Apolipoprotein (apo) E is expressed at high
levels by steroidogenic cells of the adrenal gland, ovary, and testis.
The cell surface location of apoE in adrenocortical cells suggests that apoE may facilitate the uptake of lipoprotein cholesterol by either the
endocytic or the selective uptake pathways, or both. To examine these
possibilities, the human apoE gene was expressed in murine Y1
adrenocortical cells under control of an inducible
tetracycline-regulated promoter. The results show that induction of
apoE yielded a 2-2.5-fold increase in the uptake of low density
lipoprotein-cholesteryl ester (LDL-CE) but had little effect on high
density lipoprotein-CE uptake. Analysis of lipoprotein uptake pathways
showed that apoE increased LDL-CE uptake by both endocytic and
selective uptake pathways. In terms of cholesterol delivery to the
adrenal cell, the apoE-mediated enhancement of LDL-CE selective uptake
was quantitatively more important. Furthermore, the predominant effect
of apoE expression was on the low affinity component of LDL-CE
selective uptake. LDL particles incubated with apoE-expressing cells
contained 0.92 ± 0.11 apoE molecules/apoB after gel filtration
chromatography, indicating stable complex formation between apoE and
LDL. ApoE expression by Y1 cells was necessary for enhanced LDL-CE
selective uptake. This result may indicate an interaction between
apoE-containing LDL and cell surface apoE. These data suggest that apoE
produced locally by steroidogenic cells facilitates cholesterol
acquisition by the LDL selective uptake pathway.
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INTRODUCTION |
Apolipoprotein E (apoE)1
is a prominent component of plasma lipoproteins and serves to mediate
endocytic uptake of remnant lipoproteins by members of the LDL receptor
family (1-6). In contrast to other apolipoproteins, apoE is expressed
in many peripheral tissues, including adrenal gland, ovary, testis,
brain, adipose, skin, and lung (7-15). Studies with humans, nonhuman
primates, and rats show that the apoE synthesis rate and mRNA
concentration in the adrenal gland are similar to those in liver (7, 8, 10, 16), indicating that apoE is an abundant protein product of adrenal
cells. ApoE mRNA is expressed in adrenocortical zona fasciculata
and zona reticularis cells, the sites of steroid production and
cholesteryl ester storage in rat adrenal gland (17).
The high expression of apoE in adrenocortical cells and its pattern of
regulation suggest that locally derived apoE may facilitate the
acquisition of lipoprotein cholesterol, alter cellular cholesteryl ester (CE) storage, or modulate the availability of cholesterol for
steroidogenesis (7, 16). Adrenal gland apoE expression is regulated in
direct proportion to CE stores and inversely to the level of steroid
production (16, 17). A potential role for locally produced apoE in
adrenocortical cholesterol metabolism is supported by results showing
that constitutive expression of human apoE in murine Y1 adrenocortical
cells leads to enhanced accumulation of CE (18). Immunolocalization
studies in rat adrenocortical cells show apoE intracellularly within
multivesicular bodies of the endocytic pathway and on cell surface
microvillar channels (19). Microvillar channels retain LDL and HDL
particles and have been proposed to be the site at which the selective
uptake of lipoprotein-CE occurs (20, 21). In contrast to lipoprotein uptake by endocytosis, the selective uptake pathway brings
lipoprotein-CE into the cell without the uptake and lysosomal
degradation of the lipoprotein particle (22-27). LDL-CE selective
uptake was first noted in perfused rat ovaries (28) and was later
studied in human fibroblasts (29), in the Y1-BS1 subclone of murine Y1 adrenocortical cells (29), and in human hepatoma cells (30). In ovarian
tissue (28) and in Y1-BS1 cells (29), most of the LDL-CE delivered to
the cells occurs via the selective as opposed to the endocytic
pathway.
The presence of apoE within multivesicular bodies of adrenocortical
cells and in microvillar channels may indicate that locally synthesized
apoE acts to facilitate the uptake of HDL- and/or LDL-CE by either the
endocytic or selective uptake pathways, or both (19). In the present
study, we examined these possibilities by expressing human apoE in
murine Y1 adrenocortical cells under control of an inducible
tetracycline (tet)-regulated promoter. The results show that apoE
expression yielded a 2-2.5-fold increase in the uptake of LDL-CE but
had little influence on HDL-CE uptake. Analysis of lipoprotein uptake
pathways showed that apoE increased LDL-CE uptake by both endocytic and
selective uptake pathways. These data suggest that apoE produced
locally by steroidogenic cells facilitates cholesterol acquisition from
LDL particles by two distinct pathways.
