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(Received for publication, August 10,
1994; and in revised form, September 30, 1994) From the
Macrophage scavenger receptors mediate the recognition of a wide
range of negatively charged macromolecules including acetylated low
density lipoproteins (AcLDL). Chinese hamster ovary (CHO) cells were
cultured in the presence of increasing concentrations of simvastatin, a
cholesterol biosynthesis inhibitor, and AcLDL as the sole source of
exogenous lipoproteins. The cells surviving under these conditions
specifically bound Chemically modified low density lipoproteins (LDL), ( It is becoming evident that more than one
type of receptor that can recognize chemically modified LDL may exist
in mammalian cells. The receptors that recognize chemically modified
LDL but are distinct from types I and II scavenger receptors have been
identified in mouse peritoneal macrophages using expression cloning.
These receptors include Fc Although macrophages constitutively
express scavenger receptors, it is also known that scavenger (or AcLDL)
receptor activity can be induced in other types of cell by various
stimuli. For example, platelet-derived growth factor and phorbol ester
induce type I or type II scavenger receptors in vascular smooth muscle
cells(25) , while human chorionic gonadotropin stimulates
scavenger receptor activity in rat luteal cells(26) . In the
current study, we isolated Chinese hamster ovary (CHO) cells, which
actively endocytose AcLDL, by culturing in the presence of exogenous
AcLDL as the sole cholesterol source. Evidence that the receptor on
these isolated CHO cells is distinct from other reported scavenger
receptors with respect to ligand specificity and competitor sensitivity
is presented.
Figure 3:
Binding of
Figure 1:
AcLDL requirement of CHO-AL1
cells under retardation of cholesterol synthesis. CHO-AL1 cells were
seeded on day 0 at 5
Both control and CHO-AL1 cells were
incubated with DiI-AcLDL and examined using fluorescence microscopy.
Typical fluorescence micrographs are shown in Fig. 2. Control
CHO cells exhibited no efficient fluorescence, whereas CHO-AL1 cells
accumulated a massive amount of fluorescence, appearing as small
punctate foci, suggesting that CHO-AL1 cells actively take up AcLDL.
Figure 2:
Accumulation of DiI-AcLDL by control CHO
cells and CHO-AL1 cells. CHO cells were seeded on day 0 into dishes of
medium A. On day 2, the cells were incubated with 0.5 ml of medium A
containing 5 µg/ml DiI-AcLDL for 1 h at 37 °C. The monolayers
were then washed and the accumulation of DiI-AcLDL by control CHO cells (A) and CHO-AL1 cells (B) was observed using
fluorescence microscopy. (Bar, 100
µm.)
Next, association and degradation of
Figure 4:
Time course of association and degradation
of
Figure 5:
Ability of various compounds to inhibit
the association of
Previously, we have demonstrated that
negatively charged liposomes containing acidic phospholipids such as
phosphatidylserine, phosphatidylinositol, and phosphatidic acid are
effectively taken up by cultured mouse peritoneal macrophages and that
this uptake is in part inhibited by AcLDL or OxLDL(31) . We
also examined whether CHO-AL1 cells can take up these liposomes. As
shown in Fig. 6D, upon incubation with
phosphatidylserine-containing liposome (PS-liposome) encapsulated
FITC-dextran, CHO-AL1 cells accumulated a massive amount of
intracellular fluorescence. This fluorescence was lost in the presence
of a 20-fold excess of the unlabeled liposome (data not shown). In
fact, CHO-AL1 cells actively metabolized these liposomes and
accumulated neutral lipids such as triacylglycerol and cholesteryl
ester in their cytoplasm (unpublished data). On the other hand, control
CHO cells (data not shown) and CHO-SR7 cells (Fig. 6B)
exhibited no appreciable fluorescence. These results indicate that the
receptor on CHO-AL1 cells may recognize PS-liposomes, but the type I
scavenger receptor expressed on control CHO cells does not. Liposomes
consisting of phosphatidylcholine (PC-liposome) were recognized by
neither of the receptors under these conditions (Fig. 6, A and C).
