Received for publication, February 23, 2001, and in revised form, March 22, 2001
Cells acquire lipoprotein cholesterol by
receptor-mediated endocytosis and selective uptake pathways. In the
latter case, lipoprotein cholesteryl ester (CE) is transferred to the
plasma membrane without endocytosis and degradation of the lipoprotein particle. Previous studies with Y1/E/tet/2/3 murine adrenocortical cells that were engineered to express apolipoprotein (apo) E
demonstrated that apoE expression enhances low density lipoprotein
(LDL) CE uptake by both selective and endocytic pathways. The present
experiments test the hypothesis that apoE-dependent LDL CE
selective uptake is mediated by scavenger receptor, class B, type I
(SR-BI). Surprisingly, SR-BI expression was not detected in the
Y1/E/tet/2/3 clone of Y1 adrenocortical cells, indicating the presence
of a distinct apoE-dependent pathway for LDL CE selective
uptake. ApoE-dependent LDL CE selective uptake in
Y1/E/tet/2/3 cells was inhibited by receptor-associated protein and by
activated
2-macroglobulin (
2M),
suggesting the participation of the LDL receptor-related protein/
2M receptor. Reagents that inhibited
proteoglycan synthesis or removed cell surface chondroitin sulfate
proteoglycan completely blocked apoE-dependent LDL CE
selective uptake. None of these reagents inhibited SR-BI-mediated LDL
CE selective uptake in the Y1-BS1 clone of Y1 cells in which LDL CE
selective uptake is mediated by SR-BI. We conclude that LDL CE
selective uptake in adrenocortical cells occurs via SR-BI-independent
and SR-BI-dependent pathways. The SR-BI-independent pathway
is an apoE-dependent process that involves both chondroitin
sulfate proteoglycans and an
2M receptor.
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INTRODUCTION |
LDL1 is a major carrier
of cholesterol in the plasma, and the concentration of plasma LDL
cholesterol is regulated mainly by the LDL receptor pathway in the
liver (1, 2). On the other hand, LDL receptor-independent pathways
mediate about one-third of LDL removal in normal individuals and are
responsible for most of the LDL removal in individuals homozygous for
familial hypercholesterolemia who lack functional LDL receptors (3).
The LDL receptor processes LDL via receptor-mediated endocytosis in
which the LDL particle is delivered to lysosomes where the CE is
hydrolyzed to free cholesterol for use by the cell. In addition to
receptor-mediated endocytosis, LDL CE also can be taken up by cells via
a selective uptake pathway in which lipoprotein CE is transferred into
the cell membrane without the uptake and degradation of the LDL
particle. Although the selective uptake pathway has been studied
predominantly with HDL, numerous studies have shown the selective
uptake of LDL CE in steroidogenic cells of rodents (4-9). In cultured
human ovarian granulosa cells, ~33% of total LDL CE uptake occurs
via the selective uptake pathway (10). Furthermore, LDL CE selective
uptake occurs in human fibroblasts (11), rat and human liver cells (6, 11-13), and a murine macrophage cell (11). Thus, the selective uptake
of LDL CE is a widespread process that occurs in a variety of species
and cell types.
Little is known about the mechanism of LDL CE selective uptake or the
cell surface receptors responsible for this process. In the Y1-BS1
clone of murine adrenocortical cells, SR-BI mediates a major fraction
of LDL CE selective uptake as judged by inhibition with antibodies
directed to the extracellular domain of SR-BI (14). Expression of SR-BI
in transfected COS-7 cells (14) or Chinese hamster ovary cells (15)
showed directly that SR-BI mediates LDL CE selective uptake but with a
much reduced efficiency compared with HDL CE selective uptake (14).
Thus, SR-BI may contribute to LDL CE selective uptake in some cell types.
In a previous study, we found that expression of apoE in murine Y1
adrenocortical cells enhanced the selective uptake of LDL CE by
2-2.5-fold (16). The clone of Y1 cells used in that work, Y1/E/tet/2/3, was engineered to express human apoE4 under control of a
tetracycline-regulated promoter, permitting apoE expression to be
induced some 20-fold by removal of tet from the culture medium.
Experiments in the present study were designed to explore the mechanism
by which apoE alters LDL CE selective uptake and, in particular, to
test the hypothesis that the enhancement by apoE occurred via
SR-BI-mediated LDL CE selective uptake. Surprisingly, we found that the
Y1/E/tet/2/3 adrenocortical cell line does not express detectable
levels of SR-BI. Furthermore, characterization of the LDL CE selective
uptake pathway in Y1/E/tet/2/3 cells showed its properties to be
distinct from that mediated by SR-BI and to involve cell surface
chondroitin sulfate proteoglycan, apoE, and a member of the LDL
receptor family. Thus, these findings identify a new pathway for the
selective uptake of LDL CE.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Y1-BS1 cells were maintained in a 37 °C
humidified 95% air, 5% CO2 incubator in Ham's F-10
medium supplemented with 12.5% heat-inactivated horse serum, 2.5%
calf serum, 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin as described (17). Y1/E/tet/2/3 cells were
maintained in the same fashion except that the medium also contained
100 µg/ml G418 (Geneticin, Life Technologies, Inc), 200 µg/ml
hygromycin (Calbiochem), and 2 µg/ml tetracycline (16). MEF cells
were maintained in a 37 °C humidified 95% air, 5% CO2
incubator in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum, 2 mM glutamine, 100 units/ml
penicillin, 100 µg/ml streptomycin (18).
