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INTRODUCTION |
High density lipoprotein
(HDL)1 mediates the transport
of cholesterol from peripheral tissues to the liver for excretion in bile in a process called reverse cholesterol transport (1-3). However,
the mechanisms mediating the uptake of HDL by hepatocytes and other
cells are poorly understood. Although a number of HDL binding proteins
have been identified, none so far has been shown to mediate the uptake
or degradation of HDL protein (4). Cholesterol enters the hepatocyte
from the basolateral membrane in either LDL, remnants of
triglyceride-rich lipoproteins, or HDL. HDL, and not LDL, appears to be
the major donor of free cholesterol for bile acid production (5-7).
HDL cholesterol must somehow traverse the hepatocyte from the
basolateral membrane (sinusoidal membrane) to reach the apical membrane
(bile canalicular membrane) followed by secretion into bile.
Scavenger receptor class B type 1 (SR-BI) is an authentic HDL receptor
that is highly expressed in the liver and steroidogenic tissues and
mediates the selective uptake of HDL cholesterol, i.e. the
uptake of HDL free cholesterol and cholesteryl ester without the
concomitant uptake or degradation of HDL protein (8). Based on studies
in non-polarized cells, it is widely believed that SR-BI is a
non-endocytic receptor that functions at the plasma membrane, perhaps
forming a hydrophobic channel to facilitate entry of cholesteryl ester
into the membrane (9-11). This view implies that there is a separate
process mediating holo-HDL particle uptake. Intriguingly,
SR-BI has been localized to the bile canaliculus (the apical membrane)
in mice and rats (12, 13). Because hepatocytes primarily use
transcytosis to localize membrane proteins to the apical surface
(14-16), it is tempting to speculate that SR-BI is endocytosed from
the basolateral membrane and traffics to the apical membrane. SR-BI
appears to mediate rapid transport of HDL free cholesterol and
cholesteryl ester into bile (17), suggesting that SR-BI plays an
important role in HDL cholesterol transport across the cell.
Recently, we have shown that HDL particles are internalized by
non-polarized hepatocytes and traffic to the endocytic
recycling compartment (ERC, a transferrin-positive compartment) and
that the majority of HDL particles that undergo endocytosis are
resecreted in an intact form (18), in a process previously termed
retroendocytosis (19-21). In addition, we found that selective HDL
lipid uptake occurs during HDL recycling (18). The goals of the present
study were to elucidate the route of HDL protein and cholesterol
trafficking in polarized hepatocytes and to evaluate the
role of SR-BI in HDL particle uptake and trafficking.
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EXPERIMENTAL PROCEDURES |
Hepatocyte Isolation--
Hepatocytes were isolated from
8-week-old female C57BL/6J mice (purchased from The Jackson
Laboratory, Bar Harbor, ME) as previously described (18). Hepatocyte
couplets were isolated by following the above protocol except that the
time of digestion of the liver was reduced from 18 to 12 min. The
identity of the bile canalicular vacuole was verified by staining with
filipin as previously described (18).
Lipoprotein Preparations--
Human apoE-free HDL and LDL was
isolated by buoyant density ultracentrifugation and radiolabeled with
protein and lipid tracers as previously described (18).
SR-BI-neutralizing Antibody Production--
Preimmune and immune
serum from rabbits was obtained by immunizing rabbits with a
recombinant extracellular domain of mouse SR-BI (Cardiovascular
Targets, New York, NY). IgG fraction was purified from preimmune and
immune serum by protein A chromatography. A titration experiment was
performed to determine the optimal antibody concentration to inhibit
SR-BI activity using CHO cells expressing SR-BI showed that a 1:1000
dilution of immune serum was sufficient to completely reduce HDL
association to levels measured in CHO control cells. This concentration
equaled ~10-20 µg/ml protein A-purified IgG. Preimmune serum was
without effect at 1:1000.
Pulse-chase Assays--
All cells types (freshly isolated
hepatocytes, CHO, and MDCK cells) were treated as previously described
(18). Trichloroacetic acid-soluble and -precipitable counts were
determined as a measurement of degradation and secretion, respectively.
