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Preferential ATP-binding Cassette Transporter A1-mediated Cholesterol Efflux from Late Endosomes/Lysosomes

Preferential ATP-binding Cassette Transporter A1-mediated Cholesterol Efflux from Late... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 47, Issue of November 23, pp. 43564 –43569, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Preferential ATP-binding Cassette Transporter A1-mediated Cholesterol Efflux from Late Endosomes/Lysosomes* Received for publication, August 17, 2001, and in revised form, September 13, 2001 Published, JBC Papers in Press, September 14, 2001, DOI 10.1074/jbc.M107938200 Wengen Chen‡, Yu Sun‡, Carrie Welch, Anna Gorelik, Andrew R. Leventhal, Ira Tabas, and Alan R. Tall§ From the Division of Molecular Medicine, Department of Medicine, Columbia University, New York, New York 10032 Recently, ATP-binding cassette transporter A1 phages from these animals have a profound defect in apoA-I- (ABCA1), the defective molecule in Tangier disease, has mediated cholesterol efflux (8 –10), indicating that apolipopro- been shown to stimulate phospholipid and cholesterol tein-mediated cholesterol efflux is primarily mediated by efflux to apolipoprotein A-I (apoA-I); however, little is ABCA1. In contrast, ABCA1 shows only slight interaction with known concerning the cellular cholesterol pools that act HDL and no interaction with HDL (11). Cellular cholesterol 3 2 as the source of cholesterol for ABCA1-mediated efflux. efflux mediated by HDL is thought to involve a “passive” proc- We observed a higher level of isotopic and mass choles- ess that may be diffusion-mediated or may involve an interac- terol efflux from mouse peritoneal macrophages labeled tion of HDL with scavenger receptor B-I (SR-BI) (12, 13). H]cholesterol/acetyl low density lipoprotein with [ ABCA1 is a full transporter with 12 membrane-spanning (where cholesterol accumulates in late endosomes and domains (5, 14). Transfection of ABCA1 in 293 cells reveals a H]choles- lysosomes) compared with cells labeled with [ predominant cell surface localization and suggests a direct terol with 10% fetal bovine serum, suggesting that late interaction of ABCA1 with apoA-I (11). The primary activity of endosomes/lysosomes act as a preferential source of cho- ABCA1 appears to be the translocation of phospholipid at the lesterol for ABCA1-mediated efflux. Consistent with this plasma membrane rather than direct interaction with choles- idea, macrophages from Niemann-Pick C1 mice that terol (15, 16). Phospholipid-apoA-I complexes formed by have an inability to exit cholesterol from late endo- ABCA1 may promote cholesterol efflux in a secondary fashion somes/lysosomes showed a profound defect in choles- perhaps involving distinct areas of the plasma membrane (15, terol efflux to apoA-I. In contrast, phospholipid efflux to 17). The nature of the cellular sites that donate cholesterol to apoA-I was normal in Niemann-Pick C1 macrophages, as these phospholipid-apoA-I complexes is poorly understood. was cholesterol efflux following plasma membrane cho- This may involve specific plasma membrane domains that de- lesterol labeling. These results suggest that cholesterol rive cholesterol from intracellular stores. The nature of intra- deposited in late endosomes/lysosomes preferentially acts as a source of cholesterol for ABCA1-mediated cho- cellular sites that potentially donate cholesterol to the plasma lesterol efflux. membrane for ABCA1-mediated efflux is also unclear. Ni- emann-Pick C (I and II) molecules play an essential role in intracellular cholesterol trafficking, particularly in the exit of Tangier disease (TD) is a rare condition associated with low cholesterol from late endosomes/lysosomes (18 –21). Earlier levels of plasma high density lipoproteins (HDL) and accumu- studies suggested a defect in cholesterol efflux to phospholipid lation of cholesterol and cholesteryl esters in macrophage foam vesicles in NPC1 fibroblasts (22), but the specific role of NPC1 cells in tonsils, spleen, and other tissues (1). The cellular defect in ABCA1-mediated cholesterol efflux has not been in TD involves a marked decrease in the efflux of cholesterol investigated. and phospholipid to apoA-I, the major protein of HDL (2, 3). The ABCA1 gene is up-regulated by cellular cholesterol load- Recently, TD was shown to be caused by mutations in the ing (23). The mechanism of this effect is increased gene tran- ATP-binding cassette transporter, ABCA1 (4 –7). Mice with scription mediated by the oxysterol-activated transcription fac- deficiency of ABCA1 also have low HDL. In addition, macro- tor liver X receptor (LXR) acting in a complex with retinoid X receptor (RXR) at a site on the proximal promoter of the ABCA1 gene (24). While studying cholesterol efflux from * This work was supported by Specialized Center of Research in macrophages that had been treated with the LXR/RXR ligands Atherosclerosis Grant HL-56984 (to A. R. T. and I. T) from the NHLBI, National Institutes of Health, and research grants from the Parshegian 22(R)-hydroxycholesterol and 9-cis-retinoic acid to up-regulate Foundation (to A. R. T) and Berlex Biosciences (to I. T.). The costs of ABCA1, we noticed a marked discrepancy between the magni- publication of this article were defrayed in part by the payment of page tude of ABCA1 expression and the resulting stimulation of charges. This article must therefore be hereby marked “advertisement” cholesterol efflux, depending on the method of cellular choles- in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ These authors made equal contributions to the work. terol labeling. This led to an investigation of the hypothesis § To whom correspondence should be addressed: Div. of Molecular that ABCA1 stimulates cholesterol efflux preferentially from a th Medicine, Dept. of Medicine, Columbia University, 603 W. 168 St., pool of cholesterol found in late endosomes/lysosomes. This New York, NY 10032. Tel.: 212-305-9418; Fax: 212-305-5052; E-mail: hypothesis has been evaluated by comparing cholesterol efflux [email protected]. under different labeling conditions and supported by the dem- The abbreviations used are: TD, Tangier disease; ABCA1, ATP- binding cassette transport A1; AcLDL, acetyl low density lipoprotein; onstration of a profound defect in cholesterol efflux to apoA-I apoA-I, apolipoprotein A-I; apoE, apolipoprotein E; HDL, high density using macrophages from NPC1 mice. lipoprotein; LXR, liver X receptor; RXR, retinoid X receptor; NPC1, Niemann-Pick C1; SR-BI, Scavenger receptor class B type I; FBS, fetal EXPERIMENTAL PROCEDURES bovine serum; PIPES, 1,4-piperazinediethanesulfonic acid; PBS, phos- Ribonuclease Protection Assay—Reverse transcription-polymerase phate-buffered saline; wt, wild type; DMEM, Dulbecco’s modified Eagle’s medium; BSA, bovine serum albumin. chain reaction was used to obtain a fragment of the murine ABCA1 43564 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Cholesterol Pool and ABCA1-mediated Cholesterol Efflux 43565 cDNA. Murine ABCA1 and -actin antisense riboprobes were prepared radioactivity released from the cells into the medium relative to the total radioactivity in cells and media. by in vitro transcription using murine ABCA1 -actin cDNA plasmid Cholesterol Mass Analysis—The cells in 6-well plates were [ - constructs. The protected hybrid fragments for ABCA1 and -actin were H]cho lesterol labeled by procedure a as described above. After4hof incuba- 290 and 160 base pairs, respectively. Ribonuclease protection assay was tion with 10 g/ml apoA-I (see Fig. 2) or 15 g/ml human HDL performed as described (25). In brief, 20 g of total RNA were hybrid- (see Fig. 6), medium was collected, and the cells were lysed in 0.1 M sodium ized with 5  10 cpm ABCA1 and -actin riboprobes at 48 °C overnight hydroxide and 0.1% SDS. Lipids were extracted in chloroform:methanol in 30 l of a buffer consisting of 40 mM PIPES, pH 6.0, 400 mM NaCl, 1 (2:1). The organic residue was dissolved in 0.5% Triton X-100. Choles- mM EDTA, and 80% formamide. The hybridization mixture was di- terol was determined enzymatically (Wako Chemicals USA, Richmond, gested with 20 units of T ribonuclease (Life Technologies, Inc.) at 37 °C VA). Protein was determined by the Lowry method. for 1 h, extracted with phenol/chloroform, precipitated with ethanol, H]Phospholipid Efflux Study—Macrophages in a 24-well plate and dissolved in 5 l of RNA loading buffer. The protected RNA hybrid were choline labeled for 24 h in 0.5 ml of DMEM, 10% FBS supple- fragments were resolved on a 6% polyacrylamide/urea gel and subjected mented with 1.0 Ci/ml [ H]choline (PerkinElmer Life Sciences). After to autoradiography. overnight equilibration in DMEM, 0.2% BSA with or without the LXR/ Immunoblot Analysis of ABCA1—For immunoblot analysis of RXR ligands treatment, the cells were washed twice in PBS, 0.2% BSA. ABCA1, peritoneal