Received for publication, July 21, 2000, and in revised form, September 25, 2000
Interaction of advanced glycation end products
(AGE) with AGE receptors induces several cellular phenomena potentially
relating to diabetic complications. Five AGE receptors identified so
far are RAGE (receptor for AGE), galectin-3, 80K-H, OST-48, and SRA (macrophage scavenger
receptor class A types I and II). Since SRA is
known to belong to the class A scavenger receptor family, and the
scavenger receptor collectively represents a family of multiligand
lipoprotein receptors, it is possible that CD36, although belonging to
the class B scavenger receptor family, can recognize AGE proteins as
ligands. This was tested at the cellular level in this study using
Chinese hamster ovary (CHO) cells overexpressing human CD36 (CD36-CHO
cells). Cellular expression of CD36 was confirmed by immunoblotting and
immunofluorescent microscopy using anti-CD36 antibody. Upon incubation
at 37 °C, 125I-AGE-bovine serum albumin (AGE-BSA)
and 125I-oxidized low density lipoprotein (LDL), an
authentic ligand for CD36, were endocytosed in a
dose-dependent fashion and underwent lysosomal degradation
by CD36-CHO cells, but not wild-type CHO cells. In binding experiments
at 4 °C, 125I-AGE-BSA exhibited specific and saturable
binding to CD36-CHO cells (Kd = 5.6 µg/ml). The
endocytic uptake of 125I-AGE-BSA by these cells was
inhibited by 50% by oxidized LDL and by 60% by FA6-152, an anti-CD36
antibody inhibiting cellular binding of oxidized LDL. Our results
indicate that CD36 expressed by these cells mediates the endocytic
uptake and subsequent intracellular degradation of AGE proteins. Since
CD36 is one of the major oxidized LDL receptors and is up-regulated in
macrophage- and smooth muscle cell-derived foam cells in human
atherosclerotic lesions, these results suggest that, like oxidized LDL,
AGE proteins generated in situ are recognized by CD36,
which might contribute to the pathogenesis of diabetic macrovascular complications.
 |
INTRODUCTION |
In the Maillard reaction, proteins react with glucose to form
Schiff base and Amadori products. Upon long-term incubation, these
early products are converted to advanced glycation end products (AGE),1 which are
characterized physicochemically by fluorescence, brown color, and
intra- or intermolecular cross-linking (1, 2) and biologically by
specific recognition by AGE receptors. Previous immunological
demonstration of AGE in several human tissues suggests that AGE may be
involved in aging processes, diabetic complications, and
atherosclerosis (3-11).
The physiological significance of AGE has mainly been examined from the
perspective of AGE structure(s) expressed in vivo and
AGE-binding proteins or AGE receptors, through which AGE are believed
to elicit several biological phenomena in monocytes/macrophages (12-17), endothelial cells (18, 19), and mesangial cells (20, 21).
Cellular interactions with AGE proteins are known to induce several
biological responses, not only endocytic uptake and degradation, but
also induction of cytokines and growth factors, which are likely linked
to the development of diabetic vascular complications (9). These
responses are thought to be mediated by AGE receptors, which include
RAGE (receptor for AGE) (22, 23), galectin-3 (24),
SRA (macrophage scavenger receptor class
A types I and II) (28, 29), 80K-H (25, 26) and OST-48 (25,
26). We have recently shown that SRA, which is known as a receptor for
oxidized low density lipoprotein (Ox-LDL) (27, 28), mediates the
endocytic uptake and degradation of AGE-BSA by macrophages (28, 29).
Since foam cells in the early phase of atherosclerosis are derived from
monocyte/macrophages, in which SRA is highly expressed (30, 31), SRA
may also play an important role as an AGE receptor in the early stages
of atherosclerosis.
In contrast, although functionally related to SRA, the class B
receptors differ significantly in structure. CD36, the defining member
of this class, is a highly glycosylated, single chain 88-kDa protein
that binds Ox-LDL, fatty acids, anionic phospholipids (including
phosphatidylinositol and phosphatidylserine), and the proteins collagen
and thrombospondin (32-36). As a result of the broad ligand
specificity of CD36, multiple roles for this protein have been
proposed. In vitro and in vivo studies indicate
that CD36 mediates a significant proportion of binding and
internalization of Ox-LDL by tissue-differentiated macrophages
(37-39). Antibodies to CD36 have been shown to block up to 50% of the
binding of Ox-LDL to, as well as its endocytic uptake by, normal
monocyte-derived macrophages (37, 38). In monocyte/macrophages from
donors with a human polymorphism associated with lack of CD36
expression (Naka
), the capacity to bind and internalize
Ox-LDL and the capacity to accumulate cholesteryl esters were reduced
to 50% compared with those obtained from normal subjects (39).
Similarly, the cell association of Ox-LDL at 37 °C with, and its
cell binding at 4 °C to, peritoneal macrophages obtained from CD36
null mice were reduced to 37 and 53-60%, respectively, compared with
wild-type macrophages (54). Furthermore, CD36 is expressed in
monocytes/macrophages in the core of atherosclerotic plaques (40).
Considered together, these studies suggest that the role of CD36 in
atherogenic processes might be different from that of SRA.
This study was conducted to test whether AGE proteins could be
recognized as ligands by CD36. The results obtained from cellular experiments using Chinese hamster ovary (CHO) cells overexpressing human CD36 (CD36-CHO cells) demonstrated a potential novel role for
CD36 in the pathogenesis of AGE-induced diabetic macrovascular complications.
 |
EXPERIMENTAL PROCEDURES |
Chemicals and Materials--
Penicillin G (1650 IU/mg),
streptomycin sulfate (750 IU/mg), G418, Dulbecco's modified Eagle's
medium, and Ham's F-12 medium were purchased from Life Technologies,
Inc. Na125I (3.7 GBq/ml) was purchased from Amersham
Pharmacia Biotech. FA6-152 (mouse anti-human CD36 monoclonal antibody)
was purchased from Immunotech. MO25 (mouse anti-human CD36 monoclonal
antibody) was kindly provided by Dr. G. A. Jamieson (American Red
Cross). MOPC21 (mouse IgG) was purchased from Sigma. Other
chemicals were of the best grade available from commercial sources.
Ligand Preparation and Iodination--
AGE-BSA was prepared as
described previously (41). Briefly, 2.0 g of BSA was dissolved in
10 ml of 0.5 M sodium phosphate buffer (pH 7.4) with
3.0 g of D-glucose. Each sample was sterilized by
ultrafiltration, incubated at 37 °C for 40 weeks, and dialyzed against phosphate-buffered saline (PBS) (pH 7.4). The extent of lysine
modification was 71% for AGE-BSA. The
N
-(carboxymethyl)lysine content of AGE-BSA
was 7.7 mol/mol of BSA (42). Human LDL (d = 1.019-1.063 g/ml) was isolated by sequential ultracentrifugation of
human plasma from normal lipidemic subjects after overnight fasting
(43) and dialyzed against 0.15 M NaCl and 1 mM
EDTA (43). To prepare Ox-LDL, LDL was dialyzed against PBS to remove
EDTA. LDL (0.1 mg/ml) was then incubated for 16 h at 37 °C with
5 µM CuSO4, followed by addition of 1 mM EDTA and cooling (43). The electrophoretic mobilities of
AGE-BSA preparations toward the anode were 1.4 times higher than those of unmodified BSA. Under identical conditions, Ox-LDL showed a similar
increase in electrophoretic mobilities. Thus, the modification of BSA
with glucose is associated with a significant increase in the net
negative charge (42).
