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J Biol Chem, Vol. 274, Issue 42, 29733-29739, October 15, 1999
,,,¶
From the Department of Pharmacological Sciences,
University Medical Center, State University of New York,
Stony Brook, New York 11794 and § Geriatric Research,
Education and Clinical Center, Veteran Affairs Palo Alto Health
Care System, Palo Alto, California 94304
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Scavenger receptor, class B, type I (SR-BI) is a
cell-surface glycoprotein that mediates selective uptake of high
density lipoprotein cholesteryl ester (CE) without the concomitant
uptake and degradation of the particle. We have investigated the
endocytic and selective uptake of low density lipoprotein (LDL)-CE by
SR-BI using COS-7 cells transiently transfected with mouse SR-BI.
Analysis of lipoprotein uptake data showed a
concentration-dependent LDL-CE-selective uptake when doubly
labeled LDL particles were incubated with SR-BI-expressing COS-7 cells.
In contrast to vector-transfected cells, SR-BI-expressing COS-7 cells
showed marked increases in LDL cell association and CE uptake by the
selective uptake pathway, but only a modest increase in CE uptake by
the endocytic pathway. SR-BI-mediated LDL-CE-selective uptake exceeded
LDL endocytic uptake by 50-100-fold. SR-BI-mediated LDL-CE-selective
uptake was not inhibited by the proteoglycan synthesis inhibitor,
p-nitrophenyl- Scavenger receptor class B, type I
(SR-BI)1 is a cell-surface
glycoprotein of molecular mass ~82 kDa that binds HDL, LDL, modified LDL, and VLDL (1-4). In transfected cells, SR-BI mediates the selective uptake of HDL-CE (1), a process in which HDL-CE is transferred into the cell without the concomitant uptake and
degradation of the HDL particle (5). Immunochemical analysis of SR-BI
in rodents indicates that it is expressed most abundantly in the liver
and in steroidogenic cells of the adrenal gland and ovary (1, 6, 7),
where the selective uptake of HDL-CE is greatest. In humans, SR-BI
(also referred to as Cla-1 (4)) shows a similar tissue distribution (8,
9). Direct evidence for SR-BI function is provided by studies in which
antibody to the extracellular domain of mouse (m) SR-BI blocked
HDL-CE-selective uptake and the delivery of HDL cholesterol to the
steroidogenic pathway in cultured murine adrenocortical cells (10). In
addition, inactivation of the SR-BI gene in mice increased plasma HDL
cholesterol levels and reduced neutral lipid stores in the adrenal
glands (11). Similarly, mice carrying an induced SR-BI mutation that
reduced hepatic SR-BI expression levels by 50% showed a similar
reduction in hepatic HDL-CE-selective uptake (12). These studies with reduced SR-BI expression are complemented by studies in which hepatic
SR-BI is overexpressed by either an adenovirus vector (13) or via a
transgene (14). In these studies HDL cholesterol and apoAI levels were
greatly decreased by SR-BI overexpression. Taken together, these
observations indicate that SR-BI plays a key role in mediating
HDL-CE-selective uptake in the liver and in steroidogenic cells and in
determining the plasma levels of HDL cholesterol in mice.
Although it is clear that SR-BI binds native LDL (2) and VLDL (3), the
role of SR-BI in the metabolism of apoB-containing particles is poorly
understood. The cellular metabolism of LDL particles occurs primarily
via the LDL receptor and other members of the LDL receptor family
(15-17) which process LDL via endocytic uptake and lysosomal
degradation (15, 16). Several recent in vivo studies suggest
that SR-BI may also process LDL and other apoB-containing particles.
Transgenic mice overexpressing SR-BI in the liver show reduced levels
of plasma apoB (14, 18) and LDL-CE (14) on both chow and high fat diets
and an accelerated plasma clearance of LDL apoB (18). In addition, the
sizes of the LDL/intermediate density lipoprotein particles in the
transgenic mice are reduced, a result consistent with the selective
removal of LDL core CE by hepatic SR-BI (18). Furthermore, in one study with transgenic mice, aortic root lesions were reduced by 80% in
heterozygous LDL receptor-deficient mice overexpressing hepatic SR-BI
when fed a high fat-, cholesterol-, and bile salt-containing diet (19).
In addition, mice deficient in both SR-BI and apoE show accelerated
atherosclerotic lesion development in the aortic root region and an
increased accumulation of VLDL-sized particles (20). These data
indicate that hepatic SR-BI can influence, directly or indirectly, the
metabolism of apoB-containing particles and the development of arterial lesions.