Figure 6:
Accumulation of liposomes containing
FITC-dextran by CHO-SR7 cells and CHO-AL1 cells. The cells were
incubated for 1 h at 37 °C with 0.5 ml medium A containing 100
µg/ml PC-liposome (A, C) or PS-liposome (B, D). The liposomes contained FITC-dextran. The
monolayers were then washed and the accumulation of FITC-dextran by
CHO-SR7 cells (A, B) and CHO-AL1 cells (C, D) was observed using fluorescence microscopy. (Bar,
50 µm.). PC- and PS-liposome compositions are described under
``Experimental Procedures.''
A cross-competition study between AcLDL and
PS-liposome was also performed. First, as shown in Fig. 7A, the effect of the liposome on
Figure 7:
Ability of PS-liposome to inhibit the
degradation of
Figure 8:
Effect of phospholipid composition on the
association of liposomes with control CHO cells and CHO-AL1 cells. Each
dish received 0.5 ml of medium A, which contained 100 µg/ml
liposomes composed of phosphatidylcholine, the indicated phospholipid,
dicetylphosphate, free cholesterol, and
1,2-di[1-
In the current study, we isolated CHO cells expressing a
receptor that recognizes both AcLDL and negatively charged liposomes by
culturing the cells in the presence of exogenous AcLDL as the sole
cholesterol supplement. The receptor expressed on the isolated CHO
cells (CHO-AL1) appears likely to be distinct from the macrophage type
I or type II scavenger receptor (Table 1) because: (i) dextran
sulfate, poly(I), and fucoidan, all of which are known to be effective
competitors for the scavenger receptor, do not compete for the binding
of
Previously, we have
demonstrated that negatively charged liposomes containing acidic
phospholipids such as phosphatidylserine, phosphatidylinositol, and
phosphatidic acid are effectively taken up by cultured mouse peritoneal
macrophages and that this uptake is in part inhibited by AcLDL or
OxLDL(31) . Recently, evidence against the involvement of types
I or II scavenger receptors in the uptake of negatively charged
liposomes has been documented. For example, expression of the cDNA for
the bovine types I or II scavenger receptor by CHO cells induced an
increase in the uptake of AcLDL, but not the uptake of negatively
charged liposomes (37) (this study). Moreover, in cultured
rabbit smooth muscle cells treated with phorbol ester, the uptake of
AcLDL was enhanced dramatically, but there was no effect on the uptake
of these liposomes(37) . These results indicated that types I
or II scavenger receptor cannot account for the uptake of negatively
charged liposomes by cultured mouse peritoneal macrophages, and that
other receptor(s) (denoted X in Table 1) responsible for the
uptake of negatively charged liposomes may exist on these cells. It is
possible that CD36 or Fc CHO cells expressing
high AcLDL receptor activity were obtained by culturing in a medium
containing exogenous AcLDL as the sole cholesterol supplement. The
Scatchard plot analysis of the isolated cells exhibited non linear
binding with two classes of The next challenge will
be to determine the structure and mechanism of induction of this new
type of scavenger receptor. Note Added in
Proof-Acton et al. (42) have recently
reported the cDNA cloning of a new type of scavenger receptor from the
CHO cell variant that they have established.
Volume 270,
Number 4,
Issue of January 27, 1995 pp. 1921-1927
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ISOLATION AND CHARACTERIZATION (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
I-labeled AcLDL with high affinity and
degraded them via an endocytic pathway. Unexpectedly, the association
and degradation of
I-labeled AcLDL by these CHO cells
were not inhibited by dextran sulfate, fucoidan, and polyinosinic acid,
competitors of macrophage scavenger receptors, but were completely
inhibited by maleylated bovine serum albumin. Furthermore, these cells
effectively took up negatively charged liposomes containing acidic
phospholipids such as phosphatidylserine and phosphatidic acid, whereas
CHO cells expressing macrophage scavenger receptors did not. AcLDL and
negatively charged liposomes were cross-competed with each other.
