Characterization of CLA-1, a Human Homologue of Rodent Scavenger Receptor BI, as a Receptor for High Density Lipoprotein and Apoptotic Thymocytes*

(Received for publication, December 2, 1996, and in revised form, April 25, 1997)

Koji Murao Dagger , Valeska Terpstra , Simone R. Green , Nonna Kondratenko , Daniel Steinberg and Oswald Quehenberger §

From the Department of Medicine, University of California, San Diego, La Jolla, California 92093-0682

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Recently, a murine scavenger receptor type B class I (SR-BI) was identified that binds high density lipoprotein (HDL) and mediates the selective uptake of cholesterol esters. The human CD36 and LIMPII analogous-1 (CLA-1) receptor shows high sequence homology with SR-BI, but their functional relationship has not been determined. Transfected cells expressing CLA-1 bound HDL with a Kd of about 35 µg/ml, similar to the Kd for HDL binding to rodent SR-BI. This binding resulted in an intracellular accumulation of HDL-derived [3H]cholesterol esters without internalization or degradation of 125I-apolipoprotein. CLA-1 was strongly expressed in the adrenal gland and was also abundant in liver and testis, suggesting that CLA-1, like SR-BI, could play a role in the metabolism of HDL. However, CLA-1 was also expressed in monocytes and, like SR-BI, recognized modified forms of low density lipoproteins as well as native LDL and anionic phospholipids. These findings suggest that CLA-1 might have additional physiological functions. We found that CLA-1 recognizes apoptotic thymocytes. Our results demonstrate that CLA-1, a close structural homologue of SR-BI, is also functionally related to SR-BI and may play an important role as a "docking receptor" for HDL in connection with selective uptake of cholesterol esters. An additional role in recognition of damaged cells is suggested by these studies.


INTRODUCTION

Scavenger receptor BI (SR-BI)1 was originally cloned on the basis of its ability to bind modified lipoproteins and was therefore classified as a novel scavenger receptor (1). Its ligand-binding specificity was similar to that of CD36, and thus it was included as a member of the new family of scavenger receptors, designated class B (2).

Subsequent studies showed that SR-BI has a high affinity for the binding of HDL and that it mediates the selective uptake of cholesterol esters from HDL (3). Furthermore, the tissue distribution of SR-BI, which is predominantly expressed in liver, adrenal gland, and ovary, was compatible with its playing a role in the transport of HDL-derived cholesterol esters to the liver and to steroidogenic tissues (4). More recently, it was reported that mice deficient in apoA-I overexpressed SR-BI, presumably in an effort to compensate for the decreased plasma level of HDL cholesterol and the depleted stores of adrenal cholesterol (5). In contrast, transgenic mice deficient in apolipoprotein A-II, apolipoprotein E, the LDL receptor, or cholesterol ester transfer protein did not show any changes of SR-BI expression in either adrenal gland or liver (5). Previous studies have demonstrated a strong inverse correlation between plasma HDL levels and risk of coronary heart disease (6), possibly because HDL plays a critical role in reverse cholesterol transport, i.e. transport from peripheral tissues to the liver for catabolism (7-9). Taken together, these results suggest that SR-BI may play a physiologically important role in metabolism of HDL-derived cholesterol.

Human CLA-1 was cloned from a cDNA library prepared from differentiated HL-60 cells based on the existence of regions with amino acid sequence highly conserved between CD36 and LIMPII (10). CD36 is an 88-kDa surface glycoprotein present on monocytes, platelets, and endothelial cells (11), and LIMPII, a structural homologue of CD36, is expressed mainly on lysosomal membranes. Two distinct isoforms of CLA-1 that differ by an insertion of a segment consisting of 100 amino acid residues at the N-terminal region have been identified. Analysis of the CLA-1 cDNAs predicts proteins of 409 and 509 amino acid residues with several potential N-glycosylation sites. Like CD36, which binds to thrombospondin (12, 13), collagen (14), and erythrocytes infected with Plasmodium falciparum (15, 16), CLA-1 is found mainly on the plasma membrane, underlining its potential function as a receptor.

