From the Departments of Metabolic Diseases,
** Cardiovascular Medicine, and
Infectious Diseases, Faculty of Medicine,
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655 Japan; the
¶ Laboratory of Cellular Biochemistry, The Institute of
Physical and Chemical Research (RIKEN), Wako-shi, Saitama, 351-0198 Japan; the
Departments of Metabolism, Endocrinology, and
Atherosclerosis, Institute of Clinical Medicine, University of Tsukuba,
1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8575 Japan; and the
§§ Department of Internal Medicine, Jichi
Medical School, 3311-1 Yakushiji, Minamikawachi-machi, Kawachi-gun,
Tochigi, 329-0498 Japan
Received for publication, October 4, 2002, and in revised form, December 2, 2002
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ABSTRACT |
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Advanced glycation end
products (AGEs) are nonenzymatically glycosylated proteins, which
accumulate in vascular tissues in aging and diabetes. Receptors for
AGEs include scavenger receptors, which recognize acetylated low
density lipoproteins (Ac-LDL) such as scavenger receptor class AI/AII
(SR-A), cell surface glycoprotein CD36, scavenger receptor class
B type I (SR-BI), and lectin-like oxidized low density lipoprotein
receptor-1. The broad ligand repertoire of these receptors as
well as the diversity of the receptors for AGEs have prompted us to
examine whether AGEs are also recognized by the novel scavenger
receptors, which we have recently isolated from a cDNA library
prepared from human umbilical vein endothelial cells, such as
the scavenger receptor expressed by
endothelial cells-I (SREC-I); the
fasciclin EGF-like, laminin-type EGF-like, and link domain-containing scavenger
receptor-1 (FEEL-1); and its paralogous protein, FEEL-2. At
4 °C, 125I-AGE-bovine serum albumin (BSA) exhibited high
affinity specific binding to Chinese hamster ovary (CHO) cells
overexpressing FEEL-1 (CHO-FEEL-1) and FEEL-2 (CHO-FEEL-2) with
Kd of 2.55 and 1.68 µg/ml, respectively, but not
to CHO cells expressing SREC (CHO-SREC) and parent CHO cells. At
37 °C, 125I-AGE-BSA was taken up and degraded by
CHO-FEEL-1 and CHO-FEEL-2 cells but not by CHO-SREC and parent CHO
cells. Thus, the ability to bind Ac-LDL is not necessarily a
prerequisite to bind AGEs. The 125I-AGE-BSA binding
to CHO-FEEL-1 and CHO-FEEL-2 cells was effectively inhibited by Ac-LDL
and polyanionic SR-A inhibitors such as fucoidan, polyinosinic acids,
and dextran sulfate but not by native LDL, oxidized LDL, or HDL.
FEEL-1, which is expressed by the liver and vascular tissues, may
recognize AGEs, thereby contributing to the development of diabetic
vascular complications and atherosclerosis.
Advanced glycation end products
(AGEs)1 are generated by
nonenzymatic glycosylation of proteins or lipids after prolonged
exposure to glucose (1). AGEs elicit a wide variety of cellular
responses including induction of growth factors and cytokines (2),
adhesion molecules (3), oxidant stress (4), and chemotaxis (5). These
proinflammatory responses are implicated to contribute to the
development of pathologies associated with aging, diabetes mellitus,
and Alzheimer's disease (6). Indeed, AGEs were shown to be present in
atherosclerotic lesions (7) and diabetic kidney (8).
The AGE-elicited proinflammatory reactions are mediated by its
receptors or binding proteins, which include the receptor for advanced
glycation end product (RAGE) (9, 10), OST-48 (ARE-R1)/80K-H (AGE-R2)/galectin-3 (AGE-R3) (11), scavenger receptor class AI/AII
(SR-A) (12), scavenger receptor class B type I (SR-BI) (13), cell
surface glycoprotein CD36 (14), lectin-like oxidized low density
lipoprotein receptor-1 (15), lactoferin (16), and lysozyme (16).