Northern blot analysis using the cDNA for the macrophage scavenger
receptor revealed that these CHO cells did not express this receptor.
From these observations, we conclude that the isolated CHO cells
express a novel type of AcLDL receptor, which is distinct from
macrophage scavenger receptors with respect to ligand specificity and
competitor sensitivity.
)such as acetylated LDL (AcLDL) and oxidized LDL (OxLDL),
can be rapidly taken up by cultured macrophages via receptor-mediated
endocytosis, resulting in foam cell formation (1, 2, 3) . The receptor involved in this
pathway is called the AcLDL receptor or scavenger receptor. This
receptor has been purified(4) , and its cDNA was cloned from
bovine lung(5, 6) . The homologous cDNAs of the
human(7) , murine(8, 9) , and rabbit receptor (10) have now been cloned and sequenced. In all species the
scavenger receptor sequences were predicted to encode a transmembrane
protein with multiple extracellular domains, including an 
-helical
coiled-coil domain and a collagenous domain. There are two subtypes of
scavenger receptor (type I and type II) mRNAs which are the products of
alternative splicing of the single gene(11) . These receptors
differ only by the presence in the type I receptor of an extracellular
cysteine-rich C-terminal domain and have similar ligand specificity.
Using these probes, scavenger receptors have been shown to be expressed
on macrophage cells such as monocyte-derived macrophages and Kupffer
cells. A hallmark of the scavenger receptor is its unusually broad
ligand specificity(12) . For example, AcLDL, OxLDL,
malondialdehyde-modified LDL(13, 14) , maleylated
bovine serum albumin (maleyl-BSA)(1, 15) , and
polyanionic macromolecules such as dextran sulfate, fucoidan, and
polyinosinic acid (poly(I)) are effective ligands, whereas native LDL,
BSA, and heparin are not.RII-b2 (16) and
CD36(17) . The 40-kDa Fc
RII-b2 was found to be a mouse
homologue of human class II Fc
receptor, which binds IgG only in
complexed or polymeric form. CD36 is an 88-kDa glycoprotein expressed
on the surface of monocytes, platelets(18) , and endothelial
cells(19) . An interesting feature of these receptors is that
both recognize OxLDL but not AcLDL. Endothelial cells have also been
shown to possess scavenger receptor activity by many researchers (20, 21, 22, 23) . The receptors
responsible for AcLDL binding on the endothelial cells, however, are
distinct from type I or type II scavenger receptors, since endothelial
cells show no immunoreactivity to anti-scavenger receptor
antibody(24) , and mRNAs for both types of receptor are not
detectable on them(10) .
Materials
Human AcLDL labeled with the
fluorescent probe
1,1`-dioctadecyl-3,3,3`,3`-tetramethylindocarbocyanine perchlorate
(DiI-AcLDL) was obtained from Biomedical Technologies, Stoughton, MA.
Sodium [I]iodine and
1,2-di[1-
C]palmitoylglycerophosphocholine
(100-120 mCi/mmol) were purchased from Amersham Corp. BSA,
fucoidan, poly(I), fluorescein isothiocyanate (FITC)-dextran and
mevalonic acid were purchased from Sigma. Maleyl-BSA was prepared as
described elsewhere (15) . Dextran sulfate was purchased from
Pharmacia Biotech Inc. Simvastatin, an inhibitor of
3-hydroxy-3-methylglutaryl CoA reductase, was a gift from Eisai Co.
Ltd., Japan. Lipids were purchased from Avanti Polar Lipids, Inc.,
Birmingham, AL. Mammalian expression vector, pRc/CMV, was purchased
from Invitrogen.Cells
CHO cells (CHO-K1), a gift from Dr. M.
Nishijima (National Institute of Health, Japan), were used as parental
cells to isolate cells expressing a novel type of scavenger receptor.
These CHO cells are termed control CHO cells in the following
experiments. CHO cells expressing type I human scavenger receptors were
established by the following procedure. An expression vector containing
full-length human type I scavenger receptor and the neomycin-resistance
gene, pRc/CMV SR, was constructed and transfected into CHO-K1 cells.