The in vivo studies noted above raise important questions
about the role of SR-BI in the metabolism of apoB-containing
lipoproteins. First, are the observed effects indirect via changes in
HDL metabolism or direct via processing of LDL particles by SR-BI?
Second, if SR-BI can process LDL particles, does it do so by the
selective uptake pathway or does SR-BI mediate LDL-CE uptake by
endocytic mechanisms? Third, how does the efficiency of SR-BI-mediated
CE uptake compare for LDL and HDL particles? The aim of this study was
to determine directly whether mSR-BI processes LDL particles by the
selective and endocytic uptake pathways. The results showed that mSR-BI
mediates the efficient uptake of LDL-CE via the selective uptake
mechanism in cells that normally express mSR-BI and in transfected
COS-7 cells. These findings suggest that mSR-BI may influence the
metabolism of apoB-containing lipoproteins in vivo by
mediating LDL-CE uptake into SR-BI-expressing cells.
Cell Culture and Transfections--
COS-7 cells were maintained
in Dulbecco's modified Eagle's medium, 10% calf serum, 2 mM L-glutamine, 50 units/ml penicillin, 50 µg/ml streptomycin, and 1 mM sodium pyruvate (complete
medium). At day 0, cells were seeded at a density of 2 × 106 in a 10-cm dish in 10 ml of fresh media and incubated
until approximately 80-90% confluence (~24 h). On day 1 transfections were performed with Fugene 6 (Roche Molecular
Biochemicals) as directed by the manufacturer using pSG5 vector
(Stratagene) and SR-BI-expressing pSG5 vector, pSG5(mSR-BI) (21). On
day 2, cells were trypsinized, resuspended in a total volume of 6 ml
with fresh media, and plated 1 ml to each well of a 6-well plate. On
day 3, COS-7 cells were incubated with doubly labeled LDL or HDL
particles for the indicated times after which cells were washed three
times with phosphate-buffered saline containing 0.1% bovine serum
albumin and once with phosphate-buffered saline. Cells were lysed with
1.5 ml 0.1 N NaOH, and the lysate was processed to
determine trichloroacetic acid-soluble and -insoluble 125I
radioactivity and organic solvent-extractable 3H
radioactivity as described (22) and cell protein (23). Trichloroacetic acid-insoluble 125I radioactivity represents
cell-associated apolipoprotein which is the sum of cell surface-bound
apolipoprotein and endocytosed apolipoprotein which 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 (22, 24). 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 non-saturable process (25).
Murine Y1-BS1 adrenocortical cells were maintained as described (7).
Y1-BS1 cells were treated for 24 h with 100 nM
cortrosyn (Organon), a synthetic ACTH1-24 analogue to
induce mSR-BI expression prior to incubation with labeled HDL and LDL
(10). Antibody inhibition of LDL-CE uptake was performed by incubating
Y1-BS1 cells in serum-free Ham's F-10 medium containing 6 mg/ml
non-immune IgG or anti-mSR-BI IgG for 1 h prior to addition of
labeled LDL (10).
Isolation of Y1 Subclones and Determination of Cellular SR-BI
Levels--
Subclones of the Y1 parent line obtained from ATCC were
isolated by limited dilution cloning. Cell lines were maintained as described above, stimulated with ACTH, and post-nuclear supernatants were isolated as described previously (7). Proteins (20 µg) were
resolved on an SDS-8% polyacrylamide gel, transferred to a
nitrocellulose membrane, and probed with a rabbit polyclonal IgG (2 µg/ml) raised against the extracellular domain of SR-BI (10) as
described (7). Anti-SR-BI was detected by incubation with
125I-labeled goat anti-rabbit IgG (NEN Life Science
Products, 106 dpm/ml) and quantified with a Molecular
Dynamics PhosphorImager. Each subclone was analyzed at least twice in
triplicate. Each gel also contained triplicate samples from the Y1-BS1
cell line. SR-BI levels in the subclones are expressed as a percentage
of the SR-BI content of the Y1-BS1 line.