The broad ligand repertories of these AGE-binding proteins as well as
the diversity of receptors for AGEs have prompted us to examine whether
AGEs are recognized by novel members of scavenger receptors that Adachi
et al. (17, 18) have recently cloned from a cDNA library
prepared from human umbilical vein endothelial cells as receptors for
acetylated low density lipoproteins (Ac-LDL), such as the
scavenger receptor expressed by
endothelial cells-I (SREC-I) (17) and the
fasciclin EGF-like, laminin-type
EGF-like, and link domain-containing scavenger
receptor-1 (FEEL-1) (18). These receptors are structurally
unrelated to other scavenger receptors. SREC is a protein of 830 amino
acids with five epidermal growth factor-like cysteine pattern
signatures. FEEL-1 is a protein of 2570 amino acids including 7 fasciclins, 16 EGF-like, 2 laminin-type EGF-like, and 1 link
domain near the transmembrane region. FEEL-2 is a paralogous gene of
FEEL-1 whose amino acid sequence is ~40% identical to FEEL-1.
Quantitative PCR analyses showed that both FEEL-1 and FEEL-2 are
expressed in the spleen and lymph node, whereas only FEEL-1 is
detectable in CD14+-mononuclear cells and vascular
endothelial cell lines (18).
Here we show that FEEL-1 and FEEL-2, but not SREC, are endocytic
receptors for AGEs. Because FEEL-1 is expressed by the liver, macrophages, and endothelial cells in an amount comparable with other
receptors for AGEs, FEEL-1 may play a significant role in the
elimination of AGEs from the circulation as well as in the development
of diabetic vascular complications and atherosclerosis.
Materials--
Ham's F-12 medium, Dulbecco's modified
Eagle medium (DMEM), penicillin G, streptomycin sulfate, G418, and
TriZOL were purchased from Invitrogen. Bovine serum albumin (BSA),
MOPC21 (mouse IgG), fucoidan, polyinosinic acid (poly I), and dextran
sulfate were purchased from Sigma. Heparin sodium salt was purchased
from Mitsubishi Pharma (Osaka, Japan). Glucose 6-phosphate was
purchased from Oriental Yeast (Tokyo, Japan). Endothelial cell growth
supplement was purchased from BD Biosciences.
Oligotex-dT30TM was purchased from Roche Molecular
Biochemicals. Hybond N was purchased from Amersham Biosciences.
Na-125I was purchased from Daiichi Chemical (Osaka, Japan).
IodogenTM and BCA protein assay reagent kit were purchased
from Pierce. Human umbilical vein endothelial cells (HUVECs) were
purchased from Kurabo (Tokyo, Japan). A murine monoclonal antibody
against human FEEL-1 (FE-1-1) was described previously (18).
cDNA probes for murine FEEL-1, FEEL-2 SREC, RAGE, galectin-3, SR-A,
CD36, and SR-B1 were prepared by reverse transcriptase-PCR using
primers designed based on the reported nucleotide sequences.
Ligand Preparation and Radiolabeling--
AGE-BSA was prepared
as described previously (19). 600 mg of BSA was incubated with 50 mM glucose 6-phosphate in 10 ml of sterile sodium phosphate
buffer or phosphate-buffered saline (PBS) for 10 weeks at 37 °C and
dialyzed overnight against PBS. AGE-specific fluorescence was measured
at 450 nm after excitation at 360 nm at a concentration of 1 mg/ml with
a fluorescence spectrometer (Biolumin, Amersham Biosciences).
The AGE-BSA exhibited ~38-fold higher fluorescence intensity than
BSA. AGE-BSA was labeled with 125I by using Iodogen
according to the manufacturer's instruction. Protein concentrations
were determined by BCA protein assay reagent kit. LDL
(d = 1.019-1.063 g/ml) and high density lipoprotein
(HDL) (d = 1.063-1.21 g/ml) were prepared by stepwise
ultracentrifugation from plasma obtained from healthy volunteers. LDL
was acetylated with acetic anhydrate as described previously (20) and
oxidized by incubation in a buffer containing 5 µM CuSO4
for 16 h at 37 °C (21). The lipoproteins were dialyzed against
a buffer containing 10 mM sodium phosphate, pH 7.4, and 150 mM NaCl.