Pulse-chase assays were also performed in the presence of protein
A-purified neutralizing anti-SR-BI antibody or protein A-purified
preimmune IgG (from the same rabbit used to produce the immune serum)
by preincubation of cells at 4 °C for 1 h with 20 µg/ml
antibodies followed by the pulse-chase assay as previously described
(18). The values reported for the three components in all pulse-chase
studies (i.e. association, resecretion, and degradation) are
considered specific, because they were calculated by subtracting
nonspecific background levels as measured in the presence of 100-fold
excess unlabeled HDL from total. Selective uptake was measured as
previously described (18). For pulse-chase assays using MDCK cells, 150 µg of HDL/ml was used as the lipid acceptor in the apical chamber of
Transwells (Costar).
Adenovirus Infection--
Primary hepatocytes and CHO cells were
infected with either an adenovirus harboring the DynK44A
gene or a control adenovirus expressing green fluorescence
protein (kind gifts of J. E. Pessin) according to a previous study
(22). Cells were then incubated with either 125I-labeled
HDL or LDL having specific activities of ~500 and 2300 cpm/ng, respectively.
Immunolocalization of SR-BI--
Hepatocytes were incubated for
1 h with Alexa 568-labeled human holotransferrin (Sigma Chemical
Co.). Cells were then washed with phosphate-buffered saline and fixed
in 3.7% paraformaldehyde. Immunodetection was then performed using an
anti-SR-BI antibody recognizing the carboxyl terminus of SR-BI (Novus
Biologicals). Alexa 488-labeled goat anti-rabbit IgG (Molecular Probes)
was used as the secondary antibody.
Fluorescence Confocal Microscopy--
10 mg of doubly labeled
HDL (DiI and Alexa 488) was incubated for 1 h with primary
hepatocyte couplets at 37 °C, and examined by confocal microscopy as
previously described (18). A separate sample of HDL was double-labeled
with the fluorescence cholesterol ester analog BODIPY-CE (Molecular
Probes) as previously described (18). The protein component of
BODIPY-CE-labeled HDL was then labeled with Alexa 568. This
double-labeled HDL was incubated with primary hepatocyte couplets as
described above and examined using a confocal microscopy.
MDCK Cell Manipulations--
Stable cells expressing murine
SR-BI in pCDNA3.1 (Invitrogen, Carlsbad, CA) or empty vector as
control were made using Transfectamine (Life Technologies, Inc.). A
pool of more than 300 individual clones were used for experiments.
Polarization was carried out by growing cells for 5 days in 12-mm
Transwell cell culture inserts (Corning). MDCK cell monolayer integrity
was verified by determining the basal-to-apical diffusion of
[3H]inulin (1 µCi/ml). Cells used for all experiments
had <0.1% diffusion.
Endocytosis Assays--
Measurements of endocytosis of SR-BI in
all cells types (freshly isolated hepatocytes, CHO) using a reducible
biotin cross-linker was performed according to the original method by
Bretscher et al. (23) with modifications by McGwire et
al. (24).
Immunoprecipitation--
All cell types used (freshly isolated
hepatocytes and CHO) were first lysed in radioimmune precipitation
buffer. SR-BI was immunoprecipitated overnight using the anti-SR-BI
antibody specific for the carboxyl terminus of SR-BI.
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RESULTS |
Anti-SR-BI Antibody Inhibits HDL Particle Uptake in CHO
Cells--
We previously demonstrated that HDL particles are actively
taken up by primary hepatocytes and rapidly resecreted or degraded (18). The resecreted HDL was selectively depleted of free cholesterol and cholesteryl ester. To explore the possibility that SR-BI mediates HDL particle uptake and recycling, we used an antibody to the extracellular domain of SR-BI that neutralizes SR-BI selective uptake
activity in vitro. Western blot analysis using the
neutralizing SR-BI antibody in both primary hepatocytes and
SR-BI-transfected CHO cells (25) yields a single band at the expected
molecular mass of SR-BI (~82 kDa) (Fig.
