(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 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.
Chemically modified low density lipoproteins (LDL), ()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.
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 FcRII-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) .
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 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.
Figure 1:
AcLDL requirement of CHO-AL1
cells under retardation of cholesterol synthesis. CHO-AL1 cells were
seeded on day 0 at 5 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.
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 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.
Figure 4:
Time course of association and degradation
of 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.
Figure 5:
Ability of various compounds to inhibit
the association of 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.
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 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.
Figure 7:
Ability of PS-liposome to inhibit the
degradation of 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.''
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-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.
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 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.
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 FcRII-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.
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 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.
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.