Selective Uptake of Low Density Lipoprotein-Cholesteryl Ester Is Enhanced by Inducible Apolipoprotein E Expression in Cultured Mouse Adrenocortical Cells

Abstract

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.

Full text

Selective Uptake of Low Density Lipoprotein-Cholesteryl Ester Is
Enhanced by Inducible Apolipoprotein E Expression in Cultured
Mouse Adrenocortical Cells*
(Received for publication, January 14, 1998, and in revised form, February 19, 1998)
Snehasikta Swarnakar‡, Mary E. Reyland§, Jiatai Deng‡, Salman Azhar¶,
and David L. Williams‡i
From the ‡Department of Pharmacological Sciences, University Medical Center, State University of New York,
Stony Brook, New York 11794, ¶Geriatric Research, Education and Clinical Center, Department of Veterans Affairs
Palo Alto Heath Care System, Palo Alto, California 94304, and the §Department of Basic Science and Oral Research,
University of Colorado, Denver, Colorado 80262
Apolipoprotein (apo) E is expressed at high levels by
steroidogenic cells of the adrenal gland, ovary, and tes-
tis. 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 in-
ducible tetracycline-regulated promoter. The results
show that induction of apoE yielded a 2–2.5-fold in-
crease in the uptake of low density lipoprotein-cho-
lesteryl ester (LDL-CE) but had little effect on high den-
sity lipoprotein-CE uptake. Analysis of lipoprotein
uptake pathways showed that apoE increased LDL-CE
uptake by both endocytic and selective uptake path-
ways. In terms of cholesterol delivery to the adrenal cell,
the apoE-mediated enhancement of LDL-CE selective
uptake was quantitatively more important. Further-
more, the predominant effect of apoE expression was on
the low affinity component of LDL-CE selective uptake.
LDL particles incubated with apoE-expressing cells con-
tained 0.92 60.11 apoE molecules/apoB after gel filtra-
tion chromatography, indicating stable complex forma-
tion 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 sug-
gest that apoE produced locally by steroidogenic cells
facilitates cholesterol acquisition by the LDL selective
uptake pathway.
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 reticu-
laris 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 cellu-
lar 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). Immunolocaliza-
tion studies in rat adrenocortical cells show apoE intracellu-
larly 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 lipopro-
tein 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 adreno-
cortical cells (29), and in human hepatoma cells (30). In ovarian
tissue (28) and in Y1-BS1 cells (29), most of the LDL-CE deliv-
ered to the cells occurs via the selective as opposed to the
endocytic pathway.
The presence of apoE within multivesicular bodies of adre-
nocortical 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 expres-
sion 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 facil-
* This work was supported by National Institutes of Health Grant HL
32868 and by the Office of Research and Development, Medical Re-
search Service, Department of Veterans Affairs. The costs of publication
of this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked “advertisement”inac-
cordance with 18 U.S.C. Section 1734 solely to indicate this fact.
iTo whom correspondence should be addressed: Dept. of Pharmaco-
logical Sciences, University Medical Center, State University of New
York, Stony Brook, NY 11794. Tel.: 516-444-3083; Fax: 516-444-3218;
E-mail: dave@pharm.som.sunysb.edu.
1
The abbreviations used are: apo, apolipoprotein; HDL, high density
lipoprotein; LDL, low density lipoprotein; CE, cholesteryl ester; Bt
2
-
cAMP, N
6
,29-O-dibutyryladenosine 39:59-cyclic monophosphate; ELISA,
enzyme-linked immunosorbent assay; tet, tetracycline; tTA, tet trans-
activator; TBS, Tris-buffered saline.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 20, Issue of May 15, pp. 12140–12147, 1998
Printed in U.S.A.
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itates cholesterol acquisition from LDL particles by two dis-
tinct pathways.