macrophages were washed and scraped in PBS and Efflux was performed by incubation with 10 g/ml apoA-I for4hin0.5 lysed in 10 mM Tris-HCl, pH 7.3, 1 mM MgCl , and 0.5% Nonidet P-40 ml DMEM, 0.2% BSA with or without the ligands. Then medium was g/ml leupeptin, 1 g/ml in the presence of protease inhibitors (0.5 collected and centrifuged at 6000  g for 10 min to remove cell debris. aprotinin, 1 g/ml pepstatin A; Roche Molecular Biochemicals). Post- H]Phospholipids in an aliquot of supernatant were first extracted nuclear supernatants from cell lysates were prepared by centrifugation with chloroform:methanol (2:1), and then the radioactivity was deter- at 3000  g for 10 min at 4 °C. Samples containing the indicated mined by scintillation counting. The cells were finally lysed in 0.5 ml of amounts of protein were reduced with 2-mercaptoethanol in gel loading 0.1 M sodium hydroxide, 0.1% SDS, and the radioactivity in an aliquot buffer, fractionated by 7.5% SDS-polyacrylamide gel electrophoresis, after lipid extraction was determined. The percentage of secreted and transferred to 0.22-m nitrocellulose membranes. Immunoblotting H]phospholipid was calculated by dividing the medium-derived was performed using an anti-ABCA1 antiserum (Novus, Littleton, CO) counts by the sum of the total (medium plus cell). and ECL (Amersham Pharmacia Biotech). The relative intensities of Statistical Analysis—The results are presented as the means  S.D. the bands were determined by densitometry (Molecular Dynamics, The tests for the significant differences between groups were performed model 300A). by Student’s t test. Lipoprotein Isolation—Human low density lipoprotein (LDL, 1.006d1.063) and high density lipoprotein (HDL , 1.063d1.125) RESULTS were isolated from plasma by sequential ultracentrifugation. Acetyl LDL (AcLDL) was prepared as described (26). Apolipoprotein A-I We recently showed that ABCA1 mRNA is up-regulated by (apoA-I) was purchased from Biodesign International (Saco, ME). activation of LXR/RXR (24). To determine whether this re- Isolation and Culture of Mouse Peritoneal Macrophages—Homozy- sulted in an increase in functional ABCA1 protein, we gous NPC1 were produced by intercrossing BALB/cNctr-npc1 / measured cholesterol efflux to apoA-I in mouse peritoneal (BALB-npc1 /) mice (stock number 003092; Jackson Laboratory, Bar macrophages cells treated with the LXR/RXR ligands 22(R)- Harbor, ME). Mouse peritoneal macrophages were isolated from NPC1 hydroxycholesterol and 9-cis-retinoic acid. This treatment re- and wild type (wt) littermates by peritoneal lavage with PBS 3 days sulted in a marked up-regulation of ABCA1 mRNA (not shown) after intraperitoneal injection with 1 ml of 3.85% thioglycollate (Becton Dickinson, Sparks, MD). The isolated cells were plated onto 24-well and protein levels (Fig. 1A) and an increase in cholesterol efflux plates and allowed to adhere by incubation for4hat37 °C in Dulbecco’s (Fig. 1B) as anticipated (27). Surprisingly, the level of choles- modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine terol efflux was about 2.5-fold higher in activated cells labeled serum (FBS) (Life Technologies). After removal of nonadherent cells by 3 with [ H]cholesterol AcLDL compared with cells labeled with washing with PBS, the cells were further incubated for 2 days and then [ H]cholesterol, 10% FBS (Fig. 1B, compare bars 4 and 2), used for cholesterol labeling and efflux experiments. despite comparable levels of ABCA1 expression (Fig. 1A; note [ H]Cholesterol Labeling of Cells—Mouse peritoneal macrophages were labeled with [ H]cholesterol carried by one of three delivery agents that ABCA1 protein appears as a doublet for unknown reason). to investigate cholesterol efflux from different pools: (a) AcLDL (late AcLDL is internalized by the scavenger receptor A and accu- endosomes/lysosomes pool); (b) 10% FBS/DMEM (recycling endosomes mulates primarily in late endosomes and lysosomes (28), M methyl--cyclodextrin (plasma membrane pool). For 3 pool); and (c)5m whereas the [ H]cholesterol, 10% FBS method appears to pref- procedure a, cells were labeled overnight by 1 Ci/ml [1,2- H(N)]- erentially label recycling endosomes and the trans-Golgi net- cholesterol (PerkinElmer Life Sciences) in DMEM, 0.2% BSA supple- work (29). These findings suggested the hypothesis that mented with 50 g/ml AcLDL. LXR/RXR ligands 22(R)-hydroxycholes- ABCA1 might preferentially stimulate cholesterol efflux from terol (final concentration, 10 M) and 9-cis-retinoic acid (final concentration, 10 M) (BIOMOL Research Laboratories, Plymouth late endosomes/lysosomes rather than from cellular cholesterol Meeting, PA) were added during the labeling to induce ABCA1 expres- pools labeled by [ H]cholesterol, 10% FBS. sion. After the labeling, the cells were washed with PBS and equili- To further explore this idea, cholesterol mass and isotopic brated with DMEM, 0.2% BSA for 1 h. Efflux was then performed as 3 efflux to apoA-I were measured in cells labeled with [ H]cho- described below. For procedure b, the cells were labeled with 1 Ci/ml lesterol, 10% FBS, with [ H]cholesterol/AcLDL, or with [ H]cholesterol in 0.5 ml of DMEM supplemented with 10% FBS for [ H]cholesterol/cyclodextrin. In the latter procedure, the cells 24 h. The cells were then equilibrated overnight in DMEM, 0.2% BSA are labeled briefly (15 min) with cyclodextrin:cholesterol (8:1, with or without the LXR/RXR ligands 22(R)-hydroxycholesterol and 9-cis-retinoic acid. After washing, the cells were used for efflux exper- molar ratio), and the radiolabel is thought to reside mostly in iments. For procedure c, the cells were first treated with the ligands the plasma membrane (30, 31). These experiments showed 22(R)-hydroxycholesterol and 9-cis-retinoic acid in DMEM, 0.2% BSA greater isotopic and mass efflux of cholesterol in activated cells overnight to induce ABCA1. Then the medium was replaced by 5 m 3 labeled with [ H]cholesterol/AcLDL, compared with cells la- methyl -cyclodextrin:cholesterol at molar ration 8:1 ([ H]cholesterol, 1 beled in either of the other two ways (Fig. 2), consistent with Ci/ml) for 15 min at 37 °C. After washing, the cells were used for efflux the idea that the late endosomal/lysosomal cholesterol pool is a step. [ H]Cholesterol Efflux Study—After labeling and equilibration, the preferential source for cholesterol efflux by ABCA1. Activation cells were incubated by 10 g/ml purified human apoA-I or 15 g/ml of LXR/RXR did result in a marked increase in isotopic and human HDL in 0.5 ml of DMEM, 0.2% BSA with or without the mass cholesterol efflux from cells labeled with [ H]cholesterol/ LXR/RXR ligands for 4 h. Then medium was collected and centrifuged cyclodextrin, suggesting that the plasma membrane cholesterol at 6000  g for 10 min to remove cell debris and cholesterol crystal, and also contributes significantly to ABCA1-mediated cholesterol radioactivity in an aliquot of supernatant was determined by liquid efflux. However, for the [ H]cholesterol, 10% FBS labeling scintillation counting. The cells were finally lysed in 0.5 ml of 0.1 M method, isotopic efflux was only slightly increased by LXR/RXR sodium hydroxide, 0.1% SDS and the radioactivity in an aliquot was determined. Cholesterol efflux was expressed as the percentage of the activation, whereas cholesterol mass efflux was increased in a 43566 Cholesterol Pool and ABCA1-mediated Cholesterol Efflux FIG.1. Discrepancy between the magnitude of ABCA1 expres- sion and the resulting cholesterol efflux with different cellular cholesterol labeling methods. wt peritoneal macrophages were [ H]cholesterol labeled by 10% FBS, DMEM (lanes 1 and 2)or50 g/ml AcLDL (lanes 3 and 4), with or without LXR/RXR ligands (22(R)- FIG.2. Isotopic and mass efflux of cholesterol from macro- hydroxycholesterol and 9-cis-retinoic acid) treatment as described un- phages treated with the three different [ H]cholesterol labeling der “Experimental Procedures.” After equilibration in DMEM, 0.2% methods. wt mouse peritoneal macrophages in 6-well plates were BSA, the cells were incubated with 10 g/ml of apo-A-I for4hin [ H]cholesterol labeled by 10% FBS, AcLDL, or cyclodextrin and treated DMEM, 0.2% BSA with or without the LXR/RXR ligands. A, Western with or without the LXR/RXR ligands as described under “Experimen- blot of ABCA1 following SDS-polyacrylamide gel electrophoresis of cell tal Procedures.” After equilibration in DMEM, 0.2% BSA, the cells were lysates. Representative data are from one of two independent experi- 3 incubated with 10 g/ml apoA-I for 4 h. [ H]cholesterol efflux (A) was ments. Cholesterol efflux (B) was expressed as the medium [ H]choles- expressed as the medium [ H]cholesterol radioactivity as a percentage terol radioactivity as a percentage of total [ H]cholesterol radioactivity 3 of total [ H]cholesterol radioactivity (cells plus medium). For choles- (cells plus medium). Representative data are from one of three inde- terol mass assay (B), cholesterol in the medium was first extracted and pendent experiments. The values are the means  S.D. (n  3). *, p measured enzymatically. Representative data are from one of two in- 0.01, bar 2 versus bar 4. ch, cholesterol; 22ch, 22(R)-hydroxycholesterol; dependent experiments. The values are the means  S.D. (n  3). *, p RA, 9-cis-retinoic acid. 0.01, AcLDL labeling versus 10% FBS and methyl--cyclodextrin (MCD) labelings. 