From the Departments of Molecular Pharmacology,
§ Molecular Function Analysis and ¶ Histology & Embryology, Kanazawa University Graduate School of Medical Science,
Kanazawa 920-8640 and the
Research Department, R&D Center, BML,
Inc., Saitama 350-1101, Japan
Received for publication, November 27, 2002, and in revised form, January 31, 2003
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ABSTRACT |
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Oxidation of low density lipoprotein
(LDL) is the key step for the development of atherosclerosis. The
12/15-lipoxygenase expressed in macrophages is capable of oxygenating
linoleic acid esterified to cholesterol in the LDL particle, and thus
this enzyme is presumed to initiate LDL oxidation. We recently reported
that LDL receptor-related protein (LRP) was required for the
enzyme-mediated LDL oxidation by macrophages and suggested the
selective uptake of cholesterol ester from LDL to the plasma membrane
(Xu, W., Takahashi, Y., Sakashita, T., Iwasaki, T., Hattori, H., and
Yoshimoto. T. (2001) J. Biol. Chem. 276, 36454-36459). To elucidate precise mechanisms of lipoxygenase-mediated
LDL oxidation, we investigated the intracellular localization of
12/15-lipoxygenase. The 12/15-lipoxygenase was predominantly detected
in cytosol of resting peritoneal macrophages and of macrophage-like
J774A.1 cells permanently transfected with the cDNA for the enzyme.
When the cells were treated with LDL and subjected to subcellular
fractionation, the 12/15-lipoxygenase was detected in the membranes
with a concomitant decrease in cytosol as shown by Western blot
analysis. The levels of the enzyme associated with the membrane reached
maximum in 15 min after LDL addition and then decreased. However, the
enzymatic activity of 12/15-lipoxygenase in the membrane fraction was
very weak even after LDL treatment. This fact supports the suicide
inactivation of the enzyme by the oxygenation of cholesterol ester
transferred from the LDL particle to the plasma membrane.
Immunohistochemical analysis using an antibody against
12/15-lipoxygenase revealed that the plasma membrane was the major site
of the enzyme translocation by the LDL treatment. LDL-dependent 12/15-lipoxygenase translocation was
inhibited by a blocking antibody against LRP. Furthermore, an enzyme
translocation inhibitor, L655238, inhibited the LDL oxidation caused by
the 12/15-lipoxygenase. We propose that cholesterol ester selectively transferred from the LDL particle to the plasma membrane via LRP is oxygenated by 12/15-lipoxygenase translocated to this membrane.
12/15-Lipoxygenase is a member of the lipoxygenase family, which
incorporates one molecule of oxygen in regiospecific and stereospecific
manners to unsaturated fatty acids such as arachidonic and linoleic
acids (1-4). The enzyme consists of leukocyte-type 12-lipoxygenase
found in rats, mice, cows, and pigs, and reticulocyte-type 15-lipoxygenase (15-lipoxygenase-1) expressed in humans and rabbits, oxygenating the position 12 and 15 of arachidonic acid, respectively (5). The notable feature of the 12/15-lipoxygenase is that the enzyme
directly oxygenates not only free fatty acids but also complex
substrates such as phospholipids, cholesterol ester, and the
cholesterol ester present in the low density lipoprotein
(LDL)1 particle (1-4).
Oxidation of LDL is the first key step for the development of
atherosclerosis (6, 7), and the roles of the 12/15-lipoxygenase in the
process of LDL oxidation and the progress of atherogenesis have been
extensively investigated. Recent study using
12/15-lipoxygenase-knockout mice (8) and the study using
12/15-lipoxygenase-transgenic mice (9) established that the enzyme was
involved in the development of atherosclerosis, although contrary
results were obtained using 12/15-lipoxygenase-transgenic rabbits (10,
11).
