Received for publication, December 22, 2000, and in revised form, January 16, 2001
Cellular interactions of advanced
glycation end products (AGE) are mediated by AGE receptors. We
demonstrated previously that class A scavenger receptor types I and II
(SR-A) and CD36, a member of class B scavenger receptor family, serve
as the AGE receptors. In this study, we investigated whether scavenger
receptor class B type I (SR-BI), another receptor belonging to class B
scavenger receptor family, was also an AGE receptor. We used Chinese
hamster ovary (CHO) cells overexpressed hamster SR-BI (CHO-SR-BI
cells). 125I-AGE-bovine serum albumin (AGE-BSA) was
endocytosed in a dose-dependent fashion and underwent
lysosomal degradation by CHO-SR-BI cells. 125I-AGE-BSA
exhibited saturable binding to CHO-SR-BI cells (Kd = 8.3 µg/ml). Endocytic uptake of 125I-AGE-BSA by
CHO-SR-BI cells was completely inhibited by oxidized low density
lipoprotein (LDL) and acetylated LDL, whereas LDL exerted only a weak
inhibitory effect (<20%). Cross-competition experiments showed that
AGE-BSA had no effect on HDL binding to these cells and vice versa.
Interestingly, however, SR-BI-mediated selective uptake of HDL-CE was
completely inhibited by AGE-BSA in a dose-dependent manner
(IC50 <10 µg/ml). Furthermore, AGE-BSA partially
inhibited (by <30%) the selective uptake of HDL-CE in human
hepatocarcinoma HepG2 cells (IC50 <30 µg/ml). In
addition, [3H]cholesterol efflux from CHO-SR-BI cells to
HDL was significantly inhibited by AGE-BSA in a
dose-dependent manner (IC50 <30 µg/ml). Our
results indicate that AGE proteins, as ligands for SR-BI, effectively
inhibit both SR-BI-mediated selective uptake of HDL-CE and cholesterol
efflux from peripheral cells to HDL, suggesting that AGE proteins might
modulate SR-BI-mediated cholesterol metabolism in
vivo.
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INTRODUCTION |
In the Maillard reaction, proteins react with glucose to form
Schiff base and Amadori products. After 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 inter-molecular cross-linking (1, 2), and biologically by
specific recognition by AGE receptors. The presence of AGE in several
human tissues suggests that they may be involved in the aging process,
diabetic complications, and atherosclerosis (3-11).
The physiological significance of AGE has been analyzed primarily using
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). Several AGE receptors
have been characterized (22-25), one of which is a novel 35-kDa
protein (called RAGE) from bovine lung endothelium that belongs to the
immunoglobulin superfamily (23). Two AGE-binding proteins of 60- and
90-kDa (called p60 and p90) were also identified from the rat liver
(24).Recently, galectin-3, a lectin-like protein with a high binding
affinity for galactose-containing glycoproteins, was identified as a
component of p90 (25). We have recently shown that the macrophage
scavenger receptor class AI/AII (SR-A), which is known as a receptor
for oxidized low density lipoprotein (Ox-LDL) (26), mediates the
endocytic uptake and degradation of AGE-BSA by macrophages (26,
27).
In contrast, although functionally related to SR-A, the class B
receptors differ significantly in structure. CD36, the defining member
of this class, binds Ox-LDL, fatty acids, and the proteins collagen and
thrombospondin (28-32). CD36 has a broad ligand specificity, and its
multiple potential roles have been proposed. We recently discovered
that the class B scavenger family member CD36 also serves as a receptor
for AGE-BSA (33). Our study provided novel information that in addition
to SR-A the class B scavenger receptor family seems to serve as AGE
receptors in vivo and therefore might participate in the
pathogenesis of diabetic macrovascular complications (33).
The scavenger receptor class B type I (SR-BI), a member of the class B
scavenger receptor family, was first identified as a high density
lipoprotein (HDL) receptor and was shown to mediate selective uptake of
cholesteryl esters from HDL (HDL-CE) in vitro (34). A recent
study demonstrated that SR-BI-mediated selective uptake of HDL-CE was
much more efficient than CD36-mediated uptake, despite the high
similarity in structure of SR-BI and CD36 (35). Immunohistochemical
analyses in rodents revealed abundant SR-BI expression in the liver and
steroidogenic tissues such as the adrenal glands and ovaries (34),
where a selective uptake of HDL-CE is known to predominate (36-39).
Adenovirus-mediated overexpression of SR-BI in mouse liver is
associated with reduced plasma HDL levels and increased cholesterol
secretion into bile (40). Recent studies (40-43) provided
further solid evidence for a crucial role of SR-BI in selective uptake
of HDL-CE by hepatocytes in vitro and in
vivo.
In the present study, we investigated whether AGE-proteins could be
recognized as ligands by SR-BI. Our results suggest that AGE proteins
generated in vivo could affect SR-BI-mediated cholesterol metabolism.
