doi: 10.1161/hq0901.094500
© 2001 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
LR11, a Mosaic LDL Receptor Family Member, Mediates the Uptake of ApoE-Rich Lipoproteins In Vitro
From the Department of Clinical Cell Biology, F5 (K. Taira, S.H., T.K, K. Takahashi, Y.S.), the Department of Genome Research and Clinical Application, M6 (H.B.), Graduate School of Medicine, and the Laboratory of Clinical Pharmacology (I.I.), Faculty of Pharmaceutical Sciences, Chiba University, Chiba, Japan; Kowa Research Institute (H.Y.), Kowa Co Ltd, Tokyo, Japan; the Clinical Laboratory (T.M.), Niigata University School of Medicine, Niigata, Japan; and the Department of Molecular Genetics (S.H., W.J.S.), Biocenter and University of Vienna, Vienna, Austria.
Correspondence to Dr Hideaki Bujo, Department of Genome Research and Clinical Application, M6, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail hbujo{at}intmed02.m.chiba-u.ac.jp
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Abstract |
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Abstract—— Since the molecular identification of the low density lipoprotein receptor (LDLR), an ever increasing number of related proteins have been discovered. These receptors belonging to the LDLR family are thought to play key roles in lipoprotein metabolism in a variety of tissues, including the arterial wall. We have discovered that the expression of a 250-kDa mosaic LDLR-related protein, which we termed LR11 for the presence of 11 LDLR ligand–binding repeats, is markedly induced in smooth muscle cells in the hyperplastic intima of animal models used for the study of atherosclerosis. Here, we demonstrate that the human LR11, when overexpressed in hamster cells, binds and internalizes 39-kDa receptor–associated protein (RAP), an in vitro ligand for all receptors belonging to the LDLR family. Furthermore, LR11 binds the apolipoprotein E (apoE)-rich lipoproteins, ß-very low density lipoproteins (VLDLs), with a high affinity similar to that of other members, such as the LDLR and VLDL receptor. RAP and ß-VLDL compete with each other; however, other serum lipoproteins are not able to inhibit their binding. LR11 shows specific binding of apoE-enriched HDL prepared from human cerebrospinal fluid as well as of ß-VLDL, suggesting that the apoE content of lipoproteins is most likely important for mediating the high-affinity binding to the receptor. LR11-overexpressing cells are able to internalize and degrade the bound ß-VLDL; these cells also show increased accumulation of cholesteryl esters when incubated with ß-VLDL. Incubation for 48 hours with ß-VLDL of LR11-overexpressing cells, but not of control cells, promotes the appearance of numerous intracellular lipid droplets. Taken together, LR11, a mosaic LDLR family member whose expression in smooth muscle cells is markedly induced in atheroma, has all the properties of a receptor for the endocytosis of lipoproteins, particularly for the incorporation of apoE-rich lipoproteins.
Key Words: LDL receptors • atherosclerosis • smooth muscle cells • ß-VLDL
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Introduction |
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Receptors belonging to the LDL receptor (LDLR) family are thought to play key roles in lipoprotein metabolism in a variety of tissues, including the arterial wall.1–4 Recent extensive histochemical studies have shown that receptors belonging to the LDLR family, as well as scavenger receptors mediating the incorporation of modified lipoproteins such as oxidized LDL, show marked induction of their expression during the formation of atherosclerotic lesions.5–9 For instance, 1 LDLR family member, the so-called VLDL receptor (VLDLR/LR8), has been shown to be highly expressed in smooth muscle cells (SMCs), macrophages, and endothelial cells in rabbit atherosclerotic lesions.7–9 Furthermore, VLDLR/LR8 has been shown not to be downregulated during ß-VLDL–induced foam cell formation in vitro, and overexpression of the receptor in fibroblasts causes excessive lipid droplet accumulation in the transformed cells.10
We and others have discovered and molecularly characterized a novel, unusually complex, and highly conserved member of the LDLR gene family, LR11.11–14 The predominant domain of this type I membrane protein consists of a cluster of 11 LDLR ligand–binding repeats, hence, its designation. Our recent studies using 2 experimental models of atherogenesis have demonstrated marked induction of LR11 during intimal thickening.15 The detailed immunohistochemical and in situ hybridization analyses showed that the expression of LR11 is predominantly localized to SMCs in the intima. These results suggested functional significance of LR11 in the (patho)physiology of SMCs in the atheroma.
