From The Lipoprotein & Atherosclerosis Group, University of Ottawa Heart Institute, Ottawa, Ontario K1Y 4W7, Canada and the ¶ Division of Cardiology, McGill University Health Center, Montreal, Quebec H3A 1A1, Canada
Received for publication, January 17, 2003
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ABSTRACT |
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Apolipoprotein AI (apoAI)-mediated cholesterol
efflux is a process by which cells export excess cellular cholesterol
to apoAI to form high density lipoprotein. ATP-binding cassette
protein A1 (ABCA1) has recently been identified as the key regulator of this process. The pathways of intracellular cholesterol
transport during efflux are largely unknown nor is the molecular
mechanism by which ABCA1 governs cholesterol efflux well understood.
Here, we report that, in both macrophages and fibroblasts, the
secretory vesicular transport changes in response to apoAI-mediated
cholesterol efflux. Vesicular transport from the Golgi to the plasma
membrane increased 2-fold during efflux. This increase in vesicular
transport during efflux was observed in both raft-poor and raft-rich
vesicle populations originated from the Golgi. Importantly, enhanced
vesicular transport in response to apoAI is absent in Tangier
fibroblasts, a cell type with deficient cholesterol efflux due to
functional ABCA1 mutations. These findings are consistent with an
efflux model whereby cholesterol is transported from the storage site to the plasma membrane via the Golgi. ABCA1 may influence cholesterol efflux in part by enhancing vesicular trafficking from the Golgi to the
plasma membrane.
ApoAI1-mediated
cholesterol efflux is one of the major events in "reverse cholesterol
transport," a process that generates HDL and transports excess
cholesterol from the peripheral tissues, including the arterial wall,
to the liver for biliary secretion. At the cellular level, apoAI is
capable of triggering a cascade of intracellular events, leading to
transport of stored cholesterol to the cell surface. ApoAI then
acquires lipids including cholesterol to form mature HDL.
ApoAI-mediated cholesterol efflux is thus the key step in the
maintenance of healthy levels of HDL in human. This process is absent
in a genetic disorder, Tangier disease, due to functional mutations in
ABCA1 (1). Tangier patients develop extensive lipid accumulations in
various tissues, extremely low HDL-C levels, and elevated risk for
coronary artery disease. ABCA1 is found predominantly in the
Golgi and the plasma membrane including endosomes in various cell
types. The molecular mechanism by which ABCA1 governs cholesterol
efflux is not well understood. Nor is the pathway by which cholesterol
is transported to the cell surface for efflux.
The net effect of apoAI-mediated efflux is to eliminate excess
cholesterol from the cell. Intracellular cholesterol transport during
the process is thought to be initiated from lipid droplets to the
plasma membrane (2). Cellular cholesterol plays a critical role in
several important cell functions, including protein trafficking, membrane vesiculation, and signal transduction (3). Mammalian cells
have developed highly sophisticated mechanisms to ensure adequate
cholesterol levels. Plasma membrane cholesterol content, for example,
is regulated through a feedback mechanism controlled by sterol
regulatory element binding protein-2 (SREBP-2) (4). SREBP-2 senses
membrane cholesterol levels and regulates the transcription of genes
encoding proteins required for endogenous cholesterol synthesis and
uptake of plasma lipoproteins. Under most circumstances SREBP-2 can
effectively control cellular cholesterol levels. There are, however,
situations in which large amounts of cholesterol are taken into cells
by other pathways involving receptors not regulated by SREBP-2. This
leads to accumulation of intracellular cholesterol. To maintain normal
cell function, most mammalian cells are capable of sequestering excess
cholesterol into the lipid droplets as a temporary relief. Cholesterol
may be used as additional energy source in the future. It is, however,
far more common in modern societies that the this storage system is overloaded, leading to macrophage foam cell formation with potentially lethal effects due to the build-up of unstable coronary artery plaque.
