From the Department of Biochemistry, Cornell
University Medical School, New York, New York 10021, the
§ Institut Pasteur, 75724 Paris Cedex 15, France, the
Departments of ¶ Medicine and ¶¶ Anatomy and Cell
Biology, Columbia University, New York, New York 10032, the
Department of Medicine, Mt. Sinai School of Medicine, New York,
New York 10029, the ** Gladstone Foundation for Cardiovascular Research
and University of California, San Francisco, California 94141, the
Department of Genetics, Hadassah University
Hospital, Jerusalem 91120, Israel, and the
§§ Department of Biochemistry, Dartmouth Medical
School, Hanover, New Hampshire 03755
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ABSTRACT |
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Macrophages in atherosclerotic
lesions accumulate large amounts of cholesteryl-fatty acyl esters
("foam cell" formation) through the intracellular esterification of
cholesterol by acyl-coenzyme A:cholesterol
O-acyltransferase (ACAT). In this study, we sought to
determine the subcellular localization of ACAT in macrophages. Using
mouse peritoneal macrophages and immunofluorescence microscopy, we
found that a major portion of ACAT was in a dense reticular cytoplasmic
network and in the nuclear membrane that colocalized with the luminal
endoplasmic reticulum marker protein-disulfide isomerase (PDI) and that
was in a similar distribution as the membrane-bound endoplasmic
reticulum marker ribophorin. Remarkably, another portion of the
macrophage ACAT pattern did not overlap with PDI or ribophorin, but was
found in as yet unidentified cytoplasmic structures that were
juxtaposed to the nucleus. Compartments containing labeled -very low
density lipoprotein, an atherogenic lipoprotein, did not overlap with
the ACAT label, but rather were embedded in the dense reticular network
of ACAT. Furthermore, cell-surface biotinylation experiments revealed
that freshly harvested, non-attached macrophages, but not those
attached to tissue culture dishes, contained ~10-15% of ACAT on the
cell surface. In summary, ACAT was found in several sites in
macrophages: a cytoplasmic reticular/nuclear membrane site that
overlaps with PDI and ribophorin and has the characteristics of the
endoplasmic reticulum, a perinuclear cytoplasmic site that does not
overlap with PDI or ribophorin and may be another cytoplasmic structure
or possibly a unique subcompartment of the endoplasmic reticulum, and a
cell-surface site in non-attached macrophages. Understanding possible
physiological differences of ACAT in these locations may reveal an
important component of ACAT regulation and macrophage foam cell
formation.
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INTRODUCTION |
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Macrophages enter atherosclerotic lesions at an early stage and are found at all stages of lesion development thereafter (1). Several studies have provided evidence that macrophages play an important role in both lesion initiation and late lesion complications (2, 3). A major property of lesional macrophages is their tendency to become massively loaded with cholesteryl ester, a process known as foam cell formation because the numerous cholesteryl ester droplets give the cells a foamy appearance (1). The pathway by which macrophages accumulate cholesteryl ester is complex and not completely understood. Data from several laboratories suggest that macrophages internalize cholesterol through the uptake of certain "atherogenic" lipoproteins or by direct uptake of lipoprotein cholesterol. When the cellular cholesterol content increases to a certain threshold level, mixed pools of lipoprotein-derived and cellular cholesterol gain access to an integral membrane enzyme called ACAT,1 which then catalyzes the esterification of the cholesterol to fatty acid via a fatty acyl-CoA intermediate (4).
Given the importance of macrophage foam cells in atherosclerosis, there is much interest in how the cholesterol esterification pathway is regulated. Cholesterol esterification by ACAT increases manyfold when macrophages are incubated with atherogenic lipoproteins, yet ACAT mRNA and protein do not increase under these conditions (5-7). Rather, the major regulatory mechanism appears to involve access of membrane-bound ACAT to its hydrophobic substrate cholesterol (4, 7). When access to cholesterol is provided, ACAT activity increases by an allosteric mechanism as well as by substrate availability (4, 7). Therefore, understanding how intracellular pools of cholesterol contact ACAT is an important goal in elucidating the regulation of the cholesterol esterification pathway and foam cell formation.