Cell Culture--
CHO cells overexpressing human FEEL-1
(CHO-FEEL-1), human FEEL-2 (CHO-FEEL-2), and human SREC (CHO-SREC) were
obtained as described previously (17, 18). These cells were maintained at 37 °C with 5% (v/v) CO2 in medium A (Ham's
F-12 supplemented with 100 units/ml penicillin and 100 units/ml
streptomycin) containing 10% (v/v) fetal calf serum and 0.2 mg/ml G418
(medium B). Untransfected CHO cells designated as CHO-Wild were
maintained in medium B without G418. The four lines of CHO cells were
cultured for 2 days to confluence in 12-well plates and used for the
following experiments. Thioglycolate-elicited mouse peritoneal
macrophages were prepared as described previously (22) and maintained
in DMEM containing 10% (v/v) fetal calf serum. HUVECs were cultured
with DMEM containing 20% fetal calf serum, 30 µg/ml endothelial cell
growth supplement, and 10 IU/ml heparin. Confluent dishes were
used for the studies within the 15 passages.
Northern Blot Analysis--
Total RNA was prepared by TriZOL.
Poly(A)+ RNA was purified using Oligotex-dT30 and subjected
to 1% (w/v) agarose gel electrophoresis in the presence of formalin.
The fractionated RNA was transferred to Hybond N, hybridized to
32P-labeled cDNA probes, and analyzed by BAS2000 (FUJI
XEROX, Tokyo, Japan). The expression levels of each gene were compared
between various organs after adjusting to the expression level of 36B4.
Binding of 125I-AGE-BSA at 4 °C--
After
washing twice with PBS, the confluent cells were incubated with 0.5 ml
of medium C (medium A supplemented with 3% (w/v) BSA) containing the
indicated concentrations of iodinated AGE-BSA with or without 20-fold
excess of unlabeled AGE-BSA for 2 h at 4 °C. After washing
three times with ice-cold PBS containing 2 mg/ml BSA and three times
further with PBS, the cells were dissolved with 0.1 N NaOH
and the cell-bound radioactivity and cellular proteins were determined.
Uptake and Degradation of 125I-AGE-BSA--
One hour
prior to the study, the media of the confluent cells were changed to
medium C. The media were replaced with medium C containing the
indicated concentrations of 125I-AGE-BSA with or without
20-fold excess of unlabeled AGE-BSA and incubated for 6 h at
37 °C. The amounts of 125I-AGE-BSA either degraded by or
associated with the cells were measured using trichloroacetic acid and
AgNO3 as described previously (22). The differences of the means were
compared by Student's t test.
Expression of FEEL-1, FEEL-2, and SREC in Transfected CHO
Cells--
Northern blot analyses were performed to compare the
mRNA expression of FEEL-1, FEEL-2, and SREC in CHO-FEEL-1,
CHO-FEEL-2, and CHO-SREC cells, respectively (Fig.
1). CHO-FEEL-1 and CHO-SREC expressed
comparable amounts of the respective mRNA, which was 2-fold higher
than the mRNA of FEEL-2 mRNA in CHO-FEEL-2 cells.
Binding of 125I-AGE-BSA to CHO-FEEL-1, CHO-FEEL-2, and
CHO-SREC Cells--
125I-AGE-BSA bound to both CHO-FEEL-1
and CHO-FEEL-2 cells in a saturable manner at 4 °C (Fig.
2, A and B).
Scatchard analysis (insets) showed the presence of single
binding site for AGE-BSA with an apparent Kd of 2.55 and 1.68 µg/ml for FEEL-1 and FEEL-2, respectively. CHO-FEEL-2 cells
bound ~17 times larger amounts of 125I-AGE-BSA
than CHO-FEEL-1 cells (Bmax = 315.5 versus 18.9 ng/mg). On the other hand, the specific
125I-AGE-BSA binding to CHO-SREC cells was 30% of that to
CHO-FEEL-1 cells with lower affinity (Fig. 2C).