AGE-BSA was labeled with 125I by IODO-GEN (Pierce), and
Ox-LDL was labeled as described by McFarlane (44) to specific
radioactivities of 850 and 400 cpm/ng, respectively.
Cell Culture and Isolation of a Transfected Cell
Line--
CHO-K1 cells were maintained at 37 °C in medium A (Ham's
F-12 medium supplemented with 100 units/ml penicillin and 100 units/ml streptomycin) containing 10% fetal calf serum. The cDNA of human CD36 was amplified from a human placenta cDNA library by polymerase chain reaction using the following primers: sense,
5'-CTCCAAGCTTAGAAAAATGGGCTGTGAC-3'; and antisense,
5'-GCATGCGGCCGCATTTGTGCTATTGTTACA-3'. The amplified human CD36
cDNA was subcloned into pRc/CMV (Invitrogen), sequenced, and
transfected into CHO-K1 cells by the electroporation method using a
Gene-Pulser (Bio-Rad). To select CD36-positive colonies, cells were
cultured in medium B (medium A supplemented with 10% fetal calf serum
and 0.5 mg/ml G418). Positive clones (CD36-CHO cells) detected by
immunofluorescence microscopy with anti-CD36 monoclonal antibody
FA6-152 were selected and maintained as a stock culture in medium B,
which selects against cells that spontaneously lose expression of CD36.
Immunoblotting--
Human platelets (a positive control) were
prepared using NycoPrep® (Nycomed Pharma AS, Oslo,
Norway). Blood obtained from a healthy consenting human volunteer who
had not received any medication for at least 1 week was anticoagulated
with 1 mM EDTA. Platelet-rich plasma was prepared by
centrifugation at 600 × g for 15 min at room
temperature. Washed platelets were then isolated from platelet-rich plasma using NycoPrep® 1.063. The cells were collected and
solubilized in lysis buffer (20 mM Tris and 150 mM NaCl (pH 7.4) (Tris-buffered saline) containing 2%
Triton X-100, 1 mM phenylmethanesulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1 µg/ml aprotinin).
Samples (50 µg of protein/lane) were subjected to SDS-polyacrylamide
gel electrophoresis (45) and then electrotransferred onto a
polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). The
membrane was blocked with 5% nonfat dry milk in Tris-buffered saline
containing 0.1% Tween 20 (Tris-buffered saline/Tween) for 2 h.
The membrane was then incubated with a 1:1000 dilution of mouse
anti-CD36 monoclonal antibody (MO25) in Tris-buffered saline/Tween for
1 h and washed with the same buffer three times. Immunoreactive
bands were detected by incubation with a 1:2000 dilution of horseradish
peroxidase-conjugated goat anti-mouse IgG (H + L) (Zymed
Laboratories Inc., South San Francisco, CA) for 45 min, followed
by addition of ECL reagent.
Immunofluorescence Microscopy--
For CD36 immunofluorescence
microscopy, cells were cultured in a 4-well LAB-TEK®
chamber slide (Nalge Nunc International, Naperville, IL) for 16 h,
washed three times with 0.01 M PBS (pH 7.4), fixed with 3.7% paraformaldehyde at 4 °C for 20 min, and washed again with PBS. The cells were then blocked with 5% BSA at room temperature and
incubated with anti-CD36 antibody (FA6-152) diluted 1:100 in PBS
containing 1% BSA for 1 h, followed by sequential incubation with
FluoroLink Cy2-labeled goat anti-mouse IgG (H + L) (Amersham Pharmacia
Biotech, Little Chalfont, United Kingdom) diluted 1:1000. The specimens
were mounted using Dako fluorescent mounting medium after washing and
examined with a confocal laser scanning microscope (FLUOVIEW, Olympus,
Tokyo, Japan).
Immunofluorescent Flow Cytometry--
Immunofluorescent flow
cytometric analysis was performed using a mouse monoclonal antibody
against CD36 (FA6-152). Cells (1 × 106 cells in 200 µl) were incubated with fluorescein isothiocyanate-conjugated FA6-152
(final concentration of 2.0 µg/ml) or fluorescein
isothiocyanate-conjugated mouse IgG1 (final concentration
of 2.0 µg/ml) at 4 °C for 30 min and assayed on a
fluorescence-activated cell sorter (Becton Dickinson) as previously
reported (46). Appropriate cell fractions for the analysis were
selected using a two-dimensional display of forward scatter and side
scatter of analyzed cells.
Cellular Assays--
Except for the binding study, all cellular
experiments were performed at 37 °C in a humidified atmosphere of
5% CO2 in air. Untransfected CHO cells were cultured in
medium A containing 10% fetal calf serum, and CD36-CHO cells were
cultured in medium B. Cells (8 × 104) were seeded in
a 24-well plate and cultured for 2 days in 1.0 ml of medium B (for
CD36-CHO cells) or medium A containing 10% fetal calf serum (for
untransfected CHO cells), which was then replaced by medium C
(Dulbecco's modified Eagle's medium containing 3% BSA). After
culture for 1 h, each well received 0.5 ml of medium C containing
various concentrations of 125I-Ox-LDL or
125I-AGE-BSA in the presence or absence of 20-fold excess
amounts of the unlabeled ligand to be tested. After incubation for the indicated times, the medium was taken from each well, and soluble radioactivity in trichloroacetic acid was determined as an index of
cellular degradation as described previously (3). After the cells were
washed three times with 1 ml of medium C and then three more times with
PBS, they were lysed with 1 ml of 0.1 N NaOH for 30 min at
37 °C, and the cell-associated radioactivity and cellular proteins
were determined with the BCA protein assay reagent (Bio-Rad). For the
binding study, cells that had been seeded in each well as described
above were incubated for 90 min at 4 °C in 0.5 ml of medium C with
various concentrations of 125I-AGE-BSA in the presence or
absence of 20-fold excess amounts of the unlabeled ligands. Each well
was washed three times with 1 ml of ice-cold PBS containing 3% BSA and
a further three times with PBS. The cells were lysed, and the
cell-bound radioactivity and cellular proteins were determined as
described above.
 |
RESULTS |
Overexpression of Human CD36 in CHO Cells Leads to Endocytic Uptake
of 125I-Ox-LDL--
CD36-CHO cells, which stably express
human CD36, were prepared as described above. To confirm overexpression
of CD36, we performed Western blotting with anti-CD36 monoclonal
antibody (MO25). Human platelets, which express a significant amount of
CD36, were used as a positive control. Wild-type CHO cells did not
yield any bands, whereas CD36-CHO cells showed a distinctive band (Fig.