Western Blotting--
One day before assay, medium was changed
to serum-free medium plus or minus tet containing 2 mM
Bt2cAMP where indicated. Postnuclear supernatants were
isolated as previously described (19). Proteins (10-20 µg) were
resolved by SDS, 10% polyacrylamide gel electrophoresis (PAGE) or SDS,
4-15% PAGE and transferred to nitrocellulose membranes. The membranes
were blocked for 1 h at room temperature in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.02% Tween 20 (TBST)
containing 7% nonfat milk. The blocked membranes were then incubated
with one of the following primary antibodies in TBS-Tween containing 1% nonfat milk: anti-SR-BI C-terminal tail (1:5000) (20), anti-SR-BII C-terminal tail (1:5000) (21), anti-LRP (2 µg/ml, R777) (22), anti-VLDL receptor (4 µg/ml, R2623) (23), anti-gp330 (4 µg/ml, R784) (24), anti-LDL receptor (1:500, R4526) (a generous gift from
J. Herz), and anti-LR8B/apoER2(1:1000,
-20) (a generous gift of
J. Nimpf). The membrane was washed three times with TBS containing
0.05% Tween 20 and incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature in TBS containing 1% nonfat milk and 0.05% Tween. Bands were visualized by enhanced chemiluminescence (Pierce).
Preparation of 125I-Dilactitol
Tyramine-[3H]cholesteryl Oleolyl Ether Human
HDL3 (Human 125I-Labeled
[3H]HDL3) and 125I-Dilactitol
Tyramine-[3H]cholesteryl Oleolyl Ether Human LDL (Human
125I-Labeled [3H]LDL)--
Human HDL3
(1.125 g/ml <
<1.210 g/ml) and human LDL (1.019 g/ml <
<1.063 g/ml) were double-labeled with 125I-dilactitol
tyramine and [3H]cholesteryl oleolyl ether as described
(5). The specific activity of the human 125I-labeled
[3H]HDL ranged from 46 to 70 dpm/ng of protein for
125I and from 6 to 28 dpm/ng of protein for 3H.
The specific activity of the human 125I-labeled
[3H]LDL ranged from 25 to 75 dpm/ng of protein for
125I and from 3-30 dpm/ng of protein for
3H.
Determination of Selective Uptake of LDL CE and
HDL-CE--
Y1/E/tet/2/3cells were cultured in 6-well plates in
complete medium plus or minus tet for 4 days with changes of medium on days1 and 3. On day 4, the medium was changed to serum-free medium plus
or minus tet containing 2 mM Bt2cAMP. After
24 h, 125I-labeled [3H]LDL at 50 µg/ml
protein or 125I-labeled [3H]HDL at 50 µg/ml
protein (except where indicated) was added, and the incubation was
continued for 4 h at 37 °C. For Y1-BS1 cells, 48 h after
plating, medium was changed to serum-free medium supplemented with 100 nM Cortrosyn (Organon), a synthetic (1-24) adrenocorticotropic hormone analogue. After 24 h, medium was
changed to serum-free medium lacking Cortrosyn, doubly labeled
lipoprotein particles were added, and the incubation was continued for
4 h at 37 °C. Cells were placed on ice, washed three times with
cold phosphate-buffered saline containing 1% bovine serum albumin and once with phosphate-buffered saline, and lysed with 1.5 ml of 0.1 N NaOH. The lysate was processed to determine
trichloroacetic acid-soluble and -insoluble 125I
radioactivity and organic solvent-extractable 3H
radioactivity (5) and cell protein (25). The trichloroacetic acid-soluble 125I radioactivity represents endocytosed and
degraded lipoprotein. Trichloroacetic acid-insoluble 125I
radioactivity represents cell-associated undegraded apolipoproteins. The sum of trichloroacetic acid-soluble and -insoluble 125I
radioactivity expressed as CE equivalents was subtracted from the CE
measured as extractable 3H radioactivity to determine the
selective uptake of CE from HDL or LDL (16).