Using a macrophage-like cell line J774A.1, which did not have
endogenous 12/15-lipoxygenase activity, we permanently transfected the
cells with the 12/15-lipoxyganase cDNA and demonstrated that 12/15-lipoxygenase expressed in normal macrophages at a high level was
required for LDL oxidation (12). However, the mechanism of
extracellular LDL oxidation by intracellular 12/15-lipoxygenase has not
been established. Recently, we revealed that the lipoxygenase-mediated LDL oxidation by macrophages required the binding of LDL to LDL receptor-related protein (LRP) but not to the LDL receptor, both of
which are expressed on the surface of J774A.1 cells and are capable of
binding native LDL (13).
The LDL is processed by the LDL receptor via receptor-mediated
endocytosis in which cholesterol ester in the LDL particle is delivered
to lysosomes where it is degraded (6). In contrast, the binding of LDL
to LRP has been demonstrated to selectively take up the cholesterol
ester from LDL in the plasma membrane without endocytosis and
degradation of the LDL particle (14). For the efficient enzymatic
oxygenation of the cholesterol ester transferred to the plasma membrane
via LRP, the 12/15-lipoxygenase itself should also be localized in the
plasma membrane or its neighborhood. However, the 12/15-lipoxygenase is
predominantly present in cytoplasm and not in the membranes in various
cells (15). Recent study has shown that the translocation of
12/15-lipoxygenase from the cytosol to the plasma membrane was observed
in macrophages when incubated with apoptotic cells (16). Here we
demonstrate that the binding of LDL to LRP expressed in normal
macrophages and 12/15-lipoxygenase-expressing macrophage-like J774A.1
cells translocates the enzyme from cytoplasm to the plasma membrane. The translocation is necessary for the cell-mediated oxidation of LDL.
This study reveals a novel function of LRP in the development of atherosclerosis.
Materials--
Dulbecco's modified Eagle's medium (DMEM) was
obtained from Nissui (Tokyo, Japan), fetal bovine serum from JRH
biosciences (Lenexa, KS), lipoprotein-deficient serum from Sigma (St.
Louis, MO), and 2-thiobarbituric acid and
1,1,3,3-tetramethoxypropane(bismalondialdehyde) from Wako
(Osaka, Japan), [1-14C]arachidonic acid (2.1 GBq/mmol)
and ECL Western blotting detection reagents from Amersham Biosciences
(Bucks, UK), biotinylated and peroxidase-labeled anti-rabbit IgG from
Vector (Burlingame, CA), L655238 from BIOMOL (Plymouth Meeting, PA),
silica gel thin-layer plates from Merck (Darmstadt, Germany),
polyvinylidene difluoride membranes from Millipore (Bedford, MA),
Lab-Tek chamber slide from Nalge Nunc International (Naperville, IL),
swine serum and horseradish peroxidase-conjugated streptavidin from
Dakopatts (Carpenteria, CA), and Glicidether 100 from Selva
Feinbiochemica (Heidelberg, Germany). An antiserum against
12/15-lipoxygenase was raised using purified recombinant rat pineal
12-lipoxygenase as an antigen as described previously (17). An anti-LDL
receptor antibody was raised as described and purified to IgG using
protein A-Sepharose (13). Human LDL was prepared from healthy
volunteers and dialyzed against phosphate-buffered saline at 4 °C
for 24 h before each experiment as described previously (12, 13). A murine macrophage-like cell line J774A.1 was kindly provided by Dr.
Y. Saeki of Shiga University of Medical Science. An expression vector,
pEF-BOS having a elongation factor-1 Cell Culture--
J774A.1 cells permanently transfected with the
pEF-BOS vector carrying porcine leukocyte 12/15-lipoxygenase cDNA
and mock-transfected cells were establish as describe previously (12).
The cells were cultured at 37 °C with 5% CO2 in DMEM
supplemented with 10% fetal bovine serum, 100 units/ml penicillin G
and 100 µg/ml streptomycin sulfate, and subcultured every 2-3 days
using a standard trypsin protocol. Mouse peritoneal macrophages were
collected from C57BL/6 mice as described previously (20) except that
thioglycollate was not injected before harvesting the cells.