To understand how ACAT gains access to cholesterol, it is necessary to determine the cellular location of ACAT. Studies have shown that plasma membrane cholesterol is a major source of substrate for ACAT (8, 9) and that plasma membrane vesiculation, perhaps as a means of transporting cholesterol to ACAT, is necessary for the stimulation of the cholesterol esterification pathway by atherogenic lipoproteins (10, 11). Biochemical studies employing subcellular fractionation of rat liver concluded that ACAT is located in the rough ER (12), and Chang et al. (13) published an image of melanoma cells in which the immunofluorescence pattern obtained with an anti-ACAT antibody appeared similar to the ER-like component of the pattern obtained with DiOC6, which stains mitochondria and ER. Given the inherent flaws of subcellular fractionation studies and the importance of the ACAT pathway in macrophages, we undertook a detailed examination of ACAT localization in macrophages. Herein, we report that much of ACAT in macrophages is in a dense reticular cytoplasmic network and in the nuclear membrane that colocalized with the luminal endoplasmic reticulum marker PDI and that was in a similar distribution as the membrane-bound endoplasmic reticulum marker ribophorin. A noticeable portion of ACAT in macrophages, however, resides in a perinuclear cytoplasmic site that does not overlap with PDI or ribophorin. Furthermore, using a cell-surface biotinylation protocol, we found that a portion of ACAT was on the cell surface of freshly harvested, non-attached peritoneal macrophages. These findings may have implications regarding the regulation of the cholesterol esterification pathway and macrophage foam cell formation.
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EXPERIMENTAL PROCEDURES |
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Materials--
Falcon and Corning tissue culture plasticware was
purchased from Fisher. Tissue culture media and reagents and calf serum were obtained from Life Technologies, Inc., and fetal bovine serum was
purchased from Gemini Bioproducts (Calabasas, CA). Mouse IgG2a and
anti-mouse CD12/CD32 Fc receptor antibody were from Chemicon International, Inc. (Temecula, CA) and Pharmingen (San Diego, CA),
respectively. The anti-ACAT antiserum used in our immunofluorescence studies was made by injecting rabbits with a fusion protein consisting of the amino-terminal 120 residues of murine ACAT and glutathione S-transferase (14). For the immunoblot experiments displayed in Fig. 5, we used an antiserum that was raised in rabbits against a
synthetic peptide consisting of the 40 C-terminal amino acids of human
ACAT (cf. Ref. 15). Chicken anti-PDI serum was a generous gift from Dr. Ron Raines (Department of Biochemistry, University of
Wisconsin, Madison, WI), and rabbit anti-ribophorin serum was kindly
provided by Dr. Gert Kreibich (Department of Cell Biology, New York
University Medical School, New York, NY). Rhodamine-conjugated anti-rabbit and anti-chicken IgG were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA), and Oregon green-conjugated anti-rabbit IgG was from Molecular Probes, Inc. (Eugene, OR). -VLDL
was prepared from the sera of rabbits fed a high fat, high cholesterol
diet (2% cholesterol and 10% soy bean oil) as described previously
(16) and conjugated to Texas Red (Molecular Probes, Inc.) according to
the manufacturer's instructions. All other reagents were purchased
from Sigma.
Cells--
Resident macrophages were harvested from the
peritonea of 25-30-g female ICR mice, C57BL6/129 hybrid mice from
Jackson Laboratories (Bar Harbor, ME), or ACAT knockout
(ACAT/
) mice in the C57BL6/129 hybrid background (17)
as described (18). For the immunofluorescence experiments, the
macrophages were plated on 35-mm dishes with a coverslip beneath a 7-mm
hole in the bottom (106 cells/dish) in Dulbecco's modified
Eagle's medium (high glucose) containing 10% fetal bovine serum, 292 µg/ml glutamine, 100 units/ml penicillin, and 100 µg/ml
streptomycin as described (19).
Immunofluorescence of Adherent Macrophages--
Macrophages were
washed with the supplemented Dulbecco's modified Eagle's medium
indicated above and fixed for 10 min either with cold methanol
(20 °C) on ice or with 2% paraformaldehyde in 150 mM
NaCl, 20 mM Hepes, 5 mM KCl, 1 mM
CaCl2, and 1 mM MgCl2, pH 7.4 (Medium 1), at room temperature, followed by three washes with Medium
1. After fixation, all treatments were performed at room temperature.
Paraformaldehyde-fixed cells were permeabilized for 15 min with 100 µg/ml saponin in Medium 1 containing 5 mM ammonium
chloride to react with aldehyde groups. Methanol- or paraformaldehyde-fixed cells were treated for 30 min with Medium 2 (Medium 1 plus 10% calf serum) containing 50 µg/ml purified mouse
IgG2a and 25 µg/ml purified anti-mouse CD12/CD32 Fc receptor antibody
(blocking solution) to inhibit the interactions of antibodies with Fc
receptors and to decrease nonspecific binding of antibodies. Primary
antibodies diluted in Medium 2 were applied for 1 h. The cells
were washed three times with Medium 1, incubated with
rhodamine-conjugated anti-rabbit or anti-chicken IgG at 6 µg/ml or
with Oregon green-conjugated anti-rabbit IgG at 20 µg/ml in Medium 2, and washed again three times with Medium 1. For double
immunofluorescence experiments, the labeling procedure was repeated
with the second set of antibodies.