22ch, 22(R)-hydroxycholesterol; RA, 9-cis-retinoic acid. similar fashion to [ H]cholesterol/cyclodextrin-labeled cells. This finding could arise if the radiolabel was primarily present in pools of cholesterol inaccessible to ABCA1 (i.e. recycling in NPC1 macrophages compared with wild type macrophages endosomes) (29), whereas cholesterol mass efflux reflected ef- following LXR/RXR activation (Fig. 3C). Following plasma flux from the plasma membrane where cholesterol would be membrane labeling with [ H]cholesterol/cyclodextrin, there unlabeled by this method. were identical levels of cholesterol efflux in NPC1 and wild type To further explore the hypothesis that late endosomes/lyso- cells (Fig. 3D). These experiments suggest that both lysosomal somes represent a preferred source of cholesterol for ABCA1- and plasma membrane cholesterol pools serve as a source of mediated cholesterol efflux, we next carried out efflux studies cholesterol for ABCA1 and that the lysosomal pool requires the using macrophages from Niemann-Pick C1 mice, which have a activity of the NPC1 molecule. defect in trafficking of cholesterol out of late endosomes (32). ABCA1 is thought to act as a phospholipid flippase at the Cholesterol loading was carried out using [ H]cholesterol/ plasma membrane (15, 16). This activity may lead to the for- AcLDL. Compared with macrophages from wild type mice, mation of phospholipid-apoA-I complexes that secondarily there was a profound decrease in cholesterol efflux to apoA-I in stimulate cholesterol efflux from a distinct region of plasma NPC1 macrophages, especially following induction of ABCA1 membrane (15, 17). We measured phospholipid efflux to apoA-I (Fig. 3A). Measurements of ABCA1 mRNA and protein re- in NPC1 and wt macrophages. Phospholipid efflux was stimu- vealed similar levels of induction in control and NPC1 macro- lated following activation of LXR/RXR, but there was no defect phages (not shown). In earlier studies, Liscum et al. (22) re- in phospholipid efflux in NPC1 cells (Fig. 4). This indicates that ported that human NPC1 fibroblasts had a moderate defect in the primary action of ABCA1, i.e. formation of phospholipid- cholesterol efflux to small unilamellar vesicles; this was man- apoA-I complexes, is intact in NPC1 cells. ifested as a delay in cholesterol efflux that became normal Cholesterol efflux to HDL was also significantly decreased following longer incubation periods. However, a time course in NPC1 cells loaded with [ H]cholesterol/AcLDL (Fig. 5)orby study revealed a profound 3– 4-fold decrease in cholesterol ef- the [ H]cholesterol, 10% FBS method (not shown). Because flux to apoA-I in NPC1 macrophages that was not ameliorated HDL does not interact with ABCA1 (11), this indicates a defect by prolonged incubation (Fig. 3B). If ABCA1 preferentially in cholesterol efflux via pathways not mediated by ABCA1. stimulates cholesterol efflux from late endosomes/lysosomes, Interestingly, cholesterol efflux via HDL was also induced by then it might be anticipated that there would be a less pro- LXR/RXR activation (Fig. 5). This suggests the presence of nounced defect in cholesterol efflux in NPC1 cells labeled with other LXR/RXR target genes in the HDL -mediated efflux [ H]cholesterol, 10% FBS. Accordingly, using this labeling pathway. Because apolipoprotein E (apoE) was recently iden- method, basal cholesterol efflux to apoA-I was similar in wild tified as an LXR/RXR target (33), we considered the possibility type and NPC1 cells, and efflux was only moderately decreased that increased cholesterol efflux might be due to increased Cholesterol Pool and ABCA1-mediated Cholesterol Efflux 43567 FIG.3. ABCA1-mediated cholesterol efflux in NPC1 and wild type macro- phages. NPC1 and wt peritoneal macro- phages were [ H]cholesterol labeled by AcLDL (A and B), 10% FBS (C), and cy- clodextrin (D) with or without LXR/RXR ligand treatment as described under “Ex- perimental Procedures.” After equilibra- tion in DMEM, 0.2% BSA, the cells were incubated with 10 g/ml of apo-A-I for 4 h (A, C, and D) or different time points (B) in DMEM, 0.2% BSA with or without the ligands. Cholesterol efflux was expressed as the medium [ H]cholesterol radioactiv- ity as a percentage of total [ H]cholesterol radioactivity (cells plus medium). Repre- sentative data are from one of three (A and C) or two (B and D) independent ex- periments. The values are the means S.D. (n  3). *, p  0.01, NPC1 versus wt. 22ch, 22(R)-hydroxycholesterol; RA, 9- cis-retinoic acid. FIG.4. ABCA1-mediated phospholipid efflux in NPC1 and wild FIG.5. Cholesterol efflux to HDL in NPC1 and wild type peri- type peritoneal macrophages. NPC1 and wt peritoneal macro- toneal macrophages. NPC1 and wt peritoneal macrophages were 3 3 phages were [ H]choline-labeled (1 Ci/ml of [ H]choline) and treated 3 [ H]cholesterol AcLDL-labeled and treated with or without the LXR/ with or without the LXR/RXR ligands as described under “Experimen- RXR ligands as described under “Experimental Procedures.” Efflux was tal Procedures.” [ H]Phospholipid efflux was performed by incubation performed by incubation with 15 g/ml of HDL for4hin DMEM, 0.2% with 10 g/ml of apoA-I for4hin DMEM, 0.2% BSA with or without the BSA with or without the ligands. Cholesterol efflux was expressed as ligands. [ H]phospholipid in the medium and cell lysates was deter- 3 the medium [ H]cholesterol radioactivity as a percentage of total mined following extraction in chloroform:methanol (2:1), and the radio- 3 [ H]cholesterol radioactivity (cells plus medium). Representative data activity was determined by scintillation counting. [ H]Phospholipid are from one of three independent experiments. The values are the efflux was expressed as the medium radioactivity as a percentage of means  S.D. (n  3). *, p  0.05, NPC1 versus wt. 22ch, 22(R)- total radioactivity (cells plus medium). Representative data are from hydroxycholesterol; RA, 9-cis-retinoic acid. one of two independent experiments. The values are the means  S.D. (n  3). 22ch, 22(R)-hydroxycholesterol; RA, 9-cis-retinoic acid. somes rather than cholesterol deposited at other intracellular sites. The equilibration of cell surface cholesterol with these apoE synthesis by mouse peritoneal macrophages. However, intracellular sites requires the activity of the NPC1 molecule LXR/RXR activation similarly increased cholesterol efflux to and could perhaps also involve trafficking of the ABCA1 mol- HDL in macrophages from apoE knock-out mice (not shown), ecule itself (35). ABCA1 is markedly less efficient in stimulat- eliminating this possibility. The ability of HDL to stimulate ing cholesterol efflux from cells that have been labeled with increased cholesterol efflux following LXR/RXR activation was [ H]cholesterol, 10% FBS, which probably primarily labels re- also confirmed by cholesterol mass measurements, which indi- cycling endosomes (29). A profound defect in ABCA1-mediated cated primarily an increase in HDL free cholesterol (Fig. 6). cholesterol efflux in NPC1 mutant macrophages may be an SR-BI neutralizing antibodies (34) did not affect cholesterol important factor explaining our recent observations showing efflux mediated by HDL in either basal or LXR/RXR-stimu- an increase of atherosclerosis in apoE knock-out/NPC1 mutant lated conditions (not shown). mice, compared with apoE knock-out control mice. DISCUSSION Massive cholesteryl ester accumulation in TD macrophages indicates that ABCA1 has an essential role in mediating cho- Our findings suggest that phospholipid-apoA-I complexes formed by ABCA1 initially stimulate cholesterol efflux from regions of the plasma membrane that preferentially utilize N. Sharma, G. Kuriakose, D. Zhang, I. Tabas, R. J. Deckelbaum, cholesterol deposited by modified LDL in late endosomes/lyso- A. R. Tall, and C. L. Welch, submitted for publication. 