Preparation of Labeled Lipoproteins, 125I-Dilactitol
Tyramine-[3H]Cholesteryl Oleolyl Ether hHDL3
(125I,3H-hHDL3) and
125I-Dilactitol Tyramine-[3H]Cholesteryl
Oleolyl Ether hLDL (125I,3H-hLDL)--
Human
(h) HDL3 (1.125 g/ml < Inhibition of Proteoglycan Synthesis and Sulfation--
To
inhibit proteoglycan synthesis, control or transfected COS-7 were
preincubated with 2 mM
p-nitrophenyl- SR-BI-mediated LDL-CE-selective Uptake--
In order to determine
whether mSR-BI mediates the selective uptake of LDL-CE, COS-7 cells
transfected with vector or with vector expressing mSR-BI were incubated
for 4 h with 125I,3H-hLDL after which LDL
cell association, LDL-CE-selective uptake, and LDL-CE endocytic uptake
(Fig. 1) were measured. As shown in Fig.
1A, mSR-BI-expressing cells showed a
concentration-dependent increase in the cell association of LDL
that was approximately 2.5-fold greater than vector-transfected cells.
mSR-BI-dependent cell association of LDL showed a high
affinity component with an apparent Kd of 14 µg/ml
LDL protein, which is similar to that reported for the 4 °C binding
of LDL to hamster SR-BI (Kd = 5 µg/ml) (2).
Measurement of LDL-CE-selective uptake showed a similar 2.5-fold
increase in mSR-BI-expressing cells in comparison to vector-transfected
cells (Fig. 1B). LDL-CE-selective uptake showed both low and
high efficiency components. The high efficiency component showed an
apparent Km of 9.6 µg/ml. Within the concentration
range of LDL examined, the low efficiency component showed a linear
increase in LDL-CE-selective uptake as a function of LDL concentration
(Fig. 1B) with a slope that was considerably greater than
that seen in vector-transfected cells. Thus, the low efficiency
component of LDL-CE-selective uptake, as well as the high efficiency
component, is dependent on mSR-BI expression. SR-BI expression also
increased the endocytic uptake of LDL-CE (Fig. 1C), but the
increase was significantly less than that seen for LDL-CE-selective
uptake. Importantly, the amount of SR-BI-dependent
LDL-CE-selective uptake exceeded LDL endocytic uptake by approximately
50-fold in this experiment and by as much as 100-fold in other
experiments (data not shown). These data indicate that SR-BI-mediated
cell-surface binding of LDL leads to efficient CE uptake by the
selective uptake pathway but only a marginal increase by endocytic
uptake.
To examine the relationship between SR-BI-dependent binding
and LDL-CE-selective uptake, competition experiments were performed by
preincubating transfected COS-7 cells with increasing concentrations of
unlabeled LDL prior to addition of labeled LDL. Fig.
2A shows that unlabeled LDL
competed effectively at low concentrations (<50 µg/ml) for the cell
association of 125I-LDL to both vector-transfected and
SR-BI-expressing cells. However, the mSR-BI-dependent
component of the LDL cell association showed a more sluggish or gradual
competition than occurs with the vector-transfected cells. This is
illustrated in Fig. 2B which shows the
mSR-BI-dependent component (mSR-BI values minus the vector
values at each LDL concentration) in comparison to vector-transfected
cells. The competition curve for the mSR-BI-dependent LDL
cell association is indicative of a mixture of low and high affinity
components and showed only 70% competition at the highest
concentration of competitor tested (300 µg/ml = 30-fold excess
compared with radiolabeled LDL). This pattern is even more pronounced
for the selective uptake of LDL-CE (Fig. 2C). In this case,
the mSR-BI-dependent component of
LDL-[3H]CE-selective uptake was competed gradually over a
broad LDL concentration range reaching a maximum of 45% competition at
300 µg/ml unlabeled LDL (Fig. 2D). Thus, the shallow
competition curve for mSR-BI-dependent
LDL-[3H]CE-selective uptake is indicative of a mixture of
affinities with a major fraction being of relatively low affinity. Note
in contrast that the LDL-[3H]CE-selective uptake by
vector-transfected cells was competed by 80% at 60 µg/ml unlabeled
LDL, indicating a process of higher affinity than seen for the
mSR-BI-dependent component (Fig. 2D, vector). Thus, the LDL-CE-selective uptake in
vector-transfected cells is distinct from that mediated by SR-BI. Note
also that we have been unable to detect SR-BI expression in control or
vector-transfected COS-7 cells confirming that the basal level of
LDL-CE-selective uptake is not due to SR-BI.
Similar experiments were carried out to test HDL as a competitor for
mSR-BI-mediated LDL-CE-selective uptake. Fig.