Colonies resistant to G418 were screened with DiI-AcLDL to identify the
scavenger receptor-positive colonies as previously
described(27) . One of these, named CHO-SR7, was used in the
following experiments. CHO cells were grown in Ham's F-12 medium
containing 10% fetal calf serum (medium A) unless otherwise noted.Isolation of CHO Cells Expressing AcLDL Receptor
Activity
Control CHO cells were seeded at 5 
10
cells per 150-mm dish in 15 ml of Ham's F-12 medium
containing 10% lipoprotein-deficient serum (medium B) in the presence
of 5 µg/ml AcLDL. After 2 days, the medium was exchanged for medium
B containing 250 µM mevalonic acid, 5 µM simvastatin, and 5 µg/ml AcLDL. The cells were further treated
with gradually increasing concentrations of simvastatin (increases
every 2 weeks) until the concentration finally reached 100
µM. Under culture, about half of the cells died at 25
µM simvastatin, and eventually about 95% died and were
excluded. The surviving CHO cells were cloned by limiting dilution
method. After sufficient growth (5 10
cells/cm
), the cloned cells were stored in liquid
nitrogen until use in the experiment.Lipoprotein
LDL (d =
1.019-1.050 g/ml) and high density lipoproteins (HDL) (d = 1.063-1.21 g/ml) from fresh human plasma were
isolated by preparative ultracentrifugation(28) . Iodination
and acetylation of LDL were performed as described elsewhere (28, 29) . The concentration of lipoprotein is given
in terms of its protein content. Lipoprotein-deficient serum (d > 1.21 g/ml) was prepared as previously described(30) . Liposomal Preparation
The liposomes used were
composed of
phosphatidylcholine/phosphatidylserine/dicetylphosphate/free
cholesterol (molar ratio 50:50:10:75) (PS-liposome) or
phosphatidylcholine/dicetylphosphate/free cholesterol (molar ratio
100:10:75) (PC-liposome). PS-liposomes labeled with
[C]phosphatidylcholine were composed of
phosphatidylcholine/phosphatidylserine/dicetylphosphate/free
cholesterol/1,2-di[1-C]
palmitoylglycerophosphocholine (100-120 mCi/mmol) (molar ratio
50:50:10:75:0.5). These liposomes were prepared as described
elsewhere(31) . Liposomes containing FITC-dextran were prepared
with a slight modification. Four micromoles of dried lipids were
dispersed in 0.2 ml of 0.3 M glucose containing FITC-dextran
(10 mg/ml) to obtain multilamellar vesicles. Unencapsulated
FITC-dextran was removed by chromatography on a Sepharose CL-4B column
equilibrated with 0.3 M glucose. The fractions containing
liposomes were collected and used for the following experiments.Fluorescence Microscopy
Evaluation of the
accumulation of fluorescent DiI-AcLDL was performed by the following
procedure as described by Kingsley and Krieger(32) . Cells were
incubated with 0.5 ml of medium A containing 5 µg/ml DiI-AcLDL for
1 h at 37 °C. The monolayers were then washed and fixed by soaking
in 3% formalin. Accumulation of DiI-AcLDL by the cells was observed
using fluorescence microscopy. The accumulation of liposomes containing
fluorescent FITC-dextran by the cells was performed by the same
procedure except that they were not fixed.AcLDL Requirement of the Isolated CHO
Cells
Control and the isolated CHO cells were seeded on day 0 at
5 10 cells/well in medium B. On day 2, the medium
was exchanged for 2 ml of medium B containing 250 µM mevalonic acid and various amounts of simvastatin in the absence
or presence of 5 µg/ml AcLDL. On day 4, the monolayers were washed
twice with phosphate-buffered saline to remove cell debris. Adherent
cells were then trypsinized and the cell number was counted.Assay of
Assay was
performed as previously described (1, 29) with a
slight modification. CHO cells were incubated in 1 ml of a solution of
Ham's F-12 medium, 10 mM HEPES, pH 7.4, and 10%
lipoprotein-deficient serum (medium C) and I-AcLDL Binding
I-AcLDL in the
absence or presence of 20-fold excess amounts of AcLDL for 2 h at 4
°C. After incubation the cells were washed three times with 1 ml
buffer containing 50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, and 2 mg/ml BSA (buffer A), then twice with 1 ml of buffer A
without BSA. The cells were removed from the dish by dissolution in
0.2% sodium dodecyl sulfate. Aliquots were removed from the dish for
counting in a
counter and for measurement of protein
concentration(33) . As shown in Fig. 3, the amount of
specifically bound
I-AcLDL was determined by subtracting
the radioactivity bound in the presence of a 20-fold excess amount of
AcLDL from the radioactivity bound in the absence of AcLDL. The binding
values are expressed as ng of
I-AcLDL protein bound per
mg total cell protein. Each dish contained about 100 µg total cell
protein.