43568 Cholesterol Pool and ABCA1-mediated Cholesterol Efflux Following entry of lipoprotein cholesterol into late endo- somes and lysosomes, NPC1 has an essential role in allowing cholesterol to gain access to the ABCA1 efflux pool (Fig. 3). Liscum et al. (22) reported a delay in cholesterol efflux to unilamellar vesicles in fibroblasts from NPC1 patients. How- ever, with time the efflux became normal. In contrast, the apoA-I stimulated cholesterol efflux in NPC1 mutant mouse macrophages was profoundly reduced at all time points (Fig. 3B). Recently, it has been shown that following labeling of NPC1 mutant Chinese hamster ovary cells with H-cholesteryl ester LDL, early time points of cholesterol efflux to cyclodextrin show no or little defect (31, 42). However, after the initial appearance at the plasma membrane and subsequent internal- FIG.6. Cholesterol efflux from wild type macrophages incu- ization to an intracellular pool, cholesterol shows delayed traf- bated with HDL . wt mouse peritoneal macrophages in 6-well plates ficking back to the plasma membrane and poor activation of were [ H]cholesterol/AcLDL-labeled, as described under “Experimental Procedures.” After equilibration the cells were incubated with 15 g/ml acyl-CoA-cholesterol acyltransferase in (ACAT) NPC1 mutant HDL for 4 h. Cholesterol in the medium was extracted and measured cells. This has led to the proposal that NPC1 acts on an intra- enzymatically. Representative data are from one of two independent cellular pool of cholesterol that is derived from the plasma experiments. The values are the means  S.D. (n  3). *, p  0.05, 22ch/RA versus 22ch/RA. 22ch, 22(R)-hydroxycholesterol; RA, 9-cis- membrane and is in equilibrium with ACAT. Whether NPC1 is retinoic acid; FC, free cholesterol; CE, cholesteryl ester. acting in late endosomes/lysosomes (43, 44) or on another pool of cholesterol (31), our studies suggest that this pool of choles- lesterol efflux from these cells. It is likely that cholesterol from terol represents an important source of cholesterol for ABCA1- a variety of sources, including effete red cells and modified stimulated efflux. forms of LDL, is taken up by macrophages eventuating in Recently, Leventhal et al. (45) have found a defect in basal delivery to late endosomes/lysosomes. Our studies are consist- cholesterol efflux to apoA-I in acid sphingomyelinase-deficient ent with the idea that apoA-I/ABCA1-mediated cholesterol ef- macrophages. These studies suggest that endososomal/lysoso- flux plays an essential role in removing cholesterol from these mal sphingomyelin accumulation leads to cholesterol seques- intracellular sites. The underlying mechanisms are unclear. tration and, thus, defective cholesterol trafficking and efflux. Intracellular trafficking of apoA-I has been reported in macro- The present findings extend these observations by providing phages, and this process appears to be defective in TD (36). The direct evidence that the late endosomal/lysosomal cholesterol trafficking of apoA-I has been suggested to have a role in pool represents the preferred source of cholesterol for ABCA1- mediating cholesterol efflux (37). Moreover, recent studies us- mediated efflux. Also, consistent with the present findings, ing fluorescence confocal microscopy have revealed trafficking Kojima et al. (46) recently reported that progesterone sup- of ABCA1 itself between the cell surface and intracellular sites pressed apoA-I-mediated cellular lipid release in human fibro- including late endosomes/lysosomes but not recycling endo- blasts. Progesterone has been reported to sequester cholesterol somes (35). Thus, it is conceivable that apoA-I bound to ABCA1 in lysosomes and block cholesterol trafficking to plasma mem- trafficks directly to late endosomes/lysosomes and somehow brane similar to the effects of the NPC1 mutation (47). How- mediates cholesterol efflux from these sites. However, it is ever, these earlier studies (45, 46) did not specifically compare notable that ABCA1 effectively stimulates efflux of plasma cholesterol efflux from different cellular pools under conditions membrane cholesterol, as deduced from a recently developed of high ABCA1 activities (i.e. following LXR/RXR activation plasma membrane labeling procedure (30, 31) (Fig. 2) and that and marked up-regulation of the ABCA1 (Fig. 1)) and did not this process is normal in NPC1 mutant macrophages (Fig. 3D). specifically evaluate the effect of the NPC1 molecule in ABCA1- Moreover, phospholipid efflux to apoA-I is unaffected in NPC1 mediated cholesterol efflux as in the present study. mutant cells (Fig. 4), suggesting that trafficking of ABCA1 to Our findings that NPC1 mutant macrophages have a prom- late endosomes/lysosomes is not required for formation of phos- inent defect in cholesterol efflux from the late endosomal/lyso- pholipid-apoA-I complexes. somal pool but only a moderate decrease in efflux from recy- An alternative explanation is that phospholipid-apoA-I com- cling endosomal pool (Fig. 3, A and C) may explain some earlier plexes are formed by ABCA1 at the cell surface and that these in vivo work showing that de novo synthesized cholesterol or complexes then stimulate cholesterol efflux from specialized cholesterol entering cells through the HDL/SR-BI pathway can regions of the plasma membrane that preferentially obtain be metabolized and excreted normally, whereas LDL-derived cholesterol from intracellular sources derived from late endo- cholesterol becomes sequestered in the lysosomal compartment somes/lysosomes rather than recycling endosomes. We propose and is metabolically inactive in NPC1 mutant mice (48, 49) and a model in which cholesterol trafficks from late endosomes/ in NPC1 patients (50). Labeling of cholesterol by DMEM, 10% lysosomes to the trans-Golgi in a process requiring the activity FBS might mimic more closely the trafficking of de novo syn- of NPC1 (38 – 40). Once cholesterol has arrived in the Golgi, it thesized cholesterol or cholesterol derived from the HDL/SR-BI may be formed into cholesterol/sphingolipid complexes, which pathway, whereas labeling by AcLDL is similar to LDL choles- give rise to cell surface cholesterol-enriched microdomains or terol trafficking to lysosomes. rafts. These plasma membrane domains could then act as a The HDL -mediated cholesterol efflux pathway, distinct preferential source of cholesterol for ABCA1-mediated efflux. This could explain why mass and isotopic efflux following from that mediated by ABCA1 (12, 11), was also defective in NPC1 cells. An intriguing, unexpected observation was the AcLDL labeling is even greater than following general plasma membrane labeling with cyclodextrin. This model is not neces- finding that macrophage cholesterol efflux to HDL was in- creased by treatment with LXR/RXR activators (Fig. 5), sug- sarily inconsistent with recent data, suggesting that ABCA1 stimulates cholesterol efflux from non-raft regions of the gesting a novel efflux process unrelated to ABCA1, SR-BI, or plasma membrane (41), because there could be several differ- apoE. The mechanism of HDL -mediated cholesterol efflux ap- ent types of cholesterol-enriched microdomains in the plasma pears quite distinct from apoA-I-mediated cholesterol efflux. membrane. Thus, apoA-I binds and interacts with ABCA1 to mediate cho- Cholesterol Pool and ABCA1-mediated Cholesterol Efflux 43569 FASEB J. 1, 40 – 45 lesterol efflux, whereas HDL is inactive in this regard (11). 20. Carstea, E. D., Morris, J. A., Coleman, K. G., Loftus, S. K., Zhang, D., HDL can stimulate cholesterol efflux by interacting with SR-BI Cummings, C., Gu, J., Rosenfeld, M. A., Pavan, W. J., Krizman, D. B., Nagle, J., Polymeropoulos, M. H., Sturley, S. L., Ioannou, Y. A., Higgins, (13), but this pathway does not appear to be very active in 3 M. E., Comly, M., Cooney, A., Brown, A., Kaneski, C. R., Blanchette-Mackie, and SR-BI neutralizing anti- mouse peritoneal macrophages, E. J., Dwyer, N. K., Neufeld, E. B., Chang, T. Y., Liscum, L., Tagle, D. A., bodies had no effect on the LXR/RXR-induced cholesterol efflux et al. (1997) Science 277, 228 –231 21. Naureckiene, S., Sleat, D. E., Lackland, H., Fensom, A., Vanier, M. T., to HDL . These findings suggest that there is a novel molecular Wattiaux, R., Jadot, M., and Lobel, P. (2000) Science 290, 2298 –2301 target of LXR/RXR activation in the cholesterol efflux pathway 22. Liscum, L., Ruggiero, R. M., and Faust, J. R. (1989) J. Cell Biol. 108, 1625–1636 to HDL . 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A., 96, 11992–11997 Patel, S., and Brady, R. O. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 50. Shamburek, R. D., Pentchev, P. G., Zech, L. A., Blanchette-Mackie, J., Carstea, 8247– 8251 E. D., VandenBroek, J. M., Cooper, P. S., Neufeld, E. B., Phair, R. D., 19. Pentchev, P. G., Comly, M. E., Kruth, H. S., Tokoro, T., Butler, J., Sokol, J., Brewer, H. B., Jr., Brady, R. O., and Schwartz, C. C. (1997) J. Lipid Res. 38, Filling-Katz, M., Quirk, J. M., Marshall, D. C., Patel, S., et al. (1987) 2422–2435 51. Tall, A. R., Breslow, J. L., and Rubin, E. M. (2001) The Metabolic and Molec- ular Basis of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., and 3 th Y. Sun and A. Tall, unpublished data. Valle, D., eds) 8 Ed., McGraw-Hill, New York http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Preferential ATP-binding Cassette Transporter A1-mediated Cholesterol Efflux from Late Endosomes/Lysosomes

Chen, Wengen; Sun, Yu; Welch, Carrie; Gorelik, Anna; Leventhal, Andrew R.; Tabas, Ira; Tall, Alan R.