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EXPERIMENTAL PROCEDURES |
Chemicals and Materials--
Penicillin G (1,650 IU/mg),
streptomycin sulfate (750 IU/mg), G418, Dulbecco's modified Eagle's
medium (DMEM), and Ham's F-12 medium were purchased from Life
Technologies, Inc. Na125I (3.7 GBq/ml),
[1,2-3H]cholesteryl oleoyl ether (1 mCi/ml), and
[1,2-3H]cholesterol (1 mCi/ml) were purchased from
Amersham Pharmacia Biotech. Rabbit polyclonal anti-mouse SR-BI antibody
was purchased from Novus Biologicals. Other chemicals were of the best
grade available from commercial sources.
Ligand Preparation and Iodination--
AGE-BSA was prepared as
described previously (33). 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 (pH 7.4) (PBS). 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 (33). Nonglycated BSA was prepared as described
previously (44). Briefly, 1.0 g of BSA was incubated with phenyl
boronic acid resin (PBA-60, Amicon, Beverly, MA) in 800 ml of 0.5 M glycine/NaOH buffer containing 2% MgCl2 (pH
8.5) for 2 h at room temperature. The filtrate through a glass
filter was refiltered with another portion of the same resin. The final
solution was dialyzed against PBS and used for the experiments. Human
LDL (d = 1.019-1.063 g/ml) (33) and HDL
(d = 1.063-1.21 g/ml) (45) were isolated by sequential ultracentrifugation of human plasma from normolipidemic subjects after
overnight fasting and dialyzed against 0.15 M NaCl and 1 mM EDTA (pH 7.4) (33). Traces of apoB and -E were removed
from HDL by a heparin-agarose column. Acetylated LDL (acetyl-LDL) was prepared by chemical modification of LDL with acetic anhydride as
described previously (46). 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 (46). Electrophoretic
mobility of AGE-BSA preparations toward the anode was 1.4 times higher
than that of unmodified BSA. Under identical conditions, Ox-LDL showed
a similar increase in electrophoretic mobility. Thus, modification of
BSA with glucose is associated with a significant increase in net
negative charge (33). AGE-BSA was labeled with 125I by
IODO-GEN (Pierce), and HDL was labeled as described previously (33) to
a specific radioactivity of 850 and 500 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 supplemented with 100 units/ml penicillin and 100 units/ml
streptomycin) containing 10% fetal calf serum (FCS). CHO-SR-BI cells
(47) stably expressing hamster SR-BI were maintained in medium A
supplemented with 50 µg/ml G418 (medium B). CHO-mock cells were
prepared by stably transfecting an expression vector pCR3 (Invitrogen)
without a cDNA insert (empty control vector) into CHO-K1 cells.
HepG2 cells were obtained from RIKEN cell bank (Japan) and maintained in DMEM containing 10% FCS supplemented with 100 units/ml penicillin and 100 units/ml streptomycin (medium C).
Immunoblotting--
Rat liver parenchymal cells, as a positive
control, were obtained from male Wistar rats. Isolation of liver
parenchymal cells from rats was essentially similar to the method
devised previously for preparation of rat liver cells (48). The portal
vein was cannulated with a polyethylene catheter, and the liver was
perfused with calcium-free Gey's balanced salt solution (GBSS) buffer
(pH 7.5) for 10 min at a flow of 8 ml/min and then with GBSS buffer (pH
7.5) containing 0.05% collagenase and 4 mM
CaCl2 for 3 min at 37 °C. The liver was minced and
suspended in ice-cold PBS. The cell suspension was centrifuged at
100 × g for 2 min at 4 °C. The precipitate was
suspended in ice-cold PBS, followed by centrifugation. The resulting
precipitate was solubilized in lysis buffer (20 mM Tris,
150 mM NaCl (pH 7.4) (TBS) containing 2% Triton X-100,
1 mM phenylmethanesulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1 µg/ml aprotinin). Samples (30 µg of protein/lane) were subjected to SDS-polyacrylamide gel electrophoresis (49) and then electrotransferred onto a nitrocellulose membrane (Schleicher & Schuell). The membrane was blocked for 2 h with 5%
nonfat dry milk in TBS/Tween (20 mM Tris, 150 mM NaCl, and 0.1% Tween 20 (pH 7.4)) and washed with
TBS/Tween three times. The membrane was then incubated for 1 h
with a 1:1,000 dilution of anti-SR-BI antibody in 1.5% skim milk in
TBS/Tween and then washed three times with TBS/Tween. Immunoreactive
bands were detected by incubation for 30 min with a 1:5,000 dilution of
horseradish peroxidase-conjugated goat secondary anti-rabbit IgG (H + L) (Zymed Laboratories Inc., San Francisco, CA) in
1.5% skim milk in TBS/Tween and washed three times with TBS/Tween,
followed by addition of ECL reagent.