The long (509 amino acid residues) form of human CLA-1 shares 81% sequence identity with hamster SR-BI and thus most probably represents the same gene. However, the function of CLA-1 has not been systematically studied. If the primary function of SR-BI in rodents is to facilitate selective uptake of cholesterol esters from HDL, its human homologue CLA-1 might be redundant since human tissues are supplied with cholesterol mainly via LDL. We have generated stably transfected cells to test whether CLA-1 has biological functions similar to those of rodent SR-BI. The current report provides evidence that CLA-1 can function as a receptor for HDL and can mediate selective uptake of cholesterol esters, suggesting that CLA-1 is indeed functionally related to the rodent SR-BI. We find CLA-1 primarily expressed in liver and steroidogenic tissues, like SR-BI. However, we also find it in circulating monocytes and to a lesser extent in fully differentiated macrophages. Preliminary data show also that apoptotic thymocytes can bind to cells transfected with CLA-1. Thus, in addition to its role as receptor for HDL in liver and steroidogenic tissues, CLA-1 may have alternative functions in leukocytes.


EXPERIMENTAL PROCEDURES

Materials

Cell culture media and G418 sulfate were purchased from Life Technologies Inc. Fetal calf serum was from HyClone Laboratories. Glutathione-Sepharose 4B beads were purchased from Pharmacia Biotech Inc. Carrier-free Na[125I] was obtained from Amersham and [32P]dCTP was from ICN. Nitrocellulose membranes were from Bio-Rad. The phospholipids porcine brain PS, egg yolk PC, and bovine liver PI were obtained from Avanti Polar Lipids. Cholesterol from porcine liver, PMA, and Oil Red O were purchased from Sigma.

Lipoproteins

Human HDL (d = 1.063-1.21 g/ml) and human LDL (d = 1.019-1.063 g/ml) were isolated by preparative ultracentrifugation from fresh plasma collected in EDTA (1 mg/ml) as described (17). HDL was passed through a heparin-Sepharose affinity column to remove particles containing apolipoprotein E (18). The isolated lipoproteins were iodinated by the Pierce IODOGEN method to a specific activity of 400-600 cpm/ng protein for HDL and about 200 cpm/ng protein for LDL. For specific uptake studies, the lipid moiety of human HDL was labeled with [3H]cholesterol ester ([3H]CE) and the apoA-I with 125I. The former ([3H]CE) was prepared from [3H]cholesterol and oleic anhydride as described previously (19). HDL was labeled with [3H]CE by exchange from donor liposomal particles using partially purified human CE transfer protein as the source of CE transfer activity. Donor particles were then removed from the labeled HDL by flotation at d = 1.063 g/ml. Human apoA-I was purified from HDL, labeled with 125I, and then exchanged into the [3H]HDL by a 24-h incubation at 37 °C (19). The resulting doubly labeled HDL was reisolated by flotation at d = 1.24 g/ml. OxLDL was prepared by incubating LDL for 24 h in 5 µM Cu2+, and AcLDL was prepared by treatment with acetic anhydride as described (20).

Cell Culture

Human monocyte-derived THP-1 cells (American Type Culture Collection) were grown in RPMI 1640 supplemented with 10% fetal calf serum, 100 µg/ml streptomycin, and 100 units/ml penicillin in a humidified atmosphere containing 5% CO2. Human embryonic kidney HEK 293 cells (American Type Culture Collection) were cultured in minimal essential medium with Earl's balanced salt solution containing 10% horse serum and 50 µg/ml gentamycin. Human monocytes were isolated from freshly drawn blood by centrifugation through Histopaque (Sigma), and human macrophages were prepared by plating the monocytes in RPMI 1640 supplemented with 10% fetal calf serum for 7 days (21). Differentiation of THP-1 cells was induced by culturing the cells for 2 days in the presence of various amounts of PMA as indicated. Plasma membranes were purified by a two-phase system with dextran and polyethylene glycol (22). A single cell suspension of thymocytes was obtained by disrupting thymus tissues by passing it through a stainless steel wire mesh into RPMI 1640 medium. Thymocytes were washed and cultured as described previously (23).

Apoptotic Cell Binding and Preparation of Phospholipid Liposomes

Apoptosis was induced by culturing the thymocytes in the presence of 1 µM dexamethasone for 4 h. Cell binding experiments were carried out by incubating apoptotic and viable thymocytes with transfected HEK 293 cells at 37 °C for 1 h in minimal essential medium containing 0.1% bovine serum albumin (ratio of HEK 293 cells:thymocytes = 1:10). After washing to remove unbound cells, the percentage of HEK 293 cells binding one or more thymocytes was determined. In competition experiments, the binding of thymocytes by the transfected cells was determined in the presence of unilamellar vesicles consisting of phospholipids and cholesterol. Phospholipids and cholesterol in chloroform (molar ratios: PS/PC/cholesterol = 1:1:1; PI/PC/cholesterol = 1:1:1; PC/cholesterol = 2:1) were dried under N2 flow and resuspended in phosphate-buffered saline, pH 7.4, by vortexing. Unilamellar phospholipid liposomes were formed by repeated extrusion under high pressure of N2 through polycarbonate membranes (Poretics) (24). The preparation of liposomes ranging in diameter from 80 to 100 nm were used immediately. For competition of thymocyte binding by the transfected cells, the liposomes were added to the monolayer to a final lipid concentration of 0.1 mM.

