Centre de Recherche, Hôpital Sainte-Justine, Université de
Montréal, Montréal, Québec, Canada
1 Department of Nutrition, Université de Montréal,
Montréal, Québec, Canada
2 Department of Pediatrics, Université de Montréal,
Montréal, Québec, Canada
* Author for correspondence (e-mail: levye{at}justine.umontreal.ca)
Accepted 28 August 2002
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Summary |
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Key words: Reverse cholesterol, Modified HDL, SR-BI
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Introduction |
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In contrast to LDL, elevated plasma levels of HDL lower the risk of
clinically significant coronary events
(Gordon et al., 1977;
Gordon et al., 1989
). Their
beneficial anti-atherogenic actions stem from their involvement in the removal
of excess cholesterol from extrahepatic cells for transport to and deposition
in the liver, where cholesterol is excreted in the form of bile acids
(Levy et al., 1996
;
Pieters et al., 1991
). HDL has
other potential anti-atherogenic properties, such as its ability to inhibit
LDL oxidation (Parthasarathy et al.,
1990
), cytotoxicity of oxLDL
(Hessler et al., 1979
;
Suc et al., 1997
) and monocyte
transmigration (Navab et al.,
1991
). However, HDL contains the majority of lipid peroxides in
the plasma and is also susceptible to oxidative modifications
(Bowry et al., 1992
;
Hahn and Subbiah, 1994
;
Salmon et al., 1992
). Whether
or not oxidative modification changes the antiatherogenic effects of HDL is
under debate. On the one hand, it can alter the conformation and antigenicity
of apo A-I, impair the ability of HDL to promote cholesterol efflux
(Nagano et al., 1991
;
Morel, 1994
;
Salmon et al., 1992
), reduce
HDL binding to specific receptors
(Mazière et al., 1993
)
and suppress cholesterol biosynthesis
(Ghiselli et al., 1992
). On
the other hand, various investigators have reported that oxidative
modification of HDL is less toxic than that of LDL
(Alomar et al., 1992
), reduces
TNF-
secretion from human macrophages and, thus, the inflammatory
process (Girona et al., 1997
),
and enhances cholesterol efflux (Francis
et al., 1993
; Wang et al.,
1998
). Such discrepancies in the aforementioned papers may depend
on methodological variations in the isolation, storage and dosage of HDL or
the cell model utilized.
Previously, it was emphasized that the generation of tyrosyl radicals by
myeloperoxidase represents one physiologically plausible mechanism for the
oxidation of lipoproteins in vivo. In fact, the likelihood of lipoprotein
oxidation in vivo by this model is supported by the presence of high levels of
myeloperoxidase and protein-bound dityrosine in human atherosclerotic lesions
(Daugherty et al., 1994;
Leeuwenburgh et al., 1997
;
Mazière et al., 1993
),
as well as the occurrence of micromolar concentrations of L-tyrosine in plasma
(Mitchell et al., 1995
) and
tyrosyl-radical-modified LDL in the artery wall
(Leeuwenburgh et al., 1997
).
Although tyrosyl-radical-oxidized HDL in vitro was shown to deplete
cholesterol content in human skin fibroblasts and in mouse peritoneal
macrophages (Francis et al.,
1993
; Wang et al.,
1998
), our own data demonstrated its ability to hamper in vivo
reverse cholesterol transport, that is, the transfer of cholesterol to the
liver and its transformation into bile acids
(Guertin et al., 1997
) in a
similar fashion to malondialdehyde-modified HDL
(Guertin et al., 1994
).
Therefore, it is plausible that tyrosylation changes in HDL apoA-I may be
detrimental to cholesterol efflux.
The fate of lipoproteins is largely determined by the interaction between
their constituent apolipoprotein moieties and the cellular receptors and
enzymes involved in their lipid metabolism. Although a number of HDL-binding
proteins has been described (Johnson et
al., 1991; Oram and Yokoyama,
1996
), their physiological role in reverse cholesterol transport
remains uncertain, since none has been shown to be a functional HDL receptor
that facilitates cholesterol efflux. Only recently has the scavenger receptor
type B class I (SR-BI) been convincingly shown to bind HDL with high affinity
and to mediate the selective cellular uptake and efflux of HDL cholesterol
(Acton et al., 1996
;
Ji et al., 1997
).
Interestingly, scavenger receptors are unique with respect to their ability to
interact with both native and oxidized LDL
(Rigotti et al., 1995
).
Whether or not they can act as receptors for tyrosylated HDL has never been
assessed.