Journal of Biological Chemistry – Nov 1, 2001
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 47, Issue of November 23, pp. 43564 –43569, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Preferential ATP-binding Cassette Transporter A1-mediated Cholesterol Efflux from Late Endosomes/Lysosomes* Received for publication, August 17, 2001, and in revised form, September 13, 2001 Published, JBC Papers in Press, September 14, 2001, DOI 10.1074/jbc.M107938200 Wengen Chen‡, Yu Sun‡, Carrie Welch, Anna Gorelik, Andrew R. Leventhal, Ira Tabas, and Alan R. Tall§ From the Division of Molecular Medicine, Department of Medicine, Columbia University, New York, New York 10032 Recently, ATP-binding cassette transporter A1 phages from these animals have a profound defect in apoA-I- (ABCA1), the defective molecule in Tangier disease, has mediated cholesterol efflux (8 –10), indicating that apolipopro- been shown to stimulate phospholipid and cholesterol tein-mediated cholesterol efflux is primarily mediated by efflux to apolipoprotein A-I (apoA-I); however, little is ABCA1. In contrast, ABCA1 shows only slight interaction with known concerning the cellular cholesterol pools that act HDL and no interaction with HDL (11). Cellular cholesterol 3 2 as the source of cholesterol for ABCA1-mediated efflux. efflux mediated by HDL is thought to involve a “passive” proc- We observed a higher level of isotopic and mass choles- ess that may be diffusion-mediated or may involve an interac- terol efflux from mouse peritoneal macrophages labeled tion of HDL with scavenger receptor B-I (SR-BI) (12, 13). H]cholesterol/acetyl low density lipoprotein with [ ABCA1 is a full transporter with 12 membrane-spanning (where cholesterol accumulates in late endosomes and domains (5, 14). Transfection of ABCA1 in 293 cells reveals a H]choles- lysosomes) compared with cells labeled with [ predominant cell surface localization and suggests a direct terol with 10% fetal bovine serum, suggesting that late interaction of ABCA1 with apoA-I (11). The primary activity of endosomes/lysosomes act as a preferential source of cho- ABCA1 appears to be the translocation of phospholipid at the lesterol for ABCA1-mediated efflux. Consistent with this plasma membrane rather than direct interaction with choles- idea, macrophages from Niemann-Pick C1 mice that terol (15, 16). Phospholipid-apoA-I complexes formed by have an inability to exit cholesterol from late endo- ABCA1 may promote cholesterol efflux in a secondary fashion somes/lysosomes showed a profound defect in choles- perhaps involving distinct areas of the plasma membrane (15, terol efflux to apoA-I. In contrast, phospholipid efflux to 17). The nature of the cellular sites that donate cholesterol to apoA-I was normal in Niemann-Pick C1 macrophages, as these phospholipid-apoA-I complexes is poorly understood. was cholesterol efflux following plasma membrane cho- This may involve specific plasma membrane domains that de- lesterol labeling. These results suggest that cholesterol rive cholesterol from intracellular stores. The nature of intra- deposited in late endosomes/lysosomes preferentially acts as a source of cholesterol for ABCA1-mediated cho- cellular sites that potentially donate cholesterol to the plasma lesterol efflux. membrane for ABCA1-mediated efflux is also unclear. Ni- emann-Pick C (I and II) molecules play an essential role in intracellular cholesterol trafficking, particularly in the exit of Tangier disease (TD) is a rare condition associated with low cholesterol from late endosomes/lysosomes (18 –21). Earlier levels of plasma high density lipoproteins (HDL) and accumu- studies suggested a defect in cholesterol efflux to phospholipid lation of cholesterol and cholesteryl esters in macrophage foam vesicles in NPC1 fibroblasts (22), but the specific role of NPC1 cells in tonsils, spleen, and other tissues (1). The cellular defect in ABCA1-mediated cholesterol efflux has not been in TD involves a marked decrease in the efflux of cholesterol investigated. and phospholipid to apoA-I, the major protein of HDL (2, 3). The ABCA1 gene is up-regulated by cellular cholesterol load- Recently, TD was shown to be caused by mutations in the ing (23). The mechanism of this effect is increased gene tran- ATP-binding cassette transporter, ABCA1 (4 –7). Mice with scription mediated by the oxysterol-activated transcription fac- deficiency of ABCA1 also have low HDL. In addition, macro- tor liver X receptor (LXR) acting in a complex with retinoid X receptor (RXR) at a site on the proximal promoter of the ABCA1 gene (24). While studying cholesterol efflux from * This work was supported by Specialized Center of Research in macrophages that had been treated with the LXR/RXR ligands Atherosclerosis Grant HL-56984 (to A. R. T. and I. T) from the NHLBI, National Institutes of Health, and research grants from the Parshegian 22(R)-hydroxycholesterol and 9-cis-retinoic acid to up-regulate Foundation (to A. R. T) and Berlex Biosciences (to I. T.). The costs of ABCA1, we noticed a marked discrepancy between the magni- publication of this article were defrayed in part by the payment of page tude of ABCA1 expression and the resulting stimulation of charges. This article must therefore be hereby marked “advertisement” cholesterol efflux, depending on the method of cellular choles- in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ These authors made equal contributions to the work. terol labeling. This led to an investigation of the hypothesis § To whom correspondence should be addressed: Div. of Molecular that ABCA1 stimulates cholesterol efflux preferentially from a th Medicine, Dept. of Medicine, Columbia University, 603 W. 168 St., pool of cholesterol found in late endosomes/lysosomes. This New York, NY 10032. Tel.: 212-305-9418; Fax: 212-305-5052; E-mail: hypothesis has been evaluated by comparing cholesterol efflux [email protected]. under different labeling conditions and supported by the dem- The abbreviations used are: TD, Tangier disease; ABCA1, ATP- binding cassette transport A1; AcLDL, acetyl low density lipoprotein; onstration of a profound defect in cholesterol efflux to apoA-I apoA-I, apolipoprotein A-I; apoE, apolipoprotein E; HDL, high density using macrophages from NPC1 mice. lipoprotein; LXR, liver X receptor; RXR, retinoid X receptor; NPC1, Niemann-Pick C1; SR-BI, Scavenger receptor class B type I; FBS, fetal EXPERIMENTAL PROCEDURES bovine serum; PIPES, 1,4-piperazinediethanesulfonic acid; PBS, phos- Ribonuclease Protection Assay—Reverse transcription-polymerase phate-buffered saline; wt, wild type; DMEM, Dulbecco’s modified Eagle’s medium; BSA, bovine serum albumin. chain reaction was used to obtain a fragment of the murine ABCA1 43564 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Cholesterol Pool and ABCA1-mediated Cholesterol Efflux 43565 cDNA. Murine ABCA1 and -actin antisense riboprobes were prepared radioactivity released from the cells into the medium relative to the total radioactivity in cells and media. by in vitro transcription using murine ABCA1 -actin cDNA plasmid Cholesterol Mass Analysis—The cells in 6-well plates were [ - constructs. The protected hybrid fragments for ABCA1 and -actin were H]cho lesterol labeled by procedure a as described above. After4hof incuba- 290 and 160 base pairs, respectively. Ribonuclease protection assay was tion with 10 g/ml apoA-I (see Fig. 2) or 15 g/ml human HDL performed as described (25). In brief, 20 g of total RNA were hybrid- (see Fig. 6), medium was collected, and the cells were lysed in 0.1 M sodium ized with 5  10 cpm ABCA1 and -actin riboprobes at 48 °C overnight hydroxide and 0.1% SDS. Lipids were extracted in chloroform:methanol in 30 l of a buffer consisting of 40 mM PIPES, pH 6.0, 400 mM NaCl, 1 (2:1). The organic residue was dissolved in 0.5% Triton X-100. Choles- mM EDTA, and 80% formamide. The hybridization mixture was di- terol was determined enzymatically (Wako Chemicals USA, Richmond, gested with 20 units of T ribonuclease (Life Technologies, Inc.) at 37 °C VA). Protein was determined by the Lowry method. for 1 h, extracted with phenol/chloroform, precipitated with ethanol, H]Phospholipid Efflux Study—Macrophages in a 24-well plate and dissolved in 5 l of RNA loading buffer. The protected RNA hybrid were choline labeled for 24 h in 0.5 ml of DMEM, 10% FBS supple- fragments were resolved on a 6% polyacrylamide/urea gel and subjected mented with 1.0 Ci/ml [ H]choline (PerkinElmer Life Sciences). After to autoradiography. overnight equilibration in DMEM, 0.2% BSA with or without the LXR/ Immunoblot Analysis of ABCA1—For immunoblot analysis of RXR