1D). Lower
Mr bands seen in hepatocytes were also detected
in pre-immune sera (Fig. 1D, PI), indicating specific recognition of SR-BI by immune sera (IM). Small
amounts of this antibody were shown to markedly inhibit selective
uptake of HDL cholesteryl ester (CE) by SR-BI-transfected
CHO cells (Fig. 1A). Although not previously appreciated
(26), we also found extensive uptake and recycling of HDL in CHO cells
stably expressing SR-BI (Fig. 1A); the resecreted HDL was
largely depleted in CE tracer (by ~75%, relative to protein), as
found previously in hepatocytes (17). The anti-SR-BI antibody reduced
HDL particle uptake, resecretion, and degradation in SR-BI-transfected
CHO cells to the level found in vector control-transfected CHO cells (Fig. 1A). In a further characterization of the neutralizing
antibody, CHO cells expressing SR-BI were incubated with HDL labeled
with both diI as a lipid marker and Alexa-488 as a protein marker and incubated with cells pretreated with either preimmune antibody or the
SR-BI-neutralizing antibody. Fig.
2A shows that the neutralizing SR-BI antibody (immune) blocked the uptake of both HDL lipid and protein fluorescent markers, thus confirming our findings with radiolabeled HDL. To further substantiate this finding in CHO cells and
to test if HDL traffics to the ERC as in hepatocytes (18), we examined
HDL particle uptake using confocal microscopy in SR-BI-expressing CHO
cells. Using a pulse-chase scheme, we found that HDL bound on the cell
surface at 4 °C becomes internalized during a subsequent incubation
at 37 °C (Fig. 2B), a process that was inhibited by the
SR-BI-neutralizing antibody (Fig. 2A). Moreover, the
majority of HDL protein and transferrin, a known recycling protein,
were found to colocalize within the cells seen in internal optical
confocal sections (Fig. 2C, yellow color).
Similar experiments in control CHO cells showed little uptake of
fluorescent HDL (Fig. 2A, vector). These findings
indicate that SR-BI is an endocytic receptor that traffics in a similar
route as transferrin and mediates uptake and recycling of HDL. During
the process of recycling, HDL is selectively depleted of cholesteryl
ester. These processes are very similar to HDL uptake and recycling in
hepatocytes (17). To determine the role of SR-BI in HDL trafficking in
hepatocytes, we next carried out similar experiments in primary
hepatocytes.

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Fig. 1.
Characterization of SR-BI-mediated HDL uptake
in hepatocytes and CHO cells. A and B, a
neutralizing anti-SR-BI antibody inhibits selective uptake of HDL
cholesteryl ester (CE) in both CHO cells and primary hepatocytes,
respectively. In A a comparison is made between CHO cells
expressing SR-BI to control CHO cells harboring empty vector.
PI, preimmune protein A-purified antibody; IM,
immune protein A-purified IgG (i.e. SR-BI-neutralizing
antibody). C, primary hepatocytes were preincubated for
2 h at 37 °C with or without heparinase (10 units/ml) or for
1 h with RAP (40 µg/ml), before the addition of 5 µg/ml
125I-labeled HDL for an additional 1 h. All of the
above experiments are representative of two to three independent
experiments. Error bars are S.D. D, equal amounts
of total cell extracts from CHO vector control, CHO cells expressing
SR-BI, and primary hepatocytes were analyzed by Western blot analysis
using the SR-BI-neutralizing antibody (immune serum, IM) and
preimmune serum (PI). SR-BI was detected as a single band of
~82 kDa (indicated by the arrow). Molecular mass
(kDa) markers are shown at the right of the gel.
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Fig. 2.
HDL protein uptake and localization in CHO
cells. A, CHO cells expressing SR-BI or vector
control were pretreated with either the preimmune or immune IgG
(i.e. SR-BI-neutralizing antibody), then incubated with
DiI/Alexa 488-labeled HDL (red/green color) for 1 h at
37 °C, followed by examination by confocal microscopy. B,
pulse-chase of Alexa 568-labeled HDL with CHO cells expressing SR-BI.
Cells were incubated with Alexa 568-labeled HDL at 4 °C for 1 h, then washed and chased at 37 °C for 30 min. C,
colocalization of Alexa 568-labeled HDL (shown as red) with
Alexa 488-labeled transferrin (shown as green) in internal
confocal optical sections. The merged image on the far right
panel shows colocalization as yellow.
