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J. Biol. Chem., Vol. 280, Issue 12, 11876-11886, March 25, 2005
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From the Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138
Received for publication, December 30, 2004 , and in revised form, January 14, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The mechanisms that are responsible for the formation of cholesteryl esters in atheromatous cells have only partly been resolved. It is clear that cholesteryl ester in cytoplasmic lipid droplets is produced by ER1-localized acyl-CoA:cholesterol acyltransferases (ACATs) that are allosterically activated by cholesterol (9, 10). The aberrant accumulation of cholesteryl ester in lysosomes, however, remains to be explained (8).
Cholesterol in atherosclerotic lesions is predominantly derived from serum lipoproteins such as low density lipoprotein (LDL) (11). LDL consists of a hydrophobic core containing mainly cholesteryl ester and triglycerides surrounded by a lipid monolayer and apolipoprotein B100 (12). LDL and its atherogenic derivatives are recognized by cell surface receptors, internalized, and transferred to the endosomal system. Cholesteryl ester and triglycerides are then hydrolyzed by acidic cholesteryl ester hydrolase (aCEH), generating cholesterol and fatty acids (11, 13, 14).
It has previously been proposed that lysosomal cholesteryl ester accumulation in atheromatous cells might result from a deficiency of aCEH (5, 15). Most subsequent studies on aCEH activity in atherosclerotic biopsies have found, however, that aCEH levels were unchanged or increased (14). Other hypotheses that have been proposed to explain the build-up of cholesteryl ester in lysosomes include esterification of cholesterol by a local enzyme (16), saturation of aCEH with excessive substrate (15), impaired delivery of aCEH to substrate-carrying endosomes (17), and transport of ACAT-derived cholesteryl ester to lysosomes. Direct evidence in favor of any of these models, however, has not yet been available.
To further address this issue we have studied the metabolism of cholesteryl ester and triglycerides in mouse macrophages in vivo and with intact endosomes in vitro. We show that net hydrolysis of cholesteryl ester depends on the ability of membranes to absorb cholesterol. When membrane cholesterol approaches saturation, as it does in atherosclerosis, cholesterol is re-esterified in endosomes. Endosomal esterification occurs independently of canonical ACATs. Endosomal hydrolysis of triglycerides was not affected by cholesterol, indicating that activity and transport of aCEH remained unchanged. These findings reveal a feedback connection between cellular cholesterol balance and endosomal cholesterol dispensation and provide a potential explanation for why cholesterol-saturated cells in atherosclerotic lesions accumulate cholesteryl ester in lysosomes.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-D-glucosaminide dihydrate, the sodium salt of adenosine 5'-monophosphate (A-1752), imipramine (I-0899), and progesterone (P-8783) from Sigma; U18666A from Biomol; 25-hydroxycholesterol from Steraloids; pepstatin A, leupeptin and the sodium salt of ATP from Roche Applied Science; [1,2,6,7-3H]cholesteryl oleate ([3H]cholesteryl oleate, NET-746), glyceryl tri-[9,10-3H]oleate ([3H]trioleoylglycerol, NET-431), and [1,2-3H]cholesterol ([3H]cholesterol, NET-139) from PerkinElmer Life Sciences; wheat germ agglutinin-conjugated polyvinyltoluene (PVT) beads (RPNQ 0001) from Amersham Biosciences; FBS from Invitrogen; and other cell culture reagents from Mediatech. The ACAT inhibitor CI-976 was a gift from Pfizer Global Research and Development. The Rab5 antibody used in this study was a kind gift from A. Wandinger-Ness, University of New Mexico School of Medicine, Albuquerque, NM. Lipoprotein-deficient FBS (LPDS) (18), LDL (18), and acetylated LDL (19) were prepared as described. Anionic liposomes were prepared as described (20) and contained 1 mM phosphatidylcholine, 1 mM phosphatidylserine, 0.2 mM dicetyl phosphate plus neutral lipids as follows: liposomes prepared with [3H]cholesteryl oleate contained 4 µM [3H]cholesteryl oleate, 6 µM non-radioactive cholesteryl oleate, and 1 µM non-radioactive trioleoylglycerol; liposomes prepared with [3H]trioleoylglycerol contained 1 µM [3H]trioleoylglycerol plus 10 µM non-radioactive cholesteryl oleate; liposomes prepared with [3H]cholesterol contained 1.3 mM phosphatidylcholine, 1.2 mM phosphatidylserine plus 4 µM [3H]cholesterol.