1A). We performed further flow
cytometric analysis and immunofluorescence microscopy with anti-CD36
monoclonal antibody (FA6-152). Human CD36 was expressed on the cell
surface of CD36-CHO cells, but not wild-type CHO cells (Fig. 1,
B and C). Control IgG showed no
immunofluorescence (Fig. 1C). To determine whether CD36
expressed by these cells was functional or not, the cell association
and endocytic degradation of Ox-LDL by CD36-CHO cells were compared with untransfected wild-type CHO cells. The specific cell association of 125I-Ox-LDL with, and its specific degradation by,
CD36-CHO cells exhibited a dose-dependent saturation
pattern, with specific association showing a maximal level of 175 ng/mg
of cell protein; the apparent Kd for cell
association was 0.7 µg/ml, and the maximal ligand association was
193.4 ng/mg of cell protein (Fig. 2). The specific degradation of 125I-Ox-LDL reached a plateau at
>175 ng/mg of cell protein; the apparent Kd for
degradation was 0.7 µg/ml, and the maximal ligand degradation was
232.6 ng/mg of cell protein (Fig. 2). Parallel experiments in
untransfected cells (Fig. 2) and mock-transfected CHO cells showed that
the cell association of 125I-Ox-LDL and its endocytic
degradation occurred at a negligible level (data not shown). These
results indicate that CD36 expressed on CD36-CHO cells serves as a
receptor for Ox-LDL.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 1.
Detection of CD36 protein in CD36-CHO cells
by immunoblot analysis, confocal laser microscopy, and
fluorescence-activated cell sorter analysis. A,
cell lysates from human platelets (30 µg; positive control;
first lane), CD36-CHO cells (30 µg; second
lane), or wild-type CHO cells (30 µg; third lane)
were subjected to 8% SDS-polyacrylamide gel electrophoresis and
immunoblotted with anti-CD36 antibody (MO25). B, cells were
fixed with 3.7% paraformaldehyde and incubated for 1 h with
anti-CD36 antibody (FA6-152) diluted 1:100, followed by sequential
incubation with FluoroLink Cy2-labeled goat anti-mouse IgG. The
specimens were observed using a confocal laser scanning microscope.
C, cells (1 × 106 in 200 µl) were
incubated with fluorescein isothiocyanate (FITC)-conjugated
FA6-152 (final concentration of 2.0 µg/ml) or fluorescein
isothiocyanate-conjugated mouse IgG1 (final concentration
of 2.0 µg/ml) at 4 °C for 30 min and analyzed on a
fluorescence-activated cell sorter.
|
|

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 2.
Endocytic uptake of 125I-Ox-LDL
by CD36-CHO cells. Cells were incubated for 5 h in 0.5 ml of
medium C with increasing concentrations of 125I-Ox-LDL in
the presence ( ) or absence ( ) of 20-fold excess amounts of the
unlabeled ligands. The amounts of cell-associated
125I-Ox-LDL (upper panels) and its degradation
(lower panels) were determined as described under
"Experimental Procedures." The specific cell association and
degradation ( ) were plotted after correcting for nonspecific cell
association and degradation. Data represent the means of three separate
experiments. Error bars represent S.D.
|
|
Endocytic Uptake and Degradation of 125I-AGE-BSA by
CD36-CHO Cells--
We determined the endocytic uptake and degradation
of 125I-AGE-BSA by CD36-CHO cells at 37 °C. The amount
of cell association of 125I-AGE-BSA with CD36-CHO cells
increased in a dose-dependent manner and was almost
competed away by a 20-fold excess of unlabeled AGE-BSA (Fig.
3). The specific cell association
exhibited a dose-dependent saturation pattern with a
plateau level of 140 ng/mg of cell protein, an apparent
Kd of 4.1 µg/ml, and a maximal ligand association of 215.1 ng/mg of cell protein. This level was >3.5-fold higher than
that of specific cell association with wild-type CHO cells, which
showed only a slight cell association for AGE-BSA (Fig. 3). The
degradation of 125I-AGE-BSA by the transfected cells was
also significant. The specific degradation was increased
dose-dependently, with a plateau level of 38 ng/mg of cell
protein; the apparent Kd for degradation was 3.9 µg/ml, and the maximal ligand degradation was 52.6 ng/mg of cell
protein (Fig. 3). In sharp contrast, untransfected cells did not
degrade 125I-AGE-BSA at all under the same conditions (Fig.
3). Furthermore, mock-transfected CHO cells also demonstrated no
endocytic degradation of 125I-AGE-BSA (data not shown).
This result disclosed a novel function of CD36 as a receptor for
AGE-BSA.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 3.
Dose-dependent effects of the
endocytic uptake and degradation of 125I-AGE-BSA by
CD36-CHO cells. Cells were incubated for 5 h in 0.5 ml of
medium C with increasing concentrations of 125I-AGE-BSA in
the presence ( ) or absence ( ) of 20-fold excess amounts of the
unlabeled ligands. The amounts of cell-associated
125I-AGE-BSA (upper panels) and its degradation
(lower panels) were determined as described under
"Experimental Procedures." The specific cell association and
degradation ( ) were plotted after correcting for nonspecific cell
association and degradation. Data represent the means of three separate
experiments. Error bars represent S.D.
|
|
Binding of 125I-AGE-BSA to CD36-CHO Cells--
We
attempted to determine the cellular binding of 125I-AGE-BSA
to CD36-CHO cells at 4 °C. The total binding of
125I-AGE-BSA was inhibited by 80% by an excess amount of
unlabeled AGE-BSA. The specific binding, obtained by subtracting the
nonspecific binding from the total binding, yielded a saturation
pattern. Scatchard analysis of this specific binding disclosed a
binding site with an apparent Kd of 5.6 µg/ml and
maximal surface binding of 130 ng/mg of cell protein, indicating that
CD36-CHO cells possess a high affinity binding site for AGE-BSA (Fig.
4). The Kd value of
Ox-LDL for CD36 was 3.1 µg/ml (data not shown).

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 4.