In the current study, we examined the effects of enzymatically generated tyrosylation on the physiochemical and metabolic properties of HDL. More specifically, we tested cholesterol removal from cultured mouse macrophages of the J774-A1 cell line as well as the uptake and internalization of HDL-cholesterol. We also explored the receptors involved in the binding of tyrosylated HDL and the mechanism for cell cholesteryl ester depletion.
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Materials and Methods |
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Isolation and modification of lipoproteins
Human LDL (d=1.019 to 1.063 g/ml), total HDL (d=1.063 to 1.210 g/ml) and
HDL3 fraction (d=1.125 to 1.210 g/ml) were prepared from plasma of
healthy human subjects, isolated by differential ultracentrifugation
(Havel et al., 1955) and
dialyzed intensively against PBS (pH 7.4) containing 150 mM NaCl and 0.3 mM
ethylene diaminetetraacetic acid (EDTA). HDL3 (1 mg apoA-I/ml) was
tyrosylated in the presence of horseradish peroxidase (100 nM),
H2O2 (100 µM) and L-tyrosine (100 µM) for 24 hours
at 37°C according to the method Francis et al.
(Francis et al., 1993
). Acetyl
LDL (AcLDL) was prepared by the addition of acetic anhydride to normal LDL as
described elsewhere (Basu et al.,
1976
), and its modification was verified by its mobility on
agarose gel electrophoresis (Paragon, Beckman Instruments). In order to
generate oxidized LDL (oxLDL), plasma LDL (3 mg apo B/ml) was extensively
dialyzed against PBS (pH 7.4) containing 150 mM NaCl and 5 µM EDTA and then
incubated with 10 µM CuSO4 for 18 hours at 37°C. The
peroxidation level of oxLDL was determined by the measurement of its TBARS
content (Yagi, 1987
). All
lipoproteins were filtered through a 0.2 µM Millipore membrane and stored
at 4°C. ApoB and apoA-I concentrations were determined by
immunonephelometry (Behring system) (Havel
et al., 1955
).
Labeling of lipoproteins
AcLDL was labeled with [3H]cholesteryl oleate (10 µCi/mg of
apoB) as described previously (Guertin et
al., 1994; Guertin et al.,
1997
; Terpstra et al.,
1989
). The labeling of normal and tyrosylated HDL3 with
[3H]cholesteryl hexadecyl ether (10 µCi/mg of apoA-I) was
carried out as reported previously
(Guertin et al., 1994
;
Guertin et al., 1997
;
Terpstra et al., 1989
). Normal
and tyrosylated HDL3 (10 mg apoA-I) were labeled with the
fluorescent probe Oregon Green 514 carboxylic acid, succinimidyl ester (0.5
mg) (Oregon-HDL3) according to the manufacturer's instructions
(Molecular Probes, Inc Eugene, OR). The labeling efficiency between normal and
tyrosylated HDL3 was evaluated by spectrofluorimetry, which
revealed no marked differences in their Oregon Green 514 carboxylic acid
content. Succunimidyl ester reacted with the primary amines of the two major
HDL apolipoproteins (A-I and A-II). By contrast, [3H]cholesteryl
hexadecyl ether was incorporated in HDL3 and served to measure
cellular cholesteryl ester-HDL3 uptake. All lipoproteins were
sterilized by filtration (0.2 µM Millipore membrane), stored at 4°C in
the dark and used within 2 weeks of their preparation. Native and tyrosylated
Oregon-HDL3, as well as HDL3-[3H]cholesteryl
hexadecyl ether showed no evidence of oxidative modifications compared with
non-labeled HDL3 since (a) the electerophoretic mobility of nascent
and tyrosylated HDL was similar on agarose gel; (b) there was no apparent
degradation of apolipoproteins when nascent and tyrosylated HDL were applied
on SDS-PAGE; and (C) the lipid and protein composition of nascent and
tyrosylated was not altered.