Cell-surface Biotinylation Experiments-- We utilized a modification of the procedure of Rodriguez-Boulan and co-workers (20). Resident mouse peritoneal macrophages were freshly harvested from 14 female ICR mice (see above) and washed once with PBS by low speed centrifugation. One-half of the cells were incubated with 0.5 mg/ml NHS-SS-biotin (Pierce) for 40 min at 4 °C with gentle rotation, centrifuged at 1700 rpm for 5 min, and resuspended in PBS. The cells were repelleted and then resuspended in 50 mM ammonium acetate in PBS and incubated for 10 min at 4 °C. After washing twice with PBS, the cells were resuspended in RIPA buffer (0.5% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 20 mM Tris, 150 mM NaCl, and 5 mM EDTA, pH 8) containing 0.2% bovine serum albumin and protease inhibitors. After incubation for 30 min, the lysate was centrifuged (13,000 × g for 10 min). The supernatant was precleared with unconjugated agarose (CL-6B-200, Sigma), which had been prewashed three times with RIPA buffer, for 2 h with rotating. The agarose was pelleted (13,000 × g for 10 s), and the supernatant was added to immunopure immobilized streptavidin (Pierce), which had been prewashed with RIPA buffer, and incubated with gentle rotation for 18 h. The precipitates were then centrifuged at 13,000 × g for 10 s; the supernatants were removed; and the agarose was resuspended in RIPA buffer. This washing step was repeated twice with RIPA buffer, twice with RIPA buffer containing 500 mM NaCl, and three times with PBS. The agarose was resuspended with an appropriate volume of buffer containing 1% SDS and 10 mM dithiothreitol, heated to 37 °C for 30 min, centrifuged at 13,000 × g for 45 s, and subjected to immunoblot analysis.
To assess total membrane-bound ACAT in the macrophages for comparison on the immunoblot, the other half of the macrophages were disrupted at 4 °C by sonication (Model 450, Branson Ultrasonics Corp., Danbury, CT) in PBS containing 0.5 mg/ml NHS-SS-biotin and protease inhibitors (1 mM phenylmethylsulfonyl fluoride and 10 µg/ml each aprotinin, leupeptin, and pepstatin). After 40 min of incubation at 4 °C, the macrophages were spun at 100,000 × g for 45 min at 4 °C to pellet cellular membranes. The pellet was resuspended in 50 mM ammonium acetate in PBS (quenching buffer) containing protease inhibitors, incubated for 10 min, and centrifuged again at 100,000 × g for 45 min. The final membrane pellet was resuspended with an appropriate volume of buffer containing 1% SDS and 10 mM dithiothreitol, heated to 37 °C for 30 min, centrifuged at 13,000 × g for 45 s, and subjected to immunoblot analysis.SDS-Polyacrylamide Gel Electrophoresis and
Immunoblotting--
Samples were electrophoresed on 4-20% gradient
polyacrylamide gels, electrotransferred to nitrocellulose, and
immunoblotted as described using enhanced chemiluminescence reagent
(Pierce Super Signal kit) (21). The primary antibodies used were rabbit anti-ACAT (1:1000), anti-1-integrin (1:3000),
anti-ribophorin (1:3000), and chicken anti-PDI (1:3000); the secondary
antibodies were horseradish peroxidase-conjugated goat anti-rabbit and
anti-chicken IgG (1:20,000).
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RESULTS |
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ACAT and PDI Immunofluorescence Studies in Mouse Peritoneal
Macrophages--
To determine the specificity of the anti-ACAT
antibody for our immunofluorescence studies, we used confocal
microscopy to compare the labeling pattern of adherent peritoneal
macrophages from wild-type mice with that of macrophages from
ACAT/
mice. In 12 0.6-µm-thick confocal projections,
we observed that wild-type macrophages labeled with the anti-ACAT serum
exhibited a bright pattern of staining that included the nuclear
envelope and a dense reticular network throughout the cell (Fig.
1B). In contrast, the staining
of these cells with preimmune serum generated a very dim and diffuse
fluorescent signal (Fig. 1A). The data for
ACAT
/
macrophages are shown in Fig. 1 (C
(preimmune) and D (immune)). In both cases, the staining was
dim and diffuse, similar to the preimmune staining of wild-type
macrophages. These results show that the anti-ACAT serum is specific
for the native ACAT protein and can be used in immunofluorescence
experiments to determine the localization of ACAT.