I-AcLDL by
control CHO cells and CHO-AL1 cells at 4 °C. CHO cells were seeded
on day 0 at 10
cells/well in medium A. On day 2, each dish
of CHO cells received 1 ml of ice-cold medium C containing the
indicated concentration of I-AcLDL (240 cpm/ng of
protein). The monolayers were incubated at 4 °C for 2 h. The amount
of
I-AcLDL bound to control CHO cells (open circles) and
CHO-AL1 cells (closed circles) was determined in duplicate
dishes (A). Values represent specific binding. Values of
nonspecific binding by both types of cell were nearly the same. At each
concentration of
I-AcLDL, they were less than 10% of the
values of total binding by CHO-AL1 cells. The Scatchard analysis of the
specific binding of
I-AcLDL by CHO-AL1 cells is presented
in B. The curve was culculated according to the nonlinear
least-squares method.
Assay of Proteolytic Degradation and Cell Association of
CHO cells were incubated in 0.5 ml of
medium B containing I-AcLDL
I-AcLDL in the absence or presence of
20-fold excess amounts of AcLDL at 37 °C. After washing the cells
the amount of
I-labeled acid-soluble material in the
medium (degradation) and the amount of
I-AcLDL in the
cells (association) were determined by subtracting the radioactivity
occurring in the presence of the 20-fold excess amount of AcLDL from
the radioactivity occurring in the absence of AcLDL.
Assay of Cell Association of Liposome
Association
of liposome with the cells was examined by the same procedure as the I-AcLDL association assay except that 100 µg/ml
C-labeled liposome was used as a ligand instead of I-AcLDL.
CHO Cells Exhibiting AcLDL Receptor Activity
We
isolated the CHO cells surviving in medium containing 5 µg/ml AcLDL
as the sole source of exogenous lipoproteins (see ``Experimental
Procedures''). The appearance and doubling time of the isolated
CHO cells were almost the same as those of the parental CHO cells.
Parental control cells and one typical clone of these isolated CHO
cells (denoted CHO-AL1) were used in the following experiments. When
CHO-AL1 cells were cultured in media containing different
concentrations of simvastatin for 48 h, all the cells died at a
concentration of 25 µM simvastatin. In contrast, in a
medium containing 5 µg/ml exogenous AcLDL CHO-AL1 cells were
resistant to 50 µM simvastatin and growing cells were
observed even at 100 µM (Fig. 1). Control CHO
cells, however, were sensitive to the same concentration of simvastatin
irrespective of the presence or absence of AcLDL (data not shown).
These results indicate that CHO-AL1 cells can utilize exogenous AcLDL
as a cholesterol source.
10 cells per well in 2 ml
medium B. On day 2, the medium was exchanged for medium B containing
the indicated amounts of simvastatin in the absence (closed
circles) or presence (open circles) of 5 µg/ml AcLDL.
On day 4, adherent cells were trypsinized, and the cell number was
counted. Cell number was determined in duplicate
dishes.