Enzyme Preparation and Assay--
The
12/15-lipoxygenase-expressing cells were cultured in 100-mm dishes in
DMEM with 10% lipoprotein-deficient serum for 48 h, then LDL at
400 µg/ml was added to the medium. After incubation at 37 °C for
various periods, cells were washed with ice-cold phosphate-buffered
saline at pH 7.4 and suspended in 50 mM Tris-HCl buffer at
pH 7.4 containing 1 mM EDTA. The cells were sonicated twice
on ice, each for 5 s, at 20 kHz by a Branson sonifier model 250 (Danbury, CT), followed by ultracentrifugation at 265,000 × g at 4 °C for 2 h. The supernatant was referred to
as cytosol, and the pellet resuspended in 50 mM Tris-HCl at
pH 7.4 containing 1 mM EDTA was referred to as
"membranes."
12/15-Lipoxygenase activity was determined as described previously
(12). Briefly, the cytosol and the membranes were incubated in a
200-µl reaction mixture containing 50 mM Tris-HCl buffer at pH 7.4 and 25 µM [1-14C]arachidonic acid
(1.85 kBq). The reaction was carried out at 30 °C for 10 min with
constant mixing and quenched by the addition of 1 ml of an ice-cold
mixture of diethyl ether/methanol/1 M citric acid (30:4:1,
v/v). The ether layer was spotted onto a silica gel thin layer plate,
and the plate was developed at 4 °C for 60 min with a solvent system
of diethyl ether/petroleum ether/acetic acid (85:15:0.1, v/v). The
radioactive products on the plate were detected and quantified by a
Fujix BAS 1000 imaging analyzer (Tokyo, Japan). Protein concentration
was determined by the method of Lowry et al. (21) with
bovine serum albumin as a standard.
Western Blotting--
The proteins in the cytosol and membranes
were separated by 10% SDS-polyacrylamide gel electrophoresis and
transferred to a polyvinylidene difluoride membrane, followed by
blocking with 10% (w/v) nonfat dry milk in 20 mM Tris-HCl
at pH7.4 containing 136 mM NaCl and 0.1% Tween 20 for
1 h at room temperature. The washed membranes were incubated for
1 h at room temperature with an anti-12/15-lipoxygenase antibody
at 1:1000 dilution. 12/15-Lipoxygenase band was detected using a
horseradish peroxidase-conjugated secondary antibody and ECL
chemiluminescence reagents according to the manufacturer's instruction. The density of 12/15-lipoxygenase band was quantified by
National Institutes of Health Image 1.60 analysis software (Bethesda,
MD). The intensity of the 12/15-lipoxygenase band increased linearly
with the amount of the enzyme loaded onto the gel.
Thiobarbituric Acid Reactive Substance Assay--
The
12/15-lipoxygenase-expressing cells (2 × 105) were
preincubated for 48 h in DMEM containing 10% of
lipoprotein-deficient serum followed by the addition of a translocation
inhibitor, L655238, at various concentrations. After 1 h the cells
were incubated with 400 µg/ml LDL in 100 µl of DMEM containing the
serum in the presence of L655238 for 12 h, and the culture medium
was subjected to TBARS assay as described previously (12).
Immunohistochemistry--
The light- and electron-microscopic
immunohistochemical procedures were performed as described (22).
12/15-Lipoxygenase-expressing cells and mouse resident macrophages
collected from the peritoneal cavity were cultured for 48 h in the
medium containing 10% lipoprotein-deficient serum in Lab-Tek chamber
slide. The cells were then treated with LDL for 15 min followed by
fixation in 0.1 M phosphate buffer at pH 7.4 containing 4%
paraformaldehyde on ice for 30 min and washed twice in
phosphate-buffered saline at pH 7.4. For light microscope observation,
the slides were first permeabilized by incubating with
phosphate-buffered saline containing 0.3% Tween 20 for 1 h,
treated with 3% hydrogen peroxide in methanol for 10 min, and then
incubated with 5% normal swine serum for 30 min. Subsequently, the
slides were incubated at room temperature overnight with an
anti-12/15-lipoxygenase antiserum. For the negative control, the
antibody was replaced with preimmune rabbit serum. The sites of
immunoreaction were then visualized by incubating the slides successively with biotinylated anti-rabbit IgG diluted at 1:200 for
1 h, horseradish peroxidase-conjugated streptavidin diluted at
1:300 for 1 h, and with 0.01% 3',3'-diaminobenzidine
tetrahydrochloride in the presence of 0.02% hydrogen peroxide in 50 mM Tris-HCl at pH 7.5 for 10-30 min.