Cell culture
J774-A1 cells were grown and maintained in 25 cm2 flasks with
DMEM containing 10% (vol/vol) Fetal Calf Serum, penicillin (100 U/ml) and
streptomycin (100 µg/ml) in 5% CO2 and humidified air at
37°C. For neutral cholesteryl ester hydrolase (nCEH) activity, cells were
seeded on 25 cm2 flask (2x106 cells/flask). In
order to assess cellular HDL3-apolipoprotein association and
cholesterol uptake, cells were seeded in 12-well plates at
0.2x106 cells per well. Similar experimental conditions were
applied while testing the blocking effects of anti-scavenger receptor BI and
BII antibody, except that 24-well plates were employed. On day two, all cells
were cholesterol loaded during 24 hours in DMEM containing antibiotics, 5%
LPDS and AcLDL (50 µg apoB /ml). On day three, medium was removed and cells
were washed three times with DMEM (containing 5% LPDS and antibiotics) and
then incubated in the same medium overnight to equilibrate intracellular
cholesterol pool. After two washes with DMEM (containing antibiotics and 5%
LPDS), cells were used for the different experiments.
Cholesterol efflux
J774-A1 cells were cultured in six-well plates at 0.8x106
cells per well and loaded with radiolabeled cholesterol by incubation for 24
hours in 1 ml of DMEM containing antibiotics, 5% LPDS and
7.4x105 DPM/ml [3H]cholesteryl oleate AcLDL (50
µg apoB/ml). Under the conditions used, approximately 0.6 pmol of labeled
cholesterol was incorporated per incubation dish. Cells were then treated as
mentioned above and incubated for 24 hours in DMEM supplemented with 5% LPDS
in the absence (control) or presence of different concentrations of normal or
tyrosylated total HDL or HDL3. The media were centrifuged at 4000
g for 10 minutes to remove any suspended or dead cells. The
cells were washed three times with PBS, resuspended in 1 ml of distillated
water and homogenized by sonication on ice. Cell and medium lipids were
extracted by the method of Folch (Folch et
al., 1957). The organic phase was dried down under nitrogen, and
the radioactivity resuspended in a small volume of chloroform and fractionated
by thin layer chromatography (TLC) on silica gel plates using a solvent system
composed of hexane:ethyl-ether:acetic-acid (80:20:3, vol/vol). Cholesteryl
ester and free cholesterol radioactivity in cells and media were measured in a
liquid scintillation ß counter (Beckman, LS 5000 TD). Protein
concentration was determined in cell extracts by Bio-Rad Protein Assay kit
with BSA as a standard. The net efflux of [3H] cholesterol mediated
by normal or tyrosylated HDL types was expressed as a percentage of the amount
of labeled cholesteryl ester and free cholesterol released to the medium,
relative to total label in each well (medium plus cells), per amount of cell
protein. All determinations were carried out in triplicate.
Assay of nCEH activity
An assay of nCEH activity was carried out as described by Holm and
Osterlund (Holm and Osterlund,
1999). Cells were incubated in DMEM containing antibiotics and 5%
LPDS in the absence (control) or presence of different concentrations of
normal or tyrosylated HDL3 for 24 hours. After three washes with
cold PBS, cells were homogenized in Tris-HCl buffer (50 mM, pH 7.0) containing
sucrose (250 mM), EDTA (1 mM), dithiothreitol (DTT) (1 mM), leupeptine (20
µg/ml) and pepstatine (1 µg/ml) with a Sonifier microtip at the lowest
power setting (3x10 seconds on ice). Cytosolic cell extracts were
obtained by ultracentrifugation for 30 minutes at 43,000 g at
4°C. The supernatant was used as the nCEH enzyme solution. Protein content
was determined with the Bio-Rad protein assay kit (Bio-Rad Laboratories) with
BSA as a standard. The 0.2 ml nCEH reaction contained 50 mM potassium
phosphate, pH 7.0, 0.5 mM EDTA, 0.5 mM DTT, 25 mg/ml fatty acidfree BSA, 8.04
µM cholesteryl [1-14C] oleate (2.105 dpm, 1.61 nmol),
177.5 µg/ml phosphatidylcholine/phosphatidylinisitol (3:1, wt/wt) and
freshly sonicated cytosolic cell extract (
4 mg protein/ml, 100 µl).
After incubation for 60 minutes at 37°C, the [14C] oleate
released was extracted by the addition of 3.25 ml of the mixture
methanol:chloroform:heptane (10:9:7) containing non labeled oleic acid as a
carrier (3 g/l) and 1.05 ml of 0.1 M potassium carbonate, 0.1 M boric acid, pH
10.5 (Belfrage and Vaughan,
1969
). The reaction tubes were shaken vigorously for 1 minute and
centrifuged at 1500 g for 10 minutes at room temperature.
Radioactivity of the upper layer was counted. One unit of enzyme hydrolyzes 1
µmol of cholesterol ester in 1 minute. Results are expressed as a
percentage of the control without HDL3.
