From the Institute for Medical Biochemistry and
Molecular Biology, the Department of Molecular Cell Biology and the
** Department of Internal Medicine, University Hospital
Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany and
Departament de Biologia Cellular, Institut
d'Investigacions Biomèdiques August Pi i Sunyer, Facultat de
Medicina, Universitat de Barcelona, 08036 Barcelona, Spain
Received for publication, September 4, 2002, and in revised form, February 10, 2003
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ABSTRACT |
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After receptor-mediated endocytosis of
triglyceride-rich lipoproteins (TRL) into the liver, TRL particles are
immediately disintegrated in peripheral endosomal compartments. Whereas
core lipids and apoprotein B are delivered for degradation into
lysosomes, TRL-derived apoE is efficiently recycled back to the plasma
membrane. This is followed by apoE re-secretion and association of apoE with high density lipoproteins (HDL). Because HDL and apoE can independently promote cholesterol efflux, we investigated whether recycling of TRL-derived apoE in human hepatoma cells and fibroblasts could be linked to intracellular cholesterol transport. In this study
we demonstrate that HDL3 does not only act as an
extracellular acceptor for recycled apoE but also stimulates the
recycling of internalized TRL-derived apoE. Furthermore, radioactive
pulse-chase experiments indicate that apoE recycling is accompanied by
cholesterol efflux. Confocal imaging reveals co-localization of apoE
and cholesterol in early endosome antigen 1-positive endosomes. During
apoE re-secretion, HDL3-derived apoA-I is found in these
early endosome antigen 1, cholesterol-containing endosomes. As shown by
time-lapse fluorescence microscopy, apoE recycling involves the
intracellular trafficking of apoA-I to pre-existing and TRL-derived
apoE/cholesterol-containing endosomes in the periphery. Thus, these
studies provide evidence for a new intracellular link between
TRL-derived apoE, cellular cholesterol transport, and HDL metabolism.
Chylomicrons (CM)1 and
very low density lipoproteins (VLDL) represent the two classes of
triglyceride-rich lipoproteins (TRL) that are responsible for the
transport of lipids to various cells of the body. After assembly in the
intestine, CM mediate the transport of dietary lipids, whereas VLDL are
synthesized in the liver to deliver endogenous lipids to peripheral
tissues. In the bloodstream CM are hydrolyzed by lipoprotein lipase
(LPL), resulting in the formation of chylomicron remnants (CR) (for
review see Refs. 1-3). During lipolysis these remnants become enriched
with high density lipoproteins (HDL)-derived apoprotein E (apoE) and
are rapidly cleared from the plasma in a process known to depend on
apoE and LPL via the LDL receptor-related protein and the low density
lipoprotein (4-7).
After receptor-mediated endocytosis, the intracellular fate of TRL
constituents is far more complex than the classical degradation pathway
of low density lipoproteins (LDL) described by Brown and Goldstein (8).
A number of studies revealed that the intracellular processing of TRL
first involves disintegration in peripheral endosomal compartments,
which is followed by a differential sorting of TRL components (9).
Using Because HDL is known to induce cholesterol efflux (19-21), we
investigated whether recycling of TRL-derived apoE is accompanied by
the mobilization of cellular cholesterol. In this study we demonstrate
that HDL3-inducible apoE recycling is associated with cholesterol efflux. Life cell imaging and confocal microscopy revealed
that this process involves the targeting of HDL3-derived apoA-I to EEA1-positive endosomes containing both TRL-derived apoE and
cholesterol. After re-secretion, TRL-derived apoE and cholesterol are
found associated with extracellular HDL particles. Taken together,
these findings indicate that apoE recycling provides an efficient
mechanism to re-supply plasma with apoE-containing HDL particles. This
process would ultimately accelerate the enrichment of CR with apoE and
stimulate the hepatic clearance of CR in the postprandial state.
Antibodies and Reagents--
Paraformaldehyde (PFA), filipin,
glycine, and BSA were purchased from Sigma. Mowiol®4-88
was purchased from Calbiochem. DMEM, PBS, fetal calf serum, trypsin,
penicillin, and streptomycin were from Invitrogen. Iodogen was from
Pierce. 125I and [3H]cholesterol
([3H]Chol) were from Amersham Biosciences. Heparin
(Liquemin®) was purchased from Roche Molecular Biochemicals.
Polyclonal antibody against human apoE was from Dako. Monoclonal
antibody against early endosome antigen 1 (EEA1) was from BD
Biosciences. Monoclonal antibody against adaptor protein 1 (AP1) was
kindly provided by E. Ungewickell (22). Alexa 568 nm fluorescence
labeling kit and NBD-cholesterol were from Molecular Probes. Cy3 and
Cy5 fluorescence protein labeling kit were from Amersham Biosciences.
Cy2-conjugated goat anti-rabbit F(ab')2 fragments and
horseradish peroxidase-conjugated goat anti-rabbit F(ab')2
fragments were purchased from Jackson ImmunoResearch.
Cell Culture--
Human skin fibroblasts and hepatoma cells
(HuH7) were grown in DMEM supplemented with 10% fetal calf serum and
penicillin/streptomycin at 37 °C in 5% CO2.