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SR-BI Mediates HDL Uptake and Recycling in Hepatocytes but Is Not
the Principal Receptor Mediating HDL Protein Degradation--
The
SR-BI-neutralizing antibody substantially reduced the selective uptake
of HDL CE in hepatocytes, as in transfected CHO cells (Fig.
1B). The SR-BI-neutralizing antibody markedly reduced (63%,
p < 0.001) the resecretion of HDL protein (Fig.
2B). Thus, SR-BI plays a major role in selective uptake and
resecretion of HDL taken up by hepatocytes. However, in contrast to the
CHO cells, the antibody was found to only partially inhibit (18%,
p < 0.05) HDL protein uptake by hepatocytes and not to
significantly affect HDL protein degradation (Fig. 1B).
Thus, a distinct process is responsible for the major part of the
uptake of the apoE-free HDL, in a pathway that leads to protein
degradation. To further characterize this pathway, and to evaluate a
possible role of other lipoprotein uptake pathways, such as the LDL
receptor-related protein, proteoglycans, or hepatic lipase, cells were
treated with heparinase or RAP. However, these treatments did not
inhibit HDL protein uptake (Fig. 1C), indicating that
neither proteoglycan nor LDL receptor-related protein is involved in
HDL particle uptake. Treatments with heparin, as well as heparitinase
and chondroitinase treatments were also without effect on HDL protein
uptake (data not shown).
HDL Protein and Lipid Traffic to the ERC and Sub-apical
Compartments--
Because hepatocytes in vivo are
polarized, we used fluorescence confocal microscopy to study HDL uptake
in isolated primary hepatocyte couplets. Primary hepatocyte couplets
have an intact bile canalicular membrane cordoned off by tight
junctions (27). In vivo, the canalicular space shared by two
hepatocytes is free-flowing to the bile. However, after couplet
isolation the bile canalicular space is sealed and is thus described as
a bile canalicular vacuole (27). HDL was fluorescently labeled both on
its protein component and with either a fluorescence non-degradable
cholesteryl ester analog (BODIPY-CE) or a fluorescence marker for
phospholipid (DiI). These double-labeled HDL particles were incubated
with primary hepatocytes couplets for 1 h at 37 °C, then
examined by confocal microscopy. Based on the current model of
selective uptake, HDL-derived lipid should separate from HDL protein at
the plasma membrane (9-11). On the contrary, Fig.
3, A and C, shows
that both HDL protein and BODIPY-CE colocalized at the plasma membrane,
in juxtanuclear regions previously identified as the ERC (18), and in a
region near the bile canalicular membrane, suggesting that HDL
particles (protein and lipid) traverse the hepatocyte from the
basolateral membrane to the apical canalicular region via the ERC. To
further substantiate this idea, we also examined the fate of
HDL-derived phospholipid, a major component of HDL, using the
fluorescence phospholipid analog, DiI. A similar localization pattern
was found for HDL labeled with DiI (Fig. 3, B and
C). Again upon closer examination at increased magnification
(Fig. 3B, lower panels), the HDL protein and DiI
were concentrated in sub-apical compartments beneath the canalicular
vacuole. Together the data indicate that intact HDL particles are
moving from the basolateral plasma membrane to the bile canalicular
region.

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Fig. 3.
Localization of HDL protein and lipid tracers
in primary hepatocyte couplets. A, colocalization of
both HDL protein (Alexa-apoprotein) and the cholesteryl
ester analog, BODIPY-CE, carried by HDL. Both protein and BODIPY-CE
colocalize to the ERC and bile canalicular vacuole (denoted by the
arrow) as seen in the merged image. N denotes the
nuclei. Similar patterns were observed in two independent experiments.
B, colocalization of both HDL protein
(Alexa-apoprotein) and the phospholipid tracer, DiI. The
merged image shows colocalization around the nuclei (N) and
the bile canalicular vacuole (cv). The bottom
panels show a 3× magnification of the cv. C,
additional images showing the localization of HDL protein
(Alexa-apoprotein) and BODIPY-CE or DiI around the
canalicular vacuole (denoted by the arrow) in hepatocyte
couplets.