It is thus critical that the storage capacity be efficiently renewed by
the removal of excess cholesterol from cells. ApoAI-mediated efflux is
by far the most significant process to remove excess lipid, including
cholesterol, under physiological conditions. How stored cholesterol in
the lipid droplets reaches apoAI on the cell surface during this
process is not clear at present. As cholesterol is known to actively
shuttle between the lipid droplets and the endoplasmic reticulum (ER)
(2), it is likely that stored cholesterol is first released from lipid
droplets into the ER and from there to the cell surface to interact
with apoAI. The secretory vesicular pathway is thus likely involved in
transporting lipids including cholesterol to the plasma membrane. As
many lipids are known to be transported from the Golgi to the plasma
membrane exclusively through vesicular mechanisms, we hypothesized that
the Golgi may play a role in transporting newly released cholesterol to
the plasma membrane during apoAI-mediated efflux and ABCA1 may
influence this pathway.
We therefore took advantage of recent development in green
fluorescent proteins (GFP) techniques and designed a series of experiments to quantitatively measure whether vesicular transport in
secretory pathway is affected when cells are actively pumping cholesterol to apoAI on the cell surface. We also investigated whether
vesicular transport is affected in response to apoAI in Tangier cells
with functional mutations in ABCA1. We report here that there is
enhanced vesicular transport from the Golgi to the plasma membrane in
cells in which cholesterol efflux is elicited by exposure to apoAI.
This enhanced vesicular transport was not observed in Tangier cells.
Materials--
Brefeldin A (BFA) and
phosphatidylinositol-specific phospholipase C (PI-PLC) were purchased
from Molecular Probes. Yellow fluorescent protein-vesicular stomatitis
virus glycoprotein (YFP-VSVG) and yellow fluorescent
protein-glycosylphosphatidylinositol-anchored protein (YFP-GPI) in
adenoviral vectors were gifts from Drs. P. Keller and K. Simons,
EBI. Recombinant apoAI was obtained from Drs. R. Kiss and Y. Marcel, Ottawa University Heart Institute.
Cholesterol Efflux--
Cholesterol efflux was performed using
J774 macrophages and human primary fibroblasts (from a normal
individual and a Tangier patient). The Tangier patient is a compound
heterozygote in which one allele gives the mutation C1477R and the
other one is a Gly YFP-GPI and YFP-VSVG Transport Assay--
Macrophages and
fibroblasts were loaded with cholesterol and treated with cAMP/BSA over
night. Cells were then transfected with YFP proteins using adenoviral
vectors. For the transport from the Golgi to the plasma membrane,
transfected cells were incubated at 19.5 °C for 2-4 h to accumulate
YFP protein in the Golgi. Cycloheximide was added for the last 30 min of the incubation to stop further protein synthesis. Cells were
than moved to 32 °C to allow the transport from the Golgi to the
plasma membrane in the presence or absence of apoAI. For
quantitative transport measurement, cells were fixed at the end of the
32 °C incubation with 4% paraformaldehyde and analyzed by
quantitative fluorescent microscopy.
Fluorescent Microscopy and Imaging Analysis--
To measure the
transport of YFP proteins, fluorescent images of fixed cells were
acquired on an Olympus IX70 microscope using a 40× (NA 0.75) objective
and a 12-bit CCD digital camera IMAGO. For Golgi to plasma membrane
transport, an average fluorescent intensity in the Golgi area was
measured using TILLvisION software. After background correction, Golgi
fluorescence intensity was ratioed to the average fluorescent intensity
from whole cell to give an indication of the rate of transport in that
cell. For each treatment, 50-200 individual cells were imaged and quantified.
To test whether the Golgi is indeed involved in apoAI-mediated
cholesterol efflux, we first examined the effect of BFA in the process.
BFA disassembles the Golgi and redistributes it into the ER. This leads
to a complete inhibition in secretory vesicular transport (6). In J774
macrophages, we found that cholesterol efflux is sensitive to BFA. When
treated with BFA the Golgi was dissembled within 10 min (not shown),
and there was about 50% inhibition of apoAI-mediated cholesterol
efflux during 2 h following BFA treatment (Fig.
1a). Cholesterol efflux from
human primary fibroblasts was similarly affected and, as expected,
Tangier fibroblasts failed to produce any cholesterol efflux treated
with or without BFA (Fig. 1b). Prolonged BFA treatment (>4
h) produces much more severe inhibition in both macrophages and
fibroblasts (not shown), in agreement with previous observations (7).