Untransfected CHO cells bound negligible amounts
125I-AGE-BSA (Fig. 2D).
Cellular Uptake and Degradation of 125I-AGE-BSA by
CHO-FEEL-1, CHO-FEEL-2, and CHO-SREC Cells--
To examine whether the
binding is associated with cellular uptake and degradation of
125I-AGE-BSA, we incubated the cells with
125I-AGE-BSA at 37 °C and determined the amounts of
125I-AGE-BSA bound (Fig. 3,
A-D) and degraded by the cells (Fig. 3, E-H).
Significant amounts of 125I-AGE-BSA were taken up and
degraded by both CHO-FEEL-1 and CHO-FEEL-2 cells but not by CHO-SREC
and untransfected CHO cells. The activity was larger in CHO-FEEL-2
cells than in CHO-FEEL-1 cells. These results indicate that FEEL-1 and
FEEL-2 but not SREC are endocytic receptors for AGE-BSA.
Ligand Specificity of 125I-AGE-BSA Binding to
CHO-FEEL-1 or CHO-FEEL-2 Cells--
To characterize the ligand
specificity for the AGE-BSA binding site of the FEEL-1 and FEEL-2, we
performed a competition study. First, we examined whether the binding
of 125I-AGE-BSA is inhibited by lipoproteins (Fig.
4, A and B). The binding of 125I-AGE-BSA to CHO-FEEL-1 (Fig. 4A)
and CHO-FEEL-2 (Fig. 4B) cells was inhibited by Ac-LDL but
not by LDL and HDL. It is noteworthy that Ac-LDL inhibited the binding
more potently than AGE-BSA itself in CHO-FEEL-1 cells, whereas
AGE-BSA inhibited the binding more potently than Ac-LDL in CHO-FEEL-2 cells. Table I shows
concentrations required to inhibit the binding by 50%
(IC50). Ox-LDL partially inhibited the binding only in
CHO-FEEL-1 cells (~40% reduction at 100 µg/ml). We next examined
the inhibitory effect of various materials known as SR-A inhibitors
(Fig. 4, C and D). Fucoidan, poly I, and dextran
sulfate inhibited the 125I-AGE-BSA binding almost
completely in both CHO-FEEL-1 (Fig. 4C) and CHO-FEEL-2 cells
(Fig. 4D). Heparin was also effective in suppressing the
binding, but the effect was weaker than the other three compounds
(Table I).
Inhibition of Uptake and Degradation of 125I-AGE-BSA by
CHO-FEEL-1 Cells by Anti-FEEL-1 Antibody--
We determined the
effects of anti-FEEL-1 monoclonal antibody (FE-1-1) as well as
other compounds on the endocytic uptake and degradation of
125I-AGE-BSA by CHO-FEEL-1 cells (Fig.
5). We have previously
reported that FE-1-1 effectively suppressed the cellular uptake of
Ac-LDL in CHO-FEEL-1 cells (18). AGE-BSA and Ac-LDL effectively
inhibited the cellular uptake and degradation of
125I-AGE-BSA by CHO-FEEL-1 cells. No inhibitory effect was
observed with 100 µg/ml native LDL, Ox-LDL, HDL, and 30 µg/ml
control IgG. FE-1-1 inhibited both cellular uptake and degradation of
125I-AGE-BSA by 72 and 48%, respectively. The extent of
inhibition by the antibody was slightly smaller than that by AGE-BSA or
Ac-LDL but was almost the same as observed in inhibition study for
cellular uptake of Ac-LDL (18).
Tissue Distribution of the Expression of FEEL-1 and FEEL-2--
To
estimate the contribution of FEEL-1 and FEEL-2 to the endocytosis of
AGEs in various tissues in comparison with other receptors for AGEs, we
performed Northern blot analyses (Fig.