Degradation of
125I-
2M*--
Y1/E/tet/2/3 cells were
cultured in complete medium in the absence of tet for 4 days. At day 4, the medium was changed to serum-free medium containing 2 mM
Bt2cAMP. After 24 h, cells were washed twice with
assay medium (Dulbecco's modified Eagle's medium containing
Neutridoma serum supplement, 20 mM HEPES, and 1.5% bovine serum albumin) and incubated in assay medium containing 1 nM 125I-
2M* plus or minus RAP or
2M* for different time periods. MEF cells were plated at
a density of 2 × 10 5 cells/well in 12-well plates
and grown overnight in Dulbecco's modified Eagle's medium containing
10% fetal bovine serum at 37 °C until 80% confluent. Cells were
washed twice with assay medium and incubated in assay medium containing
1 nM 125I-
2M* plus or minus RAP
or
2M*. After incubation, the medium was removed,
precipitated with trichloroacetic acid, and centrifuged, and the
supernatant was counted for the measurement of
125I-
2M* degradation (18). Degradation was
defined as the amount of radioactivity in the medium that was soluble
in 15% trichloroacetic acid. Non-cell-mediated degradation was
measured by incubating 125I-
2M* in the
absence of cells. Cellular degradation of
125I-
2M* was determined as fmol of
125I-
2M*/105 cells. Inhibition
of degradation was performed by incubating Y1/E/tet/2/3 cells with
different concentrations of RAP or
2M* for 1 h
before the addition of 125I-
2M*.
Inhibition of Proteoglycan Synthesis and Sulfation--
To
inhibit proteoglycan synthesis (26), Y1/E/tet/2/3 and Y1-BS1 cells were
pre-incubated with 1 mM or 2 mM
p-nitrophenyl-
-D-xylopyranoside (
-xyloside) for 20 h at 37 °C in serum-free medium before
lipoprotein uptake assays. To inhibit sulfation (27) of
glycosaminoglycans, cells were preincubated for 20 h at 37 °C
in serum-free medium containing 30 mM NaClO3
before the addition of lipoprotein particles. For selective removal of
cell surface heparan sulfate proteoglycans, cells were treated for
3 h at 37 °C with 3 unit/ml heparinase I (EC 4.2.2.7) or
heparinase I and III together each at 3 units/ml before the addition of
the double-labeled lipoprotein particles. For selective removal of
chondroitin sulfate and dermatan sulfate proteoglycans, cells were
treated with chondroitinase ABC (EC 4.2.2.4) for 3 h at 37 °C
with 1 unit/ml before the addition of the double-labeled particles.
Each enzyme (Sigma) was dissolved in F-10 medium and used immediately.
 |
RESULTS |
Detection of SR-BI Expression in Y1/E/tet/2/3 and Y1-BS1
Cells--
Y1/E/tet/2/3 cells were prepared from the Y1 parent cell
line by two rounds of cloning to place apoE expression under control of
the tet-off system described by Gossen and Bujard (28). Y1/E/tet/2/3 cells express apoE at 2-2.5 µg/ml in the medium in the absence of
tet and suppress apoE expression to 0.1 µg/ml in the presence of tet
(16). To examine the effect of Bt2cAMP on SR-BI expression in Y1/E/tet/2/3 cells and to compare with the level of SR-BI expression in Y1-BS1 cells, postnuclear supernatant was used for Western blot
analysis using antibody raised against the C-terminal tail of murine
SR-BI (20). Fig. 1 shows that
Y1/E/tet/2/3 cells plus or minus tet in the presence or absence of
Bt2cAMP did not show detectable SR-BI expression. As
expected, SR-BI expression was readily detected in Y1-BS1 cells and
showed the expected increase in the presence of Bt2cAMP
(19). These results indicate that Y1/E/tet/2/3 cells do not make
detectable SR-BI in the presence or absence of apoE expression or upon
activation of the protein kinase A pathway by Bt2cAMP. A
Western blot using an antibody to the C-terminal tail of SR-BII also
failed to detect this splice variant of the class B scavenger receptors
in the presence or absence of apoE expression (Fig.
2). Note that SR-BI expression was
detected in the parent Y1 cell line, from which the Y1/E/tet/2/3 clone
was generated, at ~14% of the expression level in the Y1-BS1 cell
line as determined by quantitative Western blotting (14). The basis for
the loss of SR-BI expression in the Y1/E/tet/2/3 cell line is unknown,
but a wide range of SR-BI expression levels has been observed in
unselected subclones of the parent Y1 cell line isolated by limited
dilution cloning (14).

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Fig. 1.
Immunoblotting of SR-BI in Y1/E/tet/2/3
and Y1-BS1 cells Ten µg of post-nuclear supernatant of each cell
extract was electrophoresed on a 10% SDS-polyacrylamide gel under
reducing conditions and electroblotted onto nitrocellulose. The blot
was probed with antibody raised against the C-terminal tail of murine
SR-BI. The mobilities of molecular weight markers are shown on the
right side of the gel.
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Fig. 2.
Immunoblotting of SR-BII and LDL receptor
family members Post-nuclear supernatant (20 µg of protein) of 2 mM Bt2cAMP-treated Y1/E/tet/2/3 cells grown in
the presence or absence of tet were analyzed by SDS, 4-15% PAGE under
reducing conditions and electroblotted to nitrocellulose membranes.