Cell Culture
J774 mouse macrophages were grown in monolayer culture at 37 °C in an atmosphere of 8-9% CO2 in medium A (a 1:1 mixture of Ham's F-12 medium and Dulbecco's modified Eagle's medium, 100 units/ml penicillin, 100 µg/ml streptomycin sulfate) supplemented with 10% (v/v) FBS. SRD-4 and 25RA Chinese hamster ovary cells were maintained at 37 °C in an atmosphere of 8-9% CO2 in medium A supplemented with 5% FBS and 0.1 µg/ml of 25-hydroxycholesterol.
Buffers
PBS refers to phosphate-buffered saline, pH 7.4 (catalog number 21-040-CM, Mediatech). Buffer A contains 5 mM sodium phosphate, pH 7.5, and 250 mM sucrose. Buffer B contains 50 mM Hepes, pH 7.5, 125 mM KCl, 5 mM MgCl2, 2 mM EDTA, plus protease inhibitors (10 µg/ml leupeptin and 5 µg/ml pepstatin A). Buffer C contains 10 mM Hepes, pH 7.5, 10 mM KCl, and 1 mM MgCl2 plus protease inhibitors. Buffer D refers to 10 mM Hepes, pH 7.4, plus protease inhibitors. Buffer E contains 20 mM sodium citrate, pH 4.5, 125 mM KCl, 5 mM MgCl2, and 2 mM EDTA plus protease inhibitors. Buffer F contains 100 mM Tris-HCl, pH 7.5, or 20 mM sodium citrate, pH 4.5, 12.5 mg/ml fatty acid-free bovine serum albumin, and 100 µM oleoyl-CoA plus protease inhibitors.
Cell Fractionations
Protocol 1—Three confluent 10-cm dishes of J774 cells were scraped into PBS and centrifuged for 5 min at 1000 x g. Cells were resuspended in 2 ml of buffer C, incubated for 15 min on ice, homogenized using a bent 22-gauge needle attached to a 5-ml syringe, and centrifuged for 5 min at 1000 x g. The resulting post-nuclear supernatant was mixed with 4 ml of buffer B supplemented with 62% sucrose, overlaid with a 6-ml linear gradient of 15-35% sucrose in buffer B and centrifuged at 280,000 x g for 3.5 h at 4 °C in a Beckman SW 40Ti rotor.
Protocol 2—Following in vitro reactions with membrane-covered PVT beads, samples were supplemented with 250 mg of sucrose plus 100 µl of 1 M sodium carbonate, pH 11, and vortexed to dissolve the sucrose. The mixture was then overlaid successively with 0.5 ml of 20% sucrose and 0.5 ml of 5% sucrose and centrifuged at 87,000 x g for 30 min at 4 °C in a Beckman TLA 100.4 rotor. The bead-containing fraction was collected from the 20%/5% sucrose interface.
Protocol 3—J774 cells were harvested, and a post-nuclear supernatant was prepared as described under "Protocol 1." The post-nuclear supernatant was then subjected to 30% Percoll gradient centrifugation as described previously (21).
Enzyme Assays
The activities of 
-mannosidase II, -hexosaminidase (-hex) (22), 5'-nucleotidase (23), and NADPH-cytochrome c reductase (CCR) (24) were determined as described in the indicated references.