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MATERIALS AND METHODS |
Preparation of Stably Transfected Cell Lines--
Murine Y1
adrenal cells (American Type Culture Collection) 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% heat-inactivated fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (complete medium). Y1 cell lines inducible for apoE expression were prepared with
the tet-regulated promoter system described by Gossen and Bujard (31)
using the plasmid vectors pUHD15-1, pUHD10-3, and pUHD13-3 kindly
provided by H. Bujard. The expression vector pUHD/apoE was made as
follows. The 4.2-kilobase pair BamHI-EcoRI
fragment from the vector pFE (32), which encodes the human apoE
4
genomic sequence from
8 to +4200, was ligated to a
BamHI/EcoRI adapter sequence. This fragment was
then cloned into BamHI-linearized pUHD10-3, which places it
under control of the tet-regulated promoter. Cell lines were
constructed in two steps. First, Y1 cells were co-transfected with
pUHD15-1, which encodes the tet transactivator (tTA) protein, and
pSV2neo, which encodes resistance to G418 sulfate, at a ratio of 9:1,
by calcium phosphate-mediated gene transfer essentially as described
(32). Cell clones were selected in complete media containing 200 µg/ml G418 sulfate (Geneticin, Life Technologies, Inc.) and screened
for expression of the tTA protein by transient transfection of
pUHD13-3, which encodes a luciferase reporter gene expressed from a
tTA-responsive promoter. One clone, Y1UHD/7, which expressed high
levels of the tTA protein was secondarily transfected with pUHD/apoE,
together with pCMV hygromycin (Calbiochem) at a ratio of 9:1. To make
control cell lines, Y1UHD/7 cells were transfected with the empty
pUHD10-3 vector together with pCMV hygromycin. Hygromycin-resistant
clones (Y1/E/tet or Y1/con/tet cells) were selected in complete medium
containing 200 µg/ml hygromycin B (Calbiochem) and 200 µg/ml G418
sulfate. Tetracycline (2 µg/ml) was included during selection to
suppress expression of apoE. Following selection, cell lines were
maintained in complete medium containing 100 µg/ml G418, 100 µg/ml
hygromycin, and 2 µg/ml tet. Individual Y1/E/tet cell lines were
identified by Western blotting of medium following removal of tet. Two
cell lines (Y1/E/tet/2/3 and Y1/E/tet/2/5) that showed strong induction
of apoE and two control cell lines (Y1/con/tet/1/2 and Y1/con/tet/1/6)
were used for subsequent experiments.
Western Blotting and ELISA--
Proteins were separated by 8%
SDS-PAGE, electrophoretically transferred to a nitrocellulose membrane,
and blocked for 1 h at room temperature in 20 mM
Tris-HCl, pH 7.4, 150 mM NaCl (TBS) containing 7% nonfat
milk and 0.05% Tween 20. The blocked membrane was incubated with
polyclonal goat anti-human apoE antibody (Calbiochem) (1/1000 dilution)
overnight at room temperature in TBS containing 1% nonfat milk and
0.2% Tween 20. The membrane was washed three times with TBS containing
0.05% Tween 20 and incubated with a horseradish peroxidase-conjugated
donkey anti-goat IgG (Sigma)(1/10,000 dilution) for 1 h at room
temperature in TBS containing 1% nonfat milk and 0.05% Tween 20. Bands were visualized by enhanced chemiluminescence (Amersham). ApoE
concentration in conditioned medium was determined by ELISA using an
affinity-purified goat anti-human apoE antibody (Biodesign) as
described (33). Samples were assayed in triplicate using human apoE
(PanVera) as standard.
Preparation of [125I]Dilactitol
Tyramine-[3H]Cholesteryl Oleoyl Ether hHDL3
([125I,3H]hHDL3) and
[125I]Dilactitol Tyramine-[3H]Cholesteryl
Oleoyl Ether hLDL ([125I,3H]hLDL)--
Human
(h) HDL3 (1.125 g/ml <
< 1.210 g/ml) and human
LDL (1.019 g/ml <
< 1.063 g/ml) were doubly labeled with
[125I]dilactitol tyramine and
[3H]cholesteryl oleoyl ether as described (34). The
specific activity of the [125I,3H]hHDL ranged
from 46 to 70 dpm/ng protein for 125I and from 6 to 28 dpm/ng protein for 3H. The specific activity of the
[125I,3H]hLDL ranged from 25 to 75 dpm/ng of
protein for 125I and from 3 to 30 dpm/ng of protein for
3H.