Binding of 125I-AGE-BSA to
CD36-CHO cells. CD36-CHO cells were seeded (8 × 104 cells in each well of a 24-well culture plate) in 1.0 ml of medium B and cultured for 2 days. The cells in each well were
washed with 1.0 ml of PBS, replaced with 0.5 ml of medium C containing
various concentrations of 125I-AGE-BSA, and incubated for
90 min at 4 °C in the presence ( ) or absence ( ) of excess
amounts of unlabeled ligands. The cells were then washed and lysed in
0.1 N NaOH, and cell-bound radioactivity was determined.
Specific binding ( ) was determined by subtracting nonspecific
binding ( ) from total binding ( ). Inset, Scatchard
analysis of the specific binding curve. B/F,
bound/free.
|
|
Effect of Ox-LDL on Endocytic Uptake of 125I-AGE-BSA by
CD36-CHO Cells--
To examine whether the recognition site for
AGE-BSA in CD36-CHO cells was identical to that for Ox-LDL, we studied
the effect of Ox-LDL on the cellular binding of
125I-AGE-BSA to CD36-CHO cells at 4 °C. The cellular
binding of 125I-AGE-BSA to CD36-CHO cells was effectively
(60%) replaced by unlabeled AGE-BSA, whereas unlabeled Ox-LDL had a
slightly weaker effect (30%). Unlabeled LDL (used as a negative
control) was ineffective in suppressing the cellular binding of AGE-BSA
(Fig. 5A). We further studied
the effect of Ox-LDL on the endocytic uptake of
125I-AGE-BSA by CD36-CHO cells at 37 °C. The cell
association of 125I-AGE-BSA with CD36-CHO cells was
effectively (>80%) replaced by unlabeled AGE-BSA, whereas unlabeled
Ox-LDL had a slightly weaker effect (<40%). Unlabeled LDL (the
negative control) was ineffective in suppressing the cell association
of AGE-BSA (Fig. 5B). Similarly, the endocytic degradation
of 125I-AGE-BSA at 37 °C was inhibited almost completely
by the presence of an excess amount of unlabeled AGE-BSA, whereas
unlabeled Ox-LDL had a slightly weaker effect (<55%) (Fig.
5C). Unlabeled LDL was also ineffective in suppressing the
degradation of AGE-BSA by CD36-CHO cells (Fig. 5C).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of Ox-LDL on the cellular binding of
125I-AGE-BSA to, and the endocytic uptake of
125I-AGE-BSA by, CD36-CHO cells. A, cells
were incubated at 4 °C for 90 min with 0.5 ml of medium C containing
5 µg/ml 125I-AGE-BSA in the presence of increasing
concentrations of unlabeled AGE-BSA ( ), Ox-LDL ( ), or LDL ( ).
B and C, cells were incubated at 37 °C for
5 h with 0.5 ml of medium C containing 5 µg/ml
125I-AGE-BSA in the presence of increasing concentrations
of unlabeled AGE-BSA ( ), Ox-LDL ( ), or LDL ( ). The amounts of
125I-AGE-BSA bound (A), cell-associated
125I-AGE-BSA (B), and its degradation products
(C) were determined as described under "Experimental
Procedures." Data represent the means of three separate experiments.
Error bars represent S.D. The values for 100% binding,
association, and degradation were 58 (A), 65 (B),
and 37 (C) ng/mg of cell protein.
|
|
Effect of Anti-CD36 Antibody on Endocytic Uptake of
125I-AGE-BSA by CD36-CHO Cells--
To determine the
effects of anti-CD36 monoclonal antibody (FA6-152) on the endocytic
uptake of 125I-Ox-LDL by CD36-CHO cells, we first confirmed
the capacity of this antibody to inhibit CD36-mediated cellular binding
of Ox-LDL at 4 °C. As shown in Fig.
6A, the cellular binding of
125I-Ox-LDL to CD36-CHO cells was inhibited by addition of
anti-CD36 antibody (>60%). The extent of inhibition by anti-CD36
antibody was almost the same as with unlabeled Ox-LDL (>68%), whereas
nonimmune IgG had no effect on this process. We further studied the
effect of anti-CD36 antibody on the cell association of
125I-Ox-LDL with CD36-CHO cells at 37 °C. The cell
association of 125I-Ox-LDL with CD36-CHO cells was also
effectively (>70%) inhibited by anti-CD36 antibody. The extent of
inhibition by anti-CD36 antibody was almost the same as with unlabeled
Ox-LDL (>70%), whereas nonimmune IgG had no effect on this process
(Fig. 6B). It is therefore likely that the specific cell
association of 125I-Ox-LDL with these cells was completely
inhibited by anti-CD36 antibody. This antibody was equally effective
against the endocytic degradation of 125I-Ox-LDL at
37 °C; the total endocytic degradation of 125I-Ox-LDL by
CD36-CHO cells was effectively inhibited by the antibody (>65%) and
by unlabeled Ox-LDL (68%) (Fig. 6C). These results suggest
that anti-CD36 antibody (FA6-152) recognizes a portion of CD36 that
serves as a binding site for Ox-LDL in CD36-CHO cells. Under identical
experimental conditions, we examined the effect of the same antibody on
the cellular binding of 125I-AGE-BSA to, and endocytic
uptake by, CD36-CHO cells at 4 °C. As shown in Fig.
7A, the cellular binding of
125I-AGE-BSA was inhibited by unlabeled AGE-BSA by >60%,
but only by 25% by the antibody. Similarly, the antibody could replace 40% of the total cell association of 125I-AGE-BSA at
37 °C, whereas the inhibition was more dominant when unlabeled
AGE-BSA was used (Fig. 7B). This antibody was also equally effective against the endocytic degradation of 125I-AGE-BSA
at 37 °C (Fig. 7C). These results indicate that a major part of the cell association as well as the subsequent endocytic degradation of AGE-BSA by CD36-CHO cells are mediated by CD36.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of anti-CD36 antibody FA6-152 on the
cellular binding of 125I-Ox-LDL to, and the endocytic
uptake of 125I-Ox-LDL by, CD36-CHO cells.
A, cells were incubated at 4 °C for 90 min with 0.5 ml of
medium C containing 5 µg/ml 125I-Ox-LDL in the presence
or absence of unlabeled Ox-LDL (100 µg/ml), anti-CD36 antibody
(FA6-152; 10 µg/ml), or control antibody (MOPC21; 10 µg/ml).
B and C, cells were incubated at 37 °C for
5 h with 0.5 ml of medium C containing 5 µg/ml
125I-Ox-LDL in the presence or absence of unlabeled Ox-LDL
(100 µg/ml), anti-CD36 antibody FA6-152 (10 µg/ml), or control
antibody (MOPC21; 10 µg/ml). The amounts of 125I-Ox-LDL
bound (A), cell-associated 125I-Ox-LDL
(B), and its degradation products (C) were
determined as described under "Experimental Procedures." Data
represent the means of three separate experiments. Error
bars represent S.D. The values for 100% binding, association, and
degradation were 68 (A), 58 (B), and 65 (C) ng/mg of cell protein.
|
|

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of anti-CD36 antibody FA6-152 on the
cellular binding of 125I-AGE-BSA to, and the endocytic
uptake of 125I-AGE-BSA by, CD36-CHO cells.
A, cells were incubated at 4 °C for 90 min with 0.5 ml of
medium C containing 5 µg/ml 125I-AGE-BSA in the presence
or absence of unlabeled AGE-BSA (200 µg/ml), anti-CD36 antibody
(FA6-152; 80 µg/ml), or control antibody (MOPC21; 80 µg/ml).