Ligand Preparation--
Human serum, LDL, and
apoE-deprived HDL3 (d = 1.125-1.21
g/ml) from normal healthy donors (23, 24) and TRL from an
apoCII-deficient patient were isolated as described (25). ApoE3 was
isolated by preparative SDS-PAGE (14). TRL were radiolabeled by the
iodine monochloride method, whereas apoE was radiolabeled with Iodogen (16). 100 µg of unlabeled or 125I-labeled apoE were
associated with TRL (0.5 mg of protein) to prepare apoE-TRL (final
concentration 0.28 mg of protein/ml) or 125I-apoE-TRL as
described (16). The protein concentrations of the 125I-TRL
and the 125I-apoE-TRL preparations were 0.2 ± 0.05 mg/ml, and the specific radioactivity was 100-180 cpm/ng protein,
respectively. 125I-TRL and the 125I-apoE-TRL
preparations were routinely separated by 10% SDS-PAGE, and
radiolabeling of TRL apoproteins was confirmed by autoradiography. For
the labeling of lipoproteins with [3H]Chol, 100 µl of
[3H]Chol (3.7 MBq) was dried under liquid nitrogen,
resuspended in 100 µl of DMEM + 2% BSA, and incubated overnight with
0.5-1 mg of 125I-apoE-TRL
([3H]Chol/125I-apoE-TRL) or 3-5 mg of LDL
([3H]Chol-LDL) in PBS + 10 mM EDTA at
37 °C. Non-incorporated [3H]Chol was removed by PD10
gel chromatography (Amersham Biosciences). For immunofluorescence
experiments, apoE-TRL (0.2 mg) were labeled with Cy3 (Cy3-apoE-TRL),
and HDL3 apoproteins (1 mg) were labeled with Alexa 568 nm
(Alexa-HDL3) or Cy5 (Cy5-HDL3) according to the
instructions of the manufacturers. Fluorescent label was found predominantly in apoE of Cy3-apoE-TRL (~80-90%) and apoA-I of Alexa-HDL3 or Cy5-HDL3 (~90-95%) as
determined by SDS-PAGE, respectively (data not shown). To label
Cy3-apoE-TRL with NBD-cholesterol, 0.2 mg of Cy3-apoE-TRL was incubated
with 250 µg of NBD-cholesterol in PBS, 1 mM EDTA at
37 °C overnight. Non-incorporated NBD-cholesterol was removed by
PD10 gel chromatography. For electron microscopy, HDL3 (600 µg; pI 5.5) and apoE3 (100 µg; pI 5.78) were labeled with colloidal
gold of 5 (gold-HDL) and 12 nm gold, respectively, according to Handley
et al. (26). Then apoE3 was associated with TRL
(gold-apoE-TRL) as described above. Conjugations of
gold-HDL3 and gold-labeled apoE3 were confirmed after
staining with sodium phosphotungstate by electron microscopy (data not shown).
Uptake and Recycling Assays--
For radioactive pulse-chase
experiments, cells were washed with PBS and incubated with
125I-TRL (2.5 µg/ml) for 60 min at 37 °C in 1 ml of
DMEM containing 5% BSA (pH 7.4). The cells were then washed with
ice-cold PBS, and surface-bound ligands were released with 770 units/ml
heparin (14, 23). To promote recycling, 125I-TRL labeled
cells were incubated for 60 min at 37 °C with DMEM + 0.1% BSA
supplemented with 50 µg/ml HDL3, 100 µg/ml LDL, or 10%
human serum. Then the content of trichloroacetic acid-precipitable, recycled radioactivity in the harvested media was determined. Cells
were solubilized in 0.1 N NaOH to calculate the amount of internalized and recycled radioactivity (14). In one set of experiments, recycled and radiolabeled TRL apoproteins from cell culture media were subjected to density gradient ultracentrifugation (KBr density from 1.006 to 1.21 g/ml) (23). After gradient
fractionation, the radioactivity in each fraction was determined. The
positions of lipoproteins were identified according to their density
and the cholesterol profile of the gradient (data not shown). In
another set of experiments, the recycled radiolabeled TRL apoproteins were separated by FPLC analysis with a Superdex 200 column (Amersham Biosciences). Cholesterol and protein profiles from standards (TRL,
LDL, HDL, albumin) allowed the identification of the fractions containing lipoproteins and albumin (lipoprotein-free fraction (LFF)).
Immunofluorescence and Electron Microscopy--
For
immunofluorescence experiments, fibroblasts and hepatoma cells were
incubated with apoE-TRL (1 µg/ml) in DMEM + 2% BSA for 60 min at
37 °C. Cells were washed in DMEM and treated with heparin at 4 °C
for 15 min (see above). Then cells were incubated for an additional 0, 15, or 60 min at 37 °C in DMEM (0.1% BSA) ± 50 µg/ml
unlabeled or fluorescent-labeled HDL3
(Alexa-HDL3, Cy5-HDL3). Cells were washed in
PBS and fixed in 4% PFA, and indirect immunofluorescence against apoE,
AP1, and EEA1 was performed (14). Cellular cholesterol was visualized
using 50 µg/ml filipin (27). Full cell images were taken with an
Axiovert 100 microscope equipped with a Zeiss Axiocam. Confocal images
were collected using a Zeiss LSM 510 (version 3.0) equipped with an UV
laser to detect filipin-stained cholesterol. For living cell
microscopy, fibroblasts were incubated with double-labeled
NBD-cholesterol/Cy3-apoE-TRL (1 µg/ml) in DMEM + 2% BSA for 0-30
min at 37 °C. Cells were washed in DMEM and incubated for an
additional 10 min with 50 µg/ml Cy5-HDL3. Confocal images
were taken every minute in the multitrack mode using optimized pinhole
adjustment for each fluorochrome.
For electron microscopy, a pre-embedding procedure was performed.
Hepatoma cells were incubated with 1 µg/ml gold-apoE-TRL (12 nm) in
DMEM + 2% BSA for 60 min at 37 °C. Cells were washed, treated with
heparin (see above), and incubated with 5 µg/ml gold-HDL3 (5 nm) at 37 °C for 15 min. Cells were rinsed with PBS and fixed in
2% PFA, 2.5% glutaraldehyde in PBS for 60 min at room temperature. Then fixed cells were scraped and post-fixed in 2% PFA in PBS for 2 days at 4 °C. Samples were washed, treated with 1%
OsO4, 0.8% potassium ferricyanide (Sigma) in PBS,
dehydrated with acetone, and embedded in Spurr resin (Sigma). Ultrathin
sections were obtained and counterstained with uranyl acetate and lead
citrate as described previously (28).