Characterization of AcLDL Uptake by CHO-AL1
Cells
First, the surface binding of I-AcLDL at 4
°C was examined. When CHO-AL1 cells were incubated with increasing
concentrations of
I-AcLDL at 4 °C, their specific
surface binding increased in a saturable fashion (Fig. 3A). The corresponding Scatchard plot was
indicated in Fig. 3B. Goodness of the fit was assessed
by means of the Akaike's information criterion
value(34) , and the data were fitted best to the model with two
kinds of saturable binding sites, high and low affinity. The
dissociation constants for high and low affinity binding were 3.8 and
4211 µg/ml, respectively. In contrast, control CHO cells exhibited
virtually no efficient specific binding of
I-AcLDL (Fig. 3A).
I-AcLDL by CHO-AL1 cells were examined at 37 °C (Fig. 4). In CHO-AL1 cells incubated with
I-AcLDL
at 37 °C for varying lengths of time, the cellular association of
radioactivity reached a maximum within 2 h and was maintained at
steady-state thereafter. Acid-soluble radioactivity continued to appear
in the medium at a linear rate, reflecting the continuing uptake and
degradation of
I-AcLDL. After 24 h, approximately 5 times
as much
I-AcLDL had been degraded as was contained within
the cells at steady-state. In the presence of 75 µM of the
lysosomal inhibitor chloroquine(35, 36) , degradation
was completely abolished, whereas the cellular association of
I-AcLDL increased for 4 h and reached a steady-state
plateau (data not shown). These results indicate that CHO-AL1 cells
express the receptor that recognizes AcLDL, and metabolize AcLDL
through these receptors.
I-AcLDL by CHO-AL1 cells. CHO-AL1 cells were seeded on
day 0 at 10
cells/well in medium B. On day 2, the
monolayers received 0.5 ml of medium B containing 10 µg/ml I-AcLDL. After incubation at 37 °C for the indicated
time, degradation (open symbols) and association (closed
symbols) were determined in duplicate
dishes.
Comparison of the Receptor on CHO-AL1 Cells with the
Macrophage Scavenger Receptor
As described above, it was found
that CHO-AL1 cells express a specific receptor that recognizes AcLDL.
To compare this receptor with the macrophage scavenger receptor, we
prepared CHO cells (CHO-SR7) that constitutively express human type I
scavenger receptor by transfecting its cDNA (see ``Experimental
Procedures''). First, the specificity of the receptor on CHO-AL1
cells was examined by competition with various lipoproteins. A 50-fold
excess of HDL or LDL had little effect on association and degradation
of I-AcLDL, whereas a 20-fold excess of unlabeled AcLDL
reduced them by 90% (data not shown). Similar results were obtained
with CHO-SR7 cells (data not shown), consistent with previous data from
cells expressing macrophage scavenger receptors(27) . Next, the
effects of known competitors for the macrophage scavenger receptor on
the association of
I-AcLDL to CHO-SR7 and CHO-AL1 cells
were examined. As previously reported(27) , dextran sulfate,
poly(I), maleyl-BSA and fucoidan effectively competed for the
association of
I-AcLDL by CHO-SR7 cells (Fig. 5B). Unexpectedly, however, inhibition of AcLDL
binding was not observed with dextran sulfate, poly(I) and fucoidan in
CHO-AL1 cells (Fig. 5A). Heparin was not an effective
competitor for either type of receptor. Among the competitors tested,
only maleyl-BSA inhibited
I-AcLDL association by CHO-AL1
cells. Similar results were obtained when degradation by these cells
was examined (data not shown).
I-AcLDL by CHO-AL1 cells (A)
and CHO-SR7 cells (B). Each dish received 0.5 ml of medium C
containing 10 µg/ml
I-AcLDL (240 cpm/ng of protein)
and 1 mg/ml of the following compounds: dextran sulfate (lane
2), polyinosinic acid (lane 3), fucoidan (lane
4), heparin (lane 5), and maleyl-BSA (lane 6).
After incubation for 12 h at 37 °C, association was determined in
duplicate dishes. The 100% values for association of
I-AcLDL in the absence of competing compounds (lane
1) by CHO-AL1 cells and CHO-SR7 cells were 460 and 300 ng/mg cell
protein, respectively.