Membrane strips were probed with the indicated antibody for 3 h at
room temperature followed by incubation with secondary antibody and
development via chemiluminescence. Positive controls (cont)
were provided by -SR-BII (mouse testis extract), -LR8B (extract
of COS-7 cells transfected with expression vector for LR8B), -LRP
(extract of mouse embryo fibroblast cells), -VLDLR (extract of
Y1-BS1 cells).
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Effect of RAP on LDL CE Uptake--
Since neither SR-BI nor SR-BII
seemed likely to be the basis for the apoE-mediated enhancement of LDL
CE selective uptake in Y1/E/tet/2/3 cells, we surveyed the cells for
other apoE receptors that are members of the LDL receptor superfamily.
As shown in Fig. 2, the LRP/
2M receptor and the VLDL
receptor were both detected in Y1/E/tet/2/3 cells with no reproducible
differences in expression in the presence or absence of apoE
expression. As expected, the LDL receptor was also detected in other
Western blots (data not shown). The presence of each of these receptors
in normal adrenal cells was confirmed with Western blots of extracts
from Y1-BS1 cells and from mouse adrenal glands (data not shown).
Western blots with antibody against the LR8B/apoER2 receptor failed to detect this receptor in Y1/E/tet/2/3 cells (Fig. 2), Y1-BS1 cells, or
mouse adrenal glands, although this antibody against chicken LR8B/apoER2 readily detected this receptor in extracts of mouse Neuro2A
cells (29). Antibody against gp330 detected a faint band in extracts of
Y1/E/tet/2/3 cells, but this was not seen with Y1-BS1 cells or mouse
adrenal glands (data not shown). Thus, the LDL receptor, the
LRP/
2M receptor, and the VLDL receptor were the most
prominent members of the LDL receptor family detected in adrenal cells.
To determine whether these members of the LDL receptor family might
play a role in apoE-dependent LDL CE selective uptake, Y1/E/tet/2/3 cells were incubated with RAP, which inhibits the binding
of apoE as well as other ligands to many members of this receptor
family (30, 31). As shown in Fig.
3A, increasing concentrations
of RAP blocked the apoE-dependent component of LDL CE
selective uptake in Y1/E/tet/2/3 cells but had no effect on LDL CE
selective uptake in the absence of apoE expression. In a separate
experiment with Y1/E/tet/2/3 cells induced to express apoE, RAP
inhibited LDL CE selective uptake but had no effect on HDL CE selective
uptake (Fig. 3B). Fifty percent of the
apoE-dependent LDL CE selective uptake component was
inhibited at a RAP concentration of ~50 nM.

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Fig. 3.
Effect of RAP on LDL CE and HDL CE selective
uptake in Y1/E/tet/2/3 cells. Panel A, Y1/E/tet/2/3
cells were cultured in complete medium in the presence or absence of
tet for 4 days. At day 4, medium was changed to serum-free medium
containing 2 mM Bt2cAMP plus or minus tet.
After 24 h RAP was added to the indicated concentrations and
incubated for 3 h at 37 °C, after which 50 µg/ml
125I-labeled [3H]LDL was added for another
4 h. Cells were processed to determine LDL CE selective uptake as
described under "Experimental Procedure." Panel B, in a
separate experiment after the addition of RAP for 3 h, cells were
incubated with 50 µg/ml 125I-labeled
[3H]LDL or 125I-labeled [3H]HDL
for another 4 h. Cells were processed to determine CE selective
uptake. Data are shown as the percent of CE selective uptake in the
absence of RAP. Values represent mean ± S.E. (n = 3).
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The effect of RAP on both LDL CE selective uptake and HDL CE selective
uptake was also tested in Y1-BS1 adrenocortical cells in which the
majority of LDL CE and HDL CE selective uptake is due to SR-BI (14,
32). At neither of two lipoprotein concentrations (10 µg/ml or 100 µg/ml) did RAP at 1 µM have an appreciable effect (<10% inhibition) on LDL CE selective uptake or HDL CE
selective uptake in Y1-BS1 cells (data not shown). Thus, SR-BI-mediated LDL CE and HDL CE selective uptake is not sensitive to RAP inhibition in Y1-BS1 cells, whereas apoE-dependent LDL CE selective
uptake in Y1/E/tet/2/3 cells that do not express SR-BI is completely sensitive to RAP.
Role of LRP/a2M Receptor in LDL CE Uptake--
Based
on the RAP inhibition data, we asked whether the LRP/
2M
receptor might be responsible for apoE-dependent LDL CE
selective uptake. Functional activity of the LRP/
2M
receptor in Y1/E/tet/2/3 cells in the presence of apoE was first tested
by monitoring the degradation of 125I-
2M*.
As shown in Fig. 4A,
Y1/E/tet/2/3 cells degraded 125I-
2M* at a
rate similar to that of mouse embryo fibroblast cells, which are known
to express the LRP/
2M receptor. Both RAP and
2M* blocked the degradation of
125I-
2M* in Y1/E/tet/2/3 cells. In addition,
RAP inhibited 50% of
2-M* degradation by Y1/E/tet/2/3
cells at a concentration of 50-100 nM (Fig.