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% CO
2
incubator in Ham’s F-10 medium supple-
mented with 12.5% heat-inactivated horse serum, 2.5% heat-inacti-
vated fetal calf serum, 2 mMglutamine, 100 units/ml penicillin, and 100
m
g/ml streptomycin (complete medium). Y1 cell lines inducible for apoE
expression were prepared with the tet-regulated promoter system de-
scribed 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-kilo-
base pair BamHI-EcoRI fragment from the vector pFE (32), which
encodes the human apoE
e
4 genomic sequence from 28to14200, 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 resist-
ance 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
m
g/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 re-
porter gene expressed from a tTA-responsive promoter. One clone,
Y1UHD/7, which expressed high levels of the tTA protein was second-
arily 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
m
g/ml hygromycin B
(Calbiochem) and 200
m
g/ml G418 sulfate. Tetracycline (2
m
g/ml) was
included during selection to suppress expression of apoE. Following
selection, cell lines were maintained in complete medium containing
100
m
g/ml G418, 100
m
g/ml hygromycin, and 2
m
g/ml tet. Individual
Y1/E/tet cell lines were identified by Western blotting of medium fol-
lowing 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 for1hatroom temperature in 20 mMTris-HCl, pH 7.4, 150
mMNaCl (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 tem-
perature 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) for1hatroom temperature in
TBS containing 1% nonfat milk and 0.05% Tween 20. Bands were
visualized by enhanced chemiluminescence (Amersham). ApoE concen-
tration in conditioned medium was determined by ELISA using an
affinity-purified goat anti-human apoE antibody (Biodesign) as de-
scribed (33). Samples were assayed in triplicate using human apoE
(PanVera) as standard.
Preparation of [
125
I]Dilactitol Tyramine-[
3
H]Cholesteryl Oleoyl Ether
hHDL
3
([
125
I,
3
H]hHDL
3
) and [
125
I]Dilactitol Tyramine-[
3
H]Cholesteryl
Oleoyl Ether hLDL ([
125
I,
3
H]hLDL)—Human (h) HDL
3
(1.125 g/ml ,
r
,1.210 g/ml) and human LDL (1.019 g/ml ,
r
,1.063 g/ml) were
doubly labeled with [
125
I]dilactitol tyramine and [
3
H]cholesteryl oleoyl
ether as described (34). The specific activity of the [
125
I,
3
H]hHDL
ranged from 46 to 70 dpm/ng protein for
125
I and from 6 to 28 dpm/ng
protein for
3
H. The specific activity of the [
125
I,
3
H]hLDL ranged from 25
to 75 dpm/ng of protein for
125
I and from 3 to 30 dpm/ng of protein for
3
H.
Determination of HDL and LDL Cell Association, Selective CE Up-
take, and Apolipoprotein Degradation—For all experiments, six-well
plates (Costar) were seeded with Y1/E/tet or Y1/con/tet cells at 0.8 310
6
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 [
125
I,
3
H]hHDL at 50
m
g/ml (protein) or [
125
I,
3
H]hLDL at
50
m
g/ml (protein) (except where indicated) was added, and the incu-
bation 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
125
I radioactivity and organic solvent-extractable
3
H radioactivity as described (34, 35), and cell protein (36). Trichloro-
acetic acid-insoluble
125
I radioactivity represents cell-associated apoli-
poprotein, which is the sum of cell surface-bound apolipoprotein and
endocytosed apolipoprotein that is not yet degraded. Trichloroacetic
acid-soluble
125
I radioactivity represents endocytosed and degraded
apolipoprotein that is trapped in lysosomes due to the dilactitol tyra-
mine label. The sum of the
125
I-degraded and
125
I-cell-associated unde-
graded apolipoprotein expressed as CE equivalents was subtracted
from the CE measured as extractable
3
H radioactivity to get the selec-
tive 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, P
total
5{([P
max
] [LDL])/(K
HA
1
[LDL])} 1C[LDL], where P
total
is the measured parameter, [P
max
]isthe
high affinity parameter at saturating levels of LDL, K
HA
is the apparent
high affinity K
m
, and Cis the slope of the low affinity nonsaturable
process. For each parameter, P
total
was resolved into high affinity
and low affinity components by determining Cand subtracting C[LDL]
from P
total
to generate the curve for high affinity LDL concentration
dependence (37).
Size Fractionation of hLDL—hLDL was chromatographed on Bio-Gel
A-15m (90 31.6 cm) at 6 ml/h in 5 mMNa-PO
4
, 150 mMNaCl, 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 chromatog-
raphy on Bio-Gel A-15m as above, and the fractions were assayed for
apoE by ELISA and for LDL by monitoring
125
I radioactivity.
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 me-
dium 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 follow-
ing tet removal and produced a medium apoE concentration of
2–2.5
m
g/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).
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 6S.E. (n56).
Cell line tet apoE
m
g/ml/24 h
Y1/con/tet/1/2 1ND
a
Y1/con/tet/1/2 2ND
Y1/con/tet/1/6 1ND
Y1/con/tet/1/6 2ND
Y1/E/tet/2/5 10.16 60.02
Y1/E/tet/2/5 22.5 60.07
Y1/E/tet/2/3 10.11 60.02
Y1/E/tet/2/3 22.1 60.08
a
ND, not detected.