Western Blotting--
Fibroblasts and hepatoma cells were
incubated with apoE-TRL (1 µg/ml) for 60 min at 37 °C, washed, and
incubated for an additional 0 or 60 min at 37 °C ± 50 µg/ml
HDL3 in DMEM + 0.1% BSA. The media were harvested,
filtered (inner diameter, 0.45 µm), and cleared by centrifugation at
14,000 × g for 10 min. The supernatants were subjected
to 10% SDS-PAGE and immunoblotted against apoE. After incubation with
peroxidase-conjugated secondary antibodies, the reaction product was
detected using the ECL system (Amersham Biosciences).
Cholesterol Efflux Experiments--
In the first set of
experiments, cells were incubated with double-labeled
[3H]Chol/125I-apoE-TRL (1 µg/ml) for 60 min
at 37 °C and then washed with ice-cold PBS and heparin to remove
surface-bound ligands (see above).
[3H]Chol/125I-apoE-TRL labeled cells were
incubated for additional 60 min at 37 °C in DMEM + 0.1% BSA in the
presence or absence of 50 µg/ml HDL3. The media were
harvested to quantify 125I-apoE recycling after
trichloroacetic acid precipitation (see above), whereas
[3H]Chol efflux was determined after Dole
extraction (29). In the second set of experiments, intracellular
cholesterol pools were radiolabeled overnight with
[3H]Chol-LDL (100,000 cpm/ml) at 37 °C.
Non-internalized [3H]Chol-LDL was removed by heparin
treatment (see above). Then cells were incubated with or without
apoE-TRL for 60 min at 37 °C. After heparin treatment (see above),
cells were incubated in DMEM + 0.1% BSA in the presence or absence of
50 µg/ml HDL3 for an additional 60 min at 37 °C. The
media were harvested, and cells were lysed in 0.1 N NaOH.
Dole extraction of media and cell lysates was performed to
determine [3H]Chol efflux.
HDL3 Stimulates the Recycling of TRL-derived
ApoE--
We have demonstrated previously (9, 14, 16) that the
recycling of internalized TRL-derived apoE and LPL requires the presence of an extracellular lipoprotein acceptor. To compare the
ability of different lipoproteins to mediate TRL recycling, human
hepatoma cells were preincubated with 125I-TRL, and
recycling of 125I-TRL proteins was induced with human
serum, LDL, or HDL3 (Fig. 1A). Consistent with previous
results (16), addition of 10% human serum stimulated recycling of
internalized 125I-TRL proteins 2-fold compared with the
lipoprotein-free control (Fig. 1A,
w/o). Similarly, in the presence of HDL3
(50 µg/ml protein), a 1.8-fold increase of 125I-TRL
recycling was observed. This stimulatory effect of HDL3 on
125I-TRL recycling was already detectable at very low
levels of HDL3 (2.5 µg/ml), dose-dependent,
and saturable in the presence of 50 µg/ml HDL3 (Fig.
1B). In contrast, no significant stimulation of
125I-TRL recycling was determined when LDL (100 µg/ml
protein) was utilized (Fig. 1A). These findings indicated
that HDL3 could stimulate 125I-TRL recycling
and serve as an acceptor for recycled 125I-TRL proteins. To
analyze the association of recycled 125I-TRL proteins with
lipoproteins, the media containing the lipoprotein acceptors and the
released 125I-TRL-derived radioactivity were harvested and
separated either by density gradient centrifugation (Fig.
2A) or gel filtration chromatography (Fig. 2B). Recycled 125I-TRL
proteins from the media of cells incubated with human serum identified
~60% of TRL radioactivity in the HDL fractions (fractions 7-11 in
Fig. 2A), whereas 40% was found in the lipid-free bottom of
the gradient (fractions 1-4). It is likely that lipid-free apoA-I in
human serum is responsible for the association of 125I-TRL
proteins in these bottom fractions, because lipid-free apoA-I also
induced recycling of 125I-TRL proteins into these fractions
(data not shown). When 125I-TRL-loaded cells were incubated
with HDL3, a predominant association of recycled
125I-TRL proteins with HDL was observed (fractions 7-11,
Fig. 2A). To confirm the association of recycled
125I-TRL with HDL, re-secreted 125I-TRL
proteins were separated by Superdex 200 gel filtration (Fig. 2B), which is appropriate for the resolution of small sized
lipoproteins (10-600 kDa). As expected HDL3-induced
recycling of internalized 125I-TRL resulted in the
association of 125I-labeled apoproteins with HDL (fractions
24-28; Fig. 2B). In addition 125I-TRL proteins
were found in lipid-free fractions (LFF; fractions 29-31), which were
representative of recycled 125I-TRL proteins when
lipid-free apoA-I was used as an acceptor (data not shown). Independent
of density gradient or gel filtration analysis, we never detected
significant amounts of recycled 125I-TRL proteins in the
fractions containing LDL (fractions 16-18 in Fig. 2A), TRL
(fractions 21 and 22 in Fig. 2A), or both LDL and TRL
(fractions 17-19 in Fig. 2B). These results demonstrate that TRL or LDL do not serve as acceptors for recycled
125I-TRL apoproteins. Taken together, these results
indicate that HDL in human serum is capable of stimulating TRL protein
recycling (Fig. 1, A and B) and serve as an
acceptor for recycled TRL proteins (Fig. 2, A and
B).
