Characterization of CLA-1, a Human Homologue of Rodent Scavenger Receptor BI, as a Receptor for High Density Lipoprotein and Apoptotic Thymocytes
1997; Elsevier BV; Volume: 272; Issue: 28 Linguagem: Inglês
10.1074/jbc.272.28.17551
ISSN1083-351X
AutoresKoji Murao, Valeska Terpstra, Simone R. Green, Nonna Kondratenko, Daniel Steinberg, Oswald Quehenberger,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoRecently, 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 K d of about 35 μg/ml, similar to the K d 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. 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 K d of about 35 μg/ml, similar to the K d 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. Scavenger receptor BI (SR-BI) 1The 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). was originally cloned on the basis of its ability to bind modified lipoproteins and was therefore classified as a novel scavenger receptor (1Acton S.L. Scherer P.E. Lodish H.F. Krieger M. J. Biol. Chem. 1994; 269: 21003-21009Abstract Full Text PDF PubMed Google Scholar). 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 (2Rigotti A. Acton S.L. Krieger M. J. Biol. Chem. 1995; 270: 16221-16224Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar). 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 (3Acton S. Rigotti A. Landschulz K.T. Xu S.Z. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (2030) Google Scholar). 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 (4Landschulz K.T. Pathak R.K. Rigotti A. Krieger M. Hobbs H.H. J. Clin. Invest. 1996; 98: 984-995Crossref PubMed Scopus (475) Google Scholar). 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 (5Wang N. Weng W. Breslow J.L. Tall A.R. J. Biol. Chem. 1996; 271: 21001-21004Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). 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 (5Wang N. Weng W. Breslow J.L. Tall A.R. J. Biol. Chem. 1996; 271: 21001-21004Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Previous studies have demonstrated a strong inverse correlation between plasma HDL levels and risk of coronary heart disease (6Buring J.E. O'Connor G.T. Goldhaber S.Z. Rosner B. Herbert P.N. Blum C.B. Breslow J.L. Hennekens C.H. Circulation. 1992; 85: 22-29Crossref PubMed Scopus (207) Google Scholar), possibly because HDL plays a critical role in reverse cholesterol transport, i.e. transport from peripheral tissues to the liver for catabolism (7Miller N.E. La Ville A. Crook D. Nature. 1985; 314: 109-111Crossref PubMed Scopus (160) Google Scholar, 8Franceschini G. Maderna P. Sirtori C.R. Atherosclerosis. 1991; 88: 99-107Abstract Full Text PDF PubMed Scopus (97) Google Scholar, 9Badimon J.J. Fuster V. Badimon L. Circulation. 1992; 86: III86-III94PubMed Google Scholar). 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 (10Calvo D. Vega M.A. J. Biol. Chem. 1993; 268: 18929-18935Abstract Full Text PDF PubMed Google Scholar). CD36 is an 88-kDa surface glycoprotein present on monocytes, platelets, and endothelial cells (11Greenwalt D.E. Lipsky R.H. Ockenhouse C.F. Ikeda H. Tandon N.N. Jamieson G.A. Blood. 1992; 80: 1105-1115Crossref PubMed Google Scholar), 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 (12Asch A.S. Silbiger S. Heimer E. Nachman R.L. Biochem. Biophys. Res. Commun. 1992; 182: 1208-1217Crossref PubMed Scopus (169) Google Scholar, 13Leung L.L.K. Li W.-X. McGregor J.L. Albrecht G. Howard R.J. J. Biol. Chem. 1992; 267: 18244-18250Abstract Full Text PDF PubMed Google Scholar), collagen (14Tandon N.N. Kralisz U. Jamieson G.A. J. Biol. Chem. 1989; 264: 7576-7583Abstract Full Text PDF PubMed Google Scholar), and erythrocytes infected with Plasmodium falciparum (15Ockenhouse C.F. Tandon N.N. Magowan C. Jamieson G.A. Chulay J.D. Science. 1989; 243: 1469-1471Crossref PubMed Scopus (269) Google Scholar, 16Oquendo P. Hundt E. Lawler J. Seed B. Cell. 1989; 58: 95-101Abstract Full Text PDF PubMed Scopus (412) Google Scholar), 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. 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. 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 (17Havel R.J. Eder H.A. Bragdon J.H. J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6613) Google Scholar). HDL was passed through a heparin-Sepharose affinity column to remove particles containing apolipoprotein E (18Murakami M. Horiuchi S. Takata K. Morino Y. J. Biochem. ( Tokyo ). 1987; 101: 729-741Crossref PubMed Scopus (60) Google Scholar). 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 (19Pittman R.C. Knecht T.P. Rosenbaum M.S. Taylor C.A. J. Biol. Chem. 1987; 262: 2443-2450Abstract Full Text PDF PubMed Google Scholar). 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 atd = 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 (19Pittman R.C. Knecht T.P. Rosenbaum M.S. Taylor C.A. J. Biol. Chem. 1987; 262: 2443-2450Abstract Full Text PDF PubMed Google Scholar). The resulting doubly labeled HDL was reisolated by flotation atd = 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 (20Basu S.K. Goldstein J.L. Anderson G.W. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 3178-3182Crossref PubMed Scopus (845) Google Scholar). 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 (21McNally A.K. Chisolm G.M. Morel D.W. Cathcart M.K. J. Immunol. 1990; 145: 254-259PubMed Google Scholar). 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 (22Morre D.J. Morre D.M. Biotechniques. 1989; 7: 946-958PubMed Google Scholar). 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 (23Fadok V.A. Voelker D.R. Campbell P.A. Cohen J.J. Bratton D.L. Henson P.M. J. Immunol. 1992; 148: 2207-2216Crossref PubMed Google Scholar). 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) (24Hope M.J. Bally M.B. Webb G. Cullis P.R. Biochim. Biophys. Acta. 1985; 812: 55-65Crossref PubMed Scopus (2052) Google Scholar). 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. 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 (25Mizobuchi M. Murao K. Takeda R. Kakimoto Y. J. Neurochem. 