Plasmid Construction and Stable Transfection

The full-length cDNA of the 509-amino acid residue form of CLA-1, including the consensus sequence for initiation of translation (Kozak), was amplified by PCR from THP-1 cDNA as described previously (25), and cloned into the eukaryotic expression vector pcDNA3 (Invitrogen). The sequence of CLA-1 cDNA was confirmed by double-stranded cDNA sequencing. HEK 293 cells were transfected with 5 µg of linearized plasmid DNA as described (26). Stable transfectants were selected by their resistance to G418 sulfate (0.8 mg of active drug/ml), and a clone showing high CLA-1 expression was identified by Western blot analysis.

Ligand Binding and Degradation Assay

Binding of 125I-HDL by transfected cells or THP-1 cells was assessed at 37 °C for 1 h in the presence or absence of a 30-fold excess of unlabeled HDL. Unbound ligand was removed by washing the cells twice with phosphate-buffered saline, pH 7.4, containing 0.2% bovine serum albumin and by washing an additional two times with phosphate-buffered saline alone. The washed cells were solubilized in 0.2 M NaOH, and the radioactivity was quantitated to determine cell-associated 125I-HDL. Ligand specificity was determined in competition experiments using indicated amounts of LDL, AcLDL, and OxLDL. To test the effect of phospholipids on HDL binding by CLA-1, unilamellar liposomes containing PS, PI, or PC were prepared as described above and added together with 125I-HDL to the cell monolayer to give a final total lipid concentration of 1.0 mM. Degradation assays were performed as described previously (27).

RNA Isolation and Northern Blot Analysis

Total RNA was isolated from the human THP-1 cells and several human tissues by single-step acid guanidinium thiocyanate-phenol-chloroform extraction (28) or purchased from CLONTECH. A full-length cDNA of the 509-amino acid form of human CLA-1 was synthesized by PCR using reverse-transcribed RNA from THP-1 cells and labeled with [32P]dCTP (3000 Ci/mmol) by the random priming method (Promega). Electrophoresis and hybridization were performed as described (25). Following hybridization the filter was washed in 0.2 × SSC, 0.1% SDS at 60 °C. In some instances, CLA-1 expression was also estimated by PCR analysis of the reverse-transcribed RNA. A primer pair matching the published sequence of CLA-1 but with no homology with CD36 was used to amplify an 880-bp fragment. As control, GAPDH was amplified and analyzed under identical conditions using the appropriate set of primers (29).

Generation of Antibodies and Western Blot Analysis

An antibody (AbC1) directed against the extracellular domain of CLA-1 between amino acid residues 185 and 300 of the reported sequence of the isomer containing 509 amino acid residues (10) was generated. The corresponding cDNA fragment was amplified from THP-1 cDNA by PCR. The amplified fragment was inserted into a pGEX-2T vector (Pharmacia) and sequenced, and the protein was expressed in Escherichia coli. The fusion protein was isolated with glutathione-Sepharose 4B beads (Pharmacia) and used to generate an antiserum in guinea pigs. The IgG fraction was purified and used for Western blot analysis. As second antibody we used an alkaline phosphatase-conjugated goat anti-guinea pig IgG (Sigma).

Cellular Cholesterol Assay

Cellular lipids were extracted with chloroform:methanol = 2:1 (v/v), dried under N2, and redissolved in 200 µl of isopropanol. Aliquots (20 µl) were used for determination of cholesterol mass by a modification of the enzymatic fluorometric method (30).

Other Assays

Cell suspensions were cytocentrifuged onto glass slides and stained with Oil Red O as described previously (31). Protein was determined by the method of Lowry et al. (32), and SDS-PAGE was carried out as described (33).

Statistical Analysis

Statistical comparisons were made by one-way analysis of variance (ANOVA) and Student's t test.