6). FEEL-1 was expressed in a wide
variety of organs in the following order: liver > lung = heart = white adipose tissue > aorta = kidney > spleen. FEEL-2 whose expression was much lower than that of FEEL-1 was
detectable only in the liver and spleen. In contrast to organs like
lung, spleen, and white adipose tissue in which RAGE, galectin-3, and
CD36 were most highly expressed, respectively, organs like heart,
liver, and aorta appeared to express significant amounts of FEEL-1
whose expression level was as comparable as that of other receptors for
AGEs.
Expression of FEEL-1 and FEEL-2 in Macrophages and Vascular
Endothelial Cells--
We further performed Northern blot analysis to
evaluate the expression of FEEL-1 in mouse peritoneal macrophages and
HUVECs (Fig. 7). Mouse peritoneal
macrophages and HUVECs expressed FEEL-1 to a degree that was comparable
with galectin-3 and SR-A, respectively.
We have recently reported the cloning of novel scavenger receptors
expressed on vascular endothelial cells by expression cloning strategy
using 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbo-cyanine perchlorate (DiI)-labeled Ac-LDL as the ligand, such as SREC (17), FEEL-1, and its paralogous gene, FEEL-2 (18). Structures of these receptors are unique and unrelated to other scavenger receptors. Although the precise functions of these receptors are currently unknown, in vitro studies have suggested their involvement
in cellular interaction and host defense. For example, both FEEL receptors bind to Gram-negative and Gram-positive bacteria (18). Furthermore, anti-FEEL-1 antibody inhibited the in vitro
vascular tube formation (18). On the other hand, SREC-I and its
isoform, SREC-II, showed a strong heterophilic trans-interaction
through their extracellular EGF-like repeat domains (23).
This study has first revealed that FEEL-1 and FEEL-2 are endocytic
receptors for AGEs, implicating the involvement of these receptors in
the pathologies of aging or diabetes. Some of other receptors for AGEs
are involved in these pathologies. For example, Park et al.
(24) have shown that intravenous administration of the soluble
extracellular domain of RAGE, which was originally cloned from bovine
pulmonary endothelial cells, efficiently suppressed diabetic
atherosclerosis in apolipoprotein E-deficient mice. Second, Pugliese et al. (25) have shown that diabetic
glomerulopathy was rather accelerated in mice lacking galectin-3, a
critical component of OST-48/80-K-H/galectin-3 complex (11) that is
ubiquitously expressed in mammalian cells, suggesting its protective
role. Third, Suzuki et al. (26) and Matsumoto et
al. (27) observed that peritoneal macrophages obtained from SR-A
knock-out mice had the reduced capacity for endocytic degradation of
AGE-BSA, which was 30% of that in wild-type cells. However, liver
sinusoidal endothelial cells obtained from SR-A knock-out mice had
capacity for endocytic degradation of AGE-BSA, which was
indistinguishable from that of wild-type liver sinusoidal endothelial
cells (27). These results suggest that SR-A is the primary endocytic
receptor for AGE-BSA in macrophages, whereas other receptors mediate
the uptake of AGEs in endothelial cells.
Although FEEL-1 and FEEL-2 are structurally unrelated to SR-A, their
ligand specificity is similar to that of SR-A. CHO-FEEL-1 cells bind
not only Ac-LDL but also Ox-LDL to a lesser degree (18). The binding
was competitively inhibited by SR-A inhibitors, such as maleyl-BSA and
dextran sulfate (18). Furthermore, both FEEL-1 and FEEL-2 bind AGE-BSA
(Figs. 1 and 2) as does SR-A (12). Ac-LDL and negatively charged
compounds such as fucoidan, dextran sulfate, and poly I competitively
inhibited the binding of AGE-BSA to CHO-FEEL-1 or CHO-FEEL-2 cells
(Figs. 4 and 5) as is the case for SR-A (12). These results suggest
that AGE-BSA and Ac-LDL share a same binding site on the receptors.