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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 (2tet) and B). When tet was
not removed, a faint apoE band was detected by Western blot-
ting by 36–48 h after the medium change (Fig. 1, panel A
(1tet)), 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 col-
lected 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 me-
dium at day 4 to permit secreted apoE to accumulate, and
experiments were initiated by addition of labeled lipoprotein to
the medium on day 5.
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 express-
ing apoE, tet withdrawal had little or no effect on cell associa-
tion, selective uptake, or endocytic uptake of LDL-CE. In con-
trast, 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).
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.
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 60.13-fold, n529) as
compared with selective uptake (2.04 60.10-fold, n529) 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
m
g/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).
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 compo-
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 mMBt
2
-cAMP plus or minus tet. After 24 h,
[
125
I,
3
H]hLDL or [
125
I,
3
H]hHDL was added to 50
m
g/ml (protein). After
4 h, cells were processed to determine lipoprotein cell association,
selective uptake, and endocytic uptake of CE as described under “Ma-
terials and Methods.”
Lipoprotein tet Cell association
a
Selective uptake
a
Endocytic
uptake
a
ng CE/mg cell protein
hLDL 1566 6113 2627 6415 131 630
hLDL 22295 6445 9231 61276 402 617
hHDL 184 66 806 660 28 62
hHDL 288 63 1288 6100 29 62
a
Values represent mean 6S.E. (n53).
FIG.1. Accumulation of secreted apoE. Y1/E/tet/2/3 cells were
cultured in complete medium containing 2
m
g/ml tet. Panels A and B,At
day 0, cells were plated in six-well dishes in the absence (2tet)or
presence (1tet) 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 (M5purified apoE), and panel B shows
the densitometric measurement of apoE from the Western blot in panel
A(top,2tet) (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).
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 mMBt
2
-cAMP plus or minus tet. After 24 h, [
125
I,
3
H]hLDL
was added to 50
m
g/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.”
Cell line tet Cell
association
a
Selective
uptake
a
Endocytic
uptake
a
ng CE/mg cell protein
Y1/con/tet/1/6 1240 684 2153 6606 229 663
Y1/con/tet/1/6 2292 613 2437 6129 246 615
Y1/E/tet/2/3 1495 671 1958 6155 129 64
Y1/E/tet/2/3 2719 638 4127 636 425 620
a
Values represent mean 6S.E. (n53).
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nents. These data show that, at LDL concentrations greater
than 50
m
g/ml, most of the LDL-CE selective uptake (panel A)
was due to the low affinity component; this component in-
creased further at higher LDL concentrations, whereas the
high affinity component was saturated above 20
m
g/ml LDL. A
similar result was seen with endocytic uptake of LDL-CE (pan-
el 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
m
g/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 en-
hancement by apoE expression throughout the LDL concentra-
tion range as was seen for LDL-CE selective and endocytic
uptake (Tables II and III and data not shown).
Analysis of hLDL Size Heterogeneity and hLDL-ApoE Inter-
action—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 distribu-
tion (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
m
g/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
FIG.2. Effect of apoE on selective
and endocytic uptake of [
125
I,
3
H]
hLDL. Y1/E/tet/2/3 cells were cultured in
complete medium in the presence or ab-
sence of tet for 4 days. At day 4, medium
was changed to serum-free medium con-
taining 2 mMBt
2
-cAMP plus or minus tet.
After 24 h, [
125
I,
3
H]hLDL was added to
the indicated protein concentration. After
4 h, cells were processed to determine se-
lective uptake and endocytic uptake of CE
as described under “Materials and Meth-
ods.” The insets in each panel show values
from low LDL concentrations (,16
m
g/
ml). Values represent mean 6S.E. (n5
3). Error bars not shown are within the
area of the symbol.
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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 prop-
erties of the bulk LDL population and not due to a small
fraction of large CE-enriched particles.
To address the question of whether there is a direct interac-
tion between LDL and apoE, LDL was re-isolated by gel filtra-
tion 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 molec-
ular weight peak. The profile in panel B shows that the asso-
ciation 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 incu-
bated 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 60.11 (n53) apoE mole-
cules/apoB, suggesting that each LDL particle acquired one
apoE molecule that was stable to gel filtration.