Preparation of Plasma Membrane-covered PVT Beads
Two confluent 10-cm dishes of J774 cells were incubated for 45 min on ice in the presence of 3 ml of PBS containing 5 mg/ml wheat germ agglutinin PVT beads. Cells were then washed 3 times with ice-cold PBS and scraped into 0.5 ml of ice-cold buffer D. Cells were vortexed for 15 s and disrupted for 10 s with a model W-375 Ultrasonic sonicator. Homogenates (1 ml) then received an equal volume of 60% sucrose in buffer B. The samples were overlaid with a 10-ml linear gradient of 0-30% sucrose in buffer B and centrifuged at 280,000 x g for 30 min in a Beckman SW 40Ti rotor. Plasma membrane acceptor beads were harvested from the gradient and quantified by an A600 measurement using bead suspensions of known concentrations for calibration. Protein and phospholipids were quantified as described below.
Preparation of Phospholipid-covered PVT Beads
150 µl of 7% (w/v) wheat germ agglutinin PVT beads in water were mixed with 300 µl of hexane. Phosphatidylcholine (105 µg) and phosphatidylserine (113 µg) were added and the organic solvent dried under a stream of nitrogen gas. In some cases cholesterol was added at concentrations indicated in the figure legends. Beads were washed three times with cold PBS and resuspended in 250 µl of cold buffer E. Beads were quantified as described above. Protein and phospholipid were measured as described below.
In Vitro Acidic Hydrolysis and Esterification Assays
Unless otherwise stated, each reaction contained, in a final volume of 1 ml 250 mM sucrose, 125 mM KCl, 5 mM MgCl2, 2 mM EDTA, 10 µg/ml leupeptin, 5 µg/ml pepstatin A, J774 endosomes (35-75 µg of protein), and 20 mM sodium citrate (pH 4.5 reactions) or 50 mM Hepes-KOH (pH 7.5 control reactions). Reactions were carried out in siliconized 1.5-ml microcentrifuge tubes and terminated by transfer of tubes to ice. Immediately thereafter, samples were extracted twice with chloroform: methanol (95:5) and subjected to thin layer chromatography.
ACAT Assay
One confluent 10-cm dish of J774 cells was incubated for 6 h in medium A supplemented with 10% LPDS, liposomes (containing [3H]cholesterol; 10 µCi/ml medium), 5 µg/ml 25-hydroxycholesterol, and 10 µM CI-976. Cells were then harvested, homogenized with a bent 22-gauge needle, and centrifuged at 1000 x g for 5 min. The resulting post-nuclear supernatant was centrifuged at 16,000 x g for 10 min. The membrane pellet was resuspended in 300 µl buffer F, pH 7.5, without oleoyl-CoA. ACAT activity was assayed in vitro as described (25).
Lipid and Protein Analyses
To prepare whole-cell lipid extracts, cells were washed with PBS and lysed in 0.5% Nonidet P-40 plus protease inhibitors. Lipids were extracted with chloroform:methanol (95:5) and analyzed by thin layer chromatography.
To quantify tritiated cholesteryl ester and triglycerides, lipids were separated on silica gel G plates (Analtech) using hexane:ethyl ether: acetic acid (70:30:1) as the mobile phase. Plates were calibrated with authentic standards, and lanes were split into 16 fractions and analyzed by liquid scintillation counting.
To quantify [3H]cholesterol and [3H]cholesteryl ester, lipids were separated on silica gel LK5D plates (Whatman, 150-Å pores) using hexane:ethyl acetate (90:10) as the mobile phase. Lanes were split into eight fractions, scraped, and analyzed by liquid scintillation counting. Total phospholipids were measured as described (26). Cholesterol was measured using an Amplex Red cholesterol assay kit (Molecular Probes). Protein was quantified using a bicinchoninic acid kit or with Coomassie Plus reagent (Pierce) using bovine serum albumin as a standard.