Determination of HDL and LDL Cell Association, Selective CE
Uptake, and Apolipoprotein Degradation--
For all experiments,
six-well plates (Costar) were seeded with Y1/E/tet or Y1/con/tet cells
at 0.8 × 106 cells/well in complete medium in the
presence or absence of tet. At day 1 and day 3, medium was changed and,
at day 4, medium was changed to the above medium lacking serum, plus or
minus tet, containing 2 mM dibutyryl cAMP. After 24 h,
half of the medium was removed and double-labeled
[125I,3H]hHDL at 50 µg/ml (protein) or
[125I,3H]hLDL at 50 µg/ml (protein) (except
where indicated) was added, and the incubation was continued for 4 h. Cells were washed three times with 0.1% bovine serum albumin in
phosphate-buffered saline, pH 7.4; one time with phosphate-buffered
saline, pH 7.4; 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 as described (34, 35),
and cell protein (36). Trichloroacetic acid-insoluble 125I
radioactivity represents cell-associated apolipoprotein, which is the
sum of cell surface-bound apolipoprotein and endocytosed apolipoprotein
that is not yet degraded. Trichloroacetic acid-soluble 125I
radioactivity represents endocytosed and degraded apolipoprotein that
is trapped in lysosomes due to the dilactitol tyramine label. The sum
of the 125I-degraded and 125I-cell-associated
undegraded apolipoprotein expressed as CE equivalents was subtracted
from the CE measured as extractable 3H radioactivity to get
the selective uptake of LDL-CE and HDL-CE (34, 35). Values are
expressed as nanograms of cholesterol/mg of cell protein. The LDL
concentration dependence for each of these parameters was modeled by a
simple binding isotherm composed of a high affinity saturable process
and a low affinity nonsaturable process, Ptotal = {([Pmax] [LDL])/(KHA + [LDL])} + C[LDL], where Ptotal is the measured parameter,
[Pmax] is the high affinity parameter at
saturating levels of LDL, KHA is the apparent
high affinity Km, and C is the slope of
the low affinity nonsaturable process. For each parameter,
Ptotal was resolved into high affinity and
low affinity components by determining C and subtracting
C[LDL] from Ptotal to generate the
curve for high affinity LDL concentration dependence (37).
Size Fractionation of hLDL--
hLDL was chromatographed on
Bio-Gel A-15m (90 × 1.6 cm) at 6 ml/h in 5 mM
Na-PO4, 150 mM NaCl, 0.25 mM EDTA,
pH 7.4. Pooled fractions were concentrated in a Centriprep-30 (Amicon)
concentrator and resolved by nondenaturing 2-16% gradient PAGE in
Tris borate, pH 8.3, for 2300 V-h at 4 °C as described (38). The gel
was stained with Coomassie Blue. In some experiments, medium from cells
incubated with or without LDL was resolved by chromatography on Bio-Gel
A-15m as above, and the fractions were assayed for apoE by ELISA and
for LDL by monitoring 125I radioactivity.
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RESULTS |
Characterization of Y1/E/tet and Y1/con/tet Cell Lines--
In
order to examine the effects of apoE on lipoprotein uptake, we prepared
Y1 cell lines in which apoE expression is inducible. This approach
permits the influence of apoE to be tested within a clonal cell line
and eliminates the variables inherent in selecting and comparing
lipoprotein uptake in different clones that do or do not express apoE.
With the tet-regulated system of Gossen and Bujard (31), apoE
expression is suppressed in the presence of tet and induced following
tet removal from the medium. Table I shows the apoE concentration in culture medium at 24 h after
changing to serum-free medium when cells were withdrawn from tet 4 days previously. The Y1/E/tet cell lines showed a 20-fold induction of apoE
accumulation following tet removal and produced a medium apoE
concentration of 2-2.5 µg/ml. Secreted apoE was very low but
detectable in the presence of tet, but was not detected in the
Y1/con/tet cell lines in the presence or absence of tet. Note that the
endogenous mouse apoE gene is not expressed in the Y1 cell line (32).
