©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Chinese Hamster Ovary Cells Expressing a Novel Type of Acetylated Low Density Lipoprotein Receptor
ISOLATION AND CHARACTERIZATION (*)

(Received for publication, August 10, 1994; and in revised form, September 30, 1994)

Masayoshi Fukasawa (1) Kotaro Hirota (1) Hideki Adachi (2) Keiko Mimura (3) Kimiko Murakami-Murofushi (3) Masafumi Tsujimoto (2) Hiroyuki Arai (1)(§) Keizo Inoue (1)

From the  (1)Department of Health Chemistry, Faculty of Pharmaceutical Sciences, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan, (2)Suntory Institute for Biomedical Research, Mishima, Osaka 618, Japan, and (3)Department of Biology, Faculty of Science, Ochanomizu University, Bunkyo-ku, Tokyo 112, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Chemically modified low density lipoproteins (LDL), (^1)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 alpha-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 FcRII-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.


EXPERIMENTAL PROCEDURES

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-^14C]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 times 10^5 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 times 10^4 cells/cm^2), 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 [^14C]phosphatidylcholine were composed of phosphatidylcholine/phosphatidylserine/dicetylphosphate/free cholesterol/1,2-di[1-^14C] 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 times 10^4 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 I-AcLDL Binding

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 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.


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 10^5 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 I-AcLDL

CHO cells were incubated in 0.5 ml of medium B containing 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 ^14C-labeled liposome was used as a ligand instead of I-AcLDL.


RESULTS

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.


Figure 1: AcLDL requirement of CHO-AL1 cells under retardation of cholesterol synthesis. CHO-AL1 cells were seeded on day 0 at 5 times 10^4 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.)



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).

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 10^5 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).


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 [^14C]PS-liposome association was examined in CHO-AL1 cells (Fig. 7B). Unlabeled AcLDL (1 mg/ml) completely inhibited [^14C]PS-liposome association by CHO-AL1 cells. Dextran sulfate, poly(I), fucoidan, heparin and maleyl-BSA did not suppress [^14C]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 [^14C]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 [^14C]PS-liposome and the indicated concentration of AcLDL. After incubation for 12 h at 37 °C, association of [^14C]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 [^14C]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.


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-^14C]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.




DISCUSSION

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. FcRII-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. (^2)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.


FOOTNOTES

*
This work was supported in part by a grant from the Japan Health Sciences Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Faculty of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan. Tel.: 3-3812-2111 (ext. 4720); Fax: 3-3818-3173.

(^1)
The abbreviations used are: LDL, low density lipoproteins; AcLDL, acetylated LDL; OxLDL, oxidized LDL; HDL, high density lipoproteins; maleyl-, maleylated; BSA, bovine serum albumin; poly(I), polyinosinic acid; CHO, Chinese hamster ovary; FITC, fluorescein isothiocyanate; PS, phosphatidylserine; PC, phosphatidylcholine; DiI, 1,1`-dioctadecyl-3,3,3`,3`-tetramethylindocarbocyanine perchlorate.

(^2)
M. Fukasawa, K. Hirota, H. Adachi, K. Mimura, K. Murakami-Murofushi, M. Tsujimoto, H. Arai, and K. Inoue, unpublished data.


ACKNOWLEDGEMENTS

We thank Drs. Y. Sato and Y. Kato (University of Tokyo) for helpful suggestions.

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.


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