Importantly, the binding affinity of AGE-BSA to FEEL-1 and FEEL-2 cells
appeared to exceed that to other receptors for AGEs. Calculated
Kd was 25 nM for FFEL-2; 39 nM for FEEL-1; 85 nM for CD36 (14); 100 nM for RAGE (9); 126 nM for SR-BI (13); 148 nM for lectin-like oxidized low density lipoprotein
receptor-1 (15); and 350 nM for galectin3 (11), assuming
that molecular mass of AGE-BSA is 66 kDa. The binding activity
of CHO-FEEL-1 cells was ~20-fold lower than that of CHO-FEEL-2 cells.
A similar although less significant difference in the binding activity
between the isoforms was observed for Ac-LDL (18). Together with the
fact that the mRNA expression of FEEL-2 in CHO-FEEL-2 cells was
lower than that of FEEL-1 in CHO-FEEL-1, these results indicate that a
significant proportion of the binding sites for AGEs on FEEL-1 was
inactive or masked in CHO-FEEL-1 cells. This possibility warrants
further investigation.
In many organs, the mRNA expression levels of FEEL-1 were higher
than those of FEEL-2 (Fig. 6). Mouse peritoneal macrophages and HUVECs
also expressed FEEL-1 to a degree comparable with other receptors for
AGEs (Fig. 7). Furthermore, FEEL-2 mRNA was barely detectable in
several vascular endothelial cell lines or in monocyte/macrophages at
nonstimulated conditions (18). Taken together, we propose that FEEL-1
and possibly FEEL-2 serve as functional endocytic receptors for AGEs
in vivo. This hypothesis needs to be verified by further
studies, such as gene targeting, that are currently in progress in our
laboratory. As suggested by the presence in the aorta (Fig. 6),
macrophages, and endothelial cells (Fig. 7) (18), FEEL-1 may scavenge
AGEs accumulated in vascular tissues in diabetes and aging, thereby
directly contributing to the development of diabetic vascular
complications and atherosclerosis. Because Smedsrød et al.
(28) reported that >90% of intravenously injected AGE-BSA was
distributed in the liver in rats, it is also possible that FEEL-1,
which was expressed in the liver (Fig. 6), may be involved in the
elimination of AGEs from the circulation.
In conclusion, we demonstrate that FEEL-1 and FEEL-2, but not SREC-I,
function as endocytic receptors for AGEs when overexpressed in CHO
cells. Although further studies will be needed to determine the role of
these two receptors in vivo, they may play a pivotal role in
the pathogenesis of diabetic vascular complications and could be the
promising target molecules for their prevention.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Northern blot analysis of FEEL-1, FEEL-2, and
SREC in transfected CHO cells. 3 µg of poly(A)+ RNA
was subjected to Northern blot analysis with cDNA probe for FEEL-1,
FEEL-2, and SREC. 36B4 was used as a molecular reference.
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Fig. 2.
Binding of 125I-AGE-BSA to
CHO-FEEL-1 (A), CHO-FEEL-2 (B),
CHO-SREC (C), and parent CHO cells
(D). Cells were incubated with the indicated
concentrations of 125I-AGE-BSA for 2 h at 4 °C in
the presence (open triangle) or absence (open
circles) of 20-fold excess amounts of unlabeled AGE-BSA in
triplicate wells. Specific binding (closed circle) was
determined by subtracting nonspecific binding from total binding.
Error bars represent mean ± S.D. Insets
show the Scatchard analysis of the specific binding curve.
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Fig. 3.
Cellular uptake (A-D) and
degradation (E-H) of 125I-AGE-BSA by
CHO-FEEL-1 (A and E), CHO-FEEL-2
(B and F), CHO-SREC (C
and G), and parent CHO cells (D
and H). Cells were incubated with the
indicated concentrations of 125I-AGE-BSA for 6 h at
37 °C in the presence (open triangles) or absence
(open circles) of excess amounts of unlabeled AGE-BSA in
triplicate wells. Cellular uptake (A-D) and degradation
(E-H) of 125I-AGE-BSA were determined. Specific
uptake or degradation (closed circles) was determined by
subtracting nonspecific values from total values. Error bars
represent mean ± S.D.