Western blot analysis showed that, following a medium change at 4 days
after tet removal, secreted apoE accumulated to a steady state by
24 h (Fig. 1, panel A
(
tet) and B). When tet was not removed, a faint
apoE band was detected by Western blotting by 36-48 h after the medium
change (Fig. 1, panel A (+tet)), confirming the
conclusion from the ELISA (Table I) of low level apoE expression in the
presence of tet. The time necessary to induce maximal apoE expression
upon tet removal was determined by Western blotting of medium samples
collected in 24-h intervals over a 15-day period. As shown in Fig. 1
(panel C), apoE accumulation per 24 h was maximal by
day 4 and remained stable for up to day 15. In subsequent lipoprotein
uptake studies, Y1 cells were withdrawn from tet for 4 days to induce
maximal apoE expression, switched to serum-free medium at day 4 to
permit secreted apoE to accumulate, and experiments were initiated by
addition of labeled lipoprotein to the medium on day 5.
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Table I
Accumulation of apoE secreted by Y1 cell lines
Cells were cultured in complete medium plus or minus tet for 4 days
with changes of media on days 1 and 3. On day 4, medium was changed to
serum-free medium. Medium was removed after 24 h for estimation of
apoE by ELISA. Values represent mean ± S.E. (n = 6).
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Fig. 1.
Accumulation of secreted apoE.
Y1/E/tet/2/3 cells were cultured in complete medium containing 2 µg/ml tet. Panels A and B, At day 0, cells were
plated in six-well dishes in the absence ( tet) or presence
(+tet) of tet in complete medium. At day 4, medium was
changed to serum-free medium, and aliquots of medium were removed at
the indicated times for apoE determination by Western blotting.
Panel A shows the Western blot (M = purified
apoE), and panel B shows the densitometric measurement of
apoE from the Western blot in panel A (top,
tet) (arbitrary units). Panel C, at day 0, cells were plated in the absence of tet. One day before the indicated
times, medium was changed to complete medium lacking serum, and medium
was collected for apoE determination 24 h later. ApoE was
determined by Western blotting (arbitrary units).
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Selective Uptake of CE from LDL--
The results in Table
II illustrate the effects of tet
withdrawal and apoE expression on LDL-CE uptake. With Y1/con/tet/1/6 control cells not expressing apoE, tet withdrawal had little or no
effect on cell association, selective uptake, or endocytic uptake of
LDL-CE. In contrast, with Y1/E/tet/2/3 cells that do express apoE, tet
withdrawal led to a 2-3-fold increase in LDL-CE selective and
endocytic uptake and a 1.4-fold increase in cell association of LDL
particles. These results indicate that the enhanced LDL-CE uptake and
cell association reflect the expression of apoE and not the influence
of tet. Similar results were obtained with Y1/con/tet1/2 and
Y1/E/tet/2/5 cell lines (data not shown).
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Table II
Effect of apoE and tetracycline on hLDL binding and uptake in Y1 cells
Y1 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 Bt2-cAMP plus or minus tet. After
24 h, [125I,3H]hLDL was added to 50 µg/ml
(protein). After 4 h, cells were processed to determine
lipoprotein cell association, selective uptake, and endocytic uptake of
CE as described under "Materials and Methods."
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To determine whether the apoE-mediated enhancement of LDL-CE uptake was
specific for LDL, the uptake of LDL-CE and HDL-CE were compared. The
results in Table III show that in
contrast to the marked apoE-mediated enhancement of LDL-CE cell
association and uptake, apoE expression had only a modest effect on
HDL-CE uptake. When data from seven experiments were analyzed, HDL-CE selective uptake was 30% greater in apoE-expressing cells, but this
difference was not statistically significant (p > 0.5, data not shown). This result indicates that the effect of apoE
expression is primarily on LDL-CE uptake.
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Table III
Effect of apoE on hLDL and hHDL binding and uptake in Y1 cells
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 Bt2-cAMP plus or minus tet.
After 24 h, [125I,3H]hLDL or
[125I,3H]hHDL was added to 50 µg/ml (protein).
After 4 h, cells were processed to determine lipoprotein cell
association, selective uptake, and endocytic uptake of CE as described
under "Materials and Methods."