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Localization of ACAT and -VLDL in Macrophages--
In previous
work, we showed that a portion of the atherogenic lipoprotein
-VLDL
resides in deep, wide cell-surface invaginations, called STEMs
(surface tubules for entry into
macrophages), when incubated with macrophages
for 5-20 min (25). Another foam cell-inducing lipoprotein, acetyl low
density lipoprotein, also demonstrates prolonged association with the
surface of macrophages (26, 27). Localization of
-VLDL in STEMs and
possibly acetyl low density lipoproteins in their cell-surface sites
may be important for the ability of these lipoproteins to stimulate the
cholesterol esterification pathway in macrophages (28). To determine if
-VLDL-containing structures in macrophages are in close proximity to
ACAT, we performed double-label fluorescence microscopy. Macrophages were incubated with Texas Red-conjugated
-VLDL for 10 min before fixing the cells for the immunolocalization of ACAT. We found no
overlap of the ACAT label (Fig. 4,
A and D) with the
-VLDL label (Fig. 4,
B and E), which appeared to be contained in
discrete structures (Fig. 4, C and F); in fact,
the
-VLDL-containing structures often appeared as holes in the ACAT
labeling (G, arrows). Nonetheless, labeled
-VLDL particles were often found in close proximity to the reticular
structures containing ACAT (Fig. 4, C and F).
Thus, while there was no specific concentration of ACAT in or around the
-VLDL structure, the dense network of the ACAT-containing ER
surrounds
-VLDL-containing STEMs and endosomes.
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Evidence That a Portion of ACAT is on the Cell Surface in Freshly
Harvested, Non-attached Macrophages--
Plasma membrane cholesterol
is a major source of substrate for ACAT (8, 9), and foam cell-inducing
lipoproteins demonstrate prolonged association with the surface of
macrophages (see above). Therefore, we sought to determine whether a
portion of ACAT might be localized on the cell surface. Initial
immunofluorescence studies in which the antibodies were added to
non-fixed and non-permeabilized macrophages at 4 °C were difficult
to interpret due to a high level of nonspecific staining
(i.e. staining found in ACAT/
cells). To
rectify this problem, we conducted a series of cell-surface biotinylation/immunoblot experiments (cf. Ref. 20). In this protocol, intact cells are incubated with a biotinylation reagent at
4 °C to derivatize cell-surface, but not intracellular, proteins. After the biotinylation reagent is quenched, the cells are then solubilized with detergent, precipitated with immobilized streptavidin, and subjected to immunoblot analysis. Most important, the immunoblot analysis includes antibodies against known intracellular proteins to
prove that the biotinylation reagent did not have access to the
intracellular compartment. As a means of comparison, we also performed
immunoblot analysis of biotinylated membranes from a total cell
homogenate; these membranes were biotinylated to control for any
differences in immunoreactivity due to antigen derivatization by
biotin. For some biotinylation experiments, we used the ACAT N-terminal
antibody employed in our immunofluorescence experiments (see above),
although we found that biotinylation of ACAT caused a decrease in
immunoreactivity with this antibody. Another antibody that was raised
against a C-terminal peptide of ACAT, however, reacted equally well
with underivatized and biotinylated ACAT (data not shown), and so we
used this antiserum for the experiments described below.
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DISCUSSION |
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The ACAT pathway in macrophages plays a key role in foam cell formation during atherogenesis, and previous work from our laboratories and others has suggested that determining the intracellular location of ACAT in macrophages may provide important clues to its regulation. Herein, we show that much of ACAT is in a dense, widespread reticular network throughout the cytoplasm that overlaps with the luminal ER marker PDI and that has a pattern similar to that of the membrane ER marker ribophorin. In addition, our studies have demonstrated two surprising observations. First, a portion of ACAT in most of the cells we observed was present in a perinuclear cytoplasmic site that does not overlap with PDI or ribophorin. Second, 10-15% of ACAT in non-attached macrophages was present on the cell surface.
The finding that much of ACAT is in the ER of macrophages allows us to focus our hypotheses on how atherogenic lipoproteins stimulate the cholesterol esterification pathway. ACAT esterifies mostly plasma membrane-derived cholesterol after cellular cholesterol reaches a threshold level (30), and vesicular transport is important in this process (10, 11). Taken together, these observations suggest a model in which plasma membrane-derived vesicles "percolate" through the dense network of the cholesterol-poor, ACAT-containing ER and, if the cholesterol content of these vesicles is high enough (i.e. above threshold), cholesterol is transferred to ACAT down a concentration gradient.