For electron-microscopic immunocytochemistry, the immunostained slides
were postfixed with 0.5% OsO4 for 20 min. After
block-staining with 1% uranyl acetate for 30 min, the slides were
dehydrated in graded ethanol series and embedded in an epoxy resin
based on Glicidether 100. Ultrathin sections were prepared and
subjected to observation with a Hitachi H-700 electron microscope
(Tokyo, Japan).
Membrane Translocation of 12/15-Lipoxygenase by
LDL--
12/15-Lipoxygenase is predominantly localized in cytosol but
not in the membranes (15). This was confirmed in macrophage-like J774A.1 cells overexpressing 12/15-lipoxygenase and resident peritoneal macrophages by Western blot as shown in Fig.
1A. To investigate whether the
subcellular localization of the 12/15-lipoxygenase is changed by LDL
treatment, the enzyme-expressing cells or peritoneal macrophages were
treated with LDL for various periods, and the cytosol and membranes
were subjected to Western blot analysis. Fig. 1A shows that
the band of 12/15-lipoxygenase at 75 kDa was detected not only in the
cytosol but also in the membranes with a concomitant decrease of the
enzyme level in the cytosol. Association of the enzyme to the membranes
reached maximum at 15 min after LDL addition and then decreased. After
30 min the enzyme was no longer present in the membranes. Densitometric
analysis revealed the increase in 12/15-lipoxygenase protein of the
membranes after the LDL treatment for 5 and 15 min by 23- and 33-fold,
respectively (Fig. 1B). The enzyme protein was increased by
14-fold in the membranes of macrophages by the treatment with LDL for
15 min. After 15-min treatment by LDL, the enzyme protein in the
cytosol was decreased by 38 and 47% in 12/15-lipoxygenase-expressing
cells and in macrophages, respectively. The results indicated that
12/15-lipoxygenase was transferred from cytosol to membranes by the LDL
treatment in the resident macrophages as well as
12/15-lipoxygenase-expressing J774A.1 cells.
We measured the enzyme activity in the cytosol and membranes in
12/15-lipoxygenase-expressing cells. As shown in Fig.
2, the specific activity of the enzyme in
the cytosol was decreased by 41% by the treatment with LDL for 15 min
as compared with non-treated cells. The result was in good agreement
with that from Western blot analysis (Fig. 1). The level of the
increase of the enzyme activity in the membranes was significantly
lower after LDL treatment. It is shown that cholesterol ester is
selectively transferred from the LDL particle to the plasma membrane
via LRP in Y1 murine adrenocortical cells (14) and in our
12/15-lipoxygenase-expressing cells2 and that linoleic acid
esterified to cholesterol in the LDL particle is regio- and
stereospecifically oxygenated by the 12/15-lipoxygenase-expressing cells (12). Thus, the above observations strongly support our contention that the 12/15-lipoxygenase associated with the membranes oxygenates cholesterol ester transferred to the membrane, because self-catalyzed inactivation of the 12/15-lipoxygenase, which should be
observed in the enzyme reaction with cholesterol ester in the membrane,
is known to occur (23). This would explain the much lower enzyme
activity in membranes. The results indicate that LDL brings about
translocation of 12/15-lipoxygenase from cytosol to membranes where the
oxidation of cholesterol ester from LDL takes place.