3 shows that HDL competed modestly for
LDL cell association in vector-transfected or mSR-BI-expressing cells
(Fig. 3A). HDL competed for LDL-CE-selective uptake in
SR-BI-expressing cells over a wide concentration range but showed no
competition for LDL-CE-selective uptake in vector-transfected cells
(Fig. 3B). The mSR-BI-dependent component of
LDL-[3H]CE-selective uptake was competed by HDL to a
maximum of 65% competition at 300 µg/ml unlabeled HDL. Comparison of
LDL and HDL as competitors for the mSR-BI-dependent
component of LDL-CE-selective uptake (Fig.
4) showed that both lipoproteins competed
gradually over a broad concentration range and were similarly effective on a protein basis.
The Role of Cell-surface Proteoglycans in SR-BI-mediated
LDL-CE-selective Uptake--
The interaction of apoB-containing
lipoproteins with cell-surface proteoglycans is believed to play a
major role in concentrating LDL within the space of Disse in the liver
(28), in facilitating endocytic uptake of remnant particles by the
hepatocyte low density lipoprotein receptor-related protein (29), and
in direct proteoglycan-mediated LDL endocytosis (30). To investigate
the possible significance of cell-surface proteoglycans for selective
uptake of LDL-CE, two approaches were taken. In the first, cells were
pretreated with 30 mM NaClO3 for 20 h to
inhibit sulfation of heparan sulfate and chondroitin sulfate
proteoglycans prior to measurement of LDL-CE-selective uptake. As shown
in Fig. 5A, NaClO3
treatment reduced LDL-CE-selective uptake by 50% in vector-transfected
cells but only by 20% in mSR-BI-expressing cells. After correction for the absolute level of inhibition in the vector-transfected cells, the
SR-BI-dependent component of LDL-CE-selective uptake was
inhibited by only 15%. In the second approach, COS-7 cells were
pretreated with 2 mM
p-nitrophenyl- SR-BI-mediated LDL-CE-selective Uptake in Y1-BS1 Adrenocortical
Cells--
To explore the physiological significance of the
SR-BI-mediated LDL-CE-selective uptake as seen in transfected COS-7
cells, we examined the Y1-BS1 adrenocortical cell, which naturally
expresses mSR-BI and in which mSR-BI has been shown to mediate
HDL-CE-selective uptake (10). Fig. 6
shows the cell association (Fig. 6A), selective uptake (Fig.
6B), and endocytic uptake (Fig. 6C) of LDL-CE in Y1-BS1 cells as a function of LDL concentration using double-labeled 125I,3H-hLDL particles. As was seen with the
transfected COS-7 cells, these parameters show LDL concentration
dependences indicative of both high and low affinity (or efficiency)
components. The high affinity component for cell association showed an
apparent Kd = 6.3 µg/ml, and the high efficiency
component for selective uptake showed an apparent Km = 3.2 µg/ml, values similar to those seen with the transfected COS-7
cells. Within the LDL concentration range examined, the low affinity
components for cell association (Fig. 6A), selective uptake
(Fig. 6B), and endocytic uptake (Fig. 6C) behaved
in a linear fashion.
In order to test the relationship between mSR-BI expression levels and
LDL-CE-selective uptake in Y1 cells, subclones of the parent Y1 cell
line were isolated by limited dilution cloning. Measurements of mSR-BI
levels and LDL-CE-selective uptake activity were made for 7 subclones
and for the Y1-BS1 line. Fig.
7A shows the LDL-CE-selective
uptake activities for these clones plotted versus the mSR-BI
levels, which were expressed relative to that in the Y1-BS1 set as
100%. Correlation analysis shows a strong relationship
(r2 = 0.82, p = 0.002) between
the LDL-CE-selective uptake activity and the expression level of
mSR-BI. These data suggest that the level of LDL-CE-selective uptake is
largely determined by the level of mSR-BI expression in adrenocortical
cells. A similar degree of correlation (r2 = 0.86, p = 0.001) was seen between mSR-BI levels and the
HDL-CE-selective uptake activities among the Y1 subclones (Fig.
7B).
As a further test of the role of mSR-BI in LDL-CE-selective uptake in
Y1-BS1 adrenocortical cells, we used antibody raised against the
extracellular domain of mSR-BI to block LDL-CE-selective uptake. This
antibody has been shown previously to block the selective uptake of
HDL-CE in Y1-BS1 cells (10). In comparison to control IgG, anti-mSR-BI
inhibited the cell association of LDL by 40% at either of two LDL
concentrations (Fig. 8A).