Role of Cell-associated and LDL-associated ApoE in Mediat-
ing LDL-CE Selective Uptake—ApoE accumulates in the me-
dium 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, condi-
tioned medium (from cells expressing or not expressing apoE)
was added to cells (expressing or not expressing apoE) imme-
diately 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 in-
crease 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). Par-
tial retention of the apoE-mediated increase upon addition of
FIG.3.High and low affinity compo-
nents of selective and endocytic up-
take 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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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 sam-
ple 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 me-
dium (sample 1 versus 3), and the restoration of this loss by
addition of apoE-containing medium (sample 3 versus 5, p5
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.
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 en-
hancement 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 con-
centration range examined. The concentration dependence
showed both low and high affinity components for the LDL-CE
selective uptake process. At low LDL concentrations (,50
m
g/
ml), most of the LDL-CE selective uptake occurred via the high
affinity component, but at higher LDL concentrations (.50
m
g/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
FIG.4. [
125
I,
3
H]hLDL fractionation by gel filtration chroma-
tography. Panel A, doubly labeled [
125
I,
3
H]hLDL (2 mg) was fraction-
ated on Bio-Gel A-15m as described under “Materials and Methods.”
Regions of the profile indicated by A,B, and Cwere collected and
concentrated in a Centriprep-30. Panel B, samples of each fraction and
the starting LDL (L) were analyzed by electrophoresis on a nondena-
turing 2–16% polyacrylamide gel as described under “Materials and
Methods.” The Coomassie Blue stain of the gel is shown.
FIG.5. Selective and endocytic uptake of cholesteryl ester
from subfractions of [
125
I,
3
H]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 mMBt
2
-
cAMP plus or minus tet. After 24 h, [
125
I,
3
H]hLDL was added to 20
m
g/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 6S.E. (n53)
from a representative experiment.
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humans and other species with high LDL levels, LDL-CE se-
lective uptake and the enhancement by apoE expression may
be of greater quantitative importance.
The enhancement of LDL-CE selective uptake by apoE ap-
pears to involve both secreted apoE and apoE expression by Y1
cells. Secreted apoE associated with LDL particles with ap-
proximately 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 selec-
tive 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 enhance-
ment of the LDL-CE selective uptake pathway show many
parallels to the effects of apoE on the endocytic uptake of
b
-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
b
-VLDL, an effect that involves heparan sulfate proteogly-
can and the LDL receptor-related protein (46). Conditioned
medium from apoE-expressing cells gave the full apoE en-
hancement of
b
-VLDL binding when added to nontransfected
hepatoma cells, suggesting that the enhanced binding required
secreted apoE that associated with
b
-VLDL particles (46).
However, nontransfected hepatoma cells also expressed endog-
enous rat apoE, which may have been present on the cell
surface and contributed to the enhanced uptake. Consistent
with this possibility,
b
-VLDL binding to apoE-expressing hep-
atoma cells at 4 °C was increased without the addition of ex-
ogenous 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 expres-
sion enhances the endocytic versus the selective uptake path-
way may depend on the cell type, the type of lipoprotein par-
ticle, 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 under-
stood. 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
FIG.6.Association of secreted apoE with [
125
I,
3
H]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 mMBt
2
-cAMP plus or minus tet. After 24 h,
[
125
I,
3
H]hLDL was added to 50
m
g/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
125
I 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.
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 mMBt
2
-cAMP plus or minus tet. After 24 h, culture
medium was not changed (samples 1 and 2), replaced with fresh me-
dium (samples 3 and 4), replaced with medium from apoE-expressing
cells (samples 5 and 6), or replaced with medium from cells not express-
ing apoE (sample 7). [
125
I,
3
H]hLDL was added to 50
m
g/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 6S.E. from three experiments, each with tripli-
cate determinations.
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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 possi-
bility 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 up-
take 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 path-
way in cultured adrenal cells (37). Scavenger receptor BI binds
native LDL (48), but it is not known whether scavenger recep-
tor 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 avail-
able to the selective uptake pathway, but this is a prime can-
didate 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 selec-
tive 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.
Acknowledgments—We are grateful to Dr. Lawrence L. Rudel for
helpful discussion, Penelope Strockbine for excellent technical assist-
ance, and Ramesh Shah for performing the electrophoresis.
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Williams
Snehasikta Swarnakar, Mary E. Reyland, Jiatai Deng, Salman Azhar and David L.
Inducible Apolipoprotein E Expression in Cultured Mouse Adrenocortical Cells
Selective Uptake of Low Density Lipoprotein-Cholesteryl Ester Is Enhanced by
doi: 10.1074/jbc.273.20.12140
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