Recycling of ApoE Is Associated with Cholesterol Efflux--
It is
well known that HDL can stimulate cellular cholesterol efflux (19-21).
Because HDL-dependent re-secretion of 125I-TRL
(see above) mainly involves recycling of apoE (14), we investigated
whether recycling of TRL-derived apoE could be linked to intracellular
cholesterol transport. Therefore, the distribution of TRL-derived apoE
and cholesterol in the presence or absence of HDL3 was
first studied by immunocytochemistry. In initial experiments with human
hepatoma cells, immunostaining against TRL-derived apoE interfered with
endogenous apoE expression (data not shown). Thus, human fibroblasts
with no detectable amounts of endogenous apoE were analyzed. These
cells, similar to the HuH7 hepatoma cells described above, efficiently
recycled TRL proteins through peripheral endosomal compartments (14).
In the following set of experiments, fibroblasts were incubated with
apoE-enriched TRL (apoE-TRL), which was followed by a 60-min chase ± HDL3 (Fig. 3). The cells
were then fixed, immunostained against apoE to detect TRL-derived apoE,
and incubated with filipin to visualize cellular cholesterol. In the
absence of HDL3 (Fig. 3, A and B),
significant amounts of internalized TRL-derived apoE remained
intracellularly (Fig. 3A). In these cells large amounts of
cholesterol were found in peripheral and perinuclear compartments (Fig.
3B), which appeared to co-localize with apoE mainly in the
periphery. In contrast, when cells were incubated with
HDL3, only residual amounts of internalized TRL-derived
apoE were detected intracellularly (Fig. 3A, c).
These findings suggested HDL3-induced apoE recycling, which
was confirmed by apoE Western blot analysis of the chase media (Fig.
3B, compare lanes 3 and 4 which
correspond to media from cells shown in Fig. 3A,
a and b, and c and d,
respectively). In addition, apoE recycling was accompanied by a strong
reduction of filipin staining mainly in peripheral, most likely
endosomal compartments (Fig. 3A, d).
Although filipin stains endosomal cholesterol, it is well known that
large amounts of cholesterol are found in perinuclear Golgi and
lysosomal compartments (30). In addition, analysis of full thickness
cell images could lead to the overlap of apoE and cholesterol signals
by random chance. Thus, confocal microscopy was performed to
characterize apoE and cholesterol-containing vesicles in detail (Fig.
4). First, human fibroblasts were
incubated with apoE-TRL, fixed, and stained against apoE together with
AP1, a protein predominantly found in the Golgi apparatus (22) (Fig. 4a), or EEA1, a marker for early endosomes (31) (Fig.
4b). These experiments identified large amounts of
internalized TRL-derived apoE in EEA1-positive endosomes that did not
co-localize with AP1. As shown in Fig. 4c, these
apoE-containing and EEA1-positive endosomes contain cholesterol. In
contrast, cholesterol in the perinuclear region that was not stained
with EEA1 did not contain any TRL-derived apoE. These findings
indicated that HDL3-induced apoE recycling is linked to
cholesterol efflux from endosomal compartments. However, filipin
staining is not a reliable method to estimate changes in endosomal
cholesterol. Therefore, we first tested whether
HDL3-induced recycling of apoE is accompanied by the
mobilization of radiolabeled TRL-derived cholesterol from endosomes.
Because internalization and disintegration of TRL particles could be
associated with a substantial redistribution of TRL-derived cholesterol
to multiple cellular compartments, control experiments with
NBD-cholesterol-labeled TRL were performed. In these studies TRL-derived NBD-cholesterol and apoE co-localized in EEA1-positive endosomes even after 60 min at 37 °C (data not shown, see also Fig.
11). Then cells were incubated with double-labeled
[3H]Chol/125I-apoE-TRL (Fig.
5). In these experiments HDL3
simultaneously stimulated recycling of internalized TRL-derived
125I-apoE (1.8-2.4-fold) and efflux of TRL-derived
[3H]Chol (2.8-3.2-fold). Thus HDL3
stimulates apoE recycling and cholesterol efflux from internalized TRL
particles in endosomal compartments.
To determine whether apoE recycling is associated with cholesterol
efflux from intracellular cholesterol pools in the absence of
HDL3, hepatoma cells were pre-labeled overnight with
[3H]Chol-LDL, and cell surface-bound
[3H]Chol-LDL was removed by heparin. Then cells were
incubated ± apoE-TRL for 60 min, followed by an additional
incubation ± HDL3 for 60 min. Finally the amount of
[3H]Chol in the media was determined (Fig.
6). Similar to results obtained from
hepatoma cells (32), HDL3 stimulated [3H]Chol
efflux 3-fold (compare columns 1 and 2). In the
absence of HDL3, internalized TRL-derived apoE is
concatenated by [3H]Chol efflux ~3-fold compared with
controls (compare columns 1 and 3). In the
presence of HDL3, a 6-fold induction of
[3H]Chol efflux from apoE-TRL-loaded cells was observed
(compare columns 1 and 4). These findings
indicate that apoE recycling is linked to cholesterol efflux not only
from TRL-associated pools but also from other intracellular cholesterol
pools (Figs. 5 and 6). To study the possible association of secreted
[3H]Chol with HDL, Superdex 200 gel filtration analysis
of the chase media was performed (Fig.
7). The majority of released
[3H]Chol (~80%) was found in HDL fractions, which were
shown to contain recycled TRL-derived apoE in this study (see Fig. 2,
A and B) and previous studies (16). In contrast,
only ~20% of [3H]Chol was found in fractions smaller
than HDL (LFF). Taken together, HDL3-dependent
and -independent apoE recycling is accompanied by the mobilization of
intracellular cholesterol leading to a concomitant association of
TRL-derived apoE and cholesterol with HDL particles.