B and C, cells were incubated at 37 °C for
5 h with 0.5 ml of medium C containing 5 µg/ml
125I-AGE-BSA in the presence of increasing concentrations
of unlabeled AGE-BSA (200 µg/ml), anti-CD36 antibody FA6-152 (80 µg/ml), or control antibody (MOPC21; 80 µg/ml). The amounts of
125I-AGE-BSA bound (A), cell-associated
125I-AGE-BSA (B), and its degradation products
(C) were determined as described under "Experimental
Procedures." Data represent the means of three separate experiments.
Error bars represent S.D. The values for 100% binding,
association, and degradation were 58 (A), 65 (B),
and 37 (C) ng/mg of cell protein.
|
|
 |
DISCUSSION |
Our previous studies using CHO cells overexpressing the class A
scavenger receptor (SRA) (29) and peritoneal macrophages prepared from
SRA knockout mice (28) indicated that SRA is one of the major AGE
receptors involved in the endocytic uptake of AGE proteins by
macrophages or macrophage-derived cells. The present study provided
additional and novel information that CD36, a member of the class B
scavenger receptor family, also serves as an AGE receptor in
vivo and therefore might participate in the pathogenesis of
diabetic macrovascular complications.
CD36 was identified as an Ox-LDL receptor by Endemann et al.
(32). Nozaki et al. (39) demonstrated that the endocytic uptake of Ox-LDL by monocyte-derived macrophages obtained from CD36-deficient patients was reduced by ~50% compared with that by
macrophages prepared from normal subjects and that the subsequent Ox-LDL-induced accumulation of cholesteryl esters was also reduced in
CD36-deficient macrophages. Furthermore, the experiments using CD36
null mice demonstrated that the cell association of Ox-LDL with
peritoneal macrophages obtained from CD36 null mice was reduced by 63%
compared with macrophages prepared from wild-type mice (54). Therefore,
it is generally accepted that both CD36 and SRA are major Ox-LDL
receptors in macrophages. A recent report by Nakata et al.
(40) revealed a difference in the histological distribution pattern
between CD36 and SRA; CD36 is highly expressed in macrophage-derived
foam cells in the core of atherosclerotic plaques, whereas SRA-positive
macrophage-derived foam cells tend to localize in the periphery of
atherosclerotic lesions, suggesting that CD36 might play a different
role compared with SRA in the formation of atherosclerotic plaques. The
immunohistochemical study by Kume et al. (11) using a
monoclonal antibody against N
-(carboxymethyl)lysine, a major AGE
structure, demonstrated that, in the early stage of atherosclerotic
lesions (diffuse intimal thickening and fatty streaks), AGE proteins
are localized in macrophage-derived foam cells, whereas in the advanced
stage (atherosclerotic plaques), AGE proteins are also observed in
vascular smooth muscle cell-derived foam cells and the extracellular
matrix. Other immunohistochemical studies with antibodies against
different AGE structures demonstrated similar findings (47-49),
suggesting that AGE proteins generated extracellularly are actively
endocytosed by monocyte-derived macrophages via SRA and CD36 or both,
with subsequent intralysosomal accumulation (intracellularly). In
addition, since the interaction of AGE proteins with these cells is
known to induce cellular responses such as secretion of cytokines, it
is likely that CD36 may contribute to the formation of atherosclerotic
lesions as a major AGE receptor.
RAGE is known as the main AGE receptor expressed in microvascular
endothelial cells, where the interaction of AGE proteins with RAGE
leads to enhancement of angiogenesis through induction of the vascular
endothelial growth factor (50). A recent report demonstrated, however,
that CD36 is also expressed in microvascular endothelial cells and
plays a role in apoptosis-dependent neovascularization as a
thrombospondin-1 receptor (51). Therefore, it is possible that CD36 is
also involved in diabetic microangiopathy as an AGE receptor. Further
studies are required to determine the relative contributions of CD36
and RAGE to the in vivo pathogenesis.
A competition study using anti-CD36 monoclonal antibody (52) indicated
that the binding site of CD36 for Ox-LDL resides in the region
corresponding to amino acids 155-183 of its extracellular domain.
Anti-CD36 monoclonal antibody (FA6-152) was reported to compete with
Ox-LDL for binding to CD36 (53). Specific endocytic uptake of
125I-Ox-LDL by CD36-CHO cells was completely inhibited by
FA6-152 under our experimental conditions (Fig. 6), confirming the
above notion that FA6-152 could recognize the binding domain of CD36 for Ox-LDL. Under these conditions, the endocytic degradation of
125I-AGE-BSA by CD36-CHO cells was inhibited by FA6-152 to
a slight but significant degree (40%) and also by Ox-LDL (50%), an
authentic ligand for CD36. These results clearly indicate that the
binding domain of CD36 for AGE-BSA might overlap to some extent with
the binding domain of CD36 for Ox-LDL, suggesting that binding of AGE
ligands to CD36 might occur in or near this region.
CD36 is known as a multifunctional molecule, recognizing many ligands
of different structures, including Ox-LDL (32, 39, 52, 54), apoptotic
cells (55, 56), thrombospondin-1 (57, 58), and long chain fatty acids
(54, 59-61). These ligands have, in common, a negatively charged
nature, raising the possibility that CD36 recognizes the negative
charge of AGE proteins (42). Further studies are needed to determine
the AGE structure(s) required for recognition by CD36.
It was reported that peroxisome proliferator-activated receptor-
modulates the expression of CD36 in macrophages (62-64). Recently,
Iwashima et al. (65) demonstrated that AGE/BSA induces the
expression and activation of peroxisome proliferator-activated receptor-
in cultured mesangial cells, raising the possibility that
CD36 could be up-regulated by AGE proteins through the activation of
peroxisome proliferator-activated receptor-
. In addition, CD36 was
reported to be involved in signal transduction. First, CD36 is
physically associated with Src family kinases in human platelets
(66, 67). Second, CD36 modulates signal transduction via the Src family
kinase Fyn, followed by apoptosis in microvascular endothelial cells
(51). Finally, the interaction of Ox-LDL with CD36 modulates the
activity of nuclear factor-
B in CD36-CHO cells (68). Because of the
physical and functional association of CD36 with tyrosine kinases or
nuclear factor-
B, the interaction of AGE proteins with CD36 could
modulate signal transduction.
In addition to CD36, SR-BI (scavenger receptor
class B type I) also belongs to the class B
scavenger receptor family (69). The ligand specificity of SR-BI is
essentially identical to that of CD36, except that both SR-BI and CD36
bind high density lipoprotein, but only SR-BI efficiently mediates the
cellular uptake of cholesteryl esters from high density lipoprotein
particles (70, 71). It would be an interesting issue to determine
whether SR-BI also serves as a receptor for AGE proteins.