Immunoblot Analysis
Samples were separated by 12% SDS-PAGE, transferred to Hybond C-extra nitrocellulose (Amersham Biosciences) and blotted with anti-Rab5 antibody (1:3,000). Blots were then incubated with horseradish peroxidase-conjugated anti-mouse IgG (1:40,000; Jackson ImmunoResearch Laboratories), developed with Supersignal West Pico substrate (Pierce), and exposed to Kodak X-Omat Blue XB-1 film (PerkinElmer Life Sciences) at room temperature.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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15% (data not shown). After sucrose gradient centrifugation, the bulk of the remaining esters can be collected from a sharp band that co-fractionates with the endosomal marker Rab5, well separated from the plasma membrane marker 5'-nucleotidase (Fig. 1A). By analyzing the sedimentability of the soluble endosomal enzyme -hexosaminidase we estimated that more than 90% of endosomal vesicles obtained by this method were intact and remained so during 2-h incubations at 37 °C (data not shown). Plasma membrane was purified by attachment to lectin-conjugated latex beads and equilibrium centrifugation and contained only small amounts of other organelles (Table I).
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Increased Endosomal Cholesteryl Ester Hydrolysis in the Presence of Plasma Membrane—In previous studies with cultured cells it has been estimated that about 70% of cholesterol produced by endosomal hydrolysis of cholesteryl ester is transferred to the plasma membrane (28, 29). Using our cell-free system we asked, therefore, whether the presence of plasma membrane fractions affects the degree and the kinetics of cholesteryl ester hydrolysis. When labeled endosomes were incubated without acceptors for 2 h, [3H]cholesteryl oleate was reduced by 40% (Fig. 2A). Upon addition of increasing amounts of plasma membrane [3H]cholesteryl oleate levels further decreased in a dose-dependent manner. Similar results were obtained with beads that had been covered with artificially prepared phospholipid membranes whereas naked beads were without effect (data not shown). Time curves for hydrolysis are shown in Fig. 2, B and C. In the presence of plasma membrane, hydrolysis proceeded three times faster during the first 30 min of the reaction as compared with controls (Fig. 2B). After 2 h, 40% of [3H]cholesteryl oleate was hydrolyzed in the absence of acceptors, while 82% was hydrolyzed in their presence (Fig. 2C).
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In summary, the results in Fig. 2 demonstrate that supplementary membranes promote net hydrolysis of cholesteryl ester. Since cholesterol is only poorly soluble in water (30) and a large fraction is selectively transferred to acceptors (Fig. 1C) the simplest explanation for this effect is that membranes promote hydrolysis by removing cholesterol from the endosomal "equilibrium" of cholesteryl ester metabolism (31). As one corollary of this model, the ratio of cholesterol to phospholipids in endosomal donors would be expected to remain constant. To test this prediction, [3H]cholesteryl oleate-containing endosome fractions were incubated in the absence or presence of phospholipid-covered beads. Subsequently, the reaction mixtures were again separated to quantify [3H]cholesterol, [3H]cholesteryl ester, and phospholipids on donors and acceptors. Although the total amount of [3H]cholesterol produced was significantly higher in the presence than in the absence of acceptors (Fig. 2D), the [3H]cholesterol-to-phospholipid ratio in the two populations of donor membranes remained in fact the same (Fig. 2E). The cholesterol difference was found quantitatively in the acceptor fraction, supporting the notion that the degree of net cholesteryl ester hydrolysis depends on the transfer of cholesterol to phospholipid membranes.
Modulation of Net Cholesteryl Ester Hydrolysis by Membrane Cholesterol Concentration—Studies with purified lipids in vitro have shown that the capacity of phospholipid bilayers for cholesterol becomes saturated at about 50 mol %. Above that concentration, crystals of cholesterol monohydrate begin to form and cholesterol saturation is thought to be responsible for cholesterol precipitation in advanced atherosclerotic lesions (6). The plasma membrane fractions used in our experiments contained 30% cholesterol with respect to phospholipids (Table II), which explains why these membranes are still efficient at absorbing cholesterol (Fig. 1C). To determine whether the cholesterol concentration in acceptor membranes affects the endosomal metabolism of cholesteryl ester, [3H]cholesteryl oleate-containing endosomes were incubated with membranes containing varying concentrations of cholesterol. Compared with endosomes incubated alone (Fig. 3A, Reaction 2), addition of cholesterol-free membranes more than doubled the amount of [3H]cholesteryl ester hydrolyzed (Reaction 3). When membranes with increasing amounts of cholesterol were added, net hydrolysis decreased in a concentration-dependent manner (Reactions 4-6). At the saturating concentration of 50 mol % cholesterol (6), [3H]cholesteryl ester levels remained similar to those in samples without acceptors (compare Reactions 2 and 6). These results demonstrate that saturation with cholesterol renders membranes ineffective at stimulating endosomal cholesteryl ester hydrolysis in vitro.