ligands treatment, the cells were washed twice in PBS, 0.2% BSA. ABCA1, peritoneal macrophages were washed and scraped in PBS and Efflux was performed by incubation with 10 g/ml apoA-I for4hin0.5 lysed in 10 mM Tris-HCl, pH 7.3, 1 mM MgCl , and 0.5% Nonidet P-40 ml DMEM, 0.2% BSA with or without the ligands. Then medium was g/ml leupeptin, 1 g/ml in the presence of protease inhibitors (0.5 collected and centrifuged at 6000  g for 10 min to remove cell debris. aprotinin, 1 g/ml pepstatin A; Roche Molecular Biochemicals). Post- H]Phospholipids in an aliquot of supernatant were first extracted nuclear supernatants from cell lysates were prepared by centrifugation with chloroform:methanol (2:1), and then the radioactivity was deter- at 3000  g for 10 min at 4 °C. Samples containing the indicated mined by scintillation counting. The cells were finally lysed in 0.5 ml of amounts of protein were reduced with 2-mercaptoethanol in gel loading 0.1 M sodium hydroxide, 0.1% SDS, and the radioactivity in an aliquot buffer, fractionated by 7.5% SDS-polyacrylamide gel electrophoresis, after lipid extraction was determined. The percentage of secreted and transferred to 0.22-m nitrocellulose membranes. Immunoblotting H]phospholipid was calculated by dividing the medium-derived was performed using an anti-ABCA1 antiserum (Novus, Littleton, CO) counts by the sum of the total (medium plus cell). and ECL (Amersham Pharmacia Biotech). The relative intensities of Statistical Analysis—The results are presented as the means  S.D. the bands were determined by densitometry (Molecular Dynamics, The tests for the significant differences between groups were performed model 300A). by Student’s t test. Lipoprotein Isolation—Human low density lipoprotein (LDL, 1.006d1.063) and high density lipoprotein (HDL , 1.063d1.125) RESULTS were isolated from plasma by sequential ultracentrifugation. Acetyl LDL (AcLDL) was prepared as described (26). Apolipoprotein A-I We recently showed that ABCA1 mRNA is up-regulated by (apoA-I) was purchased from Biodesign International (Saco, ME). activation of LXR/RXR (24). To determine whether this re- Isolation and Culture of Mouse Peritoneal Macrophages—Homozy- sulted in an increase in functional ABCA1 protein, we gous NPC1 were produced by intercrossing BALB/cNctr-npc1 / measured cholesterol efflux to apoA-I in mouse peritoneal (BALB-npc1 /) mice (stock number 003092; Jackson Laboratory, Bar macrophages cells treated with the LXR/RXR ligands 22(R)- Harbor, ME). Mouse peritoneal macrophages were isolated from NPC1 hydroxycholesterol and 9-cis-retinoic acid. This treatment re- and wild type (wt) littermates by peritoneal lavage with PBS 3 days sulted in a marked up-regulation of ABCA1 mRNA (not shown) after intraperitoneal injection with 1 ml of 3.85% thioglycollate (Becton Dickinson, Sparks, MD). The isolated cells were plated onto 24-well and protein levels (Fig. 1A) and an increase in cholesterol efflux plates and allowed to adhere by incubation for4hat37 °C in Dulbecco’s (Fig. 1B) as anticipated (27). Surprisingly, the level of choles- modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine terol efflux was about 2.5-fold higher in activated cells labeled serum (FBS) (Life Technologies). After removal of nonadherent cells by 3 with [ H]cholesterol AcLDL compared with cells labeled with washing with PBS, the cells were further incubated for 2 days and then [ H]cholesterol, 10% FBS (Fig. 1B, compare bars 4 and 2), used for cholesterol labeling and efflux experiments. despite comparable levels of ABCA1 expression (Fig. 1A; note [ H]Cholesterol Labeling of Cells—Mouse peritoneal macrophages were labeled with [ H]cholesterol carried by one of three delivery agents that ABCA1 protein appears as a doublet for unknown reason). to investigate cholesterol efflux from different pools: (a) AcLDL (late AcLDL is internalized by the scavenger receptor A and accu- endosomes/lysosomes pool); (b) 10% FBS/DMEM (recycling endosomes mulates primarily in late endosomes and lysosomes (28), M methyl--cyclodextrin (plasma membrane pool). For 3 pool); and (c)5m whereas the [ H]cholesterol, 10% FBS method appears to pref- procedure a, cells were labeled overnight by 1 Ci/ml [1,2- H(N)]- erentially label recycling endosomes and the trans-Golgi net- cholesterol (PerkinElmer Life Sciences) in DMEM, 0.2% BSA supple- work (29). These findings suggested the hypothesis that mented with 50 g/ml AcLDL. LXR/RXR ligands 22(R)-hydroxycholes- ABCA1 might preferentially stimulate cholesterol efflux from terol (final concentration, 10 M) and 9-cis-retinoic acid (final concentration, 10 M) (BIOMOL Research Laboratories, Plymouth late endosomes/lysosomes rather than from cellular cholesterol Meeting, PA) were added during the labeling to induce ABCA1 expres- pools labeled by [ H]cholesterol, 10% FBS. sion. After the labeling, the cells were washed with PBS and equili- To further explore this idea, cholesterol mass and isotopic brated with DMEM, 0.2% BSA for 1 h. Efflux was then performed as 3 efflux to apoA-I were measured in cells labeled with [ H]cho- described below. For procedure b, the cells were labeled with 1 Ci/ml lesterol, 10% FBS, with [ H]cholesterol/AcLDL, or with [ H]cholesterol in 0.5 ml of DMEM supplemented with 10% FBS for [ H]cholesterol/cyclodextrin. In the latter procedure, the cells 24 h. The cells were then equilibrated overnight in DMEM, 0.2% BSA are labeled briefly (15 min) with cyclodextrin:cholesterol (8:1, with or without the LXR/RXR ligands 22(R)-hydroxycholesterol and 9-cis-retinoic acid. After washing, the cells were used for efflux exper- molar ratio), and the radiolabel is thought to reside mostly in iments. For procedure c, the cells were first treated with the ligands the plasma membrane (30, 31). These experiments showed 22(R)-hydroxycholesterol and 9-cis-retinoic acid in DMEM, 0.2% BSA greater isotopic and mass efflux of cholesterol in activated cells overnight to induce ABCA1. Then the medium was replaced by 5 m 3 labeled with [ H]cholesterol/AcLDL, compared with cells la- methyl -cyclodextrin:cholesterol at molar ration 8:1 ([ H]cholesterol, 1 beled in either of the other two ways (Fig. 2), consistent with Ci/ml) for 15 min at 37 °C. After washing, the cells were used for efflux the idea that the late endosomal/lysosomal cholesterol pool is a step. [ H]Cholesterol Efflux Study—After labeling and equilibration, the preferential source for cholesterol efflux by ABCA1. Activation cells were incubated by 10 g/ml purified human apoA-I or 15 g/ml of LXR/RXR did result in a marked increase in isotopic and human HDL in 0.5 ml of DMEM, 0.2% BSA with or without the mass cholesterol efflux from cells labeled with [ H]cholesterol/ LXR/RXR ligands for 4 h. Then medium was collected and centrifuged cyclodextrin, suggesting that the plasma membrane cholesterol at 6000  g for 10 min to remove cell debris and cholesterol crystal, and also contributes significantly to ABCA1-mediated cholesterol radioactivity in an aliquot of supernatant was determined by liquid efflux. However, for the [ H]cholesterol, 10% FBS labeling scintillation counting. The cells were finally lysed in 0.5 ml of 0.1 M method, isotopic efflux was only slightly increased by LXR/RXR sodium hydroxide, 0.1% SDS and the radioactivity in an aliquot was determined. Cholesterol efflux was expressed as the percentage of the activation, whereas cholesterol mass efflux was increased in a 43566 Cholesterol Pool and ABCA1-mediated Cholesterol Efflux FIG.1. Discrepancy between the magnitude of ABCA1 expres- sion and the resulting cholesterol efflux with different cellular cholesterol labeling methods. wt peritoneal macrophages were [ H]cholesterol labeled by 10% FBS, DMEM (lanes 1 and 2)or50 g/ml AcLDL (lanes 3 and 4), with or without LXR/RXR ligands (22(R)- FIG.2. Isotopic and mass efflux of cholesterol from macro- hydroxycholesterol and 9-cis-retinoic acid) treatment as described un- phages treated with the three different [ H]cholesterol labeling der “Experimental Procedures.” After equilibration in DMEM, 0.2% methods. wt mouse peritoneal macrophages in 6-well plates were BSA, the cells were incubated with 10 g/ml of apo-A-I for4hin [ H]cholesterol labeled by 10% FBS, AcLDL, or cyclodextrin and treated DMEM, 0.2% BSA with or without the LXR/RXR ligands. A, Western with or without the LXR/RXR ligands as described under “Experimen- blot of ABCA1 following SDS-polyacrylamide gel electrophoresis of cell tal Procedures.” After equilibration in DMEM, 0.2% BSA, the cells were lysates. Representative data are from one of two independent experi- 3 incubated with 10 g/ml apoA-I for 4 h. [ H]cholesterol efflux (A) was ments. Cholesterol efflux (B) was expressed as the medium [ H]choles- expressed as the medium [ H]cholesterol radioactivity as a percentage terol radioactivity as a percentage of total [ H]cholesterol radioactivity 3 of total [ H]cholesterol radioactivity (cells plus medium). For choles- (cells plus medium). Representative data are from one of three inde- terol mass assay (B), cholesterol in the medium was first extracted and pendent experiments. The values are the means  S.D. (n  3). *, p measured enzymatically. Representative data are from one of two in- 0.01, bar 2 versus bar 4. ch, cholesterol; 22ch, 22(R)-hydroxycholesterol; dependent experiments. The values are the means  S.D. (n  3). *, p RA, 9-cis-retinoic acid. 0.01, AcLDL labeling versus 10% FBS and methyl--cyclodextrin (MCD) labelings. 