To our knowledge, there are no reports to date on the association of
CD36 with the pathogenesis of diabetes or diabetic complications. Recent results obtained in SHR/NCrj rats indirectly suggested that a
major function of CD36 is as a potential genetic factor for insulin
resistance (72). Although a role for CD36 in insulin resistance was
claimed in another recent study (73), experiments in CD36 null mice
provided direct functional evidence that CD36 is involved in fatty acid
uptake by adipocytes (54). Interestingly, CD36 null mice exhibit
hypoglycemia (the mechanism is not clear), suggesting a functional role
for CD36 in maintenance of plasma glucose (54), which raises the
possibility that CD36 might be linked to the development of diabetic
conditions. Our finding that CD36 recognizes and endocytoses AGE
proteins as ligands suggests the possible involvement of CD36 in
diabetic macrovascular complications.
We are grateful to Drs. J. A. Jamieson
and Naomasa Yamamoto (Tokyo Metropolitan Institute of Medical
Sciences) for providing anti-CD36 monoclonal antibody MO25.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M006545200
1.
|
Maillard, L. C.
(1992)
C. R. Acad. Sci. Paris Ser. III
154,
66-68
|
2.
|
Finot, P. A.
(1982)
Am. Chem. Soc.
198,
91-124
|
3.
|
Araki, N.,
Ueno, N.,
Chakrabarti, B.,
Morino, Y.,
and Horiuchi, S.
(1992)
J. Biol. Chem.
267,
10211-10214[Abstract/Free Full Text]
|
4.
|
Miyata, T.,
Oda, O.,
Inagi, R.,
Iida, Y.,
Araki, N.,
Yamada, N.,
Horiuchi, S.,
Taniguchi, N.,
Maeda, K.,
and Kinoshita, T.
(1993)
J. Clin. Invest.
92,
1243-1252[Medline]
[Order article via Infotrieve]
|
5.
|
Yamada, K.,
Miyahara, Y.,
Hamaguchi, K.,
Nakayama, M.,
Nakano, H.,
Nozaki, O.,
Miura, Y.,
Suzuki, S.,
Tuchida, H.,
Mimura, N.,
Mimura, N.,
Araki, N.,
and Horiuchi, S.
(1994)
Clin. Nephrol.
42,
354-361[Medline]
[Order article via Infotrieve]
|
6.
|
Nakayama, H.,
Mitsuhashi, T.,
Kuwajima, S.,
Aoki, S.,
Kuroda, Y.,
Itoh, T.,
and Nakagawa, S.
(1993)
Diabetes
42,
345-350[Abstract]
|
7.
|
Mitsuhashi, T.,
Nakayama, H.,
Itoh, T.,
Kuwajima, S.,
Aoki, S.,
Atsumi, T.,
and Koike, T.
(1993)
Diabetes
42,
826-832[Abstract]
|
8.
|
Makita, Z.,
Vlassara, H.,
Cerami, A.,
and Bucala, R.
(1992)
J. Biol. Chem.
267,
5133-5138[Abstract/Free Full Text]
|
9.
|
Vlassara, H.,
Bucala, R.,
and Striker, L.
(1994)
Lab. Invest.
70,
138-151[Medline]
[Order article via Infotrieve]
|
10.
|
Nakamura, Y.,
Horii, Y.,
Nishino, T.,
Shiiki, H.,
Sakaguchi, Y.,
Kagoshima, T.,
Dohi, K.,
Makita, Z.,
Vlassara, H.,
and Bucala, R.
(1993)
Am. J. Pathol.
143,
1649-1656[Abstract]
|
11.
|
Kume, S.,
Takeya, M.,
Mori, T.,
Araki, N.,
Suzuki, H.,
Horiuchi, S.,
Kodama, T.,
Miyauchi, Y.,
and Takahashi, K.
(1995)
Am. J. Pathol.
147,
654-667[Abstract]
|
12.
|
Yui, S.,
Sasaki, T.,
Araki, N.,
Horiuchi, S.,
and Yamazaki, M.
(1994)
J. Immunol.
152,
1943-1949[Abstract/Free Full Text]
|
13.
|
Miyata, T.,
Inagi, R.,
Iida, Y.,
Sato, M.,
Yamada, N.,
Oda, O.,
Maeda, K.,
and Seo, H.
(1994)
J. Clin. Invest.
93,
521-528[Medline]
[Order article via Infotrieve]
|
14.
|
Kirstein, M.,
Aston, C.,
Hintz, R.,
and Vlassara, H.
(1992)
J. Clin. Invest.
90,
439-446[Medline]
[Order article via Infotrieve]
|
15.
|
Vlassara, H.,
Brownlee, M.,
Manogue, K. R.,
Dinarello, C. A.,
and Pasagian, A.
(1988)
Science
240,
1546-1548[Medline]
[Order article via Infotrieve]
|
16.
|
Kirstein, M.,
Brett, J.,
Radoff, S.,
Ogawa, S.,
Stern, D.,
and Vlassara, H.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
9010-9014[Abstract]
|
17.
|
Schmidt, A. M.,
Yan, S. D.,
Brett, J.,
Mora, R.,
Nowygrod, R.,
and Stern, D.
(1993)
J. Clin. Invest.
91,
2155-2168[Medline]
[Order article via Infotrieve]
|
18.
|
Tezuka, M.,
Koyama, N.,
Morisaki, N.,
Saito, Y.,
Yoshida, S.,
Araki, N.,
and Horiuchi, S.
(1993)
Biochem. Biophys. Res. Commun.
193,
674-680[CrossRef][Medline]
[Order article via Infotrieve]
|
19.
|
Esposito, C.,
Gerlach, H.,
Brett, J.,
Stern, D.,
and Vlassara, H.
(1989)
J. Exp. Med.
170,
1387-1407[Abstract]
|
20.
|
Skolnik, E. Y.,
Yang, Z.,
Makita, Z.,
Radoff, S.,
Kirstein, M.,
and Vlassara, H.
(1991)
J. Exp. Med.
174,
931-939[Abstract]
|
21.
|
Doi, T.,
Vlassara, H.,
Kirstein, M.,
Yamada, Y.,
Striker, G. E.,
and Striker, L. J.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
2873-2877[Abstract]
|
22.
|
Schmidt, A. M.,
Vianna, M.,
Gerlach, M.,
Brett, J.,
Ryan, J.,
Kao, J.,
Esposito, C.,
Hegarty, H.,
Hurley, W.,
and Clauss, M.
(1992)
J. Biol. Chem.
267,
14987-14997[Abstract/Free Full Text]
|
23.
|
Neeper, M.,
Schmidt, A. M.,
Brett, J.,
Yan, S. D.,
Wang, F.,
Pan, Y. C.,
Elliston, K.,
Stern, D.,
and Shaw, A.
(1992)
J. Biol. Chem.
267,
14998-15004[Abstract/Free Full Text]
|
24.
|
Vlassara, H.,
Li, Y. M.,
Imani, F.,
Wojciechowicz, D.,
Yang, Z.,
Liu, F. T.,
and Cerami, A.
(1995)
Mol. Med.