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In Fig. 3B J774 cells were loaded with cholesterol by incubation with AcLDL and CI-976 as described above. The cells were then washed and received [3H]cholesteryl oleate in liposomes for 3 h at 15 °C. Preincubation with AcLDL had no effect on the uptake of liposomes (data not shown). But when cells were subsequently washed and moved to 37 °C, significantly more [3H]cholesteryl oleate was found in cells that had been pre-loaded with AcLDL as compared with controls (Fig. 3B). The increased cholesteryl ester levels found in response to AcLDL are unlikely to result from the action of ACAT as all incubations were performed in the presence of ACAT inhibitor. The drug effectively blocks ACAT both in cells and membrane extracts (Figs. 5 and 6, see below).
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Cholesteryl Ester Synthesis in Endosomes—The next set of experiments was designed to test whether cholesterol-loaded cells synthesize cholesteryl ester in endosomes and lysosomes. In Fig. 4 endosomes were prepared from J774 cells that had been grown with liposomes containing [3H]trioleoyl-glycerol and unlabeled cholesteryl oleate at 15 °C. Aliquots were then incubated in vitro at pH 4.5 in the presence of ACAT inhibitor. Similar to the results with living cells, no significant difference in [3H]trioleoylglycerol hydrolysis was detectable between samples incubated in the absence or presence of phospholipids (Fig. 4, A and B, upper panels) or plasma membrane (data not shown), confirming that the activity of aCEH remained unchanged.
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Neutral ACAT enzymes can be ruled out as catalysts in the esterification reactions as the experiments in Fig. 4, A and B, were performed at pH 4.5 in the presence of an ACAT inhibitor, conditions that effectively block microsomal cholesterol esterification activity even in extracts from cells that had been pretreated with 25-hydroxycholesterol (Fig. 5A), a potent activator of ACAT (10).
Contrary to results obtained with microsomes (25, 34-36), addition of 25-hydroxycholesterol to endosomes in vitro at pH 4.5 or pH 7.5 had no effect on cholesterol esterification (data not shown).
Endosomal triglyceride hydrolysis and cholesterol esterification exhibit a clear maximum at pH 4.5 (Fig. 5B). The peak at pH 4.5 for cholesteryl [3H]oleate synthesis can in part be explained by the fact that aCEH-catalyzed hydrolysis is a prerequisite for esterification. However, lowering the pH from 7.5 to 4.5 increased triglyceride hydrolysis 3-fold, while esterification was increased 22-fold, indicating that pH affects cholesteryl ester formation synergistically. Taken together, these results support the conclusion that cholesteryl ester synthesis during the in vitro reactions is catalyzed by an acidic endosomal enzyme.
In Fig. 4B endosomal cholesteryl ester synthesis was increased in the presence of membranes containing high concentrations of cholesterol. Since saturation of cellular membranes with cholesterol is a hallmark of macrophages in atherosclerotic lesions, we next performed a set of experiments to test whether cholesterol loading leads to lysosomal cholesteryl ester synthesis in vivo. First, J774 cells were grown for 3 h in the absence or presence of 50 µg/ml AcLDL and then received liposomes containing [3H]trioleoylglycerol and unlabeled cholesteryl oleate for another 4 h (Fig. 6A). In the absence of ACAT inhibitor, pretreatment with AcLDL increased cellular cholesteryl [3H]oleate levels about 2-fold. In the presence of AcLDL plus ACAT inhibitor, cholesteryl [3H]oleate levels were increased 40-fold compared with samples with ACAT inhibitor alone. Varying the AcLDL concentration indicated that ACAT-independent cholesteryl [3H]oleate synthesis was increased at AcLDL concentrations as low as 5 µg/ml (Fig. 6B).