BFA effects within 2 h are likely due to specific blockage of
vesicular transport. The inhibition by BFA thus suggests that
apoAI-mediated efflux involves a vesicular process in the secretory
pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Cys mutation at an exon/intron boundary,
causing a splice site mutation and thus a truncated mRNA (5). Cells
were cholesterol-loaded with acetyl-low density lipoprotein (75 µg/ml) for 1-2 days along with [3H]cholesterol. Cells
were then changed into medium containing 8-Br-cAMP (50 µM) and BSA (2 mg/ml) overnight. Cholesterol efflux was
induced by incubating cells with 10 µg/ml apoAI for various time. The
medium was then collected and counted for
[3H]cholesterol. In BFA experiments, cells were
preincubated 15 min with BFA (10 µg/ml) before apoAI. BFA was present
throughout the efflux period. Cells were then lysed and counted for
total cellular [3H]cholesterol. Efflux is presented as
percentage [3H]cholesterol in the medium of total
cellular [3H]cholesterol.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
ApoAI-mediated cholesterol efflux is
sensitive to BFA. Both macrophages (a) and skin
fibroblasts from normal and Tangier patients (b) were
cholesterol-loaded along with [3H]cholesterol and
incubated with apoAI for 2 h (macrophages) or 4 h
(fibroblasts). Medium was collected and counted for
[3H]cholesterol. ApoAI-specific cholesterol efflux was
obtained by subtracting nonspecific efflux induced by BSA. Data
represent the averages of triplicate experiments (mean ± S.D.).
Cholesterol is relatively enriched in the plasma membrane and in the
Golgi (8). It is believed that cholesterol/sphingomyelin-rich membrane
domains (rafts) are first formed in the Golgi and, from there,
transported to the plasma membrane. Golgi to plasma membrane vesicular
transport could be used to transport lipid including cholesterol to the
cell surface during apoAI-mediated efflux. We therefore measured the
rate of vesicular transport from the Golgi to the plasma membrane in
cells undergoing apoAI-mediated cholesterol efflux and compared it with
cells treated identically but without exposure to apoAI. In
non-polarized cells, membrane vesicles budding from the Golgi are known
to have at least two distinct populations, namely raft-rich and
raft-poor vesicles in terms of their membrane lipid compositions. Each
population of vesicles is known to carry a specific set of cargo
proteins. To accurately estimate overall membrane vesicular movement
from the Golgi to the plasma membrane, we chose two well characterized markers, a raft-associated YFP-GPI and a non-raft-associated YFP-VSVG. This allowed us to target both vesicle populations, respectively (9).
These proteins were expressed using adenovirus vectors and accumulated
in the Golgi by a 19.5 °C temperature block (Fig. 2a, top row). Upon
switching to 32 °C, YFP-GPI or YFP-VSVG entered budding vesicles,
exited the Golgi, and moved to the plasma membrane. The rate of
transport was measured in individual cells by quantifying YFP
fluorescence intensities in the Golgi as a function of time. In
cholesterol-loaded J774 macrophages, we found that, after switching temperature to 32 °C for 10 min, YFP fluorescent intensity in the
Golgi decreased more rapidly in cells undergoing efflux
(+apoAI), when compared with control cells
(apoAI). Correspondingly, there was more YFP-GPI on
the plasma membrane in cell exposed to apoAI (Fig. 2a,
bottom row, right panel). Quantitative analysis
from a large number of individual cells further confirmed that the transport of YFP-GPI from the Golgi to the plasma membrane was significantly increased in cells incubated with apoAI (Fig.
2b, gray line) relative to control cells (Fig.
2b, black line). T1/2 for YFP-GPI transport from the Golgi to the plasma membrane was about
10 min in the presence of apoAI and 20 min in the absence of apoAI.
This indicates that, when these macrophages are actively pumping lipids
including cholesterol out of the cells (+apoAI), vesicular
transport from the Golgi is up-regulated. As more vesicles are fused
with the plasma membrane, more lipids are delivered to the cell
surface.