4B), which is similar to the concentration dependence for
inhibition of apoE-dependent LDL CE selective uptake (Fig. 3B).

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Fig. 4.
Effect of RAP and
2M* on degradation of
125I- 2M* in
Y1/E/tet/2/3cells and MEF cells. Panel A, Y1/E/tet/2/3 cells
were cultured in complete medium in the absence of tet for 4 days. At
day 4, medium was changed to serum-free medium containing 2 mM Bt2cAMP. After 24 h, cells were washed
twice with assay medium and incubated with 1 nM
125I- 2M* plus or minus RAP or
2M*. After incubation, medium was removed for the
measurement of 125I- 2M* degradation. MEF
cells were plated at a density of 2 × 10 5 cells/well
in a 12-well plate and grown overnight in Dulbecco's modified Eagle's
medium containing 10% fetal bovine serum at 37 °C. Cells were then
washed twice with assay medium and used for the degradation assay as
described above. Panel B, inhibition of
125I- 2M* degradation was performed by
incubating Y1/E/tet/2/3 cells with different concentrations of RAP for
1 h before the addition of 125I- 2M*.
Cells were incubated for 12 h at 37 °C and processed to
determine 125I- 2M* degradation. Samples
containing no RAP were taken as 100% of control. Error bars
represent the range of triplicate determinations.
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Inhibition by RAP is consistent with the participation of the
LRP/
2M receptor but is not diagnostic for this receptor
alone, since RAP interacts with other members of the LDL receptor
family (30, 31). In contrast,
2M* is specific for the
LRP/
2M receptor. The experiment in Fig.
5A shows that
2M* inhibited ~30% of the total LDL CE selective
uptake activity in apoE-expressing Y1/E/tet/2/3 cells. The enhancement
of LDL CE selective uptake by apoE expression in this experiment was
~2-fold (Fig. 5B); hence, the 30% inhibition of total LDL
CE selective uptake by
2M* reflects a 60% inhibition of
apoE-dependent LDL CE selective uptake. In three such
experiments,
2M* inhibited an average of 68% of
apoE-dependent LDL CE selective uptake but inhibited less
than 10% of LDL CE selective uptake in Y1/E/tet/2/3 cells not induced
to express apoE or in Y1-BS1 cells that do not express apoE (data not
shown). These data suggest that the LRP/
2M receptor is
responsible for a substantial fraction of the
apoE-dependent LDL CE selective uptake in Y1/E/tet/2/3 cells. We attempted to confirm the role of the LRP/
2M
receptor by blocking the activity with antibody against human
LRP/
2M receptor. Under conditions where the antibody
effectively blocked 125I-
2M* uptake, we
obtained inconsistent inhibition of apoE-dependent LDL CE
selective uptake. In two experiments, 50-60% of the
apoE-dependent component was blocked by
anti-LRP/
2M receptor, but in two other experiments, no
inhibition was seen.

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Fig. 5.
Inhibition of LDL CE selective uptake by
2M*. Panel A,
Y1/E/tet/2/3 cells were cultured in complete medium in the absence of
tet for 4 days. At day 4, medium was changed to serum-free medium
containing 2 mM Bt2cAMP minus tet. After
24 h, RAP or 2M* was added to the indicated
concentration and incubated for 3 h at 37 °C. Fifty µg/ml
125I-labeled [3H]LDL was then added and
incubated for another 4 h. Cells were processed to determine
selective CE uptake. Panel B, in the same experiment, one
set of Y1/E/tet/2/3 cells was cultured in complete medium in the
presence of tet for 4 days. At day 4, medium was changed to serum-free
medium containing 2 mM Bt2cAMP plus tet. After
24 h, 50 µg/ml 125I-labeled [3H]LDL
was added for 4 h. Cells were processed to determine selective CE
uptake. Values represent mean ± S.E. (n = 3).
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Role of Cell Surface Proteoglycan in ApoE-dependent LDL
CE Selective Uptake--
Cell surface proteoglycans are known to be
important for the endocytic uptake of apoE-containing
-VLDL
particles by the LRP/
2M receptor (33, 34). In addition,
direct internalization of lipoproteins bound to the cell surface via
heparan sulfate proteoglycans, particularly syndecan and perlecan, may
occur without the participation of an LDL receptor family member (35,
36). To test the role of proteoglycan in apoE-dependent LDL
CE selective uptake in Y1/E/tet/2/3 cells, we used two approaches to
inhibit proteoglycan formation. In the first, the sulfation of
glycosaminoglycan side chains was inhibited by treatment of cells with
30 mM sodium chlorate for 20 h before the addition of
125I-labeled [3H]LDL. In three experiments,
treatment of Y1/E/tet/2/3 cells with sodium chlorate caused about 75%
inhibition of the apoE-dependent LDL CE selective uptake
(Fig. 6A) but had no effect on
SR-BI-mediated LDL CE selective uptake in Y1-BS1 cells that do not make
apoE (Fig. 6B). In the second approach, cells were treated
with 1 or 2 mM p-nitrophenyl-
-D-
xylopyranoside (
-xyloside) to inhibit the incorporation of
glycosaminoglycan side chains into cell surface proteoglycans before
incubation with 50 µg/ml 125I-labeled
[3H]LDL. The results in Fig.