Anti-mSR-BI inhibited LDL-CE-selective uptake by 30% at 6 µg/ml LDL
and by 75% at 3 µg/ml LDL (Fig. 8B). Thus, at an LDL
concentration near the apparent high efficiency Km
for LDL-CE-selective uptake, the major fraction of LDL-CE-selective
uptake in Y1-BS1 adrenocortical cells was inhibited by anti-mSR-BI
antibody.
In order to compare the efficiency of CE-selective uptake from LDL and
HDL particles, the selective uptake data of Fig. 6 were expressed on
the basis of the amount of cell-associated CE for LDL or HDL. Thus,
this analysis shows the amount of CE uptake as a function of the steady
state level of cell-associated lipoprotein during the uptake assay.
Since the selective uptake and cell association measurements were done
in replicate, the values for each individual measurement are shown
plotted in Fig. 9. These data show that for a given amount of cell-associated lipoprotein CE, HDL delivers a
much greater percent of its lipoprotein core as compared with LDL.
Similar calculations were made in a variety of experiments with the Y1
BS-1 line with similar results. When these data were pooled with those
shown in Fig. 9, the average delivery of HDL-CE was 7.4 ± 1.8 (S.D.)-fold greater than that of LDL-CE for an equivalent amount of
cell-associated lipoprotein CE. Thus, the fractional delivery of HDL-CE
was markedly greater than that of LDL-CE. When viewed on a molar
basis,2 however, an LDL
particle contains 43 times as many CE molecules per particle compared
with an HDL3 particle. Consequently, for an equivalent
steady state number of lipoprotein particles bound to the cell surface,
an LDL particle delivered 6.2 times as many CE molecules compared with
an HDL particle. Similar values were obtained when LDL-CE and HDL-CE
uptake were compared in SR-BI-expressing COS-7 cells. In this case the
fractional uptake of HDL-CE was 6.1-fold greater than the uptake of
LDL-CE (data not shown).
Although SR-BI has been characterized as an HDL receptor (1, 10),
its ability to bind LDL particles suggested that it might also play a
role in LDL metabolism. Several studies with transgenic animals have
demonstrated that SR-BI can influence plasma LDL-CE and apoB levels
in vivo (14, 18, 19), although it was not clear whether this
occurred directly or was secondary to altered HDL metabolism. In the
present study we have tested the ability of mSR-BI to mediate the
selective and endocytic uptake of LDL-CE. The results show that in
transfected COS-7 cells mSR-BI efficiently mediated LDL-CE-selective
uptake with only a minimal increase in LDL endocytic uptake.
SR-BI-mediated LDL-CE-selective uptake exceeded LDL endocytic uptake by
50-100-fold. In addition, the demonstration that mSR-BI mediates
LDL-CE-selective uptake in Y1 adrenocortical cells shows that this
activity of mSR-BI occurs in a physiological setting and is not the
result of overexpressing this receptor in a transfected cell. These
results suggest that at least some of changes in LDL metabolism
observed in SR-BI transgenic animals may result from the ability of
this receptor to mediate selective CE uptake from LDL particles.
SR-BI-mediated changes in HDL metabolism might also indirectly alter
the metabolism of apoB-containing particles in vivo.
Interestingly, the SR-BI-expressing COS-7 cells showed very little
SR-BI-dependent endocytosis of LDL, suggesting that the
increased clearance of plasma apoB in SR-BI transgenic mice (18) may be
an indirect result of SR-BI expression. Our results, which show
directly that mSR-BI mediates LDL-CE-selective uptake, are in agreement
with a recent report showing that mSR-BI expression in Chinese hamster
ovary cells stimulates the esterification of cholesterol in response to
LDL (32), a result consistent with mSR-BI mediating the uptake of either free cholesterol or cholesteryl ester, or both, from LDL particles. In the context of the transfected COS-7 cell, cell-surface proteoglycans do not appear to have a significant role in
SR-BI-mediated LDL-CE-selective uptake.
The physiological significance of SR-BI-mediated LDL-CE metabolism in
humans remains to be determined. LDL clearance in humans is primarily
determined by the LDL receptor pathway that accounts for 67-80% of
plasma LDL removal as judged by measurements of radioiodinated LDL
clearance in normal individuals and patients with homozygous familial
hypercholesterolemia (33). It is possible that SR-BI contributes to the
fraction of LDL not removed by the LDL receptor pathway or removes some
CE from particles in the VLDL-LDL cascade. Further studies will be
required to test these possibilities.