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To further verify this, we performed similar experiments using YFP-GPI
in cholesterol-loaded normal primary fibroblasts. These cells are also
capable of mobilizing cholesterol when exposed to apoAI (Fig.
1b). Under these conditions (+apoAI), we indeed observed an enhanced Golgi to plasma membrane transport in comparison with cells under control conditions (apoAI) (Fig.
3a), similar to what was
observed in macrophages. To confirm that increased vesicular transport
in the presence of apoAI is related to cholesterol efflux, we then
measured Golgi to plasma membrane transport in Tangier fibroblasts.
These cells are incapable of apoAI-mediated cholesterol efflux due to
functional ABCA1 mutations (Fig. 1b). Indeed, we could not
detect any change in vesicular transport in Tangier cells when these
cells were incubated with apoAI (Fig. 3a). This observation
was further supported by biochemical analysis. YFP-GPI on the cell
surface can be cleaved by a membrane-impermeable PI-PLC to release YFP
to the medium (10). Fibroblasts were transfected with YFP-GPI and
underwent a 19.5 °C temperature block to accumulate YFP-GPI in the
Golgi. Cells were then switched to 32 °C for 20 min. PI-PLC was
applied to cleave the GPI protein that had reached cell surface during
32 °C incubation, and media were collected to quantify YFP using a
scanning spectrofluorometer. Consistent with our imaging analysis with
individual cells, we found twice as much YFP in the medium from
normal fibroblasts exposed to apoAI relative to control
(
apoAI) (Fig. 3b). Tangier cells again showed no response to apoAI (Fig. 3b). The increased YFP-GPI
transport from the Golgi to the plasma membrane in the presence of
apoAI is therefore associated with functional ABCA1 and cholesterol efflux.
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We also analyzed the rate of transport from the Golgi to the plasma
membrane using YFP-VSVG, a non-raft protein. YFP-VSVG was again
expressed in fibroblasts and incubated at 19.5 °C to accumulate
proteins in the Golgi. We then analyzed the exit rate of YFP-VSVG in
these cells after switching to 32 °C. We found that the transport of
YFP-VSVG was similarly increased in normal fibroblasts when cholesterol
efflux was induced by apoAI (Fig. 3c). Tangier fibroblasts
did not show any changes in vesicular transport rate upon apoAI
treatment. It is thus apparent that overall vesicular transport from
the Golgi to the plasma membrane is up-regulated during apoAI-mediated
cholesterol efflux. Both raft-rich and raft-poor populations of
secretory vesicles from the Golgi are likely involved in this process.
We therefore conclude that vesicular transport from the Golgi to the
plasma membrane may participate in transporting lipids, including
cholesterol, to apoAI on the cell surface. Functional ABCA1 is required
for this up-regulation of vesicular transport in response to apoAI.
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DISCUSSION |
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In the present study, we demonstrated that vesicular transport is responsive to apoAI-mediated cholesterol efflux. In particular, Golgi to plasma membrane transport is increased during this cholesterol export process. A functional ABCA1 or active cholesterol efflux is required for this enhancement as Tangier cells fail to show any alterations in vesicular transport. This conclusion was not only supported by two independent vesicular markers, YFP-GPI and YFP-VSVG, but also by both quantitative fluorescent imaging analysis and biochemical assays. Interestingly, both raft-rich (YFP-GPI) and raft-poor (YFP-VSVG) vesicle populations budding from the Golgi are similarly accelerated by apoAI-mediated cholesterol efflux, indicating a general accelerated membrane trafficking to the plasma membrane.