7A show that
-xyloside
completely blocked apoE-dependent LDL CE selective uptake
in Y1/E/tet/2/3 cells but had no effect on LDL CE selective uptake in
Y1/E/tet/2/3 cells when apoE expression was suppressed. Similarly, this
inhibitor had no effect on SR-BI-mediated LDL CE selective uptake in
Y1-BS1 cells that do not make apoE (Fig. 7B). These results
indicate that cell surface proteoglycan is essential for
apoE-dependent LDL CE selective uptake in mouse
adrenocortical cells.

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Fig. 6.
Effect of sodium chlorate treatment on LDL CE
selective uptake in Y1/E/tet/2/3 cells and Y1-BS1 cells.
Panel A, Y1/E/tet/2/3 cells were maintained in complete F-10
medium minus tet for 4 days. At day 4, medium was changed to serum-free
medium containing 2 mM Bt2cAMP plus 30 mM sodium chlorate and incubated for 20 h at 37 °C.
The double-labeled LDL (100 µg/ml) was then added and incubated for
4 h. Cells were processed to determine LDL CE selective uptake.
The percent inhibition by chlorate of the apoE-dependent
component of LDL CE selective uptake was determined by subtracting the
apoE value from the +apoE value in both treatment groups and then
expressing the difference between the resultant values as a percent of
the apoE-dependent value for the control group. Panel
B, Y1-BS1 cells were cultured in serum-free medium plus 30 mM sodium chlorate for 20 h at 37 °C.
Double-labeled LDL (50 µg/ml) was then added and incubated for 4 h. Cells were processed to determine LDL CE selective uptake. Values
represent the mean ± S.E. (n = 3).
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Fig. 7.
Effect of
p-nitrophenyl- -D-xyloside
on selective uptake of LDL CE by Y1/E/tet/2/3 cells and Y1-BS1
cells. Panel A, Y1/E/tet/2/3 cells were maintained in
complete F-10 medium minus tet for 4 days. At day 4, medium was changed
to serum-free medium containing 2 mM Bt2cAMP
plus the indicated concentration of -D-xyloside and
incubated for 20 h at 37 °C. Double-labeled LDL (50 µg/ml)
was then added for 4 h. Cells were processed to determine LDL CE
selective uptake. Panel B, Y1-BS1 cells in serum-free medium
were incubated with the indicated concentration of
-D-xyloside for 20 h at 37 °C. Double-labeled
LDL (50 µg/ml) was then added for 4 h. Cells were processed to
determine LDL CE selective uptake. Values represent the mean ± S.E. (n = 3).
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Effect of Heparinase and Chondroitinase on
ApoE-dependent LDL CE Uptake--
To test the specific
effect of heparan sulfate proteoglycan and chondroitin sulfate
proteoglycan in apoE-dependent LDL CE selective uptake,
Y1/E/tet/2/3 cells were subjected to enzymatic digestion of
glycosaminoglycan side chains by proteoglycan lyases. When cells were
treated with 3 units/ml heparinase I or heparinase I and III together
for 3 h at 37 °C there was at most a 20% reduction of
apoE-dependent LDL CE selective uptake (Fig.
8). In addition, both heparinase II and
III had an inhibitory effect of less than 20% on
apoE-dependent LDL CE selective uptake (data not shown). However, upon treatment of Y1/E/tet/2/3 cells with 1 unit/ml
chondroitinase ABC at 37 °C for 3 h, apoE-dependent
LDL CE selective uptake was inhibited by greater than 90%, suggesting
the participation of chondroitin sulfate and/or dermatan sulfate
proteoglycans. Neither chondroitinase ABC nor the heparinases had any
effect on the apoE-independent component of LDL CE selective uptake in
Y1/E/tet/2/3 cells (Fig. 8). Note in the above studies with
proteoglycan synthesis inhibitors and proteoglycan lyases the
reductions in apoE-dependent LDL CE selective uptake were
paralleled by similar reductions in the cell association of LDL
particles (data not shown).

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|
Fig. 8.
Effect of heparinase and
chondroitinase treatment on LDL CE selective uptake in Y1/E/tet/2/3
cells. Y1/E/tet/2/3 cells were cultured in complete medium in the
absence or presence of tet for 4 days. At day 4, medium was changed to
serum-free medium containing 2 mM Bt2cAMP plus
or minus tet. After 24 h heparinase I (3 unit/ml) or
heparinase I and III (3 unit/ml each) or chondroitinase ABC (1 unit/ml)
was added for 3 h at 37 °C. Double-labeled LDL (50 µg/ml) was
then added for another 4 h, and cells were processed to determine
LDL CE selective uptake. Each data point represents the mean ± S.E. (n = 3).