I-AcLDL degradation was examined. Unlabeled PS-liposome
(100 µg/ml) inhibited
I-AcLDL degradation by CHO-AL1
cells, but not by CHO-SR7 cells. Next, the effect of AcLDL on
[
C]PS-liposome association was examined in
CHO-AL1 cells (Fig. 7B). Unlabeled AcLDL (1 mg/ml)
completely inhibited [C]PS-liposome association
by CHO-AL1 cells. Dextran sulfate, poly(I), fucoidan, heparin and
maleyl-BSA did not suppress [C]PS-liposome
association by CHO-AL1 cells, as in the case of I-AcLDL
(data not shown). These results indicate that AcLDL and PS-liposome are
recognized by the same receptor on CHO-AL1 cells, and that this
receptor is distinct from macrophage type I scavenger receptor based on
their competitor sensitivity and ligand specificity.
I-AcLDL by CHO-AL1 cells and CHO-SR7 cells (A) and ability of AcLDL to inhibit the association of
[
C]PS-liposomes by CHO-AL1 cells (B). A, each dish received 0.5 ml of medium C containing 10
µg/ml I-AcLDL (240 cpm/ng protein) and the indicated
concentration of PS-liposome. After incubation for 12 h at 37 °C,
degradation of
I-AcLDL was determined in duplicate
dishes. The 100% values for degradation in the absence of PS-liposome
by CHO-AL1 and CHO-SR7 were 1105 and 576 ng/mg of cell protein,
respectively. Open circles, CHO-SR7; closed circles,
CHO-AL1. B, each dish received 0.5 ml of medium C containing
100 µg/ml [
C]PS-liposome and the indicated
concentration of AcLDL. After incubation for 12 h at 37 °C,
association of [C]PS-liposome was determined in
duplicate dishes. The 100% value for association in the absence of
AcLDL was 13.1 µg/mg cell protein. The 0% values for A and B were based on the values for 50- and 10-fold excesses of
unlabeled ligand, respectively. The
[C]PS-liposome composition is described under
``Experimental Procedures.''
Uptake of Various Liposomes by CHO-AL1 Cells
The
effect of phospholipid composition on the association of liposomes was
further examined. The association of liposomes with CHO-AL1 cells was
found to be dependent on liposomal composition (Fig. 8).
Efficient association was observed for liposomes containing acidic
phospholipids such as phosphatidylserine, phosphatidylinositol,
phosphatidic acid, or phosphatidylethanolamine in CHO-AL1 cells.
PC-liposome which contained small amount of dicetylphosphate (see
``Experimental Procedures'') also showed a low but
significant level of association with CHO-AL1 cells. In a separate
experiment, liposomes composed of exclusively phosphatidylcholine,
which tend to aggregate, did not show appreciable binding to either
control or CHO-AL1 cells (data not shown). These results indicate that
negatively charged liposomes are taken up by the receptor for AcLDL
expressed on the CHO-AL1 cells. Even in control CHO cells, less
efficient but significant association was observed for liposomes
containing acidic phospholipids.
C]palmitoyl-glycerophosphocholine
(100-120 mCi/mmol) with a molar ratio of 50:50:10:75:0.5. After
incubation for 12 h at 37 °C, association was determined in
duplicate dishes as described under ``Experimental
Procedures.'' PE, phosphatidylethanolamine; PA,
phosphatidic acid; PI,
phosphatidylinositol.