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Fig. 4.
Competitive inhibition of the
125I-AGE-BSA binding to CHO-FEEL-1 (A and
C) or CHO-FEEL-2 cells (B and
D). A and B, cells were
incubated with 2 µg/ml 125I-AGE-BSA for 2 h at
4 °C in the presence of indicated dose of AGE-BSA (closed
circles), LDL (closed triangles), Ac-LDL (open
circles), Ox-LDL (open triangles), and HDL (open
squares) in triplicate wells. C and D, cells
were incubated with 2 µg/ml 125I-AGE-BSA for 2 h at
4 °C in the presence of indicated dose of fucoidan (closed
circles), poly I (closed triangles), dextran sulfate
(open circles), and heparin (open triangles) in
triplicate wells. Error bars represent mean ± S.D.
Concentrations of various lipoproteins and SR-A inhibitors to
inhibit the binding of 125I-AGE-BSA to CHO-FEEL-1 and
CHO-FEEL-2 cells (IC50)
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Fig. 5.
Competitive inhibition of the uptake
(A) and degradation (B) of
125I-AGE-BSA by CHO-FEEL-1 cells. Cells were incubated
with 2 µg/ml 125I-AGE-BSA for 6 h at 37 °C in the
presence of 100 µg/ml AGE-BSA, LDL, Ac-LDL, Ox-LDL, HDL, or 30 µg/ml FE-1-1 antibody or control IgG in triplicate wells. Cellular
uptake (A) and degradation (B) of
125I-AGE-BSA were determined. Error bars
represent mean ± S.D. *, p < 0.05 versus control values.
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Fig. 6.
Tissue distribution of FEEL-1 and FEEL-2 in
comparison with other receptors for AGEs. 10 µg of total RNA was
extracted from liver, kidney, heart, lung, brain, spleen, testis, white
adipose tissue (WAT), and aorta of mice and subjected to
Northern blot analysis with cDNA probe for FEEL-1, FEEL-2, RAGE,
galectin-3, SR-A, CD36, and SR-B1. 36B4 was used as a loading
control.
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Fig. 7.
Expression of FEEL-1 in HUVEC and mouse
peritoneal macrophages. 10 µg of total RNA from HUVEC and 2 µg
of poly(A)+ RNA from mouse peritoneal macrophages were
subjected to Northern blot analysis. The expression levels of
FEEL-1 were compared with those of galectin-3 in HUVEC and SR-A in
mouse peritoneal macrophages.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Addendum |
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FEEL-1 is identical to stabilin-1 (hyaluronan-scavenger receptor; HA/S-R) first identified as MS-1 antigen (29). AGE uptake by liver sinusoidal endothelial cells is partially inhibited by an antibody against HA/S-R (30).
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
¶¶ To whom correspondence should be addressed: Dept. of Internal Medicine, Division of Endocrinology and Metabolism, 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.
Published, JBC Papers in Press, December 6, 2002, DOI 10.1074/jbc.M210211200
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ABBREVIATIONS |
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The abbreviations used are: AGEs, advanced glycation end products; LDL, low density lipoprotein; Ac-LDL, acetylated low density lipoprotein; Ox-LDL, oxidized low density lipoprotein; HDL, high density lipoprotein; SR-A, scavenger receptor class AI/AII; SR-BI, scavenger receptor class B type I; SREC, scavenger receptor expressed by endothelial cells; EGF, epidermal growth receptor; FEEL-1, fasciclin EGF-like, laminin-type EGF-like, and link domain-containing scavenger receptor-1; RAGE, receptor for advanced glycation end product; BSA, bovine serum albumin; poly I, polyinosinic acid; CHO, Chinese hamster ovary; HUVECs, human umbilical endothelial cells; PBS, phosphate-buffered saline.
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REFERENCES |
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