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The concentration dependence for LDL-CE uptake in the presence and
absence of apoE expression is shown in Fig.
2. These data indicate that, in the
presence or absence of apoE, most LDL-CE uptake at all LDL
concentrations tested occurred via selective uptake as opposed to
endocytic uptake. ApoE expression enhanced LDL-CE uptake by both
endocytic and selective pathways, with the -fold enhancement of
endocytic uptake being somewhat greater (2.33 ± 0.13-fold,
n = 29) as compared with selective uptake (2.04 ± 0.10-fold, n = 29) when data in numerous experiments
were averaged. However, in terms of total LDL-CE delivery to the cell,
the major effect of apoE was on the selective uptake pathway. For
example, at 50 µg/ml LDL, apoE expression increased LDL-CE uptake by
the selective uptake pathway by about 2000 ng of CE/mg of cell protein, whereas the enhancement by the endocytic pathway was about 225 ng of
CE/mg of cell protein (Fig. 2).

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Fig. 2.
Effect of apoE on selective and endocytic
uptake of [125I,3H] hLDL.
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 Bt2-cAMP plus or minus
tet. After 24 h, [125I,3H]hLDL was added
to the indicated protein concentration. After 4 h, cells were
processed to determine selective uptake and endocytic uptake of CE as
described under "Materials and Methods." The insets in
each panel show values from low LDL concentrations (<16 µg/ml).
Values represent mean ± S.E. (n = 3). Error bars
not shown are within the area of the symbol.
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The LDL concentration dependence for selective CE uptake and for
endocytic CE uptake was indicative of both high and low affinity components. This point is illustrated in Fig.
3, which shows the LDL concentration
dependence for selective (panel A) and endocytic (panel B) uptake for apoE-expressing Y1/E/tet/2/3 cells
resolved into high and low affinity components. These data show that,
at LDL concentrations greater than 50 µg/ml, most of the LDL-CE
selective uptake (panel A) was due to the low affinity
component; this component increased further at higher LDL
concentrations, whereas the high affinity component was saturated above
20 µg/ml LDL. A similar result was seen with endocytic uptake of
LDL-CE (panel B), except that the contribution of the low
affinity component was less at lower LDL concentrations; in this case,
the low affinity and high affinity components were equivalent at about
150 µg/ml LDL. The cell association of LDL-CE, most of which is
believed to reflect cell surface bound LDL particles, showed a similar
LDL concentration dependence and a similar enhancement by apoE
expression throughout the LDL concentration range as was seen for
LDL-CE selective and endocytic uptake (Tables II and III and data not
shown).

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Fig. 3.
High and low affinity components of selective
and endocytic uptake in the presence of apoE. Data from Fig. 2 are
resolved into high and low affinity components for LDL-CE selective
(panel A) and endocytic (panel B) uptake as
described under "Materials and Methods."
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Analysis of hLDL Size Heterogeneity and hLDL-ApoE
Interaction--
Size heterogeneity within the LDL particle population
potentially could bias the selective uptake measurements if the LDL contained a significant fraction of large CE-rich particles that were
taken up in preference to the bulk of the LDL. To address this point,
LDL was fractionated by chromatography on Bio-Gel A-15m. The profile
contained no particles larger than LDL (data not shown), and, within
the LDL region of the chromatogram, the particles eluted in a near
normal distribution (Fig. 4, panel A). The LDL profile was divided into three fractions (A, B, and C)
corresponding to the leading edge, the peak, and the trailing edge,
respectively, which were analyzed by nondenaturing gradient gel
electrophoresis. As shown in panel B, the LDL contained two major species with the larger and smaller species recovered
preferentially in fractions A and C, respectively, and similar amounts
of both species recovered in fraction B. Equal amounts of LDL from each fraction (20 µg/ml protein) were compared for LDL-CE uptake with Y1/E/tet/2/3 cells with and without apoE induction. The results in Fig.
5 show that the fractions differed little
in selective (panel A) or endocytic (panel B)
uptake, with the peak fraction of the LDL profile, fraction B, being
25-50% more active than either the leading or trailing fractions of
LDL. The apoE-mediated enhancement of LDL-CE selective or endocytic
uptake was similar among the LDL fractions. Similar results were seen
with two independent preparations of LDL that were analyzed. These data
indicate that LDL-CE selective uptake by murine Y1 adrenocortical cells
and the enhancement by apoE are properties of the bulk LDL population and not due to a small fraction of large CE-enriched particles.