1,
634-646[Medline]
[Order article via Infotrieve]
|
25.
|
Li, Y. M.,
Mitsuhashi, T.,
Wojciechowicz, D.,
Shimizu, N.,
Li, J.,
Stitt, A.,
He, C.,
Banerjee, D.,
and Vlassara, H.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
11047-11052[Abstract/Free Full Text]
|
26.
|
Thornalley, P. J.
(1998)
Cell. Mol. Biol.
44,
1013-1023
|
27.
|
Doi, T.,
Higashino, K.,
Kurihara, Y.,
Wada, Y.,
Miyazaki, T.,
Nakamura, H.,
Uesugi, S.,
Imanishi, T.,
Kawabe, Y.,
and Itakura, H.
(1993)
J. Biol. Chem.
268,
2126-2133[Abstract/Free Full Text]
|
28.
|
Suzuki, H.,
Kurihara, Y.,
Takeya, M.,
Kamada, N.,
Kataoka, M.,
Jishage, K.,
Ueda, O.,
Sakaguchi, H.,
Higashi, T.,
Suzuki, T.,
Takashima, Y.,
Kawabe, Y.,
Cynshi, O.,
Wada, Y.,
Honda, M.,
Kurihara, H.,
Aburatani, H.,
Doi, T.,
Matsumoto, A.,
Azuma, S.,
Noda, T.,
Toyoda, Y.,
Itakura, H.,
Yazaki, Y.,
Horiuchi, S.,
Takahashi, K.,
Kruijt, J. K.,
van Berkel, J. C.,
Steinbrecher, U. P.,
Ishibashi, S.,
Maeda, N.,
Gordon, S.,
and Kodama, T.
(1997)
Nature
386,
292-296[CrossRef][Medline]
[Order article via Infotrieve]
|
29.
|
Araki, N.,
Higashi, T.,
Mori, T.,
Shibayama, R.,
Kawabe, Y.,
Kodama, T.,
Takahashi, K.,
Shichiri, M.,
and Horiuchi, S.
(1995)
Eur. J. Biochem.
230,
408-415[Abstract]
|
30.
|
Naito, M.,
Suzuki, H.,
Mori, T.,
Matsumoto, A.,
Kodama, T.,
and Takahashi, K.
(1992)
Am. J. Pathol.
141,
591-599[Abstract]
|
31.
|
Yla-Herttuala, S.,
Rosenfeld, M. E.,
Parthasarathy, S.,
Sigal, E.,
Sarkioja, T.,
Witztum, J. L.,
and Steinberg, D.
(1991)
J. Clin. Invest.
87,
1146-1152[Medline]
[Order article via Infotrieve]
|
32.
|
Endemann, G.,
Stanton, L. W.,
Madden, K. S.,
Bryant, C. M.,
White, R. T.,
and Protter, A. A.
(1993)
J. Biol. Chem.
268,
11811-11816[Abstract/Free Full Text]
|
33.
|
Oquendo, P.,
Hundt, E.,
Lawler, J.,
and Seed, B.
(1989)
Cell
58,
95-101[Medline]
[Order article via Infotrieve]
|
34.
|
Tandon, N. N.,
Kralisz, U.,
and Jamieson, G. A.
(1989)
J. Biol. Chem.
264,
7576-7583[Abstract/Free Full Text]
|
35.
|
Silverstein, R. L.,
Asch, A. S.,
and Nachman, R. L.
(1989)
J. Clin. Invest.
84,
546-552[Medline]
[Order article via Infotrieve]
|
36.
|
Greenwalt, D. E.,
Lipsky, R. H.,
Ockenhouse, C. F.,
Ikeda, H.,
Tandon, N. N.,
and Jamieson, G. A.
(1992)
Blood
80,
1105-1115[Medline]
[Order article via Infotrieve]
|
37.
|
Nicholson, A. C.,
Frieda, S.,
Pearce, A.,
and Silverstein, R. L.
(1995)
Arterioscler. Thromb. Vasc. Biol.
15,
269-275[Abstract/Free Full Text]
|
38.
|
Huh, H. Y.,
Pearce, S. F.,
Yesner, L. M.,
Schindler, J. L.,
and Silverstein, R. L.
(1996)
Blood
87,
2020-2028[Abstract/Free Full Text]
|
39.
|
Nozaki, S.,
Kashiwagi, H.,
Yamashita, S.,
Nakagawa, T.,
Kostner, B.,
Tomiyama, Y.,
Nakata, A.,
Ishigami, M.,
Miyagawa, J.,
Kameda-Takemura, K.,
Kurata, Y.,
and Matsuzawa, Y.
(1995)
J. Clin. Invest.
96,
1859-1865[Medline]
[Order article via Infotrieve]
|
40.
|
Nakata, A.,
Nakagawa, Y.,
Nishida, M.,
Nozaki, S.,
Miyagawa, J.,
Nakagawa, T.,
Tamura, R.,
Matsumoto, K.,
Kameda-Takemura, K.,
Yamashita, S.,
and Matsuzawa, Y.
(1999)
Arterioscler. Thromb. Vasc. Biol.
19,
1333-1339[Abstract/Free Full Text]
|
41.
|
Takata, K.,
Horiuchi, S.,
Araki, N.,
Shiga, M.,
Saitoh, M.,
and Morino, Y.
(1988)
J. Biol. Chem.
263,
14819-14825[Abstract/Free Full Text]
|
42.
|
Nagai, R.,
Matsumoto, K.,
Ling, X.,
Suzuki, H.,
Araki, T.,
and Horiuchi, S.
(2000)
Diabetes
49,
1714-1723[Abstract]
|
43.
|
Sakai, M.,
Miyazaki, A.,
Hakamata, H.,
Sasaki, T.,
Yui, S.,
Yamazaki, M.,
Shichiri, M.,
and Horiuchi, S.
(1994)
J. Biol. Chem.
269,
31430-31435[Abstract/Free Full Text]
|
44.
|
McFarlane, A. S.
(1958)
Nature
182,
53
|
45.
|
Laemmli, U. K.
(1970)
Nature
227,
680-685[Medline]
[Order article via Infotrieve]
|
46.
|
Matsumoto, K.,
Hirano, K.,
Nozaki, S.,
Takamoto, A.,
Nishida, M.,
Nakagawa-Toyama, Y.,
Janabi, M. Y.,
Ohya, T.,
Yamashita, S.,
and Matsuzawa, Y.
(2000)
Arterioscler. Thromb. Vasc. Biol.
20,
1027-1032[Abstract/Free Full Text]
|
47.
|
Sakata, N.,
Imagana, Y.,
Meng, J.,
Tachikawa, Y.,
Takebayashi, S.,
Nagai, R.,
and Horiuchi, S.
(1999)
Atherosclerosis
142,
67-77[CrossRef][Medline]
[Order article via Infotrieve]
|
48.
|
Sakata, N.,
Imagana, Y.,
Meng, J.,
Tachikawa, Y.,
Takebayashi, S.,
Nagai, R.,
Horiuchi, S.,
Itabe, H.,
and Takano, T.