Oregon-HDL3 binding and association assays
Cells were incubated for 3 hours at 37°C in 0.5 ml of DMEM supplemented
with antibiotics and 5% LPDS containing the indicated final concentrations of
native or tyrosylated Oregon-HDL3 or with 25 µg/ml of native or
tyrosylated Oregon-HDL3 for the indicated times. To determine the
level of non-specific binding, cells and Oregon-HDL3 (10 µg/ml
to 50 µg Apo A-I/ml) were incubated with a 50-fold excess of the
corresponding unlabeled HDL3. After incubation, cells were washed
twice with cold DMEM-5% LPDS and two times with cold PBS, detached from the
plate by gentle pipetting and immediately subjected to fluorescence flow
cytometry using a FACScan from Becton Dickinson. The forward-angle
light-scatter gates were established to exclude debris and cellular
aggregation. At least 10,000 cells were analyzed for each sample.
Cell-associated and bound lipoprotein were expressed as a median intensity of
fluorescence (MIF) assessed using fluorescence windows (channel numbers).
Values for Oregon-HDL3 binding and association were obtained by
subtracting non-specific background. For inhibition experiments, unlabeled
lipoproteins, polyinosinic acid (poly-I) and the labeled-HDL3 were
added simultaneously to the cells. A blocking anti-scavenger receptor BI and
BII polyclonal antibody was preincubated for 30 minutes with cells before the
addition of labeled-HDL3. Rabbit IgG was used at the same
concentration as a negative control. Experiments were done several times using
different lipoprotein preparations.
Fluorescence microscopy
Following the incubation of cells for 3 and 6 hours with 100 µg apo
A-I/ml (Oregon-HDL3) at 37°C, the coverslips were rinsed, fixed
for 15 minutes with 3% formaldehyde and photographed using a fluorescence
microscope equipped with a fluorescein isothiocyanate filter set.
Cholesterol-HDL3 uptake
Cells were incubated for 3 hours at 37°C in 0.5 ml of DMEM supplemented
with antibiotics and 5% LPDS containing the indicated final concentrations of
native or tyrosylated [3H]cholesteryl hexadecyl
ether-HDL3 or with 25 µg apoA-I/ml of native or tyrosylated
[3H]cholesteryl hexadecyl ether-HDL3 for the indicated
times. To determine the non-specific binding, cells and
labeled-HDL3 (10-50 µg/ml of apoA-I) were incubated in the
presence of a 50-fold excess of the corresponding unlabeled HDL3.
After incubation, cells were washed twice with cold DMEM containing 5% LPDS
and twice with cold PBS, detached from the plate by gentle pipetting with 1 ml
of distillated water containing 0.25% Triton and homogenized by sonication on
ice (3x10 seconds, lowest power setting). For each well, radioactivity
was counted, and protein was determined by Bio-Rad protein assay kit with BSA
as a standard. Results are expressed in DPM/mg cell protein. The specific
uptake of native or tyrosylated [3H]cholesteryl hexadecyl
ether-HDL3 by cells was calculated by subtracting the non-specific
value. For inhibition experiments, unlabeled lipoproteins, Poly-I and the
labeled-HDL3 were added to the cells. Only the blocking
anti-scavenger receptor BI and BII polyclonal antibody was preincubated for 30
minutes with cells before the addition of labeled HDL3. Experiments
were done several times using different lipoprotein preparations.
SDS-polyacrylamide gel electrophoresis
The apolipoprotein content of nascent and tyrosylated HDL was examined by
electrophoresis on sodium dodecylsulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), as described previously (Levy
et al., 1994). The gels were stained with Coomassie blue and
destained in 70% acetic acid.
Statistical analysis
Data from the experiments were analyzed by using a student's
t-test. Reported values are expressed as means±s.e.
Statistical significance was accepted at P<0.05.
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Results |
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Cholesterol efflux
Evidence suggests that HDL promotes cholesterol efflux from cells, which
constitutes the rate-limiting step in the reverse cholesterol pathway
(Pieters et al., 1991;
Levy et al., 1996
). Thus, in
the next series of experiments, we assessed the capacity of tyrosylated HDL
for cholesterol removal from J774-A1 macrophages pre-loaded with radiolabeled
AcLDL-[3H]cholesteryl oleyl ester in 5% LPDS-DMEM for 24 hours. The
findings shown in Fig. 2
indicate that tyrosine modification resulted in impaired tyrosylated
HDL3- or total HDL-mediated cholesterol efflux from J774-A1 cells.