Cellular Uptake of 125I-HDL or
125I-AGE-BSA by CHO-SR-BI Cells--
All cellular
experiments, except for the binding study, were performed at 37 °C
in a humidified atmosphere of 5% CO2 in air. CHO-SR-BI
cells and CHO-mock 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, which was then replaced by DMEM containing 3% BSA (medium D). After culture for 1 h, each well received 0.5 ml
of medium D containing various concentrations of 125I-HDL
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, medium was taken from each well, and soluble radioactivity in trichloroacetic acid was determined as an index of
cellular degradation, as described previously (33). After the cells
were washed three times with 1 ml of medium D and then three more times
with PBS, they were lysed with 1 ml of 0.1 N NaOH for 30 min at 37 °C. After incubation, 125I radioactivity of
0.1 N NaOH-soluble protein was determined (as the cell
association of 125I-HDL or 125I-AGE-BSA), and
cellular proteins were determined using 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 D with various concentrations of 125I-HDL or
125I-AGE-BSA in the presence or absence of 20-fold excess
amounts of the unlabeled ligands. Each well was washed with ice-cold
PBS containing 3% BSA and PBS. The cells were lysed, and the
cell-bound radioactivity and cellular proteins were determined as
described above.
Preparation of 125I-HDL-[3H]Cholesteryl
Oleoyl Ether--
Human HDL (d = 1.063-1.21 g/ml)
labeled with [3H]cholesteryl oleoyl ether (CE), a
nonhydrolyzable cholesteryl ester analogue, was prepared by a
modification of the procedure of Miyazaki et al. (50).
Briefly, 250 µCi of [3H]CE in toluene was evaporated
under a gentle stream of N2 at room temperature. Dried
[3H]CE was resuspended with 0.4 ml of acetone.
[3H]CE in acetone was added to 20 ml of human
lipoprotein-deficient serum (20-40 mg/ml) with gentle stirring under a
gentle stream of N2 at room temperature for 1 h. Human
HDL (15 mg) was then added to the mixture with gentle stirring at room
temperature for 30 min. After incubation and adjustment of the mixture
density with KBr (d = 1.21 g/ml), the labeled HDL was
collected by ultracentrifugation, followed by dialysis against 0.15 M NaCl and 1 mM EDTA (pH 7.4). HDL-[3H]CE was then labeled with 125I as
described above. The specific activity for
125I-HDL-[3H]CE was 158.8 cpm/ng of protein
for 125I and 11.8 dpm/ng of protein for 3H.
Selective Uptake of HDL-CE by CHO-SR-BI Cells and HepG2
Cells--
Cells (8 × 104) were seeded in a 24-well
plate and cultured for 2 days in 1.0 ml of medium B (for CHO-SR-BI and
CHO-mock cells) or medium C (for HepG2 cells), which was then replaced
by medium D. After culture for 1 h, each well received 0.5 ml of
medium C containing various concentrations of
125I-HDL-[3H]CE in the presence (nonspecific
uptake) or absence (total uptake) of 10-fold excess amounts of the
unlabeled ligand to be tested. After incubation for the indicated
times, the cells were washed three times with 1 ml of medium D and then
three more times with PBS. The washed cells were lysed with 1 ml of 0.1 N NaOH for 30 min at 37 °C. After incubation,
125I radioactivity of 0.1 N NaOH-soluble
protein (representing the cell association of 125I-HDL) was
determined, and cellular proteins were determined as described above.
We then determined the 3H radioactivity of 0.1 N NaOH-soluble protein (representing [3H]CE
uptake). HDL-CE-selective uptake was calculated by subtracting the
amount of cell-associated HDL (125I) from the amount of CE
uptake (3H) (51).
[3H]cholesterol Efflux to HDL from CHO-SR-BI
Cells--
The [3H]cholesterol efflux assay was
performed using a procedure essentially similar to that reported
previously by Ji et al. (52). Cells were labeled with
[3H]cholesterol by addition of
[3H]cholesterol in medium A containing 10% FCS. After
48 h of incubation, cells were incubated for 24 h in DMEM
containing 0.5% BSA. Cellular cholesterol efflux was determined by
measuring radioactivity released from cells into the medium upon the
addition of HDL.
Statistical Analysis--
All data were expressed as mean ± S.D. Differences between groups were examined for statistical
significance using the Student's t test. A p
value less than 0.05 denoted the presence of a statistically significant difference.
 |
RESULTS |
Expression of SR-BI in CHO Cells Overexpressing Hamster
SR-BI--
CHO-SR-BI cells that stably expressed hamster SR-BI were
established. Immunoblotting with a polyclonal anti-SR-BI antibody yielded no bands in CHO-mock cells, whereas CHO-SR-BI cells exhibited a
distinctive band. Parallel control experiments demonstrated that rat
liver parenchymal cells expressing high levels of SR-BI exhibited a
positive band with molecular weight indistinguishable from that
obtained from CHO-SR-BI cells as reported previously (47) (Fig.