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SR-BI Is Localized to the ERC and Bile Canalicular
Membrane--
The data in Fig. 1 suggest that SR-BI may mediate the
trafficking of holo-HDL particles through the endosome system. If this is the case, then SR-BI should be localized to the ERC as previously shown for HDL (18). Freshly isolated hepatocyte couplets were pulsed
with fluorescently labeled transferrin, fixed,
permeabilized, and used to immunolocalize SR-BI. Fig.
4A shows that SR-BI is found
in three locations: the basolateral plasma membrane, juxtanuclear compartments, and the bile canalicular membrane. SR-BI protein is
co-localized with transferrin in juxtanuclear compartments (Fig.
4A) indicating that SR-BI is found in the ERC, the same endosomal compartment that HDL enters (18). Transferrin is found only
at low levels near the canalicular membrane (28, 29). The localization
of SR-BI is thus consistent with its proposed role in trafficking HDL
particles to the ERC and bile canalicular region.

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Fig. 4.
Immunolocalization and endocytosis of SR-BI
in hepatocyte couplets. A, colocalization of SR-BI and
transferrin in a primary hepatocyte couplet. Arrows indicate
examples of colocalization of SR-BI antibody with transferrin in the
ERC. The arrowhead indicates the opening of the bile
canalicular vacuole and region of SR-BI localization. The secondary
fluorescent goat anti-rabbit antibody (2° ab)
showed low reactivity. Similar patterns were seen in three independent
experiments. B, distribution of fluorescence-neutralizing
SR-BI antibody (immune ab, IgG) in living primary hepatocyte
couplets. The central panel (enlarged view) shows a 3×
magnification of the bile canalicular vacuole of the couplet shown in
the left panel. The arrow indicates the bile
canalicular vacuole. N and cv denote the nuclei
and the canalicular vacuole, respectively. The far right
panel shows the lack of uptake of preimmune IgG (preimmune
ab). Similar patterns were seen in four independent
experiments.
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Anti-SR-BI Antibody Traffics to the ERC and Bile Canalicular
Region--
To test whether the localization to the ERC and bile
canalicular membrane was the result of SR-BI endocytosis from the
basolateral plasma membrane, the neutralizing SR-BI antibody was
fluorescently labeled and used as a tracer in living
non-permeabilized hepatocytes. Fig. 4B shows that
the anti-SR-BI antibody is internalized and localized in a punctate
fashion both to a juxtanuclear compartment and to the bile canalicular
membrane. At higher magnification, the antibody appears to be localized
to sub-apical compartments, and to the canalicular membrane (Fig.
4B, center panel). The fluorescently labeled
preimmune IgG showed little uptake in hepatocyte couplets. Taken
together, the data indicate that SR-BI undergoes endocytosis and
traffics to sub-apical vesicles and the canalicular region in polarized hepatocytes.
SR-BI Undergoes Endocytosis in Primary Hepatocytes--
The
results above suggest that SR-BI is an endocytic receptor, mediating
cellular uptake of HDL particles. Because SR-BI is widely considered to
be a non-endocytic receptor (9-11), we wanted to provide further
direct evidence that SR-BI undergoes endocytosis. Primary hepatocytes
were surface biotinylated with a reducible cross-linker, warmed to
37 °C for the times indicated in Fig. 5A, then cooled to inhibit
further uptake of biotinylated cell surface proteins. Biotin groups
that remained on the cell surface were subsequently removed by reducing
the disulfide bond of the cross-linker. Therefore, any SR-BI that is
internalized remains biotinylated. Fig. 5A shows a
time-dependent increase in protected, internalized SR-BI,
indicating that SR-BI does undergo rapid endocytosis from the cell
surface (~3-fold accumulation after 15 min). Fig. 5B shows
that SR-BI also rapidly undergoes endocytosis from the plasma membrane
of CHO cells. Maximum internalization of SR-BI occurred at 30 min (62%
of total biotinylated SR-BI). After 30 min, further endocytosis
(+glutathione) and total biotinylated SR-BI levels (
glutathione) were
seen to decrease. The latter indicates slow degradation of SR-BI in CHO
cells (21% of total biotinylated SR-BI degraded after 60 min).