22ch, 22(R)-hydroxycholesterol; RA, 9-cis-retinoic acid. similar fashion to [ H]cholesterol/cyclodextrin-labeled cells. This finding could arise if the radiolabel was primarily present in pools of cholesterol inaccessible to ABCA1 (i.e. recycling in NPC1 macrophages compared with wild type macrophages endosomes) (29), whereas cholesterol mass efflux reflected ef- following LXR/RXR activation (Fig. 3C). Following plasma flux from the plasma membrane where cholesterol would be membrane labeling with [ H]cholesterol/cyclodextrin, there unlabeled by this method. were identical levels of cholesterol efflux in NPC1 and wild type To further explore the hypothesis that late endosomes/lyso- cells (Fig. 3D). These experiments suggest that both lysosomal somes represent a preferred source of cholesterol for ABCA1- and plasma membrane cholesterol pools serve as a source of mediated cholesterol efflux, we next carried out efflux studies cholesterol for ABCA1 and that the lysosomal pool requires the using macrophages from Niemann-Pick C1 mice, which have a activity of the NPC1 molecule. defect in trafficking of cholesterol out of late endosomes (32). ABCA1 is thought to act as a phospholipid flippase at the Cholesterol loading was carried out using [ H]cholesterol/ plasma membrane (15, 16). This activity may lead to the for- AcLDL. Compared with macrophages from wild type mice, mation of phospholipid-apoA-I complexes that secondarily there was a profound decrease in cholesterol efflux to apoA-I in stimulate cholesterol efflux from a distinct region of plasma NPC1 macrophages, especially following induction of ABCA1 membrane (15, 17). We measured phospholipid efflux to apoA-I (Fig. 3A). Measurements of ABCA1 mRNA and protein re- in NPC1 and wt macrophages. Phospholipid efflux was stimu- vealed similar levels of induction in control and NPC1 macro- lated following activation of LXR/RXR, but there was no defect phages (not shown). In earlier studies, Liscum et al. (22) re- in phospholipid efflux in NPC1 cells (Fig. 4). This indicates that ported that human NPC1 fibroblasts had a moderate defect in the primary action of ABCA1, i.e. formation of phospholipid- cholesterol efflux to small unilamellar vesicles; this was man- apoA-I complexes, is intact in NPC1 cells. ifested as a delay in cholesterol efflux that became normal Cholesterol efflux to HDL was also significantly decreased following longer incubation periods. However, a time course in NPC1 cells loaded with [ H]cholesterol/AcLDL (Fig. 5)orby study revealed a profound 3– 4-fold decrease in cholesterol ef- the [ H]cholesterol, 10% FBS method (not shown). Because flux to apoA-I in NPC1 macrophages that was not ameliorated HDL does not interact with ABCA1 (11), this indicates a defect by prolonged incubation (Fig. 3B). If ABCA1 preferentially in cholesterol efflux via pathways not mediated by ABCA1. stimulates cholesterol efflux from late endosomes/lysosomes, Interestingly, cholesterol efflux via HDL was also induced by then it might be anticipated that there would be a less pro- LXR/RXR activation (Fig. 5). This suggests the presence of nounced defect in cholesterol efflux in NPC1 cells labeled with other LXR/RXR target genes in the HDL -mediated efflux [ H]cholesterol, 10% FBS. Accordingly, using this labeling pathway. Because apolipoprotein E (apoE) was recently iden- method, basal cholesterol efflux to apoA-I was similar in wild tified as an LXR/RXR target (33), we considered the possibility type and NPC1 cells, and efflux was only moderately decreased that increased cholesterol efflux might be due to increased Cholesterol Pool and ABCA1-mediated Cholesterol Efflux 43567 FIG.3. ABCA1-mediated cholesterol efflux in NPC1 and wild type macro- phages. NPC1 and wt peritoneal macro- phages were [ H]cholesterol labeled by AcLDL (A and B), 10% FBS (C), and cy- clodextrin (D) with or without LXR/RXR ligand treatment as described under “Ex- perimental Procedures.” After equilibra- tion in DMEM, 0.2% BSA, the cells were incubated with 10 g/ml of apo-A-I for 4 h (A, C, and D) or different time points (B) in DMEM, 0.2% BSA with or without the ligands. Cholesterol efflux was expressed as the medium [ H]cholesterol radioactiv- ity as a percentage of total [ H]cholesterol radioactivity (cells plus medium). Repre- sentative data are from one of three (A and C) or two (B and D) independent ex- periments. The values are the means S.D. (n  3). *, p  0.01, NPC1 versus wt. 22ch, 22(R)-hydroxycholesterol; RA, 9- cis-retinoic acid. FIG.4. ABCA1-mediated phospholipid efflux in NPC1 and wild FIG.5. Cholesterol efflux to HDL in NPC1 and wild type peri- type peritoneal macrophages. NPC1 and wt peritoneal macro- toneal macrophages. NPC1 and wt peritoneal macrophages were 3 3 phages were [ H]choline-labeled (1 Ci/ml of [ H]choline) and treated 3 [ H]cholesterol AcLDL-labeled and treated with or without the LXR/ with or without the LXR/RXR ligands as described under “Experimen- RXR ligands as described under “Experimental Procedures.” Efflux was tal Procedures.” [ H]Phospholipid efflux was performed by incubation performed by incubation with 15 g/ml of HDL for4hin DMEM, 0.2% with 10 g/ml of apoA-I for4hin DMEM, 0.2% BSA with or without the BSA with or without the ligands. Cholesterol efflux was expressed as ligands. [ H]phospholipid in the medium and cell lysates was deter- 3 the medium [ H]cholesterol radioactivity as a percentage of total mined following extraction in chloroform:methanol (2:1), and the radio- 3 [ H]cholesterol radioactivity (cells plus medium). Representative data activity was determined by scintillation counting. [ H]Phospholipid are from one of three independent experiments. The values are the efflux was expressed as the medium radioactivity as a percentage of means  S.D. (n  3). *, p  0.05, NPC1 versus wt. 22ch, 22(R)- total radioactivity (cells plus medium). Representative data are from hydroxycholesterol; RA, 9-cis-retinoic acid. one of two independent experiments. The values are the means  S.D. (n  3). 22ch, 22(R)-hydroxycholesterol; RA, 9-cis-retinoic acid. somes rather than cholesterol deposited at other intracellular sites. The equilibration of cell surface cholesterol with these apoE synthesis by mouse peritoneal macrophages. However, intracellular sites requires the activity of the NPC1 molecule LXR/RXR activation similarly increased cholesterol efflux to and could perhaps also involve trafficking of the ABCA1 mol- HDL in macrophages from apoE knock-out mice (not shown), ecule itself (35). ABCA1 is markedly less efficient in stimulat- eliminating this possibility. The ability of HDL to stimulate ing cholesterol efflux from cells that have been labeled with increased cholesterol efflux following LXR/RXR activation was [ H]cholesterol, 10% FBS, which probably primarily labels re- also confirmed by cholesterol mass measurements, which indi- cycling endosomes (29). A profound defect in ABCA1-mediated cated primarily an increase in HDL free cholesterol (Fig. 6). cholesterol efflux in NPC1 mutant macrophages may be an SR-BI neutralizing antibodies (34) did not affect cholesterol important factor explaining our recent observations showing efflux mediated by HDL in either basal or LXR/RXR-stimu- an increase of atherosclerosis in apoE knock-out/NPC1 mutant lated conditions (not shown). mice, compared with apoE knock-out control mice. DISCUSSION Massive cholesteryl ester accumulation in TD macrophages indicates that ABCA1 has an essential role in mediating cho- Our findings suggest that phospholipid-apoA-I complexes formed by ABCA1 initially stimulate cholesterol efflux from regions of the plasma membrane that preferentially utilize N. Sharma, G. Kuriakose, D. Zhang, I. Tabas, R. J. Deckelbaum, cholesterol deposited by modified LDL in late endosomes/lyso- A. R. Tall, and C. L. Welch, submitted for publication. 