1). We further examined whether SR-BI
expressed on the cells was functional; CHO-SR-BI cells exhibited
cellular binding of 125I-HDL with high affinity
(Kd = 6.5 µg/ml, Bmax = 72.3 µg/mg cell protein) but no significant capacity for endocytic uptake of 125I-HDL (data not shown), as reported previously
(34). Selective uptake of HDL-CE by CHO-SR-BI cells was 4-fold higher,
at least, than that of CHO-mock cells (data not shown), as reported
previously (47). These results indicated that SR-BI expressed on
CHO-SR-BI cells functioned as the HDL receptor.

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Fig. 1.
Detection of SR-BI protein in CHO-SR-BI cells
by immunoblot analysis. Cell lysates from CHO-SR-BI cells
(1st lane, 30 µg), CHO-mock cells (2nd lane, 30 µg), and rat liver parenchymal cells (3rd lane, 30 µg)
were subjected to 8% SDS-polyacrylamide gel electrophoresis and
immunoblotted with anti-mouse SR-BI antibody.
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Endocytic Uptake and Degradation of 125I-AGE-BSA by
CHO-SR-BI Cells--
We determined the endocytic uptake and
degradation of 125I-AGE-BSA by CHO-SR-BI cells at 37 °C.
The amount of cell association of 125I-AGE-BSA with
CHO-SR-BI cells increased in a dose-dependent manner and
was almost competed away by a 20-fold excess of unlabeled AGE-BSA (Fig.
2A). The specific cell
association exhibited a dose-dependent saturation pattern
with a plateau level of 640 ng/mg cell protein and an apparent
Kd = 2.3 µg/ml, and a maximal ligand association
of 738 ng/mg cell protein (Fig. 2A). Parallel experiments in
CHO-mock cells revealed that the cell association of
125I-AGE-BSA occurred at a negligible level (Fig.
2A). In sharp contrast with 125I-HDL (data not
shown), endocytic degradation of 125I-AGE-BSA by the
transfected cells was significant (Fig. 2B). Specific
degradation increased dose-dependently with a plateau level
of 230 ng/mg cell protein; the apparent Kd for degradation was 3.1 µg/ml, and the maximal ligand degradation was 297 ng/mg cell protein, whereas CHO-mock cells did not degrade 125I-AGE-BSA under the same conditions (Fig.
2B). This result indicates that SR-BI mediates the cell
association of AGE-BSA and its endocytic degradation.

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Fig. 2.
Endocytic uptake of 125I-AGE-BSA
by CHO-SR-BI cells. Cells were incubated for 5 h in 0.5 ml of
3% BSA in DMEM with increasing concentrations of
125I-AGE-BSA in the presence (closed squares) or
absence (closed circle) of 20-fold excess amounts of the
unlabeled ligands. After incubation, the medium was taken from each
well, and radioactivity soluble in trichloroacetic acid was determined
as an index of cellular degradation (B). After incubation,
the cells were washed three times with 1 ml of 3% BSA in PBS and then
three more times with PBS, lysed with 1 ml of 0.1 N NaOH
for 30 min at 37 °C, and the cell-associated radioactivity
determined (A). The specific (closed triangles)
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.
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Binding of 125I-AGE-BSA to CHO-SR-BI Cells--
We
next determined the cellular binding of 125I-AGE-BSA to
CHO-SR-BI cells at 4 °C. Total binding of 125I-AGE-BSA
was inhibited by 70% by an excess amount of the same ligand. The
specific binding, obtained by subtracting nonspecific binding from the
total binding, yielded a saturation pattern. Scatchard analysis of the
specific binding disclosed a binding site with an apparent
Kd of 8.3 µg/ml and maximal binding of 85.7 ng/mg
cell protein, indicating that CHO-SR-BI cells possess a high affinity
binding site for AGE-BSA (Fig. 3). Since
the parallel binding experiment of 125I-AGE-BSA to CHO-mock
cells failed to yield specific binding (data not shown), it is likely
that the specific binding site for AGE-BSA on CHO-SR-BI cells is
identical to SR-BI.

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Fig. 3.
Binding of 125I-AGE-BSA to
CHO-SR-BI cells. Cells were incubated for 90 min at 4 °C in 0.5 ml of 0.1% BSA in DMEM with increasing concentrations of
125I-AGE-BSA in the presence (closed squares) or
absence (closed circle) of 20-fold 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 (closed triangles) was determined by
subtracting nonspecific binding (closed squares) from total
binding (closed circle). Inset, Scatchard
analysis of the specific binding curve.
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Effect of Modified LDL on Cellular Binding of
125I-AGE-BSA to CHO-SR-BI Cells--
To examine whether
the recognition site for AGE-BSA in CHO-SR-BI cells was identical to
that for Ox-LDL, acetyl-LDL, LDL, and HDL, we determined the effect of
these lipoproteins on cellular binding of 125I-AGE-BSA to
CHO-SR-BI cells. Cellular binding of 125I-AGE-BSA to
CHO-SR-BI cells was effectively (>62%) replaced by unlabeled Ox-LDL
and acetyl-LDL and by unlabeled AGE-BSA, whereas unlabeled LDL and HDL
had a slightly weaker effect (<20%) (Fig. 4A). Non-glycated BSA, a
negative control, had no effect on this process (Fig. 4A).