RESULTS

Specific Binding of HDL to CLA-1

Western blot analysis of proteins extracted from the cells stably expressing CLA-1, using the antibody AbC1 directed against an extracellular portion of CLA-1 described under "Experimental Procedures," revealed a single band with an estimated molecular mass of 83 kDa (Fig. 1A). Mock-transfected cells also contained some CLA-1 protein, but at a much lower level. To examine the possible role of CLA-1 as a receptor for HDL, we conducted equilibrium ligand binding analyses using 125I-HDL. The transfected cells expressing CLA-1 bound HDL with high affinity (Fig. 1B). Scatchard analysis of the equilibrium binding data revealed a Kd of 35 µg of HDL protein/ml and maximal binding of 0.91 µg of HDL protein/mg of cell protein. However, binding of HDL by CLA-1 was not associated with any significant degradation of HDL protein, whereas 125I-LDL was degraded under the same conditions (see Fig. 6B).


Fig. 1. CLA-1 mediates cell association of 125I-HDL. A, Western blot analysis of membrane fraction from CLA-1-transfected and pcDNA3-transfected cells. Plasma membrane proteins were separated by SDS-PAGE, transferred to nitrocellulose, probed with 5 µg/ml anti-CLA-1 AbC1, and detected with alkaline phosphatase-conjugated IgG. Lane 1, CLA-1-transfected HEK 293 cells; lane 2, mock-transfected HEK 293 cells. B, HDL binding analysis of CLA-1-transfected HEK 293 cells. CLA-1-transfected cells (open circle ) and mock-transfected HEK 293 cells (bullet ) were incubated for 1 h at 37 °C with indicated amounts of 125I-HDL in the presence or absence of a 30-fold excess of unlabeled HDL. Shown is the specific cell-associated HDL, determined by subtracting nonspecific cell association from the total. Each point is the mean of triplicate determinations.
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Fig. 6. Binding of lipoprotein to HEK 293 cells transfected with CLA-1. A, lipoprotein inhibition of 125I-HDL binding. HDL cell association assays were carried out in the absence or presence (300 µg of protein/ml) of unlabeled HDL, Ac-LDL, native LDL, or OxLDL. The concentration of 125I-HDL used was 10 µg of protein/ml. In the absence of unlabeled competitor the transfected cells bound 225.7 ± 4.0 ng of 125I-HDL/mg of cell protein, which was taken as 100%. Results are the means ± S.E. of three experiments. B, inhibition of HDL binding by LDL. The transfected cells were incubated with 5 µg/ml 125I-HDL resulting in the binding of 181 ± 2.8 ng of protein/ml, which was taken as 100%, and increasing amounts of unlabeled HDL (open circle ) as well as LDL (bullet ) were added. The molar concentrations of the lipoproteins were estimated based on the average molecular weight of 2 × 106 containing 25% protein for LDL, and 2 × 105 containing 50% protein for HDL. Each point is the mean of triplicate determinations. C, degradation of 125I-LDL. CLA-1-transfected cells (open circle ) and mock-transfected cells (bullet ) were incubated for 5 h at 37 °C with the indicated amounts of 125I-LDL. The degradation of 125I-LDL was determined as described under "Experimental Procedures." Each point is the mean of triplicate determinations. Only the error bars exceeding the size of the symbols are shown.
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Role of CLA-1 in Cholesterol Ester Accumulation

Stably transfected HEK 293 cells expressing CLA-1 showed many vesicles in their cytoplasm when cultured in standard medium containing fetal calf serum. Oil Red O staining revealed that these represented lipid droplets. In contrast, there was no Oil Red O staining in mock-transfected cells (Fig. 2A). Expression of CLA-1 induced CE accumulation to a level that was about 4 times that in mock-transfected cells (Fig. 2B). The free cholesterol level was increased only minimally but significantly (p < 0.01). To confirm that the source of CE was exogenous, we incubated the transfected cells in lipoprotein-deficient serum. After 3 days the CE level was reduced by about 50%, suggesting that lipoproteins in the calf serum were the source for the increase in cell cholesterol content.