43568 Cholesterol Pool and ABCA1-mediated Cholesterol Efflux Following entry of lipoprotein cholesterol into late endo- somes and lysosomes, NPC1 has an essential role in allowing cholesterol to gain access to the ABCA1 efflux pool (Fig. 3). Liscum et al. (22) reported a delay in cholesterol efflux to unilamellar vesicles in fibroblasts from NPC1 patients. How- ever, with time the efflux became normal. In contrast, the apoA-I stimulated cholesterol efflux in NPC1 mutant mouse macrophages was profoundly reduced at all time points (Fig. 3B). Recently, it has been shown that following labeling of NPC1 mutant Chinese hamster ovary cells with H-cholesteryl ester LDL, early time points of cholesterol efflux to cyclodextrin show no or little defect (31, 42). However, after the initial appearance at the plasma membrane and subsequent internal- FIG.6. Cholesterol efflux from wild type macrophages incu- ization to an intracellular pool, cholesterol shows delayed traf- bated with HDL . wt mouse peritoneal macrophages in 6-well plates ficking back to the plasma membrane and poor activation of were [ H]cholesterol/AcLDL-labeled, as described under “Experimental Procedures.” After equilibration the cells were incubated with 15 g/ml acyl-CoA-cholesterol acyltransferase in (ACAT) NPC1 mutant HDL for 4 h. Cholesterol in the medium was extracted and measured cells. This has led to the proposal that NPC1 acts on an intra- enzymatically. Representative data are from one of two independent cellular pool of cholesterol that is derived from the plasma experiments. The values are the means  S.D. (n  3). *, p  0.05, 22ch/RA versus 22ch/RA. 22ch, 22(R)-hydroxycholesterol; RA, 9-cis- membrane and is in equilibrium with ACAT. Whether NPC1 is retinoic acid; FC, free cholesterol; CE, cholesteryl ester. acting in late endosomes/lysosomes (43, 44) or on another pool of cholesterol (31), our studies suggest that this pool of choles- lesterol efflux from these cells. It is likely that cholesterol from terol represents an important source of cholesterol for ABCA1- a variety of sources, including effete red cells and modified stimulated efflux. forms of LDL, is taken up by macrophages eventuating in Recently, Leventhal et al. (45) have found a defect in basal delivery to late endosomes/lysosomes. Our studies are consist- cholesterol efflux to apoA-I in acid sphingomyelinase-deficient ent with the idea that apoA-I/ABCA1-mediated cholesterol ef- macrophages. These studies suggest that endososomal/lysoso- flux plays an essential role in removing cholesterol from these mal sphingomyelin accumulation leads to cholesterol seques- intracellular sites. The underlying mechanisms are unclear. tration and, thus, defective cholesterol trafficking and efflux. Intracellular trafficking of apoA-I has been reported in macro- The present findings extend these observations by providing phages, and this process appears to be defective in TD (36). The direct evidence that the late endosomal/lysosomal cholesterol trafficking of apoA-I has been suggested to have a role in pool represents the preferred source of cholesterol for ABCA1- mediating cholesterol efflux (37). Moreover, recent studies us- mediated efflux. Also, consistent with the present findings, ing fluorescence confocal microscopy have revealed trafficking Kojima et al. (46) recently reported that progesterone sup- of ABCA1 itself between the cell surface and intracellular sites pressed apoA-I-mediated cellular lipid release in human fibro- including late endosomes/lysosomes but not recycling endo- blasts. Progesterone has been reported to sequester cholesterol somes (35). Thus, it is conceivable that apoA-I bound to ABCA1 in lysosomes and block cholesterol trafficking to plasma mem- trafficks directly to late endosomes/lysosomes and somehow brane similar to the effects of the NPC1 mutation (47). How- mediates cholesterol efflux from these sites. However, it is ever, these earlier studies (45, 46) did not specifically compare notable that ABCA1 effectively stimulates efflux of plasma cholesterol efflux from different cellular pools under conditions membrane cholesterol, as deduced from a recently developed of high ABCA1 activities (i.e. following LXR/RXR activation plasma membrane labeling procedure (30, 31) (Fig. 2) and that and marked up-regulation of the ABCA1 (Fig. 1)) and did not this process is normal in NPC1 mutant macrophages (Fig. 3D). specifically evaluate the effect of the NPC1 molecule in ABCA1- Moreover, phospholipid efflux to apoA-I is unaffected in NPC1 mediated cholesterol efflux as in the present study. mutant cells (Fig. 4), suggesting that trafficking of ABCA1 to Our findings that NPC1 mutant macrophages have a prom- late endosomes/lysosomes is not required for formation of phos- inent defect in cholesterol efflux from the late endosomal/lyso- pholipid-apoA-I complexes. somal pool but only a moderate decrease in efflux from recy- An alternative explanation is that phospholipid-apoA-I com- cling endosomal pool (Fig. 3, A and C) may explain some earlier plexes are formed by ABCA1 at the cell surface and that these in vivo work showing that de novo synthesized cholesterol or complexes then stimulate cholesterol efflux from specialized cholesterol entering cells through the HDL/SR-BI pathway can regions of the plasma membrane that preferentially obtain be metabolized and excreted normally, whereas LDL-derived cholesterol from intracellular sources derived from late endo- cholesterol becomes sequestered in the lysosomal compartment somes/lysosomes rather than recycling endosomes. We propose and is metabolically inactive in NPC1 mutant mice (48, 49) and a model in which cholesterol trafficks from late endosomes/ in NPC1 patients (50). Labeling of cholesterol by DMEM, 10% lysosomes to the trans-Golgi in a process requiring the activity FBS might mimic more closely the trafficking of de novo syn- of NPC1 (38 – 40). Once cholesterol has arrived in the Golgi, it thesized cholesterol or cholesterol derived from the HDL/SR-BI may be formed into cholesterol/sphingolipid complexes, which pathway, whereas labeling by AcLDL is similar to LDL choles- give rise to cell surface cholesterol-enriched microdomains or terol trafficking to lysosomes. rafts. These plasma membrane domains could then act as a The HDL -mediated cholesterol efflux pathway, distinct preferential source of cholesterol for ABCA1-mediated efflux. This could explain why mass and isotopic efflux following from that mediated by ABCA1 (12, 11), was also defective in NPC1 cells. An intriguing, unexpected observation was the AcLDL labeling is even greater than following general plasma membrane labeling with cyclodextrin. This model is not neces- finding that macrophage cholesterol efflux to HDL was in- creased by treatment with LXR/RXR activators (Fig. 5), sug- sarily inconsistent with recent data, suggesting that ABCA1 stimulates cholesterol efflux from non-raft regions of the gesting a novel efflux process unrelated to ABCA1, SR-BI, or plasma membrane (41), because there could be several differ- apoE. The mechanism of HDL -mediated cholesterol efflux ap- ent types of cholesterol-enriched microdomains in the plasma pears quite distinct from apoA-I-mediated cholesterol efflux. membrane. Thus, apoA-I binds and interacts with ABCA1 to mediate cho- Cholesterol Pool and ABCA1-mediated Cholesterol Efflux 43569 FASEB J. 1, 40 – 45 lesterol efflux, whereas HDL is inactive in this regard (11). 20. Carstea, E. D., Morris, J. A., Coleman, K. G., Loftus, S. K., Zhang, D., HDL can stimulate cholesterol efflux by interacting with SR-BI Cummings, C., Gu, J., Rosenfeld, M. A., Pavan, W. J., Krizman, D. B., Nagle, J., Polymeropoulos, M. H., Sturley, S. L., Ioannou, Y. A., Higgins, (13), but this pathway does not appear to be very active in 3 M. E., Comly, M., Cooney, A., Brown, A., Kaneski, C. R., Blanchette-Mackie, and SR-BI neutralizing anti- mouse peritoneal macrophages, E. J., Dwyer, N. K., Neufeld, E. B., Chang, T. Y., Liscum, L., Tagle, D. A., bodies had no effect on the LXR/RXR-induced cholesterol efflux et al. (1997) Science 277, 228 –231 21. Naureckiene, S., Sleat, D. E., Lackland, H., Fensom, A., Vanier, M. T., to HDL . These findings suggest that there is a novel molecular Wattiaux, R., Jadot, M., and Lobel, P. (2000) Science 290, 2298 –2301 target of LXR/RXR activation in the cholesterol efflux pathway 22. Liscum, L., Ruggiero, R. M., and Faust, J. R. (1989) J. Cell Biol. 108, 1625–1636 to HDL . 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Published: Nov 1, 2001

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