In the present investigation, we studied the receptor function of LR11 for binding and internalization of plasma lipoproteins and of 39-kDa receptor–associated protein (RAP) by using LR11-overexpressing cells generated by transfection of human LR11 cDNA into a hamster cell line. In addition, lipid accumulation in these cells was analyzed after incubation with the apoE-rich lipoprotein, ß-VLDL. We also assessed binding activity of another apoE-rich lipoprotein, ie, HDL, which was prepared from human cerebrospinal fluid (CSF).
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Expression of LR11 cDNA in ldlA-7 Cells
The recombinant expression plasmid pBKCMhLR11 was constructed by cloning a 7-kb EcoRI-EcoRI fragment carrying the entire protein coding sequence of the human LR11 cDNA,13 obtained by multiple ligations, into the EcoRI site of the vector pBKCMV (Stratagene). All ligations were subjected to sequence analysis. Transfection into ldlA-7 cells, an LDLR-deficient Chinese hamster ovary (CHO) cell line (a kind gift of Dr M. Krieger, Massachusetts Institute of Technology, Cambridge), was carried out with 20 µg (per 6x106 cells) of pBKCMhLR11 or pBKCMV (mock) by using the calcium phosphate method.16 Stable transfectants were selected in medium supplemented with 500 µg/mL G418 (Life Technologies). The cells were cultured in medium A (Ham’s F-12 medium containing 100 U/mL penicillin, 100 µg/mL streptomycin, 500 mmol/L glutamine, and 500 µg/mL G418) supplemented with 5% FBS.
Antibody Production
The recombinant glutathione-S-transferase (GST) fusion protein expression plasmid, pGEX/HB305S, was constructed by cloning a 1.2-kb polymerase chain reaction–amplified fragment corresponding to residues 287 to 679 of the human LR11 cDNA into the expression vector pGEX-4T-2 (Amersham Pharmacia Biotech) cleaved by NotI and EcoRI.13 Fusion protein expressed in Escherichia coli XL2-Blue MRF', transformed with the recombinant vector, was purified and emulsified with Freund’s complete adjuvant, according to the manufacturer’s instructions (Toyobo), and used for immunization of BALB/c mice.10 P3U1 murine myeloma cells were used to produce hybrid cells, and anti-fusion protein antibody–secreting cells were cloned by limiting dilution; 1 of the clones, 5-4-30-19-2 (IgG1, 
), was used for the present experiments.
Immunocytochemistry
Cells stably expressing LR11 were plated on glass chamber slides and were grown to a confluence of 50% to 70%. After washing twice with PBS at 23°C, cells were fixed for 15 minutes by using 3% paraformaldehyde in PBS, washed again, and permeabilized with PBS containing 0.2% Triton X-100 for 15 minutes. The permeabilized cells were washed again with PBS, incubated with the primary antibody, 5-4-30-19-2, at a dilution of 1:2 in PBS for 1 hour, washed, and incubated with biotinylated horse anti-mouse IgG for an additional 30 minutes. For immunostaining, we used a Pathostain ABC-POD (M) kit (Wako) according to the manufacturer’s instructions. The same monoclonal antibody that was used for Western blot analysis was applied in immunostaining the cells. Cells incubated with a monoclonal anti-GST antibody were used as controls.