To determine the subcellular localization of the cholesteryl [3H]oleate that accumulates in the presence of ACAT inhibitor, J774 cells were grown plus or minus AcLDL and then received liposomes containing [3H]trioleoylglycerol and cholesteryl oleate as described above. ACAT inhibitor was present throughout the experiment. Cell homogenates were then subjected to sucrose gradient centrifugation and fractions were analyzed for organelle markers and radiolabeled lipids. Due to the presence of the ACAT inhibitor, only minimal quantities of cholesteryl [3H]oleate were seen in the ER fractions under all conditions (Fig. 6C, upper panel). In lysosomes from cells grown with AcLDL, cholesteryl [3H]oleate levels were 14-fold higher compared with the same fraction from control samples.
The results in Fig. 6 show that elevated cholesterol levels can promote cholesteryl ester synthesis in lysosomes of living cells, which mirrors the results we obtained with endosomes and cholesterol-saturated membranes in vitro (Fig. 4B).
Inhibition of Cholesterol Transport Promotes Cholesteryl Ester Synthesis in Lysosomes in Vivo—The results in Figs. 3, 4, 5, 6 show that endosomes esterify cholesterol when phospholipid acceptors are absent or when available membranes are saturated with cholesterol. These data support the conclusion that endosomal cholesteryl ester metabolism is coupled to the transfer of cholesterol to phospholipids. As another verification of this concept in vivo, we tested whether cholesteryl ester levels are increased when cholesterol export from the endosomal system is blocked pharmacologically.
In Fig. 7A J774 macrophages were initially starved for cholesterol and then loaded with [3H]cholesteryl oleate plus unlabeled trioleoylglycerol at 15 °C to accumulate the esters in endosomes. The cells were then shifted to 37 °C in the absence or presence of the cholesterol transport inhibitor progesterone (37, 38). At 3-10 µg/ml progesterone, cellular [3H]cholesteryl ester levels were 50-75% higher as compared with controls (upper panel).
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Both experiments in Fig. 7 were performed in the absence of an ACAT inhibitor as several studies have shown that progesterone blocks transport of cholesterol to the ER (37). To further verify whether cholesteryl ester synthesis can occur in endosomes we performed a series of experiments that are shown in Fig. 8. First, J774 cells were allowed to endocytose [3H]trioleoylglycerol together with non-radioactive cholesteryl oleate for 4 h. Some cells also received CI-976, progesterone, U18666A, or imipramine. U18666A and imipramine, similar to progesterone, block cholesterol release from lysosomes (37, 39, 40). In the absence of cholesterol transport inhibitors, cholesteryl [3H]oleate was produced and this was largely blocked by the ACAT inhibitor CI-976 (Fig. 8A, upper panel). In the presence of progesterone, U18666A, or imipramine, however, cholesteryl [3H]oleate levels remained high even in the presence of CI-976.
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Inspection of [3H]triglycerides in Fig. 8, A and B, indicates that the cholesterol transport inhibitors did not interfere with aCEH activity per se (lower panels). Inhibition of cholesteryl ester synthesis, however, consistently led to a compensatory increase of [3H]triglyceride levels.
To determine the subcellular localization of the cholesteryl [3H]oleate that accumulates upon inhibition of cholesterol transport, J774 cells were incubated with [3H]trioleoylglycerol and non-radioactive cholesteryl oleate plus or minus U18666A and ACAT inhibitor as described above. Cells were then fractionated by density gradient centrifugation to separate lysosomes from ER and cytosolic lipid droplets. In homogenates from control-treated cells, most of the cholesteryl [3H]oleate was found in cytosolic lipid droplets and a sizable amount was found in the ER (Fig. 8C, upper panel). In the presence of ACAT inhibitor, accumulation of cholesteryl [3H]oleate in lipid droplets and in the ER was blocked as expected (10, 44). Addition of U18666A also prevented accumulation of cholesteryl [3H]oleate in lipid droplets and in the ER but led to a 10-18-fold increase of cholesteryl [3H]oleate levels in lysosomes. In summary, the data in Figs. 7 and 8 demonstrate that pharmacological inhibition of cholesterol transport leads to ACAT-independent cholesterol esterification in lysosomes.