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Fig. 4.
[125I,3H]hLDL
fractionation by gel filtration chromatography. Panel A,
doubly labeled [125I,3H]hLDL (2 mg) was
fractionated on Bio-Gel A-15m as described under "Materials and
Methods." Regions of the profile indicated by A,
B, and C were collected and concentrated in a
Centriprep-30. Panel B, samples of each fraction and the
starting LDL (L) were analyzed by electrophoresis on a
nondenaturing 2-16% polyacrylamide gel as described under
"Materials and Methods." The Coomassie Blue stain of the gel is
shown.
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Fig. 5.
Selective and endocytic uptake of cholesteryl
ester from subfractions of
[125I,3H]hLDL. 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 Bt2-cAMP plus or minus tet. After 24 h,
[125I,3H]hLDL was added to 20 µg/ml
(protein). After 4 h, cells were processed to determine LDL-CE
selective uptake (panel A) and endocytic uptake (panel
B) as described under "Materials and Methods." Results are the
mean ± S.E. (n = 3) from a representative
experiment.
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To address the question of whether there is a direct interaction
between LDL and apoE, LDL was re-isolated by gel filtration chromatography after incubation with cells expressing apoE. The elution
profile in Fig. 6 (panel A)
shows that apoE eluted with the LDL peak as well as in a second lower
molecular weight peak. The profile in panel B shows that the
association of apoE with LDL was dependent upon apoE expression by the
Y1 cells and not due to apoE contamination in the purified LDL. The
profile in panel C shows that the presence of apoE in the
LDL fraction required the addition of LDL. These data indicate that LDL
particles acquired apoE when incubated with apoE-expressing Y1 cells.
The stoichiometry of this association was estimated by comparing the
quantity of apoE recovered in the LDL fraction as determined by ELISA
with the apoB content as determined from the apoB radioactivity. These measurements gave a value of 0.92 ± 0.11 (n = 3)
apoE molecules/apoB, suggesting that each LDL particle acquired one
apoE molecule that was stable to gel filtration.

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Fig. 6.
Association of secreted apoE with
[125I,3H]hLDL. 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 Bt2-cAMP plus or minus tet. After 24 h,
[125I,3H]hLDL was added to 50 µg/ml
(protein). After 4 h, culture medium was removed and analyzed by
chromatography on Bio-Gel A-15m. The elution profile of LDL was
monitored by measurement of 125I radioactivity. ApoE was
determined by ELISA. Panel A, elution profile of LDL and
apoE after LDL incubation with apoE-expressing cells. Panel
B, elution profiles of apoE after LDL incubation with cells
expressing or not expressing apoE. Panel C, elution profiles
of apoE after apoE-expressing cells were incubated with or without
LDL.
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Role of Cell-associated and LDL-associated ApoE in Mediating LDL-CE
Selective Uptake--
ApoE accumulates in the medium and associates
with LDL particles, but is also present on the surface of
apoE-expressing Y1 cells (data not shown) and adrenocortical cells
in vivo (19). Thus, both LDL-associated or cell
surface-associated apoE, or both, could account for the increased
LDL-CE selective uptake. To test this point, conditioned medium (from
cells expressing or not expressing apoE) was added to cells (expressing
or not expressing apoE) immediately before LDL particles were added for the 4 h uptake assay. As shown in Fig.
7, with the controls, for which the
medium was not changed, apoE expression gave a 3-fold increase in
LDL-CE selective uptake (compare samples 1 and 2). Adding fresh
unconditioned medium prior to LDL reduced but did not eliminate the
apoE-enhancement of LDL-CE selective uptake in the apoE-expressing
cells (sample 3 versus 1) and gave a slight increase in
cells not expressing apoE (sample 4 versus 2) that was not
statistically significant (p > 0.08). Partial
retention of the apoE-mediated increase upon addition of fresh medium
in sample 3 could be due to cell surface apoE or to secreted apoE that
would accumulate to some extent during the 4-h uptake assay (about 20%
of the steady state level; see Fig. 1, panel B). When medium
from apoE-expressing cells was added back to apoE-expressing cells,
full restoration of LDL-CE selective uptake was seen (sample 5 versus 1). However, the addition of medium from
apoE-expressing cells to cells not expressing apoE (sample 6) did not
increase LDL-CE selective uptake (sample 6 versus 4). The
level of LDL-CE selective uptake in sample 6 was the same as seen when
cells not expressing apoE received either fresh medium (compare sample
6 versus 4) or medium from cells not expressing apoE
(compare sample 6 versus 7). These data indicate that apoE
expression by the cells is necessary for the apoE-mediated enhancement
of LDL-CE selective uptake. The partial loss in the apoE-mediated
enhancement upon addition of fresh medium (sample 1 versus
3), and the restoration of this loss by addition of apoE-containing
medium (sample 3 versus 5, p = 0.001)
suggest that LDL-associated apoE also contributes to the enhancement of
LDL-CE selective uptake. However, the enhancement of selective uptake
by LDL-associated apoE only occurred when cells also were expressing
apoE.