Electrophoresis and Western and Ligand Blotting
Membrane and cytosol fractions from cultured cells and rabbit brain were prepared as previously described.17 Protein concentrations were determined with the BCA Protein Assay Reagent (Pierce). Five percent SDS-PAGE was performed according to the method of Laemmli (Sambrook et al)16 by using a minigel system (Nihon Eido). Samples were prepared in the absence of dithiothreitol and without heating (nonreducing conditions).18 Electrophoresis was performed at 30 mA for 60 minutes. Calibration was with Rainbow-colored protein molecular weight markers (Amersham Pharmacia Biotech). Electrophoretic transfer of the proteins to polyvinylidine difluoride membranes (pore size 0.45 mm, Millipore) was performed in transfer buffer (100 mmol/L Tris, 192 mmol/L glycine, and 20% methanol) for 1 hour at room temperature and 110 mA by using the Atto Horizeblot System AE-6670. Western blotting was performed by using the supernatant at 1:2 dilution from the cultured 5-4-30-19-2 cells or control monoclonal antibodies to GST, followed by peroxidase-conjugated anti-mouse IgG (heavy and light chain, Promega) and the chemiluminescence detection method (ECL system, Amersham Pharmacia Biotech). The membranes were exposed for 5 to 15 minutes on Hyperfilm-ECL (Amersham Pharmacia Biotech). The relative intensities of the signals were determined by densitometric scanning with NIH image software. Ligand blotting with 125I-labeled RAP-GST and lipoprotein assessment with SDS-PAGE under nonreducing conditions were performed as reported.19
Preparation and Radiolabeling of Ligands
Recombinant human RAP was produced as a GST fusion protein by using a pGEX 2T-derived (Amersham Pharmacia Biotech) expression plasmid in E coli, DH5
.20 Rabbit ß-VLDL was prepared as described in a previous publication.11 Human VLDL, LDL, and HDL were prepared from the plasma of normal subjects by sequential ultracentrifugation.17 Human HDL purified from CSF was prepared as previously described.21 CSF was collected by lumbar puncture from neurologically free subjects. After centrifugation at 3700 rpm for 5 minutes, 
50 mL CSF from 50 subjects was pooled. CSF-HDL (density >1.210) was isolated by ultracentrifugation. Then, the top fraction was further concentrated with the membrane ultrafiltration (molecular mass limit 50 kDa; Centricut, Kurabo). High-performance liquid chromatography with cholesterol monitoring revealed that >90% of cholesterol was recovered from the HDL peak, which was a little larger than the serum HDL peak. All lipoproteins were labeled with 125I to a specific activity of 250 to 400 cpm/ng by using the iodine monochloride methods described previously.18 Purified RAP-GST was iodinated by using chloramine T according to the method of Sambrook et al.16 Labeled ligands were separated from free 125I by passing them over a PD10 column (Amersham Pharmacia Biotech). After extensive dialysis against TBS (50 mmol/L Tris and 150 mmol/L NaCl, pH 8) and 0.1 mmol/L EDTA, they were stored at 4°C for up to 2 weeks.
Assays of Surface Binding, Internalization, and Degradation of 125I-RAP and 125I-Lipoproteins in Monolayers of Cultured Cells
To determine cell binding of RAP and lipoproteins, monolayers of LR11-expressing ldlA-7 cells and control cells (transfected with pBKCMV) were incubated for 3 hours at 4°C in medium A containing 2 mg/mL BSA and the concentrations of radioiodinated and unlabeled ligands as indicated in the figure legends. The medium was then removed, and the monolayers were carefully washed to remove unbound ligand as described previously.17 Cell-associated radioactivity was determined by a 
-counter after solubilization of the cells in 1 mL of 0.1N NaOH. Cell protein was determined from an aliquot of the solution by the method described above. For the assays of cell-bound and internalized activities of 125I-labeled ligands, monolayers of cultured cells were incubated for 3 hours at 37°C. Assays of proteolytic degradation of 125I-labeled ligands in monolayers of cultured cells were determined according to the standard protocol for LDL.17 Briefly, each monolayer received medium A containing 5% lipoprotein-deficient serum and 125I-ß-VLDL (10 µg/mL, specific activity 350 cpm/ng) in the absence or presence of excess unlabeled ß-VLDL as indicated. The cells were incubated at 37°C for 3 hours and then incubated at 4°C. The medium was immediately removed, and the radioactivity in the medium was determined as degradation activity of the cells. For the time course of surface binding, internalization, and degradation, the ß-VLDL concentrations of 80, 20, and 10 µg/mL were used for their incubations, respectively.