Fig. 2. Cholesterol accumulation in CLA-1-transfected cells. A, photomicrographs of CLA-1-transfected (top) and mock-transfected (bottom) HEK 293 cells. The cells were fixed, and intracellular lipid droplets were visualized by staining with Oil Red O. B, determination of cellular cholesterol. Mock-transfected (Control cells) and CLA-1-transfected HEK 293 cells grown in medium containing 10% fetal calf serum were replated and grown for an additional 3 days either in complete serum or in lipoprotein-deficient serum (LPDS). Cellular cholesterol was extracted, and free (hatched bars) and esterified (open bars) cholesterol were measured. Results are the means ± S.E. of three experiments.
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The mechanisms by which cells take up cholesterol ester from HDL are not clearly understood, but results from a series of reports favor a selective, nonendocytotic pathway for delivery of HDL-associated CE (19). We tested whether CLA-1 could participate in such a pathway by examining the kinetics of the uptake of 125I-apoA-I and of [3H]cholesteryl oleate in experiments using doubly labeled HDL. The binding of 125I-HDL to transfected cells expressing CLA-1 reached a maximum within 30 min at 37 °C and then remained constant to 180 min (Fig. 3). In contrast, cell-associated [3H]cholesterol ester increased continuously and was still rising at 180 min, indicating continuous transfer of CE into the cell. As pointed out above, no degradation products of 125I-apoA-I were detected in the medium. The mock-transfected cells displayed only very low levels of either CE or apolipoprotein uptake.


Fig. 3. Uptake of cholesterol ester and apoA-I from HDL by transfected HEK 293 cells. Doubly labeled HDL ([3H]cholesteryl oleate, 125I-apoA-I) was prepared as described under "Experimental Procedures." Cells were incubated at 37 °C with 15 µg of HDL protein/ml of doubly labeled HDL for the indicated time periods, and cell-associated 3H or 125I was quantified. CLA-1-transfected cells (open circle , square ) and mock-transfected cells (bullet , black-square) were harvested and assayed as outlined under "Experimental Procedures." Uptake of [3H]cholesteryl oleate (open circle , bullet ) and of 125I-apoA-I (square , black-square) was estimated. The values shown are expressed as the percent of total HDL-associated radioactivity added to the incubation medium. Each point is the mean of triplicate determinations. Only error bars exceeding the size of the symbols are shown.
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CLA-1 Tissue Distribution

Based on the considerable sequence homology, CLA-1 is believed to belong to the same gene family as CD36 and LIMPII (10, 34). However, its biological function and tissue distribution is still unknown. We examined the expression of CLA-1 message in several human tissues by Northern blot analysis (Fig. 4A). The message was detected as a 2.9-kilobase species, consistent with previous reports (10). CLA-1 was strongly expressed in the adrenal gland, and was also expressed in liver, testis and monocyte. Little expression was seen in adipose tissue and lung, and no CLA-1 mRNA was detected in the pancreas. These findings are consistent with the possibility that CLA-1 in humans has a role in selective uptake of CE from HDL in nonplacental steroidogenic tissues and perhaps in reverse cholesterol transport (3, 4, 35).


Fig. 4. Tissue distribution of CLA-1. A, Northern blot. Total RNA (20 µg) from the indicated human tissues was separated by agarose gel electrophoresis and transferred to nitrocellulose. The blot was hybridized with 32P-labeled full length CLA-1 probe (top panel). As control for equal loading and transfer of RNA in each lane, methylene blue staining of 28 and 18 S ribosomal RNA was performed (bottom panel). Lane 1, liver; lane 2, lung; lane 3, adipose tissue; lane 4, testis; lane 5, adrenal gland; lane 6, pancreas; lane 7, monocyte. B, reverse transcription-PCR analysis of CLA-1 mRNA expression. Total RNA was isolated from human monocyte and macrophage, and reverse transcription-PCR was performed as described under "Experimental Procedures." For PCR amplification, primers were used to give a ~880-bp fragment for CLA-1, corresponding to the region between nucleic acid residues 150 and 1030, and a ~520-bp fragment for GAPDH, which was included as internal control. Lanes 1 and 4, THP-1 cells; lanes 2 and 5, monocytes; lanes 3 and 6, macrophages. Lanes 1-3 and 4-6 indicate CLA-1 and GAPDH, respectively.
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The function of CLA-1 in monocytes, however, is less clear. It could have a role as a scavenger receptor, like its homologue, CD36. To test this possibility, we examined the expression of CLA-1 in human monocytic cell lines. Of the cell lines tested (THP-1, U937, and Mono Mac 6 cells), THP-1 cells expressed the highest level of CLA-1. The level of expression in these cell lines was comparable to that in freshly prepared human monocytes. However, expression was greatly down-regulated in human monocyte-derived macrophages (Fig. 4B). Incubation of THP-1 cells for 2 days with 100 nM PMA resulted in a loss of detectable CLA-1 mRNA (Fig. 5A). This reduction of CLA-1 expression paralleled the expected PMA-induced phenotypic changes. THP-1 cells typically grow in suspension, and addition of PMA at concentrations above 10 nM induces cell adhesion to the tissue culture flask. Western blot analysis using the antibody AbC1 revealed that CLA-1 was a plasma membrane-associated protein (Fig. 5B), which was confirmed by fluorescence-activated cell sorting analysis (data not shown). Parallel with the reduction of mRNA, CLA-1 protein was also reduced during the PMA-induced differentiation of THP-1 cells (Fig. 5B). In HDL-binding experiments conducted on undifferentiated and differentiated THP-1 cells, we found that monocytic THP-1 cells bound HDL with an affinity similar to that of CLA-1 expressed in transfected cells. Treatment of cells with 10 nM PMA reduced the number of HDL binding sites by about 50% with little or no change of binding affinity (Fig. 5C). The HDL binding experiments using PMA-treated THP-1 cells confirmed that CLA-1 could in principle serve as a receptor for HDL in monocytes, although it is unclear whether this is its primary function.