|
|
 |
DISCUSSION |
The major finding in this study is the identification of an LDL CE
selective uptake pathway in murine adrenocortical cells that requires
apoE but is independent of SR-BI. The apoE-dependent LDL CE
selective uptake pathway involves both an
2M receptor and cell surface proteoglycans. As judged by quantitative comparisons of apoE-dependent LDL CE selective uptake in Y1/E/tet/2/3
cells with SR-BI-dependent LDL CE selective uptake in
Y1-BS1 cells (Figs. 6 and 7, for example), both pathways have the
capacity to take up similar amounts of LDL CE (3-6 µg of CE/4 h/mg
of cell protein at 50 µg/ml LDL). Interestingly, with both the
apoE-dependent pathway (16) and the
SR-BI-dependent pathway (14) the majority of LDL CE is
taken up by cultured adrenal cells by the selective pathway as opposed
to endocytic processing of LDL particles. LDL CE selective uptake has
been observed in steroidogenic cells and the liver in vivo
(4, 6) as well as in a variety of cultured cells such as human
fibroblasts (11), rat and human liver cells (6, 11-13), and a murine
macrophage cell (11). The contributions of the
SR-BI-dependent versus
apoE-dependent LDL CE selective uptake pathways in each of
these cell types in culture and in vivo is currently
unknown. However, many of these cell types including macrophage,
hepatocytes, and steroidogenic cells synthesize substantial quantities
of apoE (37-48), raising the possibility that
apoE-dependent LDL CE selective uptake is a widespread
process in LDL metabolism.
The finding that RAP inhibited the apoE-dependent component
of LDL CE selective uptake suggested that a member of the LDL receptor
family might be involved. Western blots showed that Y1/E/tet/2/3 cells
expressed three members of the LDL receptor family, two of which
(LRP/
2M receptor and VLDL receptor) are inhibited by RAP
concentrations similar to those that inhibit
2M*
degradation. Furthermore, approximately two-thirds of the
apoE-dependent LDL CE selective uptake was inhibited by
2M*. Since the LRP/
2M receptor is the
only known receptor for
2M*(30, 31), these data provide strong evidence that the LRP/
2M receptor participates in
the apoE-dependent selective uptake of LDL CE. These
results, however, do not eliminate the potential participation of other
LDL receptor family members, some of which have only recently been
identified (49).
The mechanism through which the LRP/
2M receptor
participates in LDL CE selective uptake is unclear. Results of studies
with inhibitors of proteoglycan biosynthesis clearly show that
proteoglycans are necessary for apoE-dependent LDL CE
selective uptake. This result is further supported by the finding that
treatment of cells with chondroitinase ABC completely abolished
apoE-dependent LDL CE selective uptake. Thus, the
participation of the LRP/
2M receptor in the LDL CE
selective uptake process requires cell surface proteoglycans. In
addition it appears likely that a portion of the
apoE-dependent LDL CE selective uptake occurs as a result
of proteoglycan interactions independently of the LRP/
2M
receptor since
2M* inhibited only 68% of the
apoE-dependent LDL CE uptake, whereas RAP and inhibitors of
proteoglycan synthesis completely blocked apoE-dependent CE uptake. RAP binds with high affinity to most LDL receptor family members but also binds with lower affinity to cell surface proteoglycan with a KD of greater than 200 nM (50,
51). The more gradual inhibition of LDL CE selective uptake at higher
RAP concentrations (100-2000 nM, Fig. 3) may reflect the
inhibition of LDL CE selective uptake due to direct interaction of
apoE-enriched LDL with proteoglycan.
Previous studies showed that an LDL particle acquires approximately one
molecule of apoE upon incubation with Y1/E/tet/2/3 cells (16). In
addition, the enhancement of LDL CE selective uptake by apoE also
requires apoE expression by Y1/E/tet/2/3 cells (16), suggesting that
both LDL-bound apoE and cell surface-bound apoE are necessary. A likely
scenario is that apoE serves a bridging function to localize LDL
particles to the cell surface via interactions with proteoglycans via
the LRP/
2M receptor or to a ternary complex of
apoE-enriched LDL, the LRP/
2M receptor, and
proteoglycans. The observation that inhibition of proteoglycan
synthesis or treatment of cells with chondroitinase ABC blocks
apoE-dependent LDL cell association as well as LDL CE
selective uptake suggests that the primary role of apoE is at the step
of LDL binding to the cell surface. The findings reported here on
apoE-dependent LDL CE selective uptake have striking
parallels to previous studies on the apoE-mediated enhancement of
chylomicron remnant uptake into liver cells in response to apoE
enrichment of
-VLDL particles (33, 34, 51-54). Very similar
proposals have been made to explain the roles of the
LRP/
2M receptor and proteoglycans in chylomicron remnant uptake by hepatocytes. In that case, however, the uptake of
-VLDL CE
is believed to occur primarily via endocytic uptake of intact
-VLDL
particles (33).