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Fig. 5.
Endocytosis of endogenous SR-BI in primary
hepatocytes. A, endocytosis of biotinylated SR-BI from
the cell surface of primary hepatocytes. At the times indicated, cells
were treated with glutathione to remove cell surface biotin, and
internalized SR-BI (protected from glutathione treatment) was detected
by immunoprecipitation and immunodetection against biotin using
streptavidin-HRP. B, endocytosis of SR-BI in CHO cells
having stable expression of SR-BI (CHO-SR-BI). The
experiment was performed as in A. Cells that were not
treated with glutathione ( ) represent the total level of biotinylated
SR-BI throughout the experiment. Experiments shown in A and
B are representative of two independent experiments.
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Mechanism of HDL Endocytosis--
To determine whether the uptake
of HDL protein occurs via clathrin pits, primary hepatocytes and
CHO-SR-BI cells were infected with an adenovirus expressing the
dominant negative dynamin-1 mutant DynK44A, which inhibits the
formation of clathrin-coated vesicles (30). Control cells were infected
with an adenovirus expressing green fluorescence protein (GFP). Cells
were then incubated with protein-labeled HDL under steady-state
conditions for 1 h. Cells were also incubated for 1 h with
protein-labeled LDL as a positive control for DynK44A activity. It was
found that DynK44A was unable to inhibit HDL protein uptake but did
inhibit LDL uptake ~40% in both hepatocytes and CHO-SR-BI cells
(Table I). This level of inhibition of
LDL uptake is similar to what was reported for uptake of transferrin,
another protein that is internalized through clathrin-coated pits (22,
30). Furthermore, we tested whether a dominant negative mutant of
caveolin-1, CavKSF, could inhibit HDL protein uptake, because it has
been shown to inhibit uptake of SV40 (31, 32). Overexpression of CavKSF
did not inhibit HDL nor LDL protein uptake by CHO-SR-BI cells (data not shown). Together, these data indicate that HDL is endocytosed through a
non-clathrin-coated pit pathway, not requiring caveolin-1.
SR-BI Facilitates Transcytosis of HDL Cholesterol in Polarized MDCK
Cells--
Hepatocytes represent a polarized epithelium but do not
readily form tight junctions when grown on filters. Thus, we next examined transport of HDL cholesterol and protein in polarized MDCK
cells, an established model of polarized epithelium (33). To determine
the membrane distribution (i.e. apical versus
basal) of SR-BI, MDCK cells expressing SR-BI (MDCK-SR-BI) were
polarized on filters and either basolateral or apical membranes were
selectively biotinylated, followed by immunoprecipitation of SR-BI.
Fig. 6A shows that SR-BI is
localized to both the basolateral and apical membranes with a ratio of
~4:1. Polarized MDCK cells expressing SR-BI or vector control cells,
which express extremely low levels of SR-BI (data not shown; SR-BI
expression is ~5-fold above vector control cells) were pulse-chased
with protein and lipid-labeled HDL in the basolateral compartment and
the amount of lipid and protein tracer that enters the cells and is
secreted into the apical compartment was measured. The apical
compartment contained unlabeled HDL, which acts as an acceptor for
cellular cholesterol (34). Fig. 6B shows that MDCK cells
expressing SR-BI have greatly enhanced uptake of HDL protein (4-fold),
increased resecretion of intact HDL protein from basal membranes
(6-fold), but very little apical secretion of HDL protein. In contrast,
there was substantial apical secretion of HDL FC and CE (Fig.
6C). The HDL that is resecreted from the basal membrane is
depleted in CE and FC tracer (~22% less CE and FC compared with
starting material). SR-BI expression results in increased selective
uptake of HDL CE and FC, as expected (Fig. 6D). Our results
show that "selective uptake" comprises both selective cell
association of HDL CE and FC, as well as selective apical secretion of
CE and FC compared with HDL protein. To determine if efflux of
cholesterol from the apical membrane was SR-BI-dependent,
MDCK cells were labeled with [3H]cholesterol tracer, and
the percentage of tracer efflux to HDL was compared between
SR-BI-expressing cells and control. We found that, after a 6-h chase
period, SR-BI-expressing cells effluxed ~2-fold more tracer
(15.0 ± 0.92 versus 7.0 ± 0.22%;
p < 0.001). Therefore, efflux from the apical membrane
was dependent on SR-BI.