Recycling of ApoE Is Associated with the Internalization of
HDL3-derived apoA-I--
Most current models favor the
localization of HDL at the plasma membrane to promote cholesterol
efflux. We suggested that HDL is located at the cell surface to serve
as an acceptor for recycled apoE (9, 14). Although the pulse-chase
experiments described above (Figs. 2 and 7) demonstrated the
association of recycled apoE and cholesterol with HDL, these
experiments did not provide information on the question whether complex
formation of TRL-derived apoE and cholesterol with HDL could already
occur intracellularly. To address this matter, apoE recycling in
apoE-TRL-loaded cells was induced with fluorescent-labeled
HDL3 proteins (Alexa-HDL3) containing
predominantly labeled apoA-I (see "Experimental Procedures"). Cells
were fixed and analyzed for the distribution of apoE, cholesterol, and
Alexa-HDL3. Similar to the results described in Fig. 3,
a and b, TRL-derived apoE accumulates in
peripheral compartments (Fig.
8a), which could also be
stained with filipin (Fig. 8c), indicating co-localization
of apoE and cholesterol in endosomes. Because complete recycling of
TRL-derived apoE is observed after 60 min (Fig. 3A,
c and d), only a 15-min induction of apoE
recycling with Alexa-HDL3 was performed (Fig. 8,
d-f). This enabled us to detect simultaneously
TRL-derived apoE, cholesterol, and Alexa-HDL3. As expected,
Alexa-HDL3 stimulated apoE recycling and cholesterol efflux
as shown by a reduced endosomal apoE and cholesterol staining (Fig. 8,
d and f). In these experiments the
reduction in cholesterol staining in the periphery was not as prominent
as described in Fig. 3, which could in part be explained by the shorter
exposure time to HDL3. In addition, filipin staining of
full cell images is not suitable to detect minimal changes in endosomal
cholesterol concentrations. Most strikingly, co-localization of
apoE/cholesterol and Alexa-HDL3 was visible in peripheral
compartments (compare arrows in Fig. 8,
d-f) indicating the formation of endosomal
apoE/cholesterol and apoA-I complexes. This observation was confirmed
by confocal microscopy, which identified the co-localization of apoE
and apoA-I in EEA1-positive endosomes during apoE recycling (Fig.
9).
To support these findings, electron microscopy of apoE-TRL-loaded
hepatoma cells ± HDL3 was performed. Incubation of
cells with gold-labeled apoE-TRL (12 nm gold) resulted in the
association of gold-apoE with endocytic structures (data not shown). To
promote apoE recycling, gold-apoE-loaded cells were
incubated with gold-labeled HDL3 (5 nm gold) for an
additional 15 min (Fig. 10). Subsequent analysis of EM sections identified gold-labeled protein complexes consisting of apoE- and HDL3-derived apoA-I to be
associated with peripheral endosomes and membranous structures at the
plasma membrane.
These findings suggested that HDL3-derived apoA-I is
internalized to pre-existing apoE/cholesterol-containing endosomes to promote apoE recycling and cholesterol efflux. To confirm this hypothesis we performed time-lapse confocal microscopy to study intracellular localization of apoA-I during early stages of apoE recycling (Fig. 11). Cells were first
incubated for 30 min with double-labeled NBD-cholesterol/Cy3-apoE-TRL.
Then Cy5-HDL3 was added for an additional 10 min. An
example of a mobile NBD-cholesterol/apoE-containing endosome
(frames 6-15) that interacts with another mobile
apoA-I-containing endosome is shown (frames 16-20). The
merged images (frames 1-5) revealed co-localization of
NBD-cholesterol and apoE indicating that these TRL components remained
associated even 30 min after TRL internalization (frames 2 and 3). Most importantly, immediately after addition of
Cy5-HDL3, endosomal apoA-I is targeted to the NBD-cholesterol/apoE-containing vesicle (frames 4 and
5). Thus, the intracellular formation of
apoE/cholesterol/apoA-I-containing complexes is involved in apoE
recycling.
The aim of this study was to identify a possible intracellular
link between the recycling of apoE, cholesterol efflux, and HDL
metabolism. It is well documented that internalized TRL-derived apoE is
efficiently recycled in liver cells (13, 14, 16-18), but relatively
little is known about the intracellular events that accompany apoE
recycling. Here we demonstrate that apoE recycling is linked to
HDL3-dependent and -independent cholesterol
efflux. In the presence of HDL the re-secretion of apoE and cholesterol leads to the formation of apoE/cholesterol-enriched HDL particles. Detailed confocal microscopy and live cell imaging revealed that after
internalization TRL-derived apoE and cholesterol remain associated in
EEA1-positive endosomes. Most importantly, apoE recycling involves the
internalization of HDL3-derived apoA-I and its targeting to
apoE/cholesterol-containing endosomes. These findings indicate that
endosomal HDL3-derived apoA-I can mobilize pre-existing
apoE/cholesterol complexes.
In the light of these findings, our previous model that HDL acts as an
extracellular acceptor for recycled TRL-derived apoE (9) deserves to be
reconsidered and extended. Concurrent cholesterol efflux and apoE
recycling would be in agreement with models favoring the binding of HDL
to the plasma membrane. At the cell surface, HDL could interact with
ATP-binding cassette transporter A1 (ABCA1), scavenger receptor class B
type I (SR-BI), or other HDL-binding proteins (for reviews see Refs.