Once bound to mSR-BI, lipoprotein CE is transferred to the cell in a
process that appears to occur with different efficiencies for HDL and
LDL. In either Y1 cells or mSR-BI-expressing COS-7 cells, the
fractional transfer of lipoprotein CE was approximately 6-7-fold
greater for HDL than LDL (Fig. 9). Whether this reflects differences in
how LDL and HDL particles interact with mSR-BI or is due to inherent
differences in the transfer of CE from the particles is not known.
Interestingly, because the CE core of the LDL particle is so much
larger than that of an HDL3, the absolute uptake of CE from
the LDL particle is about 6-fold greater than from an HDL.2
Thus, in terms of the absolute delivery of CE molecules,
mSR-BI-mediated selective uptake of LDL-CE has the potential to provide
for substantial cholesterol delivery to SR-BI-expressing cells. In
previous work Reaven and colleagues (22) showed that LDL was used
efficiently by the selective uptake pathway of rat ovarian granulosa
cells to support steroid production in vivo (34) and in cell
culture. Since these cells have now been shown to express high levels
of SR-BI (6, 35), it is likely that the ovarian uptake of LDL-CE is
largely due to SR-BI.
The present data show that in addition to SR-BI-mediated
LDL-CE-selective uptake, COS-7 cells show an SR-BI-independent pathway for LDL-CE-selective uptake. The experiments in Figs. 1B,
2B, and 3B show that vector-transfected cells
exhibit LDL-CE-selective uptake at levels of 10-40% that seen in
mSR-BI-expressing cells. We have been unable to detect SR-BI expression
by COS-7 cells indicating that this level of LDL-CE-selective uptake is
unlikely due to low levels of endogenous SR-BI in untransfected cells. Furthermore, the lipoprotein competition data showed that the LDL-CE-selective uptake in vector-transfected COS-7 cells could be
competed by LDL (Fig. 2, C and D) but not by HDL
(Fig. 3B). In contrast, the mSR-BI-dependent
component of LDL-CE-selective uptake was competed equally well by LDL
and HDL (Fig. 4). Thus, the LDL-CE-selective uptake seen in
untransfected COS-7 cells is not due to SR-BI. The physiological
significance of this SR-BI-independent pathway for LDL-CE-selective
uptake is unknown at present.
An interesting feature of mSR-BI-mediated LDL-CE-selective uptake is
that the LDL concentration dependence shows two components, one
indicative of a high efficiency (or affinity) process and the other of
a low efficiency process. This was seen in both mSR-BI-expressing COS-7
cells (Fig. 1B) and in Y1-BS1 adrenocortical cells (Fig. 6B). Consistent with the interpretation of multiple
mSR-BI-dependent components, competition of
LDL-[3H]CE-selective uptake by unlabeled LDL occurred
gradually over a broad LDL concentration range reaching a maximum of
only 45% competition at 300 µg/ml unlabeled LDL (Fig.
2D). One interpretation of these data is that mSR-BI occurs
in two forms that exhibit high and low efficiencies of LDL-CE-selective
uptake, possibly depending on the state of receptor multimerization or
on its localization to different domains of the plasma membrane. An
alternative interpretation is that mSR-BI expression alters the lipid
domain organization of the plasma membrane in such a way as to promote
low affinity LDL-cell membrane interactions that, while requiring
mSR-BI expression, do not reflect direct mSR-BI-LDL binding and, thus,
are not effectively competed by unlabeled LDL. Support for the idea
that SR-BI expression alters membrane lipid organization is provided by
recent studies showing that SR-BI expression in COS-7 cells increased
the fraction of membrane-free cholesterol that was sensitive to
oxidation by extracellular cholesterol oxidase (36). In addition,
mSR-BI expression markedly enhanced the efflux of membrane-free
cholesterol to cyclodextrins, extracellular acceptors that do not bind
to mSR-BI (37). Although speculative, one possibility is that these changes reflect an altered plasma membrane domain with an increased capacity for free cholesterol flux and possibly for low affinity LDL
interactions that lead to LDL-CE-selective uptake. We cannot distinguish these possibilities with the available evidence, but additional studies will serve to test these hypotheses.