LRP Mediates the Translocation of
12/15-Lipoxygenase--
We previously reported the
essential requirement of LRP for the cell-mediated oxidation of LDL in
macrophages (13). To examine whether the LRP is also involved in the
translocation of the enzyme, we employed an anti-LRP antibody that
blocked the binding of LDL to LRP (19). The 12/15-lipoxygenase
expressing cells and mouse resident peritoneal macrophages were
preincubated in the presence of an anti-LDL receptor antibody or an
anti-LRP antibody for 2 h. After 15-min incubation with LDL, the
cytosol and membranes were subjected to Western blot analysis. As shown
in Fig. 3 (A and
C), the 12/15-lipoxygenase band in the membranes of the
cells preincubated with an anti-LRP antibody was faint after LDL
treatment as compared with that from the control. Consistent with this
observation, the density of the 12/15-lipoxygenase band in the cytosol
of the cells preincubated with an anti-LRP antibody was not
significantly different from that of the cells that were not treated
with LDL. Preincubation with an anti-LDL receptor antibody did not
significantly affect the enzyme translocation by the LDL treatment
(Fig. 3). The results indicate that the translocation of
12/15-lipoxygenase is mediated by binding of LDL to the LRP but not to
the LDL receptor.
Immunohistochemical Staining of
12/15-Lipoxygenase--
To determine the intracellular
localization of the 12/15-lipoxygenase after LDL treatment, the
enzyme-expressing cells were subjected to immunohistochemical analysis
using an antibody against the enzyme (Fig.
4). A different staining pattern of the
12/15-lipoxygenase was observed between the cells treated with and
without LDL under light microscopy. In the non-treated cells, the
enzyme was predominantly stained in cytoplasm of
12/15-lipoxygenase-expressing cells (Fig. 4, C and
D). When the cells were treated with LDL for 15 min, the
positive staining of 12/15-lipoxygenase was observed not only in
cytoplasm but also in the plasma membrane of the enzyme-expressing cells (Fig. 4, A and B). Essentially the same
results were obtained with LDL-treated resident peritoneal macrophages.
The control experiments with preimmune rabbit serum in place of the
antiserum against 12/15-lipoxygenase exhibited negative immunostaining
(data not shown). The results indicate that the plasma membrane is at least one of the major sites where 12/15-lipoxygenase translocates after LDL treatment. To investigate the precise localization of the
enzyme in the LDL-treated cells, we observed the immunostained cells
with an electron microscopy. As shown in Fig. 4F,
non-treated cells showed diffuse staining pattern in cytoplasm. In
contrast, the plasma membrane was the major site where the
12/15-lipoxygenase was localized in LDL-treated cells, although
membranes of some other intracellular organelles were also stained in
addition to cytoplasm (Fig. 4E). It should be noted that the
nuclear envelope was essentially not stained in LDL-treated cells.
Enzyme Translocation Is Required for LDL Oxidation--
To examine
whether enzyme association with the membranes is required for the LDL
oxidation, we employed a translocation inhibitor, L655238. This
compound was first found to inhibit translocation of 5-lipoxygenase
(24) but later shown to inhibit translocation of other lipoxygenases
without affecting the enzyme activity per se (25). As shown
in Fig. 5 (A and
B), L655238 inhibited translocation of 12/15-lipoxygenase in
the LDL-treated cells in a dose-dependent manner without
affecting the enzyme activity. Fig. 5C shows that LDL
oxidation determined as TBARS generation in the medium was blocked in a
dose-dependent manner by the translocation inhibitor. The
inhibitor at 10 µM, which completely suppressed the
enzyme translocation (Fig. 5, A and B), inhibited
the LDL oxidation to the level of mock-transfected cells (Fig.
5C). The results clearly indicate that the association of
the enzyme with the plasma membrane is required for the LDL
oxidation.
We demonstrate here that 12/15-lipoxygenase is translocated from
cytosol to the membranes by LDL treatment in
12/15-lipoxygenase-expressing macrophage-like cells and resident
peritoneal macrophages (Figs. 1 and 2). The translocated enzyme is
preferentially localized in the plasma membrane (Fig. 4), strongly
suggesting that the enzyme directly oxygenates cholesterol ester
selectively transferred from the LDL particle to the plasma membrane.