Fig. 5. Regulation of CLA-1 expression in human monocytic THP-1 cells. A, Northern blot analysis. Ten µg of total RNA isolated from THP-1 cells treated for 2 days with 0 nM (lane 1), 0.1 nM (lane 2), 1 nM (lane 3), 10 nM (lane 4), 100 nM (lane 5), and 1000 nM (lane 6) of PMA were separated by agarose gel electrophoresis and transferred to nitrocellulose. The blots were hybridized with the full-length coding region of CLA-1 [32P]cDNA (top panel) and human GAPDH [32P]cDNA that was used as a control for equal RNA loading and transfer (bottom panel). B, Western blot analysis. Plasma membrane proteins (lane 1) and total cell protein extracted with 1% Nonidet P-40 (lane 2) of untreated THP-1 cells and total cell protein extract from THP-1 cells treated for 2 days with 10 nM PMA (lane 3) were separated by SDS-PAGE electrophoresis, transferred to nitrocellulose membrane, and blotted with anti-CLA-1 antibody AbC1. Each lane contained 50 µg of protein. C, HDL cell association of PMA-treated (bullet ) and untreated (open circle ) THP-1 cells. THP-1 cells were treated for 2 days with 10 nM PMA, and their HDL binding activity was compared with that of untreated cells. The cells were incubated for 1 h at 37 °C with the indicated concentrations of 125I-HDL in the presence or absence of a 30-fold excess of unlabeled HDL. Specific HDL association was determined as described in Fig. 1B. Each point is the mean of triplicate determinations.
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Ligand Binding Specificity of CLA-1

A previous study suggested that both native LDL and modified LDL (AcLDL and OxLDL) bind to hamster SR-BI (1). We, therefore, further characterized the ligand binding specificity of human CLA-1. The long form of CLA-1 (509 amino acid residues) stably expressed in HEK 293 cells bound HDL, but it also interacted with native and modified LDL (Fig. 6A). OxLDL inhibited the binding of HDL to CLA-1 by about 60%. The inhibitory effects of AcLDL and native LDL were less pronounced. However, the competing lipoproteins were all added at an equal concentration of 300 µg/ml based on their protein content. The results from a more detailed competition experiment indicated that LDL, when present at similar particle concentration, inhibited binding of 125I-HDL by the CLA-1-transfected cells as efficiently as unlabeled HDL (Fig. 6B). In direct binding analysis we confirmed that CLA-1 recognized native LDL with a Kd of about 5 µg of LDL protein/ml. However, in contrast to the apoprotein in HDL, the apoprotein of native LDL was both internalized and degraded (Fig. 6C). The control-transfected cells were also able to mediate some LDL degradation, possibly through uptake via the apoB/E receptor. However, expression of CLA-1 in the same cell line increased that basal level substantially by about 4 fold. Our findings that CLA-1 is expressed on monocytes prompted us to extend our ligand binding studies to include phospholipids that are known ligands for scavenger receptors B (36). Phosphatidylinositol inhibited the binding of HDL by about 60% which was very similar to the inhibition by PS (Fig. 7). In contrast, PC had no effect on HDL binding. One pathway by which phagocytes recognize apoptotic cells is through binding to PS exposed on the outer leaflet of the plasma membrane (37). To test whether CLA-1 can recognize apoptotic cells via the PS-dependent mechanism, we incubated CLA-1-transfected cells with apoptotic thymocytes. Analysis of the binding studies showed that CLA-1 recognized apoptotic thymocytes, but not viable thymocytes (Table I). The extent of binding of apoptotic cells was statistically highly significant (p < 0.001). However, only about 20% of the transfected cells showed binding of apoptotic thymocytes. The CLA-1-transfected cells did not bind oxidized red blood cells (data not shown). The binding of apoptotic thymocytes to CLA-1 was completely prevented by unilamellar liposomes containing PS or PI. In contrast, PC had no effect on the binding, indicating that the recognition of apoptotic cells by phagocytes might be through a PS-dependent mechanism possibly involving CLA-1. However, further studies are needed to assess this scavenger-like function.