It was evident that at all concentrations between 50-200 µg apoA-I/ml a
diminished efflux occurred regardless of whether the tyrosylated total HDL
(Fig. 2A) or HDL3
(Fig. 2B) subfraction was used
as the cholesterol acceptor. Starting from 11.2±1.4% (control values in
the absence of lipoprotein acceptors), the maximal efficacy was obtained at
nearly 100 µg/ml of apoA-I for both native (28.86±0.51%) and
tyrosylated (21.14±0.84%) HDL3. For the same apoA-I
concentration, native and tyrosylated total HDL appeared to extract more
cholesterol. Calculated from the specific activity of cholesterol loading of
the cells, the difference in the efflux rate between native and modified total
HDL at 100 µg apoA-I/ml was 0.12 pmol cholesterol efflux per 24 hours,
whereas that between native and tyrosylated HDL3 was 0.17 pmol
cholesterol efflux per 24 hours. Therefore, only the HDL3 fraction
was used for the following studies. From these experiments, we concluded that
the capacity of tyrosylated HDL for cholesterol efflux from J774-A1
macrophages was weaker than that of native HDL.
|
In our experiments, both free (FC) and esterified (EC) cholesterol were measured in the medium for efflux studies. The average ratio of CE/FC was 0.21 for controls (absence of lipoprotein acceptors), 0.15 for native HDL and 0.07 for tyrosylated HDL.
HDL3 cell association
Since interaction of apolipoprotein with specific membrane receptors is
required for the mobilization of cellular cholesteryl ester, we verified
whether tyrosylation altered HDL3 recognition by J774-A1 macrophage
membrane-binding domains. Indeed, defective interaction between tyrosylated
HDL3 and cells could affect the effluxing capacity of
HDL3. To this end, we covalently labeled apolipoprotein moieties of
native and tyrosylated HDL3 with the fluorescent dye Oregon Green
514 to study HDL3 cell association by flow cytometry, after
cellular cholesterol loading with AcLDL.
Fig. 3A represents the results
of the time course studies carried out with J774-A1 macrophages incubated with
25 µg apoA-I/ml of Oregon-HDL3. Under our experimental
conditions, the estimation of HDL3 cell association by the Oregon
technique revealed an increased association of tyrosylated HDL3
with cells compared with native HDL3 at the time points analyzed.
In fact, two- to fourfold enhancement occurred at all incubation periods. Cell
association was also tested as a function of increasing HDL3
concentrations (Fig. 3B) and
found to be directly proportional to Oregon intensity. Again, similar results
were obtained showing that tyrosylated HDL3 cell association was
two- to threeold increased in comparison with native HDL3, which
confirmed the pronounced association of tyrosylated particles with
macrophages. In addition, fluorescence microscopy was used to visualize the
association of Oregon-HDL3 with J774-A1 cells after the incubation
periods of 3 and 6 hours and confirmed the augmented association of
tyrosylated HDL3 monitored with flow cytometry. Therefore,
tyrosylation did not impair the cell-tyrosylated HDL3
interaction.
|
HDL3 cholesteryl ether cell uptake
With regard to these data, cholesterol ether-HDL3 uptake was
monitored in a dose- and time-dependent manner. Cells were incubated with 25
µg apoA-I/ml of [3H]cholesteryl hexadecyl ether-HDL3
for up to 300 minutes at 37°C (Fig.
4A) or for 3 hours with increasing concentrations of the labeled
HDL3 (Fig. 4B). As
for the Oregon studies, the uptake of radiolabeled tyrosylated HDL3
was more pronounced than that of native [3H]cholesterol
ether-HDL3 (Fig. 4).
We concluded that, in contrast to its reduced ability to remove cholesterol
from cholesterol-enriched cells, tyrosylated HDL3 displayed both a
more efficient interaction with and more cholesteryl ether transfer to J774-A1
macrophages than native HDL3. Therefore, low ability to extract
cholesterol could not be attributed to defective cell association.