In fact, the LDL oxidation was inhibited by a translocation inhibitor,
L655238 (Fig. 5). Regio- and stereospecific oxygenation of linoleic
acid esterified to cholesterol in LDL by 12/15-lipoxygenase-expressing cells indicates that cholesterol ester is enzymatically oxygenated by
the cells (12). The enzymatic oxygenation is presumed to be the first
key step for the generation of the completely oxidized LDL, which is
made in the subsequent steps, including non-enzymatic radical chain
reaction (12, 23). The fact that 12/15-lipoxygenase translocation takes
place in a very short period such as 5-15 min supports this notion
(Fig. 1). Furthermore, such a short time course minimizes the
oxygenation of phospholipids in plasma membrane, which may cause the
cell injury. The weak activity of the membrane-associated enzyme after
LDL treatment strongly suggests that the enzyme in the plasma membrane
reacts with colocalized substrates, including cholesterol ester and
then suicides. However, the reduction of the enzyme activity may be due
to other mechanisms unrelated to suicide inactivation such as poor
substrate availability or conformational changes of the enzyme. It is
reported that 12/15-lipoxygenase preferentially oxygenates cholesterol
ester in the LDL particle, whereas phospholipids or even free fatty
acids are not oxygenated, although they are present on the surface of
the LDL particle (23). These results suggest that the specific
oxygenation of cholesterol ester transferred to the plasma membrane by
12/15-lipoxygenase could take place. LRP is an LDL-binding receptor
that selectively transfers cholesterol ester in the LDL particle
(14).2 We show here that binding of LDL to LRP is also
required for 12/15-lipoxygenase translocation (Fig. 3).
12/15-Lipoxygenase-expressing J774A.1 cells have both LRP and the LDL
receptor, although the expression level of the LDL receptor is low
(13). In contrast, normal macrophages express high level of LRP but do
not express the LDL receptor (26, 27). The contribution of LRP but not of the LDL receptor to 12/15-lipoxygenase translocation supports the
notion that the LDL-dependent translocation is also
mediated by LRP in normal macrophages. In fact, the enzyme
translocation is inhibited in mouse peritoneal macrophages lacking the
LDL receptor by the anti-LRP antibody (Fig. 3, C and
D). However, we cannot completely exclude a role for the LDL
receptor in the translocation, because either type of cells used in our
experiments express a little or no LDL receptor where an anti-LDL
receptor antibody would not be expected to have an effect. Coupling of
selective uptake of cholesterol ester with 12/15-lipoxygenase
translocation would cause efficient oxygenation of linoleic acid
esterified to cholesterol. The mechanisms of the efflux of oxygenated
cholesterol ester to the LDL particle are now under extensive
investigation in our laboratory. The cholesterol ester in the high
density lipoprotein is selectively transferred to the plasma membrane
by scavenger receptor class B type I (28). The same receptor is shown
to mediate cholesterol efflux to high density lipoprotein (29, 30). It
may be possible that LRP also mediates the efflux of oxygenated
cholesterol ester from the plasma membrane to the LDL particle.
The mechanism of translocation of 12/15-lipoxygenase in macrophages is
not known (16), although the N-terminal C2-like domain in the enzyme is
proposed to be responsible for the enzyme binding to the membrane
phospholipids in a calcium dependent way (31). In fact, our finding
that the translocation of 12/15-lipoxygenase is inhibited by L655238
suggests the similar translocation mechanism to that of 5-lipoxygenase.