Cholesterol Esterification Assay
Approximately 3x104 to 4x104 cells were seeded into 60-mm Petri dishes and grown in medium A supplemented with 5% FBS. On day 7, each monolayer received a final volume of 2 mL of medium A supplemented with 1 mmol/L ß-mercaptoethanol, 5% FBS, and ß-VLDL as indicated in the legends. After the indicated time, cells were pulse-labeled with 0.1 mmol/L [14C]oleate bound to albumin at a specific activity of 8600 to 10 600 dpm/nmol and harvested for measurement of cholesteryl [14C]oleate as described previously.22
Lipid Staining
Cells (5x104) were preincubated on chamber slides for 2 days in medium A supplemented with 5% lipoprotein-deficient serum. On day 3, the medium was removed, and fresh medium with 100 µg/mL ß-VLDL was added. After a 3-day incubation, the cells were washed with 1x PBS, fixed with 1.25% glutaraldehyde, and stained with oil red O for 15 minutes, followed by hematoxylin counterstaining described previously.10 Cells were washed in tap water, and lipid staining was evaluated by light microscopy.
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LR11 Overexpression in LDLR-Deficient CHO Cells
To characterize the potential function(s) of LR11, we first stably expressed human LR11 in the LDLR-deficient CHO cells, ldlA-7. Immunocytochemical studies showed strong expression of LR11 in these cells (Figure 1A, a and d). Particularly, the microscopical studies of stained CHO cells expressing LR11, KT38, demonstrated the presence of LR11 in the cytoplasm as well as on the cell surface (Figure 1A, d). No positive reaction was observed in the mock-transfected cells, KT2 (Figure 1A, b and e). We then investigated the localization of LR11 in the cells expressing the receptor by using immunoblot analysis (Figure 1B). The expressed receptor displayed the same migration as found in the rabbit brain and was predominantly present in the membrane fraction but also in the cytosol. On the other hand, control CHO cells, KT2, did not show any detectable signals.
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Binding Activity of LR11 for RAP
To functionally assess the competence of the recombinant receptor to interact with possible ligands, we measured LR11-mediated binding and internalization of RAP, which has been shown to bind to native LR11 in the human brain.12 Triton X-100 extracts of LR11-expressing cells were subjected to ligand blot experiments with 125I-labeled RAP, and as seen in Figure 2A, LR11 expressed in ldlA-7 cells indeed bound RAP (lane 4), demonstrating the binding competence of the recombinant receptor expressed in the cells. An identically migrating protein of 250 kDa could be observed in all sections prepared from rabbit brains, as well as other RAP-binding proteins (500, 130, and 105 kDa), which are probably present in other receptors belonging to the LDLR gene family.12,20 We then used monolayers of these cells to study further the binding and internalization activities of LR11 for RAP. As Figure 2B shows, KT38 cells bind and internalize RAP dose-dependently, and the maximum binding is 8-fold higher than that in mock-transfected cells, KT2 (Figure 2B, a). The Kd of RAP for receptor binding in KT38 was calculated as 1.5 nmol/L. Mock-transfected CHO cells bind small amounts of RAP, most likely mediated by other LDLR gene family members.12,20 Most significant, compared with the binding affinity of cell lines expressing VLDLR/LR8, LDLR-related protein 2MR (LRP), or LR8B, the binding affinity of LR11 for RAP is similar to that of other receptors of the gene family.20,23–28 In addition, KT38 cells mediate internalization of RAP 7-fold more efficiently than do KT2 cells (Figure 2B, b). These results show that LR11 binds and internalizes RAP.
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Binding Activities of LR11 for Lipoproteins
Having demonstrated that 125I-labeled RAP binds to human LR11, we tested whether this ligand would interfere with the receptor binding of ß-VLDL, which has previously been shown by ligand blot analysis.11 For the first set of experiments (Figure 3A), we used labeled ß-VLDL as a ligand in the presence of RAP and an excess amount of ß-VLDL or of unlabeled lipoproteins. As shown in lane 1, ß-VLDL binds to LR11 of KT38 cells (see also Figure 1B). Binding is totally abolished by an excess of unlabeled ß-VLDL (lane 2). RAP also competed for ß-VLDL in a concentration-dependent manner (lanes 3 and 4), reducing the signal produced by labeled ß-VLDL to the background level at 500 µg/mL. We then used apoE-rich HDL from human CSF, which has been shown to be rich in apoE and to bind LRP in vitro,22 and evaluated competition by RAP or ß-VLDL. 125I-labeled HDL-CSF binding (Figure 3B, lane 1) is abolished by an excess amount of unlabeled HDL-CSF, ß-VLDL, and RAP (lanes 2 to 4). Next, we studied the effects of divalent cations on the binding of 125I-labeled ß-VLDL to LR11 (Figure 3C). The binding of ß-VLDL was Ca2+ sensitive, inasmuch as the addition of EDTA (final concentration 20 mmol/L) abolished it (lane 3). Finally, we assessed the binding of other lipoproteins to LR11. Human LDL, VLDL, or HDL did not bind LR11 under the same conditions as in the experiments that used ß-VLDL and HDL-CSF (not shown).