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Fig. 6.
Transport of HDL cholesterol and cholesteryl
ester across polarized MDCK cells. A, distribution of
SR-BI in MDCK cells having stable expression of SR-BI on the
basolateral and apical membrane. Polarized MDCK cells were biotinylated
on either the basolateral (B) or apical (A)
membrane, and biotinylated SR-BI was then immunoprecipitated and
immunodetected using streptavidin-HRP. B, pulse-chase
analysis of triple-labeled HDL (125I-HDL protein,
3H-CE, 14C-FC) in polarized MDCK cells having
stable expressing of SR-BI or vector control. The amount of HDL protein
resecreted intact from the cells after the chase (sec) or
degraded (deg) is shown for both the apical and basolateral
chambers. The amount of HDL protein remaining in the cell after the
chase (cell) is also shown. C, levels of HDL
cholesteryl ester (CE) and free cholesterol (FC)
remaining in the cells, in the apical chamber, and in the basolateral
chamber after the chase period in the experiment in A. D, selective HDL CE and FC uptake was calculated by
subtracting the amount of HDL protein uptake remaining in the cells in
A from the amount of HDL CE or FC uptake remaining in the
cells in B at the end of the chase period. All error
bars are S.D. The experiments in A through D were
repeated three times with similar results.
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DISCUSSION |
This study begins to elucidate the pathways for HDL protein and
cholesterol trafficking in hepatocytes. ApoE-free HDL particles are
taken up by two distinct processes. One process is mediated by SR-BI
and leads to trafficking of HDL through the ERC and sub-apical region,
resecretion of a FC and CE-depleted HDL particle from the basal cell
surface, and polarized secretion of HDL-derived cholesterol through the
apical membrane. We term this process selective transcytosis of
lipoprotein cholesterol. The other hepatocyte-specific uptake pathway
leads to degradation of HDL protein and appears to involve a novel,
hepatocyte-specific process not mediated by known lipoprotein receptors.
It is believed that SR-BI mediates the selective uptake of HDL
cholesterol at the plasma membrane and does not mediate the uptake of
holo-HDL particles (9-11). This hypothesis has been based primarily on
studies using pharmacological inhibitors of clathrin-coated
pit-mediated endocytosis, which do not inhibit HDL-selective uptake
(35). Our experiments employed several different approaches, such as
transfection of SR-BI cDNA, specific SR-BI-neutralizing antibodies,
confocal microscopy, and cell surface biotinylation of SR-BI, to show
that SR-BI is an endocytic receptor that mediates uptake and recycling
of HDL particles in transfected CHO cells, MDCK cells, and hepatocytes.
A dominant negative dynamin-1 mutant K44A did not inhibit HDL protein
uptake or HDL-selective uptake, indicating that HDL enters the cell
through a non-clathrin pathway, possibly explaining the previous
results (9-11). In agreement with this finding, the amino acid
sequence of SR-BI does not contain obvious signals for selective
inclusion in clathrin-coated vesicles, such as NPXY,
di-leucine-, or tyrosine-based motifs (33). In addition, HDL uptake may
not be through caveolae, because it was not inhibited by a dominant
negative caveolin-1 mutant, even though SR-BI has been reported to
partially colocalize with caveolin-1 in CHO cells (36). Examples of
clathrin- and caveolae-independent endocytosis have been reported
(37-39), although a mechanism for these pathways has yet to be elucidated.