|
nCEH activity in the presence of HDL3
Previous evidence supported an association between nCEH activity and
cholesterol ester accumulation in macrophages. Therefore, we intended to
determine whether the limited cholesterol efflux by tyrosylated
HDL3 could be accounted for by a corresponding reduction in the
activity of nCEH, the main enzyme responsible for the bulk hydrolysis of
cytosolic CE droplets (Contreras and
Lasuncion, 1994; Graham et
al., 1996
). For this purpose, experiments were carried out under
similar conditions of cholesterol efflux measurement. Cells were first
cholesterol loaded with AcLDL-[3H]cholesteryl oleyl ester before
their incubation for 24 hours in the presence or absence of native or
tyrosylated HDL3 (0-200 µg apoA-I/ml). Then, nCEH activity in
cell homogenate supernatant was assayed. As seen in
Fig. 5, the incubation of
J774-A1 macrophages with tyrosylated HDL3 resulted in lower nCEH
activation compared with native HDL3. Furthermore, no significant
differences were observed when nCEH activity was measured in the presence or
absence of tyrosylated HDL3, indicating that the latter did not
enhance nCEH activity.
|
HDL3 cell association competition studies
By which mechanisms does HDL3 interact with J774-A1? In order to
answer this question, we first defined the ability of lipoproteins to compete
for native or tyrosylated Oregon-labeled HDL3 cell association. To
this end, 25 µg apoA-I/ml of Oregon-HDL3 was co-incubated with
increasing concentrations of unlabeled native HDL3, tyrosylated
HDL3, AcLDL or oxLDL for 3 hours at 37°C. The non-specific
binding was obtained by the addition of a 50-fold excess of corresponding
unlabeled HDL3 and was subtracted from the other values. As shown
in Fig. 6, native
HDL3 cell association was markedly (70 and 90%) inhibited by
unlabeled native HDL3 (125 and 250 µg apoA-I/ml, respectively).
A complete inhibition (98%) was reached with 375 µg apoA-I/ml. A similar
pattern was observed with modified lipoproteins in decreasing competition
efficiency order: tyrosylated HDL>oxLDL>AcLDL. Tyrosylated
Oregon-HDL3 was also incubated with increasing concentrations of
unlabeled native HDL, tyrosylated HDL3, AcLDL or oxLDL for 3 hours
at 37°C. The most competitive effect was obtained with AcLDL: 49, 94 and
100% at 125, 250 and 375 µg apoA-I/ml, respectively. The competition
efficiency declined from AcLDL to native HDL3 and OxLDL. Additionally, we
noted in few studies that native LDL (at the concentrations of 250-375 µg
apo B/ml) was able to compete with tyrosylated HDL and to inhibit its cell
association by 30-40%. These findings suggest that (1) tyrosylated
HDL3 efficiently interacts with cell-surface receptors for native
HDL3 and modified LDL and (2) the involved receptors have a broad
ligand specificity.
|
HDL3-cholesteryl ether cell uptake competition
studies
In a second step, we evaluated the transfer of [3H]cholesteryl
ether-labeled native and tyrosylated HDL3 (10 µg/ml apoA-I)
co-incubated with 50, 100 and 150 µg/ml apoA-I of unlabeled native
HDL3 and tyrosylated HDL3, as well as with AcLDL or
OxLDL for 3 hours at 37°C. As illustrated in
Fig. 7, a decrease in the
cellular uptake of labeled cholesteryl ether was noted when the lipoproteins
were incubated with native HDL3
(Fig. 7A) or tyrosylated
(Fig. 7B). Only tyrosylated HDL
was unable to compete with native HDL3. Overall, our data confirm
the cell association competition studies and suggest common binding site(s)
for cholesterol deposition in macrophages.
|
Identification of tyrosylated HDL3 cell receptor
We attempted to define the mechanism of receptor-mediated cell association
with and lipid uptake from tyrosylated HDL3. We first examined the
class B type I/II scavenger receptors (SR-BI/BII) that are expressed in
various mammalian cells, bind HDL with high affinity and also serve as
receptors for modified lipoproteins (Acton
et al., 1996; Ji et al.,
1997
). Using an antibody raised against the residues 230-380 of
the extracellular domain of SR-BI/BII, we could detect about 50% inhibition of
cell association with native or tyrosylated Oregon-HDL3
(Fig. 8A). Similarly,
SR-BI/BII-specific blocking antibody profoundly inhibited the cellular uptake
of native and tyrosylated [3H]cholesteryl ether-HDL3
(Fig. 8B). These studies,
therefore, support the suggestion that the association and the CE
internalization of tyrosylated HDL3 are substantially mediated by
SR-BI/BII.