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Fig. 7.
Role of secreted apoE and apoE expression in
the apoE-mediated enhancement of selective LDL-CE uptake.
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 Bt2-cAMP plus or minus
tet. After 24 h, culture medium was not changed (samples 1 and 2),
replaced with fresh medium (samples 3 and 4), replaced with medium from
apoE-expressing cells (samples 5 and 6), or replaced with medium from
cells not expressing apoE (sample 7).
[125I,3H]hLDL was added to 50 µg/ml
(protein). After 4 h, cells were processed to determine LDL-CE
selective uptake as described under "Materials and Methods." The
apoE expression state of the cells due to regulation by tet is
indicated at the top of the figure. Results are the mean ± S.E.
from three experiments, each with triplicate determinations.
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DISCUSSION |
The major finding in this study is that apoE expression markedly
increased LDL-CE uptake into adrenocortical cells via both endocytic
and selective uptake pathways. The enhancement of LDL-CE uptake by apoE
expression may play a quantitatively important role in LDL-CE uptake
into adrenal cells in vivo since apoE expression occurs in
adrenal cells of all mammalian species examined (7, 8, 10, 13, 16).
ApoE also is expressed by steroidogenic cells of the ovary and the
testis (14, 39-41), suggesting that apoE may play a general role in
LDL-CE uptake in steroidogenic cells.
Expression of apoE increased LDL-CE selective uptake by 2-2.5-fold
over a broad range of LDL concentrations. ApoE markedly enhanced LDL-CE
selective uptake, but this process does not, in itself, appear to
require apoE since it also occurred in control Y1 cells that do not
express apoE (Table II) (29). The LDL concentration dependence for CE
selective uptake was similar in the presence and absence of apoE
expression, with apoE increasing LDL-CE selective uptake throughout the
concentration range examined. The concentration dependence showed both
low and high affinity components for the LDL-CE selective uptake
process. At low LDL concentrations (<50 µg/ml), most of the LDL-CE
selective uptake occurred via the high affinity component, but at
higher LDL concentrations (>50 µg/ml), the low affinity component
predominated and increased linearly as a function of LDL concentration
(Fig. 3). This result suggests that the low affinity component may
provide greater cholesterol delivery than the high affinity component
in vivo in species with high LDL levels. In the mouse, which
has very low LDL levels (42, 43) and relies on HDL-CE selective uptake
to provide cholesterol to steroidogenic cells (22, 23, 26), LDL-CE
selective uptake would not be expected to play a major role in steady
state cholesterol delivery to adrenal cells. However, in humans and
other species with high LDL levels, LDL-CE selective uptake and the
enhancement by apoE expression may be of greater quantitative
importance.
The enhancement of LDL-CE selective uptake by apoE appears to involve
both secreted apoE and apoE expression by Y1 cells. Secreted apoE
associated with LDL particles with approximately one molecule of apoE
recovered per LDL particle after gel filtration chromatography. This is
a minimal estimate because more weakly associated apoE molecules may
not have been stable to chromatography. Interestingly, addition of
apoE-containing medium to Y1 cells did not enhance LDL-CE selective
uptake unless the cells also were expressing apoE. This suggests that
cell surface apoE or the continuous production of apoE was required to
enhance LDL-CE selective uptake.