I-AcLDL (Fig. 5). (ii) Both AcLDL and
negatively charged liposomes containing acidic phospholipids are
recognized by the same receptor on the CHO cells, whereas CHO-SR7 cells
transfected with cDNA for the human type I scavenger receptor do not
endocytose negatively charged liposomes (Fig. 5)(6, 37) . (iii) CHO-AL1 cells expressed
no detectable scavenger receptor mRNA by Northern blot analysis (data
not shown). To identify the receptor molecule, we also performed ligand
blot analysis for CHO-AL1 cells with
I-AcLDL or
I-maleyl-BSA using the methods of Daniel et al.(38) and Kodama et al.(4) . Under these
conditions, where the band corresponding to the scavenger receptor
could be detected in CHO-SR7 cells, the specific band for the receptor
that binds to
I-AcLDL or
I-maleyl-BSA was
not observed for CHO-AL1 cells. This may indicate that the AcLDL
receptor on CHO-AL1 cells became irreversibly inactivated during the
ligand blot experiment, or that it has properties different from those
of type I or type II scavenger receptors. Fc
RII-b2 (16) and CD36(17) , both of which are expressed on
macrophages and bind OxLDL, are also not candidates as the receptor on
CHO-AL1 cells, since these receptors cannot recognize AcLDL
appreciably. Endothelial cells also express AcLDL
receptors(20, 21, 22, 23) , but
these receptors appear to be different from types I or II scavenger
receptors. Although the receptors on endothelial cells have not yet
been clearly identified(39) , the fact that polyanionic
macromolecules such as poly(I) compete for the binding of
I-AcLDL by endothelial cells suggests (40, 41) that the receptor on CHO-AL1 cells is also
distinct from those on endothelial cells (Table 1). All of these
data support the idea that the receptor on the isolated CHO-AL1 cells
seems to be a new class of scavenger receptor with respect to ligand
specificity and competitor sensitivity.
RII-b2 may be involved in the uptake of
negatively charged liposomes, but the fact that their uptake was
inhibited significantly by AcLDL (31) cannot be explained by
the involvement of these receptors. The binding of negatively charged
liposomes to the receptors on mouse peritoneal macrophages was
effectively suppressed by polyanionic sugars(31) . Unlike the
binding of liposomes to macrophages, binding of AcLDL and negatively
charged liposomes to the receptor on the CHO-AL1 cells was not
inhibited by polyanionic sugars (data not shown). A further difference
in the nature of the receptors between CHO-AL1 cells and mouse
peritoneal macrophages is the extent of the uptake of
phosphatidylethanolamine-containing liposomes (Fig. 8). Mouse
peritoneal macrophages did not take up significant amounts of
phosphatidylethanolamine-containing liposomes(31) , whereas
CHO-AL1 cells take up these liposomes as effectively as liposomes
containing phosphatidylserine, phosphatidic acid, or
phosphatidylinositol under the present conditions. Since
phosphatidylethanolamine exhibits a weakly acidic nature in the neutral
pH range, the receptor on CHO-AL1 cells may be able to recognize these
liposomes as well. Although our data demonstrate the presence of a
receptor that recognizes both AcLDL and negatively charged liposomes in
CHO cells, this receptor may not be identical to the receptor detected
on macrophages that recognizes the liposomes.
I-AcLDL binding sites (high
and low affinity). However, this does not necessarily represent the
expression of two distinct receptor proteins, since the CHO cells that
received transfection of the murine scavenger receptor cDNA (types I or
II) also showed high and low affinity binding of
I-AcLDL(9) . The mechanism by which receptor
expression occurs is at present totally unknown. The expression of the
receptor is likely to be reversible in CHO cells, since incubation of
CHO-AL1 cells in medium without simvastatin caused the gradual
reduction of receptor activity. (
)The parental control CHO
cells might have had a low level of receptor activity for the uptake of
negatively charged liposomes, since a very low but significant level of
association of these liposomes was observed even for the control cells (Fig. 8). Liver parenchymal cells also exhibit very low level
AcLDL uptake activity and that this uptake is not inhibited by
poly(I)(40) , indicating that AcLDL receptor may be expressed
on many types of cell at a low level. Certain signals may induce
increase of the expression of the receptor for AcLDL and negatively
charged liposomes under some conditions. An interesting hypothesis is
that a physiologic stimulus that induces the increased expression of
this type of receptor may exist in animals.
))
We thank Drs. Y. Sato and Y. Kato (University of
Tokyo) for helpful suggestions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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