Parallel experiments demonstrated that cellular binding of
125I-HDL to CHO-SR-BI cells was effectively (60%) replaced
by unlabeled HDL, whereas AGE-BSA had little effect (<15%) (Fig.
4B). These results suggest that the binding site of SR-BI
for AGE-BSA might overlap with that for Ox-LDL and acetyl-LDL but not
that for LDL and HDL.

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Fig. 4.
Effect of modified LDLs and native
lipoproteins on cellular binding of 125I-AGE-BSA to
CHO-SR-BI cells. A, 125I-AGE-BSA binding.
Cells were incubated at 4 °C for 90 min with 0.5 ml of 0.1% BSA in
DMEM containing 5 µg/ml 125I-AGE-BSA in the presence or
absence of a 20-fold amount of unlabeled AGE-BSA, Ox-LDL, acetyl-LDL,
LDL, HDL, or nonglycated BSA. B, 125I-HDL
binding. Cells were incubated at 4 °C for 90 min with 0.5 ml of
0.1% BSA in DMEM containing 5 µg/ml 125I-HDL in the
presence or absence of a 20-fold amount of unlabeled HDL, AGE-BSA, or
non-glycated BSA. The amounts of 125I-AGE-BSA bound
(A) and 125I-HDL bound (B) were
determined as described under "Experimental Procedures." Data
represent the means of three separate experiments. Error
bars represent S.D.
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Effect of Modified LDL on Endocytic Uptake of
125I-AGE-BSA by CHO-SR-BI Cells--
We determined the
effect of Ox-LDL, acetyl-LDL, LDL, and HDL on endocytic uptake of
125I-AGE-BSA by CHO-SR-BI cells. The cell association of
125I-AGE-BSA was effectively (>85%) replaced by unlabeled
Ox-LDL and acetyl-LDL and by unlabeled AGE-BSA, whereas the effect of unlabeled LDL and HDL was almost negligible (<20%), and nonglycated BSA, a negative control, had no effect on this process (Fig.
5A). Similarly, the endocytic
degradation of 125I-AGE-BSA was effectively inhibited
(>85%) by the presence of unlabeled Ox-LDL, acetyl-LDL, and unlabeled
AGE-BSA, whereas unlabeled LDL and HDL also exerted a much weaker
effect (<25%) (Fig. 5B). The results of endocytic
experiments are consistent with those of the binding experiments (Fig.
4).

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Fig. 5.
Effect of modified LDLs and native
lipoproteins on endocytic uptake by CHO-SR-BI cells. Cells were
incubated at 37 °C for 5 h with 0.5 ml of 3% BSA in DMEM
containing 5 µg/ml 125I-AGE-BSA in the presence or
absence of a 20-fold amount of unlabeled AGE-BSA, Ox-LDL, acetyl-LDL,
LDL, HDL, or non-glycated BSA. The amounts of cell-associated
125I-AGE-BSA (A) and its degradation products
(B) were determined as described under "Experimental
Procedures." Data represent the means of three separate experiments.
Error bars represent S.D.
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Effect of AGE-BSA on Selective Uptake of HDL-CE by CHO-SR-BI
Cells--
We next examined the effects of AGE-BSA on the selective
uptake of HDL-CE by CHO-SR-BI cells. AGE-BSA inhibited the selective uptake of HDL-CE by CHO-SR-BI cells in a dose-dependent
manner, whereas non-glycated BSA had no effect (Fig.
6). SR-BI-mediated selective uptake of
HDL-CE, which was obtained by subtracting the amount of selective
uptake by CHO-mock cells from that by CHO-SR-BI cells, was inhibited
completely by 100 µg/ml AGE-BSA (Fig. 6). This inhibitory effect of
AGE-BSA (IC50 <10 µg/ml) was more effective than that of
unlabeled HDL (IC50 = 60 µg/ml) (Fig. 6). AGE-BSA was
ineffective in suppressing selective uptake by CHO-mock cells (data not
shown). These data suggest that SR-BI-mediated selective uptake of
HDL-CE is sensitively inhibited by AGE-BSA.

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Fig. 6.
Effect of AGE-BSA on the selective uptake of
HDL-CE by CHO-SR-BI cells. CHO-SR-BI cells were incubated for
5 h in 0.5 ml of 3% BSA in DMEM containing 10 µg/ml
125I-HDL-[3H]cholesteryl oleate ether in the
presence or absence of increasing concentrations of unlabeled HDL
(closed squares), AGE-BSA (closed circle), or
non-glycated BSA (closed triangles). Under the identical
conditions, CHO-mock cells (open circle) were incubated with
10 µg/ml 125I-HDL-[3H]cholesteryl oleate
ether in the absence of competitors. Selective uptake of HDL-CE was
determined as described under "Experimental Procedures." Data
represent the means of three separate experiments. Error
bars represent S.D.