-D-xylopyranoside or by the sulfation inhibitor sodium chlorate, indicating that SR-BI-mediated LDL-CE uptake occurs independently of LDL interaction with cell-surface proteoglycan. Analyses with subclones of Y1 adrenocortical cells showed
that LDL-CE-selective uptake was proportional to the level of SR-BI
expression. Furthermore, antibody directed to the extracellular domain
of SR-BI blocked LDL-CE-selective uptake in adrenocortical cells. Thus,
in cells that normally express SR-BI and in transfected COS-7 cells
SR-BI mediates the efficient uptake of LDL-CE via the selective uptake
mechanism. These results suggest that SR-BI may influence the
metabolism of apoB-containing lipoproteins in vivo by
mediating LDL-CE uptake into SR-BI-expressing cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
< 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 oleolyl ether as described (24). 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
protein for 125I and from 3 to 30 dpm/ng protein for
3H. For 125I-VLDL rabbit VLDL was
prepared as described (26) except all protease inhibitors were omitted.
VLDL was iodinated with Na125I using the modified ICl
method (27). Specific activities were 600-1000 dpm/ng protein.
-D-xylopyranoside (-D-xyloside) for 20 h in complete medium prior to
lipoprotein uptake assays. To inhibit sulfation of glycosaminoglycans,
COS-7 cells were preincubated for 20 h in complete medium
containing 30 mM NaClO3 prior to the addition
of lipoprotein particles.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Concentration dependence of LDL-CE cell
association, selective CE uptake, and endocytic CE uptake in
mSR-BI-expressing COS-7 cells. COS-7 cells transiently transfected
with pSG5 (vector) or pSG5(mSR-BI) were incubated
with the indicated protein concentrations of
125I,3H-hLDL for 4 h at 37 °C. Cells
were processed to determine LDL-CE cell association (A),
LDL-CE-selective uptake (B), and LDL-CE endocytic uptake
(C) per mg of cell protein as described under
"Experimental Procedures." Data represent the mean ± S.E.
(n = 3) from a representative experiment.
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Fig. 2.
Inhibition of mSR-BI-mediated LDL-CE cell
association and selective uptake in mSR-BI-expressing COS-7 cells by
unlabeled hLDL. COS-7 cells transiently transfected with pSG5 or
pSG5(mSR-BI) vector were incubated with indicated concentrations of
unlabeled hLDL, which were added 1 h before the addition of
125I,3H-hLDL (10 µg/ml protein) and incubated
for 4 h at 37 °C. The cells were then processed for
determination of LDL-CE cell association (A) and selective
CE uptake (C). Error bars represent the range of
triplicate measurements. The percent inhibition of LDL-CE cell
association (B) and selective CE uptake (D) was
calculated as a percent of the value obtained with no competitor added
for the vector-transfected cells. The percent inhibition of the
mSR-BI-dependent component was calculated after subtraction
of values obtained with vector alone from values obtained with
mSR-BI-expressing cells at each concentration of unlabeled hLDL.
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Fig. 3.
Inhibition of LDL-CE cell association and
selective CE uptake in mSR-BI-expressing COS-7 cells by unlabeled
hHDL3. COS-7 cells transiently transfected with pSG5
or pSG5(mSR-BI) vector were incubated with indicated concentrations of
unlabeled hHDL3, which were added 1 h before addition
of 125I,3H-hLDL (10 µg/ml protein) and
incubated for 4 h at 37 °C. The cells were then processed for
determination of LDL-CE cell association (A) and
LDL-CE-selective uptake (B). Values represent the mean ± S.E. (n = 3).
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Fig. 4.
Comparison of hLDL and hHDL3 for
inhibition of mSR-BI-mediated LDL-CE-selective uptake. Competition
of the mSR-BI-dependent component of LDL-CE-selective
uptake by hLDL and hHDL3 are shown for comparison. Data for
competition by hLDL are from Fig. 2D. Data for competition
by hHDL3 are from Fig. 3B. These values show
competition for the mSR-BI-dependent component and were
obtained by subtracting the values obtained with vector-transfected
cells from the values obtained with mSR-BI-expressing cells at each
concentration of hHDL3.
-D-xylopyranoside for 20 h
to inhibit the addition of glycosaminoglycan side chains during
proteoglycan biosynthesis.
p-Nitrophenyl--D-xylopyranoside is a potent
inhibitor of chondroitin sulfate proteoglycan expression but has little effect on heparan sulfate proteoglycans (31). As shown in Fig. 5B,
p-nitrophenyl--D-xylopyranoside failed to
inhibit LDL-CE-selective uptake in vector-transfected or
mSR-BI-expressing COS-7 cells. To ensure that these inhibitors had
effectively reduced cell-surface proteoglycans, untransfected COS-7
cells were pretreated with inhibitors as above and then incubated at
4 °C with rabbit 125I-VLDL since previous studies
showed that the cell-surface binding of VLDL is mediated by
proteoglycans (29). The results showed that the binding of
125I-VLDL was reduced by 41 and 55% by
NaClO3 and
p-nitrophenyl--D-xylopyranoside, respectively, indicating that the inhibitors were effective in reducing
proteoglycan biosynthesis (data not shown). We conclude that
mSR-BI-mediated selective uptake of LDL-CE occurs independently of
cell-surface proteoglycan.