Irrespective of the details of how the apoE-enriched LDL particle is
bound to the cell surface, it seems likely that the
apoE-dependent selective uptake of LDL CE differs in
important ways from the process mediated by SR-BI. For example, SR-BI
is believed to be localized to cholesterol/sphingolipid-enriched
membrane microdomains or caveolae that appear to be the site of CE
uptake from HDL particles (9, 55, 56). In contrast, the
LRP/
2M receptor and other members of the LDL receptor
family are targeted for endocytosis via coated pits (57). As recently
discussed (33), cell surface binding of remnant-like particles in the
livers of LDL receptor-deficient mice is very rapid, but endocytosis
via an apoE-dependent pathway (presumably the
LRP/
2M receptor) is very slow compared with that mediated by the LDL receptor (58). This suggests that the
LRP/
2M receptor is relatively inefficient at endocytosis
and that the residence time on the cell surface for an apoE-enriched
LDL bound to the LRP/
2M receptor may be very long. It is
also of note from other studies that LDL bound to cell surface
proteoglycan occurs in two kinetic pools, one of which appears to be a
sequestered pool that is internalized slowly, if at all (59). Although
speculative, we suggest that a prolonged residence time of
apoE-enriched LDL on the cell surface, whether bound to the
LRP/
2M receptor or proteoglycan, will lead to selective
CE transfer to the cell membrane. Whether this occurs because some of
the bound LDL is directed to caveolae or this process is independent of
these membrane domains is unknown. Interestingly, the recently reported
enhancement of LDL CE selective uptake by lipoprotein lipase (60) also
requires cell surface proteoglycan as well as interaction of
lipoprotein lipase with both LDL and the proteoglycan. These data as
well as previous results (16) and the results of the present study suggest the hypothesis that bridging proteins such as apoE or lipoprotein lipase or hepatic lipase facilitate the selective uptake of
lipoprotein CE by holding the lipoprotein particle on the cell surface
for prolonged periods of time.
The present results on apoE-dependent LDL CE selective
uptake show an interesting difference in comparison to the
secretion/capture hypothesis for apoE-mediated chylomicron remnant
uptake into hepatocytes due to the LRP/
2M receptor
and/or proteoglycans (33). In the case of
-VLDL uptake into
hepatocytes, heparan sulfate proteoglycans appear to be of major
importance for the enhancement by apoE (34). In contrast, in the
Y1/E/tet/2/3 adrenal cells, lyases specific for heparan sulfate
proteoglycans had only modest effects, whereas chondroitinase
completely abolished apoE-dependent LDL CE selective uptake. This result suggests that apoE-enriched LDL interacts with
chondroitin sulfate or dermatan sulfate proteoglycan on adrenocortical cells. This difference with the results of
-VLDL uptake in hepatoma cells (33) could reflect cell-type differences in proteoglycan composition or differences in how apoE-enriched LDL versus
apoE-enriched
-VLDL interact with different proteoglycans.
Interestingly, Burgess et al. (61) noted that a major pool
of HepG2 cell surface apoE is associated with chondroitin sulfate
proteoglycans. Similarly, there is ample documentation of binding of
chondroitin/dermatan sulfate proteoglycan to LDL both in
vivo and in vitro (62-64). In this regard, it is of
interest that a basal level of LDL CE selective uptake occurs in the
Y1/E/tet/2/3 cells in the absence of apoE expression (Figs.
3A, 5B, 6A, 7A, and 8).
Although the nature of this pathway is not understood, we have observed
that heparin blocks this apoE-independent process (data not shown). This result is consistent with the possibility that the
apoE-independent LDL CE selective uptake in the Y1/E/tet/2/3 cells is
due to an apoB100/proteoglycan interaction that serves to localize LDL
particles to the cell surface.
In summary, we report here that delivery of LDL CE by the selective
uptake process occurs via multiple pathways in mouse adrenocortical cells. One pathway is apoE-dependent and involves the
LRP/
2M receptor and proteoglycans. A second pathway is
SR-BI-dependent, occurs in the absence of apoE, and does
not require cell surface proteoglycan. Additional studies will be
required to assess the contributions of these pathways to LDL CE
selective uptake in various cell types both in culture and in
vivo.
We thank J. Herz and J. Nimpf for
providing antibodies and Margery Connelly for purification of
cholesteryl ester transfer protein.
The abbreviations used are:
LDL, low density
lipoprotein;
VLDL, very LDL;
HDL, high density lipoprotein;
apo, apolipoprotein;
SR-BI, scavenger receptor class B type I;
CE, cholesteryl ester;
2M,
2-macroglobulin;
2M*, methylamine-activated
2M;
LRP/
2M receptor, LDL receptor-related
protein/
2M receptor;
tet, tetracycline;
RAP, receptor-associated protein;
Bt2cAMP, dibutyryl
cyclic AMP;
TBS, Tris-buffered saline;
human 125I-labeled
[3H]HDL3, 125I-dilactitol
tyramine-[3H]cholesteryl oleolyl ether human
HDL3;
human 125I-labeled [3H]LDL, 125I-dilactitol tyramine-[3H]cholesteryl
oleolyl ether human LDL.
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