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Effect of AGE-BSA on Selective Uptake of HDL-CE by HepG2
Cells--
To investigate this notion further, we examined the effect
of AGE-BSA on selective uptake of HDL-CE by human hepatocarcinoma cells, HepG2, model cells for human hepatocytes; the expression of
human SR-BI (CLA-1) by these cells was confirmed by immunoblotting (data not shown). Selective uptake of HDL-CE by HepG2 cells was inhibited by HDL by 70% in a dose-dependent manner. The
effect of AGE-BSA on this process was partial but significant
(~30%), whereas nonglycated BSA had no effect (Fig.
7). When the maximal effect of AGE-BSA is
compared with that of HDL, it might follow that AGE-BSA may inhibit the
specific selective uptake of HDL-CE by more than 40%. Thus, it is
highly likely that selective uptake of HDL-CE by the liver could be
inhibited by plasma AGE proteins in vivo, if it is actually
present in the circulation as reported previously (53).

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Fig. 7.
Effect of AGE-BSA on the selective uptake of
HDL-CE by HepG2 cells. Under identical conditions to those
described in Fig. 6, HepG2 cells were incubated for 5 h in 0.5 ml
of 3% BSA in DMEM containing 10 µg/ml
125I-HDL-[3H]cholesteryl oleate ether in the
presence or absence of increasing concentrations of unlabeled HDL
(closed circle), AGE-BSA (closed square), or
non-glycated BSA (closed triangles). Selective uptake of
HDL-CE was determined as described under "Experimental Procedures."
Data represent the means of three separate experiments. Error
bars represent S.D.
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Effect of AGE-BSA on [3H]Cholesterol Efflux from
CHO-SR-BI Cells to HDL--
Finally, we examined the effect of AGE-BSA
on SR-BI-mediated HDL-dependent cholesterol efflux from
CHO-SR-BI cells. CHO-SR-BI cells showed a 3-4-fold increase in
[3H]cholesterol efflux from these cells to HDL in a time-
and dose-dependent manner compared with CHO-mock cells
(data not shown), as reported previously (52). Under the present
conditions, the time-dependent [3H]cholesterol efflux from CHO-SR-BI cells to HDL (50 µg/ml) was inhibited by 40% by AGE-BSA (100 µg/ml), whereas
non-glycated BSA had no effect (Fig.
8A). Cholesterol efflux from
CHO-SR-BI cells to 50 µg/ml of HDL was inhibited
dose-dependently by AGE-BSA (10-200 µg/ml), whereas
AGE-BSA had no effect on cholesterol efflux from CHO-mock cells to HDL
(Fig. 8B). Comparison based on the SR-BI-mediated
HDL-dependent cholesterol efflux, which was obtained by
subtracting the efflux level of CHO-mock cells from that of CHO-SR-BI
cells, showed that it was inhibited by AGE-BSA
dose-dependently with 50% inhibition at <30 µg/ml of
AGE-BSA, whereas non-glycated BSA had no effect (Fig. 8B).
Taken together, these results suggest that AGE-BSA inhibits
SR-BI-mediated HDL-dependent cholesterol efflux.

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Fig. 8.
Effect of AGE-BSA on
[3H]cholesterol efflux to HDL from CHO-SR-BI cells.
A, time course of [3H]cholesterol efflux from
CHO-SR-BI. CHO-SR-BI cells were labeled overnight with
[3H]cholesterol (1 µCi/ml), incubated for 1 day in 0.5 ml of 0.5% BSA in DMEM, washed, and incubated with HDL (50 µg/ml)
(closed circle) in the presence or absence of AGE-BSA (100 µg/ml) (closed triangles) or nonglycated BSA (100 µg/ml)
(open squares), or incubated without HDL (open
circle). Efflux values are expressed as
[3H]cholesterol released to the medium. Data represent
the means of three separate experiments. Error bars
represent S.D. B, cholesterol efflux to HDL from cells
incubated with different concentrations of competitors. CHO-SR-BI and
CHO-mock cells were incubated for 5 h with HDL (50 µg/ml) in the
presence or absence of different concentrations of AGE-BSA (CHO-SR-BI,
closed circle; CHO-mock, open circle) or
nonglycated BSA (CHO-SR-BI, closed squares; CHO-mock,
open squares). Values are averages of duplicates
representative of two separate experiments.
|
|
 |
DISCUSSION |
Our previous studies using CHO cells overexpressing the class A
scavenger receptor (SR-A) (27) and peritoneal macrophages from SR-A
knockout mice (26) and our recent study using CHO cells overexpressing
the class B scavenger receptor CD36 (33) demonstrated that AGE ligands
are effectively taken up by these scavenger receptors by a typical
receptor-mediated endocytosis mechanism. These findings raised the
general hypothesis that the scavenger receptor as the AGE receptor is
not confined to the class A but also class B scavenger receptor family
(33). We have now demonstrated this to be the case with another class B scavenger receptor, SR-BI, using CHO cells overexpressing hamster SR-BI. The unique function of SR-BI (not found in other scavenger receptors such as SR-A and CD36) is its major role as the HDL receptor
in reverse cholesterol transport, from cholesterol efflux from
peripheral cells into HDL particles to selective uptake of HDL-CE by
the liver. Therefore, the novel findings of our present study were that
SR-BI mediated the endocytic degradation of AGE ligands in a manner
similar to SR-A and CD36, and also that the selective uptake of HDL-CE
and cholesterol efflux mediated uniquely by SR-BI as an HDL receptor
was inhibited by AGE ligands. These findings suggest a novel idea in
AGE research that AGE proteins generated in situ could
regulate SR-BI-mediated cholesterol metabolism in vivo.