Fig. 7. Competition analysis of 125I-HDL binding with anionic phospholipids. HDL association with transfected HEK 293 cells expressing CLA-1 was determined in the absence or presence of phospholipids. Phospholipid liposomes prepared as described under "Experimental Procedures" were added to a final lipid concentration of 1.0 mM. Binding of 125I-HDL (10 µg of protein/ml) in the absence of competitor was 241.9 ± 7.3 ng/mg of cell protein and was taken as 100%. Results are the means ± S.E. of three experiments. *, significantly different from control (p < 0.001).
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Table I. Binding of apoptotic thymocytes to CLA-1-transfected cells

Viable or apoptotic thymocytes were added to the transfected HEK 293 cells expressing CLA-1 at a ratio of HEK 293:thymocytes = 1:10 and incubated for 1 h at 37 °C. In the competition experiments, the phospholipids were added as unilamellar liposomes (average diameter = 80-100 nm), prepared as described under "Experimental Procedures" at a final lipid concentration of 0.1 mM. Unbound cells were removed, and the thymocytes attached to the transfected cells were counted. The results are expressed as the percentage of mock- or CLA-1-transfected cells binding one or more thymocytes. Values represent the mean ± S.E. of five individual experiments. *, significantly different from the binding to mock transfected cells (p < 0.001). **, significantly different from the binding to CLA-1 transfected cells without competitor (p < 0.001).
Cell/competitor Percentage of cells binding one or more thymocytes
Viable thymocyte Apoptotic thymocyte

mean ± S.E. mean ± S.E.
Mock-transfected cells
  None 4.04  ± 0.6 4.7  ± 0.7
  PI 2.5  ± 0.9 4.5  ± 0.9
  PS 2.6  ± 1.1 4.3  ± 0.7
  PC 3.8  ± 0.8 4.1  ± 1.0
CLA-1-transfected cells
  None 4.9  ± 1.5 19.7  ± 2.7*
  PI 3.8  ± 1.1 8.7  ± 2.6**
  PS 4.5  ± 0.7 7.9  ± 2.2**
  PC 4.7  ± 1.5 18.5  ± 2.4


DISCUSSION

These studies show that the 509-amino acid residue isoform of CLA-1, like its very close homologue, rodent SR-BI, binds HDL in a specific, saturable fashion and can mediate the selective uptake of cholesterol esters from HDL. Whether or not this role of the receptor is important in lipid transport in humans remains to be determined. Many studies suggest that the major source of cholesterol for steroidogenesis in humans is LDL, but it is difficult to assess the relative importance of LDL and HDL under in vivo conditions. Certainly in situations where LDL concentrations are very low (e.g. in hypobetalipoproteinemia), adrenal function remains adequate although less able to respond to stress. Under these situations selective cholesterol ester transport from HDL via CLA-1 might become physiologically important. The role of SR-BI in the selective uptake of HDL-derived cholesterol esters has been demonstrated in recent animal studies (4, 5). CLA-1 not only displays a high sequence homology, but as our results suggest, it is also functionally related to SR-BI. Both receptors display a very similar ligand-binding specificity and show essentially an identical tissue distribution, indicating that in humans CLA-1 may have a function similar to that of SR-BI in rodents. Ligand binding analysis clearly demonstrated that CLA-1 can function as receptor for HDL. The binding of HDL was associated with accumulation of HDL-derived CE without any significant degradation of apoA-I. In contrast, the transfected cells not only bound LDL, but also internalized and degraded its apoB. CLA-1 binds LDL with relative high affinity (Kd = 5 µg of protein/ml), but its importance as a receptor for LDL is questionable. In humans, defects in the apoB receptor lead to a significant increase in plasma LDL cholesterol, suggesting that the apoB receptor is the major LDL receptor (38) and that CLA-1 might not be importantly involved in LDL metabolism.