|
Since class A scavenger receptors are mainly expressed in macrophages and
recognize and internalize modified lipoproteins, we assessed their
contribution to cell association and cholesterol ester uptake of tyrosylated
HDL3. Thus, we employed polyinosinic acid (poly-I), an established
inhibitor of class A scavenger receptors, that bind tightly to SR-AI/II
because it is capable of forming base quarted-stabilized four-stranded
helices. J774-A1 cells were incubated for 3 hours at 37°C with 10 µg
apoA-I/ml of native or tyrosylated Oregon HDL3
(Fig. 9A) or with native or
tyrosylated [3H]cholesterol ether-HDL3
(Fig. 9B) in the presence of
increasing concentrations of Poly-I. As shown in
Fig. 9A, this ligand slightly
reduced the cell association with native Oregon-HDL3 (20% of
control), whereas that of tyrosylated Oregon-HDL3 was dramatically
hampered (50-60% of control). By contrast, Poly-I enhanced
[3H]cholesterol ether cell uptake from native HDL3,
whereas tyrosylated [3H]cholesterol ether-HDL3 cell
uptake was inhibited by Poly-I in a concentration-dependent manner
(Fig. 9B). These findings
indicated clearly that type I and II class A scavenger receptors mediated
macrophage binding to and cholesterol ester uptake from tyrosylated
HDL3.
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Discussion |
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J774 cells represent a remarkable macrophage model that are frequently
employed for the investigation of cholesterol metabolism. They have largely
been used to study cholesterol efflux mechanisms triggered by HDL particles
and their apolipoprotein content. Indeed, this cell line does not express apo
E (Langer et al., 2000), nor
caveolin-1 (Matveev et al.,
1999
), which constitutes an evident advantage for the assessment
of cholesterol efflux by native or tyrosylated HDL given that apo E may
interfere and disguise the effects of tyrosalated apo A-I by compensatory
mechanisms
Previously, Francis et al. reported that oxidative tyrosylation of HDL
enhanced cholesterol removal from cultured fibroblasts and macrophage foam
cells (Francis et al., 1993).
Possible explanations for the discrepancy between our observations and those
of Francis et al. include the use of different cell lines, the degree of
oxidative tyrosylation that can produce intermolecular dytirosine crosslinks
of apo A-I and apo A-II as well as the size, density, apolipoprotein
composition and lipid content of the lipoprotein acceptor, which were shown to
independently affect cholesterol release
(Johnson et al., 1991
). Our
data are consistent with numerous studies reporting a lessened effect of
oxidized HDL on cholesteryl ester depletion in foam cells by various agents,
such as hypochlorite (Bergt et al.,
1999
), malondialdehyde
(Guertin et al., 1994
;
Salmon et al., 1992
),
Cu2+ (Gesquière et al.,
1997
) or even cell-HDL association
(Cogny et al., 1996
).
We attempted to elucidate the mechanisms linked to the impaired cholesterol
efflux abilities of tyrosylated HDL. One hypothesis raised in our studies was
that tyrosylation might be involved in reducing the binding affinity of HDL,
resulting in the reduced capacity of the particle to remove cholesterol from
J774-AI macrophages. However, tyrosylation of HDL particles did not compromise
their association nor their CE transfer to J774-A1 macrophages. This indicates
that the reduction in cholesterol efflux was not due to the disruption of
tyrosylated HDL interaction with regulatory domains of plasma membrane lipids
or critical receptors. On the contrary, our observations unambiguously
demonstrated that the interaction between cells and tyrosylated HDL, as well
as the uptake of tyrosylated HDL by J774-A1 macrophages, is increased. In the
second step, it was reasonably assumed that the hydrolysis and mobilization of
intracellular cholesteryl ester stores, leading to cellular cholesterol efflux
through an HDL cell-surface receptor (Oram
et al., 1991; Slotte et al.,
1987
), were affected. We thus focused on neutral cholesterol
esterase involved in the degradation of CE, since several investigators have
suggested that the mobilization of CE in foam cells by HDL is mediated by this
cytosolic enzyme (Contreas and Lasuncion, 1994;
Goldstein et al., 1983
).
Indeed, we found that incubation with native HDL raised nCEH activity in
J774-A1 cells. The post stimulation values were slightly lower than those
described before by Miura et al. (Miura et
al., 1997
), probably because J774-AI cells were cholesterol loaded
with AcLDL prior to the addition of HDL3. Accordingly, an increase
in intracellular cholesterol was previously shown to downregulate nCEH
(Tomita et al., 1997
). On the
other hand, the exposure of J774-A1 macrophages to tyrosylated HDL resulted in
reduced nCEH activity, which may be related to the decrease in cholesterol
efflux. Since CE stored in the cytoplasm undergoes a continual cycle of
hydrolysis and re-esterification, additional studies are necessary to assess
the contribution of acyl CoA:cholesterol acyltransferase in the slow
desorption of cholesterol from the plasma membrane by tyrosylated HDL.