SR-BI was first identified as an HDL receptor by Acton et
al. (34). Immunohistochemical analyses of SR-BI in rodents showed its major sites of expression to be the liver and in steroidogenic cells such as adrenal glands and ovaries (34), where selective uptake
of HDL-CE is significant (36-39). Although belonging to the class B
scavenger receptor family, SR-BI is functionally characterized by
mediating selective uptake of HDL-CE in vitro (34) and
in vivo (40-43). The present study showed that this
phenomenon mediated by CHO-SR-BI cells was effectively (almost
completely) inhibited by AGE-BSA (Fig. 6), suggesting that AGE ligands
could inhibit selective CE uptake in the liver and steroidogenic
tissues. This finding in CHO-SR-BI cells was supported by results
obtained from HepG2 cells, model cells for human hepatocytes; selective
uptake of HDL-CE by these cells was partially but significantly
inhibited by AGE-BSA (Fig. 7). Although immunoblot analysis showed that SR-BI was positively expressed in these cells (data not shown), the
extent of contribution of human SR-BI to selective uptake of HDL-CE by
HepG2 cells is unknown at present. Rhainds et al. (54)
reported that SR-BI and/or CD36 of HepG2 cells are involved, at least
in part, in this process. Therefore, the extent of inhibition by
AGE-BSA of SR-BI-mediated-CE uptake by HepG2 cells could be lower than
estimated. A recent study using transiently expressing cells
demonstrated that selective uptake of HDL-CE by SR-BI is of more
functional significance than that by CD36 (35). Therefore, it is likely
that the extent of inhibition by AGE-BSA of selective uptake of HDL-CE
by HepG2 cells could be significant. Thus, it is possible that AGE
proteins generated in vivo may inhibit SR-BI-mediated selective uptake of HDL-CE, the final step of reverse cholesterol transport, implicating AGE ligands in regulation of plasma cholesterol levels.
In addition to selective uptake of HDL-CE by SR-BI, this receptor is
also involved in cholesterol efflux from peripheral cells to HDL, the
first step of reverse cholesterol transport (52). The present study
using CHO-SR-BI cells showed that AGE-BSA effectively inhibited
[3H]cholesterol efflux from CHO-SR-BI cells to HDL (Fig.
8). Although Hirano et al. (55) reported that human SR-BI is
expressed and markedly up-regulated in differentiated human
macrophages, the lack of in vivo experimental results means
that the functional involvement of SR-BI in cholesterol efflux remains
uncertain. It has been proposed, however, that SR-BI facilitates
bidirectional cholesterol flux depending on the cholesterol gradient
between HDL particles and the cell plasma membrane (52, 56). Therefore, the present results suggest that AGE proteins inhibit both selective CE
uptake and cholesterol efflux. Recent studies using genetically engineered mice, however, have shown that SR-BI plays an important role
in the control of biliary cholesterol secretion (40, 41). This notion
was supported by the fact that SR-BI was expressed on the canalicular
membrane of mouse hepatocytes overexpressing SR-BI (40, 58). In
addition, the immunohistochemical study by Ling et al. (59)
using a monoclonal antibody against fluorolink, one of the AGE
structures, demonstrated that AGE proteins are localized
intracellularly and extracellularly in human hepatocytes. Therefore, it
is possible that AGE proteins formed in hepatocytes may inhibit
SR-BI-mediated biliary cholesterol secretion in vivo.
SR-BI is known to be a multifunctional molecule that recognizes many
ligands of different structures including acetyl-LDL, Ox-LDL, LDL,
maleylated BSA (60), HDL (34), VLDL (61), anionic phospholipids
(62), and apoptotic cells (63). These findings do not provide a clear
picture of the molecular basis for the recognition of these ligands by
SR-BI. However, among these ligands, modified LDLs have in common a
negatively charged nature, suggesting that SR-BI recognizes the
negative charge of AGE proteins. Further studies are necessary to
determine the AGE structure(s) required for recognition by SR-BI.
Several attempts were made to characterize the ligand binding domain of
SR-BI. Temel et al. (64) prepared anti-SR-BI antiserum raised against the extracellular domain of mouse SR-BI (amino acid
residues 174-356) and demonstrated that it not only inhibited HDL
binding and selective lipid uptake but also inhibited LDL binding and
selective lipid uptake (65). Gu et al. (57) reported that a
double substitution of arginine for glutamine at positions 402 and 418 (Q402R/Q418R) of mouse SR-BI led to a loss of ability to bind HDL but
not LDL. Another mutant of mouse SR-BI with a Met-to-Arg substitution
at position 158 (M158R) exhibited diminished binding ability to HDL and
LDL, but no change in its binding capacity to acetyl-LDL (57).