The rather high level of expression of CLA-1 in circulating human monocytes (levels comparable to that found in human liver) was surprising. At first glance it is not clear why monocytes would require selective uptake of HDL cholesterol esters, since they do not, as far as we know, metabolize cholesterol to any important extent. However, during hematopoiesis and development of mature monocytes, the requirement for cholesterol is high and might exceed the amount available from LDL. Conceivably the progenitor cells in the bone marrow, when they are dividing rapidly, may require delivery of cholesterol at very high rates, and HDL delivery via selective cholesterol ester uptake might become quantitatively important. The other possibility is that CLA-1, shown here to bind apoptotic thymocytes, may play a role in the scavenging of damaged cells. Speaking against a function of CLA-1 as a scavenger receptor is the fact that its expression is down-regulated when the monocyte differentiates into a macrophage. However, low levels of CLA-1 mRNA were detected in human monocyte-derived macrophages (Fig. 4B), some CLA-1 expression and HDL binding was seen in THP-1 cells differentiated with PMA (Fig. 5, B and C), and selective uptake of cholesterol esters was demonstrated in plated human macrophages (39). In contrast, expression of SRA, CD36, and macrosialin/CD68, a recently identified receptor for OxLDL and possibly apoptotic cells, is up-regulated in the macrophage, which is teleologically rational for receptors that are involved in clearance of damaged cells (40-44). One might speculate that CLA-1 on the developing monocyte might play a role in the selective removal of cells differentiating along undesirable lines within the bone marrow, but there is no direct evidence for this.

One of the mechanisms by which phagocytes recognize apoptotic cells is mediated through putative receptors for PS (37). Senescent cells and cells undergoing apoptosis lose the normal membrane asymmetry in which anionic phospholipids are confined to the inner layer of the plasma membrane. As a consequence, PS appears on the outer leaflet of the membrane. CLA-1-transfected cells, like SR-BI- and CD36-transfected cells, bound PS and also recognized apoptotic thymocytes. Oxidized red blood cells, which are phagocytosed by macrophages through a presumably PS-dependent mechanism (45), were not recognized by the transfected cells (data not shown), indicating that several distinct PS-dependent pathways for phagocytosis might exist. Also, the mechanisms for internalizing oxidized red blood cells is more complex and depends on modifications of the plasma membrane in addition to increased PS exposure on the outer leaflet (45). The receptor(s) required for the recognition of these additional modifications may not be present on the CLA-1-transfected cells, which therefore do not bind oxidized red blood cells. Anionic phospholipids not only blocked the binding of apoptotic thymocytes by CLA-1, but they are also very effective inhibitors for the binding of HDL. The negative charges on the phospholipids as well as their spacing on the liposomes have been suggested to play an important role in that interaction (2).

Taken together, these data suggest that several mechanisms exist by which monocytes could recognize apoptotic cells, one of which involves CLA-1. The initial interaction does not induce phagocytosis, but may trigger a CLA-1 mediated cell signaling, resulting in cell differentiation and expression of components necessary for phagocytosis. A similar role has been suggested for CD36, which may contain a signal-transducing element able to trigger activation of monocytes and thus may have functions extending beyond adherence and phagocytosis (46).

In summary, we have demonstrated that, like SR-BI, CLA-1 can function as a receptor for HDL and mediate the selective uptake of the HDL cholesterol esters. It is expressed mainly in liver and nonplacental steroidogenic tissues. In addition, CLA-1 shows, like SR-BI, a ligand specificity pattern that is characteristic for scavenger receptors; however, its expression is drastically down-regulated in fully differentiated macrophages. The role of CLA-1 in cholesterol metabolism, as well as its function as a scavenger/signaling receptor on monocytes, deserves further investigations.


FOOTNOTES

*   This work was supported in part by Grant HL 14197 (SCOR on Arteriosclerosis) from NHLBI, National Institutes of Health.
Dagger    Supported in part by the Uehara Memorial Foundation in Japan.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: Dept. of Medicine, 0682, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093. Tel.: 619-534-4401; Fax: 619-534-2005; E-mail: oquehenberger{at}ucsd.edu.
1   The abbreviations used are: SR-BI, scavenger receptor type B class I; CLA-1, CD36 and LIMPII analogous-1 receptor; HDL, high density lipoprotein; apoA-I, apolipoprotein A-I; apoB, apolipoprotein B; LDL, low density lipoprotein; LIMPII, lysosomal integral membrane protein II; PS, phosphatidylserine; PC, phosphatidylcholine; PI, phosphatidylinositol; PMA, phorbol 12-myristate 13-acetate; CE, cholesterol ester; OxLDL, oxidized LDL; AcLDL, acetylated LDL; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s).

ACKNOWLEDGEMENTS

We thank Drs. W. Palinski and J. L. Witztum for helpful advice and F. Almazan, E. Miller, and J. Pattison for their expert technical assistance.


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