L655238 was first developed as an inhibitor of
5-lipoxygenase-activating protein, which was later shown to function as
a substrate transfer protein promoting the use of arachidonic acid and
other unsaturated fatty acids (32). In 5-lipoxygenase, the N-terminal
C2-like domain is demonstrated to be a calcium-dependent
membrane-targeting domain without requirement of any special docking
protein (33). We have reported that 12-lipoxygenase in human platelets
is activated by membrane translocation when stimulated by collagen or
thrombin and that a translocation inhibitor of 5-lipoxygenase, L655238,
inhibits production of 12-HETE from platelets without affecting the
enzyme activity (25). The results clearly indicate that L655238 is not
a 5-lipoxygenase-specific inhibitor but a general translocation
inhibitor of lipoxygenases. The structural difference of the C2-like
domain in the enzyme has been proposed to determine the enzyme
preference of the membrane type (34). For example, the C2-like domain
in 5-lipoxygenase and cytosolic phospholipase A2 binds preferentially
to the nuclear envelope (33, 35), whereas that in protein kinase C LRP is known to couple with a Gi class of
heterotrimeric GTPases when it binds to apoE4 to induce apoptosis of
neuronal cells (38). We demonstrate here that the LDL binding to LRP
but not to the LDL receptor is required for the translocation of
12/15-lipoxygenase (Fig. 3). Although LDL contains apoB but not apoE as
an apolipoprotein, some signal transduction pathways may be activated
after the binding of LDL to LRP rather than simple membrane association
via the N-terminal C2 domain-like structure of the 12/15-lipoxygenase in the cells treated by LDL. Further investigations are necessary to
elucidate the mechanism of LRP-mediated membrane association of the
12/15-lipoxygenase.
LRP is a multifunctional receptor capable of binding a wide variety of
ligands and postulated to participate in a number of pathophysiological
processes ranging from atherosclerosis, fibrinolysis, neuronal
degeneration, to apoptosis (39). As a role in homeostasis of plasma
lipoproteins, LRP expressed in liver has been established as a remnant
receptor using LRP-disrupted mice in a liver-specific manner (40).
However, they demonstrate that more than 70% of the remnant is cleared
from plasma by the LDL receptor expressed in liver, and LRP in liver is
a compensating receptor for the clearance of the remnant. We have
proposed the dual functions of LRP expressed in macrophages, selective
uptake of cholesterol ester from LDL, and 12/15-lipoxygenase
translocation. Thus, an LRP antagonist may be anti-atherogenic by
inhibition of LDL oxidation in the two ways: blocking of the binding of
native LDL to macrophages followed by the selective transfer of
cholesterol ester to the plasma membrane and by inhibiting the
association of the plasma membrane with 12/15-lipoxygenase, which
oxygenates the cholesterol ester in the plasma membrane.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
promoter (18) was kindly
provided by Dr. S. Nagata of Osaka University. An anti-LRP antibody
(19) was a generous gift from Dr. Joachim Herz of University of Texas
Southwestern Medical Center.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Membrane translocation of 12/15-lipoxygenase
in the enzyme-expressing cells and macrophages treated with LDL.
A, a representative Western blot of 12/15-lipoxygenase
protein in the cytosol and membranes in the
12/15-lipoxygenase-expressing J774A.1 cells and resident peritoneal
macrophages in the presence or absence of 400 µg/ml LDL for indicated
periods. The amounts, 10 µg of protein for the enzyme-expressing
cells and 2 µg for peritoneal macrophages, were separated by SDS-PAGE
and subjected to Western blot analysis as described under
"Experimental Procedures." Arrows indicate the positive
12/15-lipoxygenase band at 75 kDa. B, densitometric analysis
of 12/15-lipoxygenase protein in the cytosol and the membranes as
compared with the density of the control cells. Ratios over the density
for the cytosol of the cells incubated without LDL for 5 min are shown.
Data represent means of three separate experiments, and the
bars denote standard errors. Asterisks show
significant difference from the cells treated without LDL at each time
by Welch's t test (p < 0.01).
LOX, lipoxygenase.
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Fig. 2.
Change of 12/15-lipoxygenase activity in
cytosol and membranes by LDL treatment. The
12/15-lipoxygenase-expressing cells were treated with 400 µg/ml LDL
for 5, 15, and 30 min and subjected to subcellular fractionation as
described under "Experimental Procedures." The cytosol (50 µg of
protein) and the membrane (500 µg of protein) were incubated with 25 µM [1-14C]arachidonic acid, and the
products were separated by thin layer chromatography. Data represent
the means of three separate experiments, and bars denote
standard error. Asterisks show significant difference from
the cells treated without LDL at each time by Welch's t
test (p < 0.01).
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Fig. 3.
Inhibition of the 12/15-lipoxygenase
translocation by an anti-LRP antibody.