The present results showing an apoE-mediated enhancement of the LDL-CE
selective uptake pathway show many parallels to the effects of apoE on
the endocytic uptake of
-VLDL in hepatoma cells. ApoE is localized
to the cell surface of adrenocortical cells (19) and hepatocytes (44)
and is known to associate with cell surface proteoglycan when secreted
from hepatoma cells in culture (45, 46). In hepatoma cells, apoE
expression enhances cell surface binding and endocytic uptake of
-VLDL, an effect that involves heparan sulfate proteoglycan and the
LDL receptor-related protein (46). Conditioned medium from
apoE-expressing cells gave the full apoE enhancement of
-VLDL
binding when added to nontransfected hepatoma cells, suggesting that
the enhanced binding required secreted apoE that associated with
-VLDL particles (46). However, nontransfected hepatoma cells also
expressed endogenous rat apoE, which may have been present on the cell
surface and contributed to the enhanced uptake. Consistent with this
possibility,
-VLDL binding to apoE-expressing hepatoma cells at
4 °C was increased without the addition of exogenous apoE, again
suggesting a role for cell surface apoE (46). This is similar to the
present results with adrenocortical cells in which enhancement of
LDL-CE selective uptake by apoE required apoE expression by the cells.
The current data in adrenal cells and the results with hepatoma cells
(46) may indicate that apoE-enriched lipoprotein particles interact with cell surface apoE to facilitate CE uptake by both endocytic and
selective uptake pathways. The extent to which apoE expression enhances
the endocytic versus the selective uptake pathway may depend
on the cell type, the type of lipoprotein particle, and the spectrum of
lipoprotein receptors expressed by the cells.
The biochemical mechanism of LDL-CE selective uptake and the manner in
which apoE enhances uptake are poorly understood. The LDL concentration
dependence showed that apoE enhanced selective uptake throughout the
concentration range tested (Fig. 2). Thus, both the high and low
affinity uptake processes were increased. This was also true for the
uptake of LDL-CE via the endocytic pathway (Fig. 2). Similarly, the
cell association of LDL particles (data not shown), most of which is
believed to reflect cell surface LDL binding, was also increased by
apoE throughout the LDL concentration range. Although we cannot rule
out that apoE expression enhances each of these parameters by a
different mechanism, we consider that possibility unlikely. We
speculate that the primary effect of apoE is to enhance cell surface
LDL binding, thereby increasing the local surface concentration of LDL
particles available to both the low and high affinity components of the
endocytic and selective uptake pathways.
The cell surface receptors responsible for the selective uptake of
lipoprotein CE are not well understood. In the case of HDL, scavenger
receptor BI can mediate HDL-CE selective uptake in transfected cells
(47) and is the major route for high affinity HDL-CE uptake and
delivery to the steroidogenic pathway in cultured adrenal cells (37).
Scavenger receptor BI binds native LDL (48), but it is not known
whether scavenger receptor BI mediates high or low affinity selective
uptake from LDL particles. Recent studies show that cell surface
proteoglycans can mediate the endocytosis of LDL particles via a
bridging molecule of lipoprotein lipase (49). A major component of this
LDL endocytosis appears to occur via direct endocytosis of a syndecan
proteoglycan without the participation of other cell surface receptors
(50). Interestingly, proteoglycan-bound LDL occur in two kinetic pools,
one which is internalized rapidly and the other which appears to be a
sequestered cell surface LDL pool with a prolonged residence time (49). It is not known whether this sequestered pool of LDL, by virtue of its
prolonged cell surface residence time, might make LDL particles available to the selective uptake pathway, but this is a prime candidate to test in future studies. We speculate that a "blanket" of apoE on the cell surface (19) and LDL-associated apoE act as a
bridging mechanism to localize LDL particles to the cell surface
proteoglycans.
In summary, the results of this study showed that inducible apoE
expression in Y1 adrenocortical cells enhanced the selective uptake of
LDL-CE by 2-2.5-fold over a broad range of LDL concentrations. Endocytic uptake of LDL was also increased by apoE expression, but this
was quantitatively less important in cholesterol delivery to adrenal
cells. ApoE expression had little effect on the cell association or
selective uptake of HDL-CE. ApoE expression under control of an
inducible tet-regulated promoter system provided a sensitive means of
evaluating the effects of apoE without the complications of clonal
variation inherent in comparing among individual cell lines.
We are grateful to Dr. Lawrence L. Rudel for
helpful discussion, Penelope Strockbine for excellent technical
assistance, and Ramesh Shah for performing the electrophoresis.