1994; 62: 322-328Crossref PubMed Scopus (51) Google Scholar), 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 (26Prossnitz E.R. Quehenberger O. Cochrane C.G. Ye R.D. Biochem. Biophys. Res. Commun. 1991; 179: 471-476Crossref PubMed Scopus (43) Google Scholar). 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. Binding of125I-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 mNaOH, 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 (27Sparrow C.P. Parthasarathy S. Steinberg D. J. Biol. Chem. 1989; 264: 2599-2604Abstract Full Text PDF PubMed Google Scholar). Total RNA was isolated from the human THP-1 cells and several human tissues by single-step acid guanidinium thiocyanate-phenol-chloroform extraction (28Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (64501) Google Scholar) 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 (25Mizobuchi M. Murao K. Takeda R. Kakimoto Y. J. Neurochem. 1994; 62: 322-328Crossref PubMed Scopus (51) Google Scholar). 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 (29Hanauer A. Mandel J.L. EMBO J. 1984; 3: 2627-2633Crossref PubMed Scopus (174) Google Scholar). 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 (10Calvo D. Vega M.A. J. Biol. Chem. 1993; 268: 18929-18935Abstract Full Text PDF PubMed Google Scholar) 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 inEscherichia 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 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 (30Heider J.G. Boyett R.L. J. Lipid Res. 1978; 19: 514-518Abstract Full Text PDF PubMed Google Scholar). Cell suspensions were cytocentrifuged onto glass slides and stained with Oil Red O as described previously (31Brown M.S. Goldstein J.L. Krieger M. Ho Y.K. Anderson R.G. J. Cell Biol. 1979; 82: 597-613Crossref PubMed Scopus (393) Google Scholar). Protein was determined by the method of Lowry et al. (32Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar), and SDS-PAGE was carried out as described (33Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (212375) Google Scholar). Statistical comparisons were made by one-way analysis of variance (ANOVA) and Student's ttest. 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.1 A). 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. 1 B). Scatchard analysis of the equilibrium binding data revealed aK d 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. 6 B).Figure 6Binding of lipoprotein to HEK 293 cells transfected with CLA-1. A, lipoprotein inhibition of125I-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 of125I-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 (○) as well as LDL (•) were added. The molar concentrations of the lipoproteins were estimated based on the average molecular weight of 2 × 106containing 25% protein for LDL, and 2 × 105containing 50% protein for HDL. Each point is the mean of triplicate determinations. C, degradation of 125I-LDL. CLA-1-transfected cells (○) and mock-transfected cells (•) were incubated for 5 h at 37 °C with the indicated amounts of125I-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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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. 2 A). Expression of CLA-1 induced CE accumulation to a level that was about 4 times that in mock-transfected cells (Fig. 2 B). 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. 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 (19Pittman R.C. Knecht T.P. Rosenbaum M.S. Taylor C.A. J. Biol. Chem. 1987; 262: 2443-2450Abstract Full Text PDF PubMed Google Scholar). 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. Based on the considerable sequence homology, CLA-1 is believed to belong to the same gene family as CD36 and LIMPII (10Calvo D. Vega M.A. J. Biol. Chem. 1993; 268: 18929-18935Abstract Full Text PDF PubMed Google Scholar, 34Calvo D. Dopazo J. Vega M.A. Genomics. 1995; 25: 100-106Crossref PubMed Scopus (104) Google Scholar). 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.4 A). The message was detected as a 2.9-kilobase species, consistent with previous reports (10Calvo D. Vega M.A. J. Biol. Chem. 1993; 268: 18929-18935Abstract Full Text PDF PubMed Google Scholar). 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 (3Acton S. Rigotti A. Landschulz K.T. Xu S.Z. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (2030) Google Scholar,4Landschulz K.T. Pathak R.K. Rigotti A. Krieger M. Hobbs H.H. J. Clin. Invest. 1996; 98: 984-995Crossref PubMed Scopus (475) Google Scholar, 35Glass C. Pittman R.C. Civen M. Steinberg D. J. Biol. Chem. 1985; 260: 744-750Abstract Full Text PDF PubMed Google Scholar). 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.4 B). Incubation of THP-1 cells for 2 days with 100 nm PMA resulted in a loss of detectable CLA-1 mRNA (Fig. 5 A). 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. 5 B), 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. 5 B). 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. 5 C). 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. A previous study suggested that both native LDL and modified LDL (AcLDL and OxLDL) bind to hamster SR-BI (1Acton S.L. Scherer P.E. Lodish H.F. Krieger M. J. Biol. Chem. 1994; 269: 21003-21009Abstract Full Text PDF PubMed Google Scholar). 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.6 A). 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. 6 B). In direct binding analysis we confirmed that CLA-1 recognized native LDL with aK d 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. 6 C). 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 (36Pearson A.M. Curr. Opin. Immunol. 1996; 8: 20-28Crossref PubMed Scopus (255) Google Scholar). 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 (37Savill J. Fadok V. Henson P. Haslett C. Immunol. Today. 1993; 14: 131-136Abstract Full Text PDF PubMed Scopus (989) Google Scholar). 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 (TableI). 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
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