(1998)
Atherosclerosis
141,
61-75[CrossRef][Medline]
[Order article via Infotrieve]
|
49.
|
Uchida, K.,
Khor, O. T.,
Oya, T.,
Osawa, T.,
Yasuda, Y.,
and Miyata, T.
(1997)
FEBS Lett.
410,
313-318[CrossRef][Medline]
[Order article via Infotrieve]
|
50.
|
Yamagishi, S.,
Yonekura, H.,
Yamamoto, Y.,
Katsuno, K.,
Sato, F.,
Mita, I.,
Ooka, H.,
Satozawa, N.,
Kawakami, T.,
Nomura, M.,
and Yamamoto, H.
(1997)
J. Biol. Chem.
272,
8723-8730[Abstract/Free Full Text]
|
51.
|
Jimenez, B.,
Volpert, O. V.,
Crawford, S. E.,
Febbraio, M.,
Silverstein, R. L.,
and Bouck, N.
(2000)
Nat. Med.
6,
41-48[CrossRef][Medline]
[Order article via Infotrieve]
|
52.
|
Puente Navazo, M. D.,
Daviet, L.,
Ninio, E.,
and McGregor, J. L.
(1996)
Arterioscler. Thromb. Vasc. Biol.
16,
1033-1039[Abstract/Free Full Text]
|
53.
|
Calvo, D.,
Gomez-Coronado, D.,
Suarez, Y.,
Lasuncion, M. A.,
and Vega, M. A.
(1998)
J. Lipid Res.
39,
777-788[Abstract/Free Full Text]
|
54.
|
Febbraio, M.,
Abumrad, N. A.,
Hajjar, D. P.,
Sharma, K.,
Cheng, W.,
Pearce, S. F.,
and Silverstein, R. L.
(1999)
J. Biol. Chem.
274,
19055-19062[Abstract/Free Full Text]
|
55.
|
Savill, J.,
Hogg, N.,
Ren, Y.,
and Haslett, C.
(1992)
J. Clin. Invest.
90,
1513-1522[Medline]
[Order article via Infotrieve]
|
56.
|
Ren, Y.,
Silverstein, R. L.,
Allen, J.,
and Savill, J.
(1995)
J. Exp. Med.
181,
1857-1862[Abstract]
|
57.
|
Asch, A. S.,
Barnwell, J.,
Silverstein, R. L.,
and Nachman, R. L.
(1987)
J. Clin. Invest.
79,
1054-1061[Medline]
[Order article via Infotrieve]
|
58.
|
Silverstein, R. L.,
Baird, M.,
Lo, S. K.,
and Yesner, L. M.
(1992)
J. Biol. Chem.
267,
16607-16612[Abstract/Free Full Text]
|
59.
|
Abumrad, N. A.,
el-Maghrabi, M. R.,
Amri, E. Z.,
Lopez, E.,
and Grimaldi, P. A.
(1993)
J. Biol. Chem.
268,
17665-17668[Abstract/Free Full Text]
|
60.
|
Sfeir, Z.,
Ibrahimi, A.,
Amri, E.,
Grimaldi, P.,
and Abumrad, N.
(1999)
Mol. Cell. Biochem.
192,
3-8[CrossRef][Medline]
[Order article via Infotrieve]
|
61.
|
Abumrad, N.,
Coburn, C.,
and Ibrahimi, A.
(1999)
Biochim. Biophys. Acta
1441,
4-13[Medline]
[Order article via Infotrieve]
|
62.
|
Tontonoz, P.,
Nagy, L.,
Alvarez, J. G.,
Thomazy, V. A.,
and Evans, R. M.
(1998)
Cell
93,
241-252[Medline]
[Order article via Infotrieve]
|
63.
|
Nagy, L.,
Tontonoz, P.,
Alvarez, J. G.,
Chen, H.,
and Evans, R. M.
(1998)
Cell
93,
229-240[Medline]
[Order article via Infotrieve]
|
64.
|
Huang, J. T.,
Welch, J. S.,
Ricote, M.,
Binder, C. J.,
Willson, T. M.,
Kelly, C.,
Witztum, J. L.,
Funk, C. D.,
Conrad, D.,
and Glass, C. K.
(1999)
Nature
400,
378-382[CrossRef][Medline]
[Order article via Infotrieve]
|
65.
|
Iwashima, Y.,
Eto, M.,
Horiuchi, S.,
and Sano, H.
(1999)
Biochem. Biophys. Res. Commun.
264,
441-448[CrossRef][Medline]
[Order article via Infotrieve]
|
66.
|
Hirao, A.,
Hamaguchi, I.,
Suda, T.,
and Yamaguchi, N.
(1997)
EMBO J.
16,
2342-2351[Abstract/Free Full Text]
|
67.
|
Huang, M. M.,
Bolen, J. B.,
Barnwell, J. W.,
Shattil, S. J.,
and Brugge, J. S.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
7844-7848[Abstract]
|
68.
|
Lipsky, R. H.,
Eckert, D. M.,
Tang, Y.,
and Ockenhouse, C. F.
(1997)
Recept. Signal Transduct.
7,
1-11[Medline]
[Order article via Infotrieve]
|
69.
|
Acton, S. L.,
Scherer, P. E.,
Lodish, H. F.,
and Krieger, M.
(1994)
J. Biol. Chem.
269,
21003-21009[Abstract/Free Full Text]
|
70.
|
Gu, X.,
Trigatti, B.,
Xu, S.,
Acton, S.,
Babitt, J.,
and Krieger, M.
(1998)
J. Biol. Chem.
273,
26338-26348[Abstract/Free Full Text]
|
71.
|
Connelly, M. A.,
Klein, S. M.,
Azhar, S.,
Abumrad, N. A.,
and Williams, D. L.
(1999)
J. Biol. Chem.
274,
41-47[Abstract/Free Full Text]
|
72.
|
Aitman, T. J.,
Glazier, A. M.,
Wallace, C. A.,
Cooper, L. D.,
Norsworthy, P. J.,
Wahid, F. N.,
Al-Majali, K. M.,
Trembling, P. M.,
Mann, C. J.,
Shoulders, C. C.,
Graf, D.,
St. Lezin, E.,
Kurtz, T. W.,
Kren, V.,
Pravenec, M.,
Ibrahimi, A.,
Abumrad, N. A.,
Stanton, L. W.,
and Scott, J.
(1999)
Nat. Genet.
21,
76-83[CrossRef][Medline]
[Order article via Infotrieve]
|
73.
|
Gotoda, T.,
Lizuka, Y.,
Kato, N.,
Osuga, J.,
Bihoreau, M. T.,
Murakami, T.,
Yamori, Y.,
Shimano, H.,
Ishibashi, S.,
and Yamada, N.
(1999)
Nat. Genet.
22,
226-228[CrossRef][Medline]
[Order article via Infotrieve]
|