺/⫺ macrophages showed a further reduction in
the specific uptake (49%) and degradation of 125I-Ac-LDL (59%).
Based on the values for 20 ␮g/ml degraded Ac-LDL, the contribution of SR-A and SREC-I to the overall degradation of 125I-AcLDL was calculated to be 90 and 6%, respectively. LPS increased
the absolute contribution of SR-A and SREC-I by 1.9- and 2.3fold, respectively. On the other hand, LPS decreased the absolutecontribution of other pathways by 31%.
DISCUSSION

In the present study, we have first shown that SREC-I, anovel member of the scavenger receptor family that recognizes
modified lipoproteins, is expressed in a wide variety of tissues
including macrophages and aortas, implicating its involvementin the development of atherosclerosis. The expression of
SREC-I was not significantly different between normal andatherosclerotic aortas, although it was robustly induced by LPS
in macrophages, a major cell type that is present in foam celllesions. To define the precise role of SREC-I, we have generated
wild-type, SR-A⫹/⫹;SREC-I⫺/⫺, SR-A⫺/⫺;SREC-I⫹/⫹, and
SR-A⫺/⫺;SREC-I⫺/⫺ mice and compared the uptake and degradation of Ac-LDL in macrophages between these mice. Results show that the contribution of SREC-I to the overall up-

take and degradation of Ac-LDL was 5% in the non-stimulated
condition and 6% in the LPS-stimulated condition. Although
the involvement of SREC-I was relatively small compared withthat of SR-A, LPS increased the SREC-I mediated degradation
by 2.3-fold, which accounted for 60% of the amounts of Ac-LDL
degraded by the pathway independent of SR-A.
Because the responses of scavenger receptors to LPS arevariable, we did not expect that LPS induced the expression of
SREC-I in macrophages. LPS induces SR-A expression inmouse macrophages, which was confirmed in our experiments(Figs. 2 and 3) but not in THP-1 monocyte/macrophages (24)and human monocyte-derived macrophages (25). Because SR-A
is able to bind LPS (26), the induction of SR-A by LPS may
have a protective role against endotoxemia. This notion is inline with the susceptibility of SR-A knock-out mice to endotoxin shock (4, 27, 28). On the other hand, LPS down-regulates SR-B1 expression (29), which was confirmed in our
experiments (Fig. 2).
The mechanism by which LPS induces SREC-I is intriguing.
It is well known that LPS modulates gene expression though
the activation of NF-␬B signaling (30), and some of the effects
are mediated by proinflammatory cytokines, whose expression
is stimulated by LPS. However, there is no NF-␬B binding site
in the 5⬘-flanking region of the human SREC-I gene 1 kb
upstream of the transcription initiation site (31). Furthermore,SREC-I-mediated Uptake of Ac-LDL in LPS-treated Macrophages
LPS did not induce SREC-I expression in human umbilical vein
endothelial cells, and tumor necrosis factor-␣ did not haveincreasing effects on SREC-I expression in mouse macrophages(data not shown). Further studies are warranted to decipher
how LPS induces SREC-I expression.
Although SREC-I is expressed in macrophages, particularly
when stimulated with LPS, there was no significant differencein the expression levels between normal and atherosclerotic
aortas (Fig. 4). This suggests that other cell types such as
endothelial cells and smooth muscle cells in the aortas express
comparable levels of SREC-I.
SR-A is the major pathway for the uptake and degradation of
Ac-LDL, accounting for 80% of the total activity (4, 5). Because
other scavengers are expressed in macrophages (Fig. 2) and areable to bind Ac-LDL (2), the question is which scavenger receptor is the second most important in the uptake and degradationof Ac-LDL. Our results have revealed that the role of SREC-I isrelatively minor in the non-stimulated macrophages, which islargely consistent with the recent report by Kunjathoor et al.
(22). According to them, SR-A and CD36 account for 75–90% of
the total amounts of chemically modified LDL degraded bymacrophages. In SR-A-deficient macrophages stimulated with
LPS, however, the absolute contribution of SREC-I was significantly increased by 2.3-fold, accounting for 60% of the SR-Aindependent uptake and degradation of Ac-LDL (Fig. 7B). The
degree of increase in the SREC-I mediated uptake and degradation of Ac-LDL is largely comparable with that in SREC-I
protein expression (Fig. 6B). It is interesting to note that LPS
decreased the absolute contribution of the other endocytic pathway, which is independent of either SR-A or SREC-I, by 31%.
This finding is consistent with the Northern blot results thatshow that LPS did not significantly increase the expression of
the other members of scavenger receptor family such as
MARCO, SR-BI, CD36, and FEEL-1 (Fig. 3). Thus, SREC-I isthe second most important receptor mediating the uptake of
Ac-LDL, at least in macrophages stimulated with LPS. Given
the activated state of macrophages in rupture-prone unstableplaques (8), particularly in plaques infected with microorganisms such as Chlamydia, which is associated with an increased
prevalence of coronary events (32), SREC-I may take a significant part in the foam cell formation in these pathologicalconditions. Other aspects of LPS may be involved in the atherogenesis. For example, Baranova et al. (29) and Khovidhunkit etal. (33) have recently reported that LPS inhibits high density
lipoprotein-mediated cholesterol efflux via down-regulation of
the expression of ABCA1 and ABCG1. These observations are
consistent not only with the ability of LPS to stimulate lipid
accumulation in macrophages in vitro (34) but also with theproatherogenic effects of LPS (9) and its cognate receptor,
Toll-like receptor 4, in vivo (11).
Functions of adhesion molecules have been assigned to both
SR-A and SREC-I/II. Chinese hamster ovary cells overexpressing SR-A have an increased ability to adhere to plastic surfaces(35). Likewise, intense aggregation was observed when SRECI-expressing fibroblast L-cells were mixed with those expressing SREC-II (7). Thus, it is reasonable to speculate that SRECI⫺/⫺ mice have some phenotypes with regard to cell adhesion.
However, there were no obvious abnormalities in the pathologies (data not shown).
The precise roles of scavenger receptors in atherogenesis
have been tested only for SR-A and CD36. With regard to SR-A,
we (4, 36, 37) and Babaev et al. (38) have reported that SR-A
deficiency protects against the development of atherosclerosis
in either apoE, LDL receptor-deficient, or wild-type mice. deWinther et al. (39), however, recently reported apparently opposite results, i.e. SR-A deficiency leads to more complex le-30943

sions in the APOE3Leiden mice. The same group reported the
reduction in atherosclerosis in LDL receptor knock-out mice inwhich SR-A was overexpressed in a macrophage-specific manner (40). These contradictory results could be attributed to thebroad repertoire of functions and the widespread expression of
SR-A (41). With regard to CD36, Febbraio et al. have reported
that CD36 deficiency protects against atherosclerosis in an
apoE-deficient background (42). Availability of the SR-A⫺/⫺;
SREC-I⫺/⫺ mice should allow us to determine the role of
SREC-I in the development of atherosclerosis by crossing withthe genetically hyperlipidemic mice, for example. If the hypothesis is correct, SREC-I should be a new target for preventing
atherosclerosis.
Acknowledgments—We thank Kimiko Saito, Megumi Herai, Mihoko
Kusubae, and Rie Tamura for excellent technical assistance. We also
thank Tetsuya Kitamine, Stephane Perrey, Michiyo Amemiya-Kudo,
and Takanari Gotoda for helpful comments and discussion.
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