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Binding, Internalization, and Degradation Activities of LR11 for ß-VLDL
To assess internalization competence of LR11 toward lipoproteins, we used the LR11-expressing CHO cells, KT38, to study LR11-mediated binding and internalization of ß-VLDL in detail. As shown in online Figure I (please see http://www.atvb.ahajournals.org), cells expressing LR11 specifically and saturably bound labeled ß-VLDL (Figure IA). On the other hand, the cells did not bind LDL, VLDL, or HDL, which is consistent with the results described above. Internalization studies using KT38 cells demonstrated that LR11-bound ß-VLDL is internalized in a dose-dependent manner (Figure IB). The Kd of ß-VLDL for receptor binding was calculated as 28 µg/mL; LDL, VLDL, and HDL were not internalized to any significant extent. Binding and internalization showed high affinity and saturation kinetics; at 40 µg/mL ß-VLDL, maximum internalization amounted to 1000 ng of 125I-labeled ß-VLDL per milligram cell protein in 3 hours. The mock-transfected cells, KT2, showed one ninth of maximum internalization of 125I-labeled ß-VLDL per milligram cell protein (not shown). These results indicate that LR11 binds and internalizes ß-VLDL, but not LDL, VLDL, or HDL. Online Figure II (please see http://www.atvb.ahajournals.org) shows the time course of binding, uptake, and degradation of ß-VLDL with the use of KT38 and KT2 cells. As shown in Figure IIA and IIB, KT38 cells bound and internalized labeled ß-VLDL in a time-dependent fashion; degradation reached 1550 ng of 125I-labeled ß-VLDL per milligram protein in 120 minutes, which was 7.5-fold higher than in KT2 cells (Figure IIC).
LR11-Mediated Cholesterol Accumulation
The availability of the KT38 cells provided the opportunity to study LR11-mediated lipoprotein metabolism in the cells. To measure receptor-mediated uptake of ß-VLDL, we used the cholesterol esterification assay.22 This assay measures the stimulation of [14C]oleate incorporation into cholesteryl [14C]oleate, an event that requires hydrolysis of lipoprotein-derived cholesteryl esters in lysosomes. As shown in online Figure III (please see http://www.atvb.ahajournals.org), when KT38 cells were incubated with ß-VLDL, there was a dose-dependent increase in the ability of ß-VLDL to stimulate cholesteryl [14C]oleate formation. The maximum response (5.2 nmol of cholesteryl [14C]oleate per hour per milligram protein) was observed at 80 µg of ß-VLDL/mL, 10-fold more than in KT2.
Finally, we stained LR11-expressing cells with oil red O after incubation with ß-VLDL for 48 hours. As shown in Figure 4, many lipid droplets could be detected in the cytoplasm of LR11-expressing cells (Figure 4B and 4C), but only few were detected in mock-transfected cells (Figure 4D). These results with LR11-expressing cells show that LR11 overexpression leads to excess amounts of intracellular lipid accumulation via uptake of ß-VLDL.
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The present study demonstrates that human LR11 overexpressed in hamster cells binds and internalizes RAP, a common ligand for the LDLR family. Furthermore, LR11 binds the apoE-rich lipoprotein, ß-VLDL, with an affinity similar to that for other family members. The receptor binding of RAP and ß-VLDL are competitive with each other, but other serum lipoproteins did not bind or inhibit their receptor binding. LR11 showed specific binding to HDL-CSF as well as to ß-VLDL, suggesting that enriched apoE content of lipoproteins is important for binding to the receptor with high affinities. LR11 internalized and degraded the bound ß-VLDL. The LR11-overexpressing cells showed progressive accumulation of cholesteryl esters when incubated with increasing amounts of ß-VLDL, and intracellular lipid droplets appeared. These results suggest that LR11 functions, at least in part, in the endocytosis of lipoproteins, particularly apoE-rich lipoproteins.