19-21 and 33-36). However, the evidence of HDL-derived apoA-I
internalization during apoE/cholesterol re-secretion (Figs. 8-11) is
in agreement with a number of reports describing the intracellular
localization of HDL receptors during HDL-mediated cholesterol transport
(for reviews see Refs. 20, 33, and 36). Similarly, results presented in
this work identified HDL-derived apoA-I in EEA1-positive early
endosomes. Oram (20) recently proposed that ABCA1 and apoA-I are
internalized to intracellular lipid vesicles, where ABCA1 could deliver
lipids into the lumen of exocytic vesicles before re-secretion. This is
based on the following observations. First, ABCA1 recycles between the
plasma membrane and endosomes (37). Second, ABCA1 promotes cholesterol efflux from endosomal compartments (38). Third, apoA-I is internalized and re-secreted during cAMP-inducible and ABCA1-dependent
cholesterol efflux (39-42).
Although these observations would favor a role for ABCA1 in the
HDL-induced and concurrent apoE recycling and cholesterol efflux, the
potential contribution of SR-BI cannot be excluded. SR-BI is localized
predominantly in caveolae and not in clathrin-coated pits (43, 44).
However, detailed studies revealed that a significant proportion of
SR-BI proteins are rapidly internalized (45). This is accompanied by
endocytosis and re-secretion of HDL (46). The concept that SR-BI could
mediate uptake and recycling of HDL particles is consistent with a
punctate, endosomal localization of fluorescent-labeled HDL immediately
(10 min) after cell surface ligand binding in SR-BI-overexpressing
cells (47) (see also Figs. 8-11). In addition, Silver et
al. (45) observed the internalization and recycling of HDL
particles through an endocytic recycling compartment in non-polarized
hepatocytes. These results are in agreement with earlier studies
(48-51) describing receptor-mediated endocytosis and retroendocytosis
of HDL.
Co-localization of apoA-I, apoE, and cholesterol in TRL-loaded cells
indicates that apoE recycling and cholesterol efflux results in the
intracellular assembly of apoA-I with apoE/cholesterol-containing complexes. Similarly, a number of reports (52, 53) hypothesized an
interaction between apoA-I and apoE and concluded that apoA-I concurrently stimulates secretion of endogenous apoE and cholesterol efflux from lipid-loaded macrophages. In addition secretion of apoE
from macrophages is at least in part associated with cell-derived cholesterol (54, 55). This stimulatory role of apoA-I and endogenous
apoE for cholesterol efflux in peripheral cells is the presupposition
for efficient reverse cholesterol transport to the liver for bile
secretion (33, 56). In our study we identified the concomitant
re-secretion of endosomal TRL-derived and/or intracellular cholesterol
with internalized TRL-derived apoE in hepatoma cells. These findings
suggest that intracellular association of cholesterol with TRL-derived
apoE and concurrent re-secretion could provide a mechanism allowing
dietary cholesterol to escape bile secretion.
Most importantly we demonstrate that the intracellular association of
apoA-I with apoE-cholesterol complexes could lead to the formation of
apoE-enriched HDL particles. Once secreted, these particles would
represent a pool of apoE proteins associated with HDL. In the
postprandial state HDL-derived apoE is the major source for apoE
enrichment of CR to ensure the rapid clearance of CR into the liver (1,
2). To avoid a rapid decrease of HDL-derived apoE during the
postprandial state, the recycling of TRL-derived apoE and subsequent
generation of apoE-containing HDL particles would secure the
maintenance of high HDL-associated apoE levels in plasma. Therefore,
recycled apoE could serve as a continuous donor for intravascular apoE
transfer from HDL to CR during lipolysis and hepatic clearance (9, 16).
Future in vivo experiments will have to clarify whether
newly formed HDL particles play a physiological role in CR enrichment
with apoE.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-VLDL as a "TRL model particle," Maxfield and co-workers
(10-12) identified
-VLDL-derived lipids in lysosomal compartments
of mouse macrophages, whereas apoE was present in peripheral vesicles.
Consistent with these findings, lysosomal targeting of TRL proteins and
apoE was markedly reduced compared with the degradation of cholesteryl
oleate in mouse hepatocytes (13). In human hepatoma cells and
fibroblasts, the majority of TRL lipids are targeted to the lysosomal
compartment, whereas TRL-derived apoE is found in peripheral recycling
endosomes, which are distinct from the perinuclear transferrin
recycling compartment (14, 15). Subsequently substantial amounts of
TRL-derived apoE are recycled back to the cell surface and re-secreted
(14). Recently, several studies (13, 16-18) on the hepatic TRL
metabolism in vivo confirmed the disintegration of TRL
components within the endosomal compartment and the targeting of TRL
lipids to lysosomes. Most remarkably, TRL-derived apoE is recycled and
found associated with newly synthesized or exogenous lipoproteins (16).
Fazio and co-workers (17) observed an association of recycled apoE with
nascent VLDL particles in liver Golgi fractions. Alternatively, we
recently identified (14, 16) that HDL serves as a potent extracellular
acceptor for re-secreted apoE in hepatocytes in vitro and
in vivo.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
HDL-induced recycling of TRL-associated
apoproteins in hepatoma cells. Pulse-chase experiments were
performed by preincubating human HuH7 hepatoma cells with
125I-TRL for 60 min at 37 °C (see "Experimental
Procedures"). Cells were washed at 4 °C, and cell-bound
radiolabeled material was removed by heparin. After incubation for
additional 60 min at 37 °C, cells were incubated with media only
(0.1% BSA in DMEM; w/o, white bar) or
media supplemented with 10% human serum (hatched bar), 100 µg/ml LDL (black bar), or 50 µg/ml HDL3
(gray bar) (A); or with increasing amounts of
HDL3 (0-250 µg/ml) (B). The media were
collected to calculate the amount of recycled, intact (trichloroacetic
acid-precipitable) 125I-TRL-derived apoproteins. The
remaining cells were lysed, and protein content was determined. The
data are given in ng of apoprotein recycling/mg of cell protein and
represent the mean ± S.D. of five (A) and four
(B) independent experiments with duplicate samples.