Furthermore, Acton et al. (60) demonstrated that the binding
of 125I-acetyl-LDL to COS cells transfected with hamster
SR-BI was completely inhibited by Ox-LDL. Similarly, Calvo et
al. (61) also showed that Ox-LDL and LDL effectively replaced
binding of DiI-HDL to Sf9 cells transfected with human SR-BI.
Taken together, these results support the notion that the region
corresponding to amino acids 174-356 of the extracellular domain of
SR-BI is necessary for its HDL binding and selective CE uptake, and
that the binding domain of Ox-LDL and acetyl-LDL to SR-BI might overlap
with amino acids 174-356 of its extracellular domain. In addition, it
is likely that amino acids 402 and 418 in the extracellular domain of
mouse SR-BI is important for binding HDL but not LDL. In the present
study, Ox-LDL and acetyl-LDL completely inhibited endocytic uptake of
125I-AGE-BSA by CHO-SR-BI cells, indicating that the
binding domain of AGE-BSA to SR-BI might overlap with amino acids
174-356 of its extracellular domain (Figs. 4 and 5). However, the
extent of inhibition of HDL and LDL for 125I-AGE-BSA
binding was weak (Figs. 4 and 5), suggesting that the main binding site
of AGE-BSA to SR-BI would be within amino acids 174-356 of its
extracellular domain but not its binding domain to HDL and LDL. The
most novel finding in the present study was that AGE-BSA completely
(>90%) inhibited selective uptake of HDL-CE by CHO-SR-BI cells (Fig.
6). In addition, AGE-BSA exhibited a significant inhibitory effect on
HDL-induced cholesterol efflux from these cells (Fig. 8). It is not
clear whether the HDL binding domain of SR-BI is involved in selective
CE uptake. Williams and co-workers (66, 67) recently proposed that
SR-BI may form "a lipid channel" that facilitates transfer of lipid
between cells and lipoproteins. Therefore, it is possible that AGE-BSA
could inhibit SR-BI-mediated CE uptake by binding to the lipid channel rather than by binding to the HDL binding domain. Further studies are
needed to elucidate the binding domain of SR-BI to AGE ligands.
Recently, it has been reported that CD36 and SR-BI are localized in
caveolae of the plasma membrane and that Ox-LDL binding to CD36 or HDL
binding to SR-BI affects the lipid composition of caveolae, which in
turn modulates activation of endothelial nitric-oxide synthase in human
microvascular endothelial cells (68). Stitt et al. (69) also
reported that the R1 (OST-48), R2 (80K-H), and R3 (galectin-3)
components of the AGE receptor complex were localized in caveolae of
retinal microvascular endothelial cells. Therefore, it is possible that
interaction of AGE ligands with CD36 and SR-BI modulates endothelial
nitric-oxide synthase activity in the caveolae. Further studies are
required to determine whether CD36 and SR-BI, acting as AGE receptors
in caveolae, directly regulate the generation of nitric oxide.
Chinetti et al. (70) recently showed that CLA-1/SR-BI
expressed on macrophages is induced by ligands for PPAR
and PPAR
in human atherosclerotic lesions. Recent reverse
transcriptase-polymerase chain reaction analyses by Iwashima et
al. (71) demonstrated that AGE-BSA induced PPAR
expression and
activation in cultured mesangial cells, and that SR-BI, SR-A, CD36, and
Lox-1 were also up-regulated by AGE-proteins through activation of
PPAR
in phorbol 12-myristate 13-acetate-treated THP-1 cells
(72).
In conclusion, our finding that SR-BI-mediated functions were inhibited
by AGE proteins suggests a potential link between SR-BI-mediated
cholesterol metabolism and development of diabetic vascular complications.
We are grateful to Dr. Akihiko Kuniyasu of
our laboratory for discussions. We are also grateful to Drs. Kenshi
Matsumoto and Hideki Hakamata in the Department of Biochemistry,
Kumamoto University School of Medicine, for helpful discussions.
Published, JBC Papers in Press, January 17, 2001, DOI 10.1074/jbc.M011613200
The abbreviations used are:
AGE(s), advanced
glycation end products;
BSA, bovine serum albumin;
LDL, low density
lipoprotein;
HDL, high density lipoprotein;
Ox-LDL, oxidized LDL;
acetyl-LDL, acetylated LDL;
CE, cholesteryl oleoyl ether;
SR-A, scavenger receptor class AI/AII;
SR-BI, scavenger receptor class B type
I;
PBS, phosphate-buffered saline;
CHO, Chinese hamster ovary;
PPAR
, peroxisome proliferator-activated receptor
;
VLDL, very low density
lipoprotein.
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