The accumulation of oxidised LDL has been related to the transformation of
monocyte-derived macrophages into foam cells
(Brown and Goldstein, 1984;
Steinberg et al., 1989
). The
uptake of these modified LDLs is mediated by scavenger receptors (SR) located
on the surface of macrophages (Acton et
al., 1996
; Ji et al.,
1997
; Stangl et al.,
1998
). The major function of SR-BI is thought to allow cholesterol
transport, and in particular the selective uptake of HDL cholesterol ester by
the liver and steroidogenic tissues, without having to internalize the entire
HDL macromolecule (Acton et al.,
1996
; Ji et al.,
1997
; Rigotti et al.,
1995
). Our results clearly demonstrated that, besides recognizing
native HDL and oxidatively modified LDL, J774-A1 macrophage cells interacted
with tyrosylated HDL particles. The latter were shown to be effective
competitors of scavenger-receptor-mediated binding of native HDL as well as
acetylated and oxidized LDL, all ligands ascribed to SR-BI/BII
(Acton et al., 1996
;
Ji et al., 1997
;
Rigotti et al., 1995
;
Stangl et al., 1998
). Direct
evidence for SR-BI/II involvement is provided by our in vitro experiments in
which the antibody to the extracellular domain of SR-BI/II blocked HDL-CE
uptake and delivery to cultured J774-A1 cells. In view of the comparable
competence of SR-BI/BII to bind native and tyrosylated HDL3, noted
with the specific antibody, it is possible to exclude its participation in
defective cholesterol efflux in J774-A1 cells.
With regard to the scientific literature, SR-BI recognizes native and
modified LDL in addition to HDL (Acton et
al., 1996; Ji et al.,
1997
; Rigotti et al.,
1995
). However, acetylated LDL are preferentially captured by
SR-AI/AII, whereas oxidized LDL are probably taken by SR-BI
(Lougheed et al., 1997
;
Lougheed et al., 1999
).
Furthermore, according to Murao et al. and Fluiter et al., the affinity of
SR-BI respects the following pattern HDL>LDLox>LDL>LDLac (Murao et
al., 1999; Fluiter et al., 1997). The data in
Fig. 8 fit this profile
especially when we consider the important role of SR-BI in the recognition of
native HDL (Fig. 8).
SR-AI/AII was originally identified on the basis of its ability to mediate
the internalization of chemically modified LDL and cause massive lipid
accumulation in macrophages (Brown et al.,
1980; Goldstein et al.,
1979
). It is present on the surface of macrophages, Kupffer cells
and endothelial cells. Polynucleotides can bind tightly to the SR-AI/II given
their ability to form base quartet-stabilized four-stranded helices
(Pearson et al., 1993
). Using
polyionsinic acid in our in vitro experiments, we observed that cell
association with tyrosylated HDL was reduced by 50% compared with that of
native HDL. Similar results were obtained with regard to
[3H]cholesteryl ether uptake from native and tyrosylated HDL.
These data suggest that SR-AI/II may be responsible for the enhanced uptake
of CE from tyrosylated HDL. Since the main function ascribed to the SR-AI/II
involves promoting the clearance of microbial pathogens, senescent cells or
altered plasma proteins by phagocytic cells
(Pearson et al., 1993;
Pearson, 1996
;
Platt et al., 1996
;
Terpstra et al., 1997
), it is
possible that the modification of apos A-I and A-II by tyrosylation makes them
more suitable for recognition and internalization by SR-AI/AII. Moreover, the
affinity of SR-AI/AII for tyrosylated HDL would favor cholesterol uptake,
which could explain the decreased cholesterol efflux by these lipoprotein
particles. Finally, there may be additional, as yet unidentified, cell-surface
molecules that bind to tyrosylated HDL and underlie some of the actions
mentioned in this study.
In conclusion, the current data highlight the efficiency of tyrosylated HDL for competing with oxidatively modified LDL, binding to scavenger receptors AI/II and BI/II and transferring their cholesterol moiety to J774-A1 macrophages. By contrast, tyrosylated HDL particles were less efficient in effluxing cholesterol and more competent in accumulating cholesterol in J774-A1 cells, two processes that were accompanied by diminished nCEH activity and enhanced SR-AI/II recognition and cholesterol ester uptake.
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Acknowledgments |
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References |
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