Depending on the specific activity of 125I-TRL preparations
(100-180 cpm/ng protein, see "Experimental Procedures"),
background 125I-TRL recycling in the absence of
HDL3 was in the range of ~20-60 ng/mg cell
protein.
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Fig. 2.
Density gradient and FPLC analysis of
recycled 125I-TRL-derived apoproteins. A, the
non-degraded radioactivity of media from cells analyzed in Fig.
1A, which were incubated with media alone ( ,
w/o), with 50 µg/ml HDL3 (
,
HDL3) or 10% human serum (
, serum) were separated by
KBr density gradient (see "Experimental Procedures"). Fractions
from bottom to top (500 µl) were collected, and the radioactivity was
determined in each fraction. The gradient distribution (mean values of
duplicate samples) of recycled 125I-TRL apoproteins
(counts/min) of a representative experiment is shown (n = 3). The fractions containing TRL (fractions 21 and 22), LDL
(fractions 16-18), HDL (fractions 7-11), and the lipid-free bottom
(fractions 1-4) are indicated and were identified by their respective
density and cholesterol content. B, the non-degraded
radioactivity of media from cells analyzed in Fig. 1A, which
were incubated with media alone (
, w/o) or
with 50 µg/ml HDL3 (
, HDL3) were separated
by a Superdex 200 column (see "Experimental Procedures"); 500-µl
fractions were collected, and the radioactivity was determined in each
fraction. The distribution of recycled 125I-TRL apoproteins
(counts/min) for a representative experiment is shown
(n = 3). The fractions containing TRL/LDL (fractions
17-19) and HDL (fractions 24-28) are indicated. The LFF (fractions
29-31) contain proteins in the range of 20-80 kDa. The positions of
lipoproteins were identified by the comparison of cholesterol profiles
from the analyzed samples with isolated preparations of TRL, LDL, and
HDL, respectively.
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Fig. 3.
Recycling of TRL-derived apoE from
cholesterol-containing endosomes. A, human
fibroblasts were preincubated with apoE-TRL for 60 min at 37 °C.
After removal of cell-bound material with heparin, cells were
incubated ± HDL3 (50 µg/ml) for an additional 60 min at 37 °C as indicated. Media were collected to identify recycled
apoE (see B). Cells were fixed and analyzed by fluorescence
microscopy for apoE (green, a and c)
and cholesterol (blue, b and d).
Arrows point to apoE staining in cholesterol-rich endosomes
labeled with filipin (see "Experimental Procedures").
Bar is 10 µm. B, pulse-chase experiments were
performed by preincubating human fibroblasts (lanes 1-4) or
hepatoma cells (lanes 5-8) with apoE-TRL for 60 min at
37 °C. Cell-bound material was removed with heparin, and cells were
incubated ± HDL3 (50 µg/ml) for 0 or 60 min at
37 °C as indicated. Then cell culture media were harvested, and the
presence of recycled, intact apoE was determined by Western blot
analysis (see "Experimental Procedures"). The position of apoE is
indicated. Molecular mass is given in kDa.
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Fig. 4.
Internalized TRL-derived apoE co-localizes
with cholesterol in EEA1-positive endosomes. Human fibroblasts
were incubated with apoE-TRL for 60 min at 37 °C. After removal of
cell-bound material with heparin, cells were fixed, and sections ( 1
µm) were analyzed by confocal fluorescence microscopy. Fibroblasts
were stained against apoE (red) and AP1 (green)
in a or apoE (red) and EEA1 (green) in
b as indicated in the lower panel. The merged
images and co-localization of apoE and EEA1 (yellow) are
shown in the upper panels. c, the merged image of
fibroblasts stained against EEA1 (red), apoE
(green), and cholesterol (blue) is shown in the
upper panel (N = nucleus). In the
lower panel the highlighted area is enlarged and
shown separately for each channel. Arrowheads point to EEA1
and apoE staining in cholesterol-rich endosomes labeled with filipin.
Arrows mark endosomes only containing EEA1, apoE or
cholesterol, respectively. Bar is 10 µm.
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Fig. 5.
HDL3-induced recycling of
TRL-derived apoE and cholesterol. Pulse-chase experiments were
performed by preincubating human HuH7 hepatoma cells with
[3H]Chol/125I-apoE-TRL for 60 min at
37 °C. Cells were washed with heparin and incubated for additional
60 min at 37 °C with media in the presence or absence of 50 µg/ml
HDL3 as indicated. The media were harvested, and the amount
of re-secreted 125I-apoE (gray bar) and
[3H]Chol (white bar) was determined. The
remaining cells were lysed, and protein content was determined. The
radioactivity is given (cpm × 103/mg of cell protein)
and represents the mean ± S.D. of four independent experiments
with triplicate samples.
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Fig. 6.
Recycling of apoE is associated with efflux
of cellular cholesterol. Human hepatoma cells were pre-loaded with
[3H]Chol-LDL (100,000 cpm/ml) for 24 h at 37 °C.
Cell surface-bound [3H]Chol-LDL was removed with heparin,
and cells were incubated in the presence or absence of apoE-TRL for 60 min at 37 °C. After incubation with or without HDL3 (50 µg/ml) as indicated, aliquots of the media were harvested to
determine [3H]Chol efflux. The remaining media were
analyzed by FPLC (see Fig. 7). Cells were lysed, and the internalized
amount of [3H]Chol was determined. Values of
[3H]Chol efflux are given as a percentage of internalized
radioactivity and represent the mean ± S.D. of four independent
experiments with triplicates.
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Fig. 7.