The LR11 protein overexpressed in CHO cells was localized dominantly in the cytoplasm as well as on the cell surface. The localization profile is similar to that of other LDLR family members. For instance, LRP was found in ribosomes, lysosomes, multivesicular bodies, and lipofuscin granules, as well as on the plasma membrane in neurons, monocytes, and fibroblasts.29,30 LR8B localizes to the cell surface and vesicular structures within the cell.20 Immunocytochemistry with the VLDL receptor/LR8 (VLDLR)-specific antibodies of endothelial cells demonstrated punctate cell-surface staining, as well as staining of large and small cytoplasmic vesicles.31 Immunoblot analysis showed LR11 association predominantly with cellular membranes, consistent with the biochemical studies by Jacobsen et al,12 who have purified the protein from membrane fraction of human brain.
RAP is localized predominantly in the endoplasmic reticulum and Golgi apparatus and is thought to prevent aggregation by premature binding of ligands to the LDLR family members.32 RAP, which binds class A repeats of the LDLR family, is now known to bind the neurotensin receptor, Sortilin.33 Sortilin harbors a VPS10 domain, which is well conserved in domain II of LR11.11–14 Our binding experiments revealed the high binding affinity of LR11 for RAP (1.5 nmol/L), which is compatible with values for VLDLR, LR8B, LRP, and gp330.20,23–28 In fact, the LR11 protein has been purified from human brain by using RAP affinity chromatography.12 Thus, LR11 has 2 characteristic sequence motifs that are both known to be able to bind RAP with high affinities.
We have already shown that rabbit LR11 binds ß-VLDL with the use of ligand blot analysis.11 In the present study, the specific binding of ß-VLDL to LR11 was inhibited by RAP. Competition of ß-VLDL by RAP might indicate that RAP binds to the class A repeats, known to be binding sites for ß-VLDL. On the other hand, RAP binding to the receptor was not inhibited by an excess amount of ß-VLDL (not shown); thus, RAP may bind not only to class A repeats but also to other sites, such as the VPS10 domain in LR11. The LDLR family members are known to bind apoE-rich lipoproteins with high affinities. In addition to ß-VLDL, the specific binding of HDL-CSF, but not LDL, VLDL, or HDL, strongly suggests that the binding of lipoproteins is mediated by the presence of apoE molecules. Furthermore, the dependence on divalent cations of the ligand binding of LR11 is entirely consistent with the properties of receptors belonging to the LDLR family.34
LR11 bound, internalized, and degraded ß-VLDL in a dose- and time- dependent manner. The binding affinity was 28 µg/mL, similar to that for VLDLR, apoER2, gp330, and LRP.22,35–40 It has been reported that VLDLR-overexpressing CHO cells show a foam cell–like appearance with many lipid droplets in the cytoplasm after incubation with ß-VLDL.10 We observed significant accumulation of cholesteryl esters and a lipid droplet–rich appearance when the LR11-expressing cells were incubated with ß-VLDL. These excessive accumulations of lipids in the cytoplasm raise the possibility that LR11-mediated incorporation of apoE-enriched lipoproteins causes foam cell formation in atheromata.
In conclusion, we demonstrate that the LR11 receptor functions in the binding, internalization, and degradation of apoE-rich lipoproteins in vitro. Our recent study in neuroblastoma cells identified transcriptional regulation of LR11 mRNA and protein induction during rapid proliferation of those cells in vitro.41 Recently, novel functional roles of certain LDLR family members in the process of cortical foliation of neurons have been revealed.42 The present and previous observations suggest that LR11 plays a role in the process of atheroma formation via lipoprotein metabolism associated with cellular proliferation and migration. Further extensive studies of the receptor function of LR11 is expected to clarify the (patho)biological significance of the marked induction of LR11 in atheroma, in conjugation with increased expression of other LDLR gene family members in vascular cells.
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Acknowledgments |
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This study was supported by grants from the Japanese Ministry of Education, Science, and Culture to Y.S. and H.B., and from the Austrian Science Foundation to W.J.S. (FWF-mp-13940).
Received February 22, 2001; accepted May 25, 2001.
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