The nature of the ACAT-containing perinuclear site that is distinct from PDI and ribophorin remains to be identified. Although this site could be a specialization of the ER, almost all subcompartments of the ER, including ACAT-rich mitochondrion-associated membranes (31), contain PDI (23).2 Therefore, it is possible that the site is one of several non-ER organelles that concentrate near the nucleus, such as the Golgi apparatus, the endocytic recycling compartment, late endosomes, or lysosomes. As mentioned above, most of the cholesterol substrate for ACAT is derived from the plasma membrane; recent data, however, indicate that a smaller portion may be delivered directly from lysosomes or late endosomes following the endocytosis of lipoproteins (32, 33). It is therefore possible that perinuclear ACAT, which is in close proximity to perinuclear lysosomes, may be a subpopulation of the enzyme that esterifies lysosomal free cholesterol. This idea and other possible functions of ACAT in the perinuclear site will be further evaluated once this compartment is definitively identified.
The finding that a portion of cellular ACAT is on the cell surface of non-attached macrophages, which may be consistent with the findings of Green et al. (34) using Xenopus oocytes transfected with ACAT cDNA, raises two important questions: what is the mechanism whereby cell-surface ACAT disappears upon macrophage attachment, and what is the functional importance of ACAT in this site? The disappearance of cell-surface ACAT upon attachment is not due to redistribution of the protein to the basal surface of the cell (see "Results"). One possibility is that one or more signaling pathways known to be activated by cellular attachment to the matrix (e.g. Ref. 35) lead to the internalization of cell-surface ACAT. In terms of function, it will be important to determine if ACAT stimulation by a variety of stimuli (cf. Refs. 10 and 36) differs between attached and non-attached macrophages. Another critical issue related to our finding is whether the active site of ACAT faces the intracellular or extracellular space; studies in rat liver led to the conclusion that the active site of microsomal ACAT faces the cytoplasm (37). Although the proportion of cell-surface ACAT is only 10-15% in non-attached macrophages, ACAT in this site may be functionally important since most of the cholesterol esterified by ACAT is derived from plasma membrane pools (see above), and thus, cell-surface ACAT may provide a direct route for cholesteryl ester synthesis under certain conditions.
Several studies from our (16, 25, 26) and other (27) laboratories have
shown that foam cell-inducing lipoproteins undergo prolonged contact
with the surface of macrophages, an event that may be important in the
stimulation of the cholesterol esterification pathway (28). In this
report, we examined the relationship in location between one such
lipoprotein, -VLDL, and ACAT. We know from previous work that a
10-min incubation of mouse peritoneal macrophages with
-VLDL results
in much of the
-VLDL being contained in deep, wide cell-surface
invaginations called STEMs (25). Herein, we show that "reticular"
ACAT (see above) is not contained in these
-VLDL-containing
structures, but rather seems to "wrap" around them. From these
data, it is tempting to speculate that
-VLDL-derived free
cholesterol may be transferred from these structures to nearby ACAT,
analogous to and perhaps complementary with the plasma membrane-derived vesicle hypothesis mentioned above. Future fluorescence studies will be
directed at determining the trafficking of cholesterol derived from
-VLDL and other atherogenic lipoproteins to ACAT in the cellular
sites described in this work.
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ACKNOWLEDGEMENTS |
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We thank Dr. Ron Raines for the anti-PDI antibody and Dr. Gert Kreibich for the anti-ribophorin antiserum.
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FOOTNOTES |
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* This work was supported by NHLBI Grant HL-57560 from the National Institutes of Health (to I. T. and F. R. M.) and by Grant-in-Aid 95-239 from the American Heart Association (to R. V. F.). The Columbia University Confocal Microscope Facility used for this study was established by NIH Shared Instrument Grant #1S10 RR10506 and is supported by NIH Grant #5 P30 CA13696 as part of the Herbert Irving Cancer Center at Columbia University.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 and reprint requests should be addressed: Dept. of Medicine, Columbia University, 630 West 168th St., New York, NY 10032. Tel.: 212-305-9430; Fax: 212-305-5052; E-mail: iat1{at}columbia.edu.
1
The abbreviations used are: ACAT, acyl-coenzyme
A:cholesterol O-acyltransferase; ER, endoplasmic reticulum;
PDI, protein-disulfide isomerase; -VLDL,
-very low density
lipoprotein; PBS, phosphate-buffered saline; RIPA, radioimmune
precipitation assay.
2 J. E. Vance, personal communication.
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REFERENCES |
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