View larger version (16K):
[in a new window]
Fig. 5.
Effect of sodium chlorate and
p-nitrophenyl- -D-xyloside
on LDL-CE-selective uptake in mSR-BI-expressing COS-7 cells. COS-7
cells transiently transfected with vector alone or mSR-BI expression
vector were incubated in serum-free media containing 30 mM
sodium chlorate (A) or 2 mM
p-nitrophenyl--D-xyloside (B) for
20 h at 37 °C. Double-labeled LDL at a concentration of 10 µg/ml was then added and incubated for another 4 h. Cells were
then processed for determination of LDL-CE-selective uptake. Values
represent the mean ± S.E. (n = 3).
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[in a new window]
Fig. 6.
Concentration dependence of LDL-CE cell
association, selective CE uptake, and endocytic CE uptake in Y1-BS1
adrenocortical cells. Y1-BS1 cells were incubated with the
indicated concentrations of 125I,3H-hLDL for
4 h at 37 °C, after which cells were processed to determine
LDL-CE cell association (A) selective CE uptake
(B) and endocytic CE uptake (C) as described
under "Experimental Procedures." Error bars
represent the range of triplicate measurements.
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[in a new window]
Fig. 7.
mSR-BI-mediated LDL-CE and HDL-CE-selective
uptake in Y1 subclones. 125I,3H-hLDL (30 µg/ml protein) and 125I,3H-hHDL (10 µg/ml
protein) were incubated for 4 h at 37 °C with Y1-BS1 cells and
with the different Y1 subclones isolated as described under
"Experimental Procedures." Cells were processed to determine
selective uptake of LDL-CE (left panel) and HDL-CE
(right panel). Each data point represents the mean of
duplicate (LDL) or triplicate (HDL) determinations of CE-selective
uptake for an individual subclone. The selective uptake values are
plotted as a function of the mSR-BI level in each subclone expressed as
a percentage of that present in the Y1-BS1 clone set as 100%.
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Fig. 8.
Effect of anti-mSR-BI IgG on LDL-CE-selective
uptake and LDL cell association in Y1-BS1 adrenocortical cells.
Y1-BS1 cells were incubated at 37 °C for 6 h with 6 and 3 µg/ml 125I,3H-hLDL in medium containing 6 mg/ml anti-SR-BI IgG or non-immune IgG which were added 1 h prior
to addition of labeled LDL. Cells were processed to determine LDL-CE
cell association (A) and LDL-CE-selective uptake
(B). Data represent the mean ± S.E. (n = 3).
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Fig. 9.
Fractional uptake of lipoprotein CE from LDL
and HDL. Individual values for HDL- or LDL-selective CE uptake
from Fig. 7 are plotted against the level of cell-associated
lipoprotein determined in each sample.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
| |
ACKNOWLEDGEMENT |
|---|
We thank Ronald DeMattos for providing the
rabbit -VLDL.
| |
FOOTNOTES |
|---|
* This work was supported by National Institutes of Health Grants HL 32868, HL 58012, and DK 49705 and by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 516-444-3083; Fax: 516-444-3218; E-mail: dave@pharm.sunysb.edu.
2 In comparing the delivery of CE from HDL and LDL on a molar basis, we assume that the molecular weight of an LDL particle is 2.35 × 106 and that of an HDL3 is 1.55 × 105. An LDL particle having 42% CE by mass contains 985 kDa of CE or 1513 molecules of CE using a molecular weight of 651 for an average CE. An HDL3 particle having 15% CE by mass contains 22.5 kDa of CE or 35 molecules of CE.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: SR-BI, scavenger receptor class B, type I; m, mouse; h, human; HDL, high density lipoprotein; LDL, low density lipoprotein; VLDL, very low density lipoprotein; CE, cholesteryl ester; apo, apolipoprotein; ACTH, adrenocorticotropic hormone.
| |
REFERENCES |
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|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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