FPLC analysis of re-secreted cellular
[3H]cholesterol. [3H]Chol-containing
media (see Fig. 5) obtained from cells incubated with ( ,
HDL3) or without HDL3 (
,
w/o) were separated by a Superdex 200 column;
500-µl fractions were collected, and the radioactivity was determined
in each fraction. The association of re-secreted [3H]Chol
(counts/min) with HDL and LFF for a representative experiment is shown.
The fractions containing TRL/LDL (fractions 17-19), HDL (fractions
24-28) and LFF (fractions 29-31) are indicated. The positions of
lipoproteins were determined as described above (Fig.
2B).
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Fig. 8.
Internalized apoA-I co-localizes with
TRL-derived apoE and cholesterol. Human fibroblasts were
preincubated with apoE-TRL for 60 min at 37 °C. After removal of
cell-bound material with heparin, cells were incubated with
HDL3 (50 µg/ml) for 0 (a-c) or 15 min
(d-f) at 37 °C as indicated. Cells were fixed and
analyzed by fluorescence microscopy for apoE (green,
a and d), HDL3 (red,
b and e) and cholesterol (blue, c
and f). Arrowheads point to apoE and
cholesterol-containing endosomes (compare a and
c). In the presence of fluorescent-labeled HDL3,
HDL3-derived apoA-I is found almost exclusively in
apoE-containing and cholesterol-rich endosomes (compare
arrows, d-f). Bar is 10 µm.
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Fig. 9.
TRL-derived apoE co-localizes with apoA-I in
EEA1-positive endosomes during apoE recycling. Human fibroblasts
were incubated with apoE-TRL for 60 min at 37 °C. After removal of
cell-bound material with heparin, cells were incubated for 15 min with
50 µg/ml Cy5-HDL3. Cells were fixed, and sections ( 1
µm) of a representative area in the cellular periphery were analyzed
by confocal fluorescence microscopy. Fibroblasts were analyzed for EEA1
(red) (a), apoE (green)
(b), and apoA-I (blue) (c). The merged
image is shown in d. Arrows point to
EEA1-positive endosomes containing apoE and apoA-I.
Arrowheads mark endosomes only containing EEA1, apoE, or
apoA-I, respectively. Bar is 10 µm.
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Fig. 10.
Co-localization of apoA-I with TRL-derived
apoE. Human hepatoma cells were preincubated with gold-apoE-TRL
(12 nm) for 60 min at 37 °C. After removal of cell-bound material
with heparin, cells were incubated with gold-HDL3 (5 nm)
for 15 min at 37 °C. Cells were fixed, prepared, and analyzed by
electron microscopy for apoE (arrowheads) and
HDL3 (arrows). In the presence of
HDL3, complexes containing gold-labeled apoE and
HDL3 are found associated with endosomal structures or at
the plasma membrane. Bar is 200 nm.
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Fig. 11.
Dynamic association of apoA-I with
pre-existing apoE- and cholesterol-containing complexes. Human
fibroblasts were incubated with double-labeled
NBD-cholesterol/Cy3-apoE-TRL for 0-30 min at 37 °C. Cells were
washed in DMEM and incubated for an additional 0-10 min with 50 µg/ml Cy5-HDL3. Time-lapse confocal image acquisition of
apoE (red), NBD-cholesterol (green), and
HDL3-protein (blue) in a representative area of
the cellular periphery was performed at 37 °C (see "Experimental
Procedures"). The merged image is shown in the upper panel
(image numbers 1-5). Each channel is shown separately in
the lower panels as indicated. Arrowheads mark
and follow the movement of a complex in the merge (image numbers
2-5) and in each lower panel: apoE (image
numbers 7-10), NBD-cholesterol (image numbers 12-15),
and apoA-I (image numbers 19 and 20).
Bar is 2 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank D. Wendt for excellent technical assistance. We are most grateful to M. Calvo (Institut d'Investigacions Biomèdiques August Pi i Sunyer/Fons d'Investigació Sanitària) for assistance in the preparation of gold probes and to Serveis Científico-tècnics, Universitat de Barcelona, for the electron microscopy facilities.
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FOOTNOTES |
---|
* This work was supported in part by the Deutsche Forschungsgemeinschaft Grants Be 829/5-1, Ja 421/3-1, and Ri 436/8-1 and Ministerio de Ciencia y Tecnologia Grants PM99-0166, Acciones Integradas HA98-0007, and Generalitat de Catalunya BE2002 (to C. E.).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: Institute for Medical Biochemistry and Molecular Biology, Dept. of Molecular Cell Biology, University Hospital Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany. Tel.: 49-40-42803-3917; Fax: 49-40-42803-4592; E-mail: heeren@uke.uni-hamburg.de.
¶ Recipient of a fellowship from the Studienstiftung des Deutschen Volkes.
Recipient of Fellowship GRK 336 from the Deutsche
Forschungsgemeinschaft-financed Graduiertenkolleg.
Published, JBC Papers in Press, February 12, 2003, DOI 10.1074/jbc.M209006200
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ABBREVIATIONS |
---|
The abbreviations used are: CM, chylomicrons; AP1, adaptor protein 1; apo, apoprotein; ABCA1, ATP-binding cassette transporter A1; BSA, bovine serum albumin; Chol, cholesterol; CR, chylomicron remnants; EEA1, early endosome antigen 1; FPLC, fast performance liquid chromatography; HDL, high density lipoprotein; HuH7, human hepatoma cell line 7; LDL, low density lipoprotein; LFF, lipoprotein-free fraction; LPL, lipoprotein lipase; NBD-cholesterol, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)23,24-bisnor-5-cholen-3B-ol; TRL, triglyceride-rich lipoproteins; PBS, phosphate-buffered saline; PFA, paraformaldehyde; VLDL, very low density lipoproteins; DMEM, Dulbecco's modified Eagle's medium; SR-BI, scavenger receptor class B type I; w/o, without.
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