12/15-Lipoxygenase-expressing cells (A) and mouse peritoneal
macrophages (C) were pretreated with 50 µg/ml of an
anti-LDL receptor antibody or 10 µl/ml of an anti-LRP antiserum at
37 °C for 2 h. LDL was added at 400 µg/ml LDL for 15 min
followed by separation of cytosol and membranes. The amounts, 10 µg
of protein from each fraction of the enzyme-expressing cells and 2 µg
of from that of the macrophages, were subjected to Western blot
analysis. An arrow indicates 12/15-lipoxygenase protein at
75 kDa. The densitometric analysis was carried out, and the ratios over
the density for the cytosol of the enzyme-expressing cells
(B) and macrophages (D) incubated without LDL are
shown. Data represent the means of three separate experiments, and
bars denote standard error. Asterisks show
significant difference from the cells treated without LDL in each
preparation by Welch's t test (p < 0.01).
LOX, lipoxygenase; LDLR, LDL receptor.
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Fig. 4.
Immunohistochemical analysis of
12/15-lipoxygenase in the enzyme-expressing cells. The
12/15-lipoxygenase-expressing cells were incubated for 15 min
with (A, B, and E) or without LDL
(C, D, and F). The cells were stained
with an anti-12/15-lipoxygenase antibody as described under
"Experimental Procedures" and observed under light (A
through D) or electron (E and F)
microscopy. Arrows indicate the localization of the enzyme
at the plasma membrane. m, mitochondrion; n,
nucleus.
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Fig. 5.
Translocation inhibitor L655238 inhibits LDL
oxidation. A, 12/15-lipoxygenase-expressing cells were
preincubated with L655238 at indicated concentrations for 1 h and
treated with LDL for 15 min. Cytosol and membranes were subjected to
Western blot analysis. B, the densitometric analysis was
carried out, and the ratios over the density for the cytosol of the
cells incubated without LDL are shown. Data represent the means of
three separate experiments, and bars denote standard error.
Asterisks show significant difference from the cells treated
without LDL in each preparation by Welch's t test
(p < 0.01). Open circles show the
12/15-lipoxygenase activity in the cytosol prepared from the
enzyme-expressing cells measured with [1-14C]arachidonic
acid as a substrate in the presence of indicated concentrations of
L655238. Data show the means of triplicate experiments, and
bars denote standard errors. C, LDL at 400 µg/ml was added to the culture medium of
12/15-lipoxygenase-expressing cells (closed circles) and
mock-transfected cells (an open circle) preincubated with
indicated concentrations of L655238 for 1 h. The oxidized LDL in
the medium was measured as TBARS after 12 h. Data are shown as
means of quadruplicate experiments after subtraction of no-cell
control, and bars denote standard errors.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and phospholipase C
1 prefers targeting to the plasma membrane (36,
37) in a calcium-dependent way. In fact, our preliminary
results using 12/15-lipoxygenase-expressing cells suggested that the
calcium ionophore A23187 caused translocation of the enzyme (data not shown), although different results have been reported (35).
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ACKNOWLEDGEMENTS |
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We are indebted to Dr. J. Herz of the University of Texas Southwestern Medical Center for the generous gift of an anti-LRP antibody, Dr. Y. Saeki of Shiga University for providing J774A.1 cells, and Dr. S. Nagata for providing the pEF-BOS vector. We thank Dr. M. R. Waterman of Vanderbilt University for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, the Honjin Foundation, the Hokkoku Foundation for Cancer Research, and the Ono Medical Research Foundation.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. Tel.: 81-76-265-2186; Fax: 81-76-234-4227; E-mail: yoshimot@med.kanazawa-u.ac.jp.
Published, JBC Papers in Press, February 3, 2003, DOI 10.1074/jbc.M212104200
2 W. Xu, Y. Takahashi, T. Murakami, T. Iwasaki, H. Hattori, and T. Yoshimoto, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: LDL, low density lipoprotein; DMEM, Dulbecco's modified Eagle's medium; LRP, LDL receptor-related protein; TBARS, thiobarbituric acid reactive substance.
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