As lipids are the most abundant component of these transporting vesicles, increased vesicular transport means that more lipids including cholesterol are delivered to the plasma membrane during apoAI-mediated cholesterol efflux. Our results thus suggest that vesicular transport from the Golgi to the plasma membrane is one of the means for cells to move stored cholesterol to the cell surface where it can be transferred to the cholesterol acceptor, apoAI. It is unlikely, however, that this vesicular transport is the sole pathway responsible for cholesterol export, since BFA only partially inhibited cholesterol efflux. This suggests that other pathways must be working in parallel to supply lipid including cholesterol to the plasma membrane during efflux. Cholesterol, for instance, can traffic through the cytoplasm to different cellular locations via carrier proteins such as sterol-carrier protein 2 (SCP2) (11) or caveolin (12). This would provide a non-vesicular transport pathway to translocate intracellular stored cholesterol to the cell surface. This scenario is consistent with what is known for newly synthesized cholesterol transport (13). The relative contribution from different transport components, such as non-vesicular verses vesicular, may vary to facilitate the cholesterol flow during apoAI-mediated cholesterol efflux at a time when cells are transporting more cholesterol to the cell surface.
It is interesting that ABCA1 has a direct impact on the up-regulation of Golgi to plasma membrane trafficking during apoAI-mediated cholesterol efflux. Non-functional mutations in ABCA1 as in Tangier fibroblasts not only abolish cholesterol efflux, but also diminish apoAI-induced enhancement in vesicular transport from the Golgi to the plasma membrane. It is not clear how ABCA1 up-regulates vesicular transport from the Golgi. It is likely, however, that apoAI acquires lipids, including cholesterol, from the cell surface and this action depends on a functional ABCA1. Lipid depletion on the cell surface may initiate intracellular processes that signal cells to increase the delivery machinery required to supply more lipids including cholesterol to the plasma membrane. Vesicular transport from the Golgi is one of components of this process. Without effective cholesterol depletion on the cell surface as in Tangier cells, there may be no need to up-regulate the transport system. Alternatively, ABCA1 may directly influence membrane vesiculation process at the Golgi, especially when cells are under stimulated conditions, such as apoAI-mediated efflux. This would imply that apoAI might be able to initiate a signal transduction process from the cell surface to regulate ABCA1 function in the Golgi. ABCA1 has been identified as a phosphatidylserine flippase (14) and shown to influence vesiculation processes on the plasma membrane (15). Golgi membrane, especially the membrane in trans-Golgi network, is thought to share many characteristics, such as lipid content, with the plasma membrane. Thus, it would not be entirely surprising if ABCA1 actually functions in the Golgi to directly influence vesicular transport to the plasma membrane, even though the mechanism may not be identical as on the plasma membrane. This notion is further supported by the fact that a morphologically abnormal Golgi has been frequently observed in Tangier cells (15).
In summary, we have identified the involvement of the Golgi in
ABCA1-mediated cholesterol efflux. We demonstrate for the first time
that membrane trafficking from the Golgi is regulated under physiological conditions, such as apoAI-mediated efflux. As enhanced Golgi to plasma membrane trafficking likely represents only part of a
complex intracellular process, it will be now important to investigate
other cellular transport events that may interplay with the Golgi to
facilitate cholesterol transport to the cell surface during efflux.
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ACKNOWLEDGEMENTS |
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We acknowledge Drs. P. Keller and K. Simons for providing YFP-GPI and YFP-VSVG viral constructs. We are grateful to P. Lau for technical assistance. We also thank Dr. R. Milne for his critical comments.
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FOOTNOTES |
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* This work was supported by Canadian Institutes of Health Research (CIHR) Grant MOP-44360 (to R. M.) and by a Wyeth-Ayerst/CIHR Chair in Cardiovascular Disease (to R. M.).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 may be addressed. E-mail: rmcpherson@ottawaheart.ca or xzha@ohri.ca.
§ Present address: Ottawa Health Research Inst., 725 Parkdale Ave., Ottawa K1Y 4W9, Canada.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.C300024200
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ABBREVIATIONS |
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The abbreviations used are: apoAI, apolipoprotein AI; ABCA1, ATP-binding cassette protein A1; HDL, high density lipoprotein; SREBP, sterol regulatory element binding proteins; YFP, yellow fluorescent protein; VSVG, vesicular stomatitis virus glycoprotein; GPI, glycosylphosphatidylinositol; PI-PLC, phosphatidylinositol-specific phospholipase C; ER, endoplasmic reticulum; GFP, green fluorescent protein; BSA, bovine serum albumin; BFA, brefeldin A.
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