Our data show that resecreted HDL after uptake via SR-BI is
cholesterol-depleted, indicating that selective uptake occurs during
the process of recycling, either at the plasma membrane or at
intracellular sites. The physiological significance of the SR-BI
pathway for HDL uptake is indicated by the finding of elevated HDL
cholesterol and increased atherosclerosis in mouse models of SR-BI
deficiency (40, 41). Furthermore, HDL recycling and selective uptake
are dramatically reduced in hepatocytes of ob/ob mice, in
association with depletion of sterol in the ERC and elevated plasma HDL
cholesterol levels. Together the studies suggest that SR-BI-mediated
HDL recycling plays a role in delivering cholesterol to the ERC and
regulates plasma HDL cholesterol levels (18). SR-BI protein and
mRNA levels are not altered in ob/ob mice, suggesting that SR-BI is dysfunctional.
The endocytic recycling pathway for uptake of HDL protein and
cholesterol by hepatocytes contrasts with the well defined process of
LDL particle uptake, which terminates in the degradation of LDL
cholesteryl ester and protein in lysosomes (42). In contrast, HDL
uptake and trafficking is more analogous to the process of iron
delivery via transferrin. Iron-saturated transferrin undergoes receptor-mediated endocytosis and enters into the early endosome system
where transferrin unloads its iron. Iron-depleted transferrin remains
bound to its receptor and recycles back to the plasma membrane through
the ERC (43). In a similar fashion, we found that HDL particles undergo
SR-BI-mediated endocytosis into the ERC and sub-apical compartments
followed by the recycling of HDL protein back to the basolateral plasma
membrane. However, unlike the trafficking of transferrin and its
receptor, the removal of HDL cholesterol involves a further sorting
event leading to the polarized secretion of HDL cholesterol from the
apical membrane.
The transcytosis of the well characterized polymeric IgA receptor (44)
involves its movement through sorting endosomes and apical recycling
compartments followed by vesicular transport to the apical membrane
where secretory component is cleaved and released into bile. The
transcytosis of SR-BI may be partly analogous to the polymeric IgA
receptor. However, in contrast to IgA, the trafficking of HDL likely
involves the selective sorting of cholesterol away from the apoprotein
component. Our analysis of the movement of fluorescent lipid and HDL
protein tracers in primary mouse hepatocytes showed that both HDL
protein and cholesterol traffic together to the ERC and sub-apical
compartments (Fig. 4, A and C). It is likely that
SR-BI brings HDL particles into the ERC and sub-apical regions, where a
sorting event occurs. This could involve release of a lipid-depleted
HDL remnant from SR-BI, while cholesterol bound to SR-BI traffics in a
vesicle to the apical membrane, where the HDL-derived cholesterol is
taken up by biliary micelles. Cholesterol secretion from the apical
surface of the hepatocyte may be facilitated by SR-BI as demonstrated
in polarized MDCK cells. The level of HDL apoproteins in bile is quite
low, despite substantial levels of HDL-derived free cholesterol (45). Our model of SR-BI-mediated trafficking and selective sorting of HDL
protein from cholesterol in the ERC and sub-apical region suggests a
mechanism to explain the selective exclusion of HDL apoprotein from the
bile despite the enrichment with HDL cholesterol.
These studies provide the first clear-cut evidence for a HDL protein
catabolic process in hepatocytes that is not mediated by SR-BI. The
finding that acute inhibition of SR-BI function using a SR-BI antibody
in vitro does not substantially inhibit HDL protein uptake
and degradation is consistent with the fact that SR-BI-deficient mice
have similar HDL apoprotein levels (46) and hepatic apoprotein
catabolism (47) as wild-type mice. However, the previous in
vivo findings did not exclude a role of SR-BI in mediating HDL
protein degradation, because there could be compensation by other
pathways, such as receptors that could clear apoE-rich HDL particles in
SR-BI-deficient mice (48, 49); furthermore, SR-BI overexpression is
associated with a marked increase in HDL protein degradation in liver
and kidney (50). Thus, our results provide the first direct evidence
for the existence of a distinct, novel pathway of apoE-free HDL
particle uptake leading to protein degradation in hepatocytes. Because
there is a marked defect in clearance of HDL apoA-I and apoA-II in
ob/ob mice (51), this pathway may be down-regulated. Further
studies on the mechanisms of SR-BI-mediated selective transcytosis and
the molecular characterization and regulation of the HDL protein
degradation pathway may be rewarding.