In Fig. 9 we asked whether lysosomal accumulation of cholesteryl ester in response to inhibition of cholesterol transport is reversible. J774 cells were allowed to accumulate cholesteryl [3H]oleate in lysosomes after growth with non-radioactive cholesteryl oleate, [3H]trioleoylglycerol, ACAT inhibitor, and imipramine. When imipramine was subsequently washed out, hydrolysis resumed and cholesteryl [3H]oleate levels decreased by 96% after 4 h (Fig. 9, upper panel). In cells that continued to grow in the presence of imipramine, cholesteryl [3H]oleate levels remained high. [3H]Triglycerides decreased with similar kinetics with or without imipramine, indicating that the intrinsic activity of aCEH remained unchanged (Fig. 9, lower panel). In addition to demonstrating that cholesteryl [3H]oleate accumulation in response to imipramine is reversible, these results provide an illustration for how cholesterol release from lysosomes promotes net hydrolysis of cholesteryl ester.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In support of this scheme, we found that isolated endosomes hydrolyzed cholesteryl ester only to a limited extent, whereas addition of cholesterol-poor membranes allowed selective cholesterol release and promoted cholesteryl ester reduction (Figs. 1 and 2). Equivalent results were obtained in living cells; upon pharmacological inhibition of cholesterol export from lysosomes, cholesteryl ester levels remained high but then declined when the transport block was removed (Figs. 7 and 9).
Our studies indicate that, under physiological conditions, net hydrolysis of cholesteryl ester is determined by the cholesterol concentration in acceptor membranes. Overloading cells with cholesterol in the absence of serum and in the presence of an ACAT inhibitor reduced net hydrolysis of cholesteryl ester. Similarly, experiments with cell-free extracts demonstrated that endosomal cholesteryl ester levels remain high in the presence of membranes already saturated with cholesterol (Fig. 3).
A number of papers reported increased cell death in macrophages exposed to modified lipoproteins for prolonged periods of time (45-47). Under the conditions used here, however, no increased apoptosis was detected as judged by caspase activation (data not shown).
The conclusion that endosomes esterify cholesterol under conditions of limited cholesterol release is based on experiments in which cells were loaded with non-radioactive cholesteryl oleate plus [3H]trioleoylglycerol. If cholesteryl oleate was re-synthesized in endosomes, we reasoned, hydrolysis of [3H]trioleoylglycerol should cause the occasional incorporation of a tritiated fatty acid molecule to produce cholesteryl [3H]oleate. Cholesteryl [3H]oleate was in fact produced in endosomes in vitro when net cholesterol release was limited due to lack of acceptors or because acceptor membranes were saturated with cholesterol (Figs. 4 and 5). Saturation of membranes with cholesterol also led to a build-up of lysosomal cholesteryl [3H]oleate in vivo (Fig. 6). Whether cholesteryl ester sequestration under these conditions results from changes in cholesterol trafficking or whether the lysosomal cholesterol pool remains in dynamic equilibrium with other membranes remains to be determined.
Increased amounts of cholesteryl [3H]oleate were synthesized in lysosomes of cells that had been grown in the presence of drugs that block the export of cholesterol from lysosomes, supporting the notion that endosomal cholesteryl ester metabolism is coupled to cholesterol release (Figs. 7, 8, 9). The current results on the effects of progesterone, U18666A, and imipramine might seem paradoxical in light of the fact that these drugs were described as cholesterol transport inhibitors based in part on their ability to cause the accumulation of unesterified cholesterol in lysosomes (37). In these studies, cells were exposed to inhibitors for one to several days, while our experiments were terminated after 4 h. We noted that the synthesis of cholesteryl [3H]oleate in the presence of cholesterol transport inhibitors started to diminish after about 8 h (data not shown), possibly because endosomes start to accumulate phospholipids, increasing their capacity for unesterified cholesterol.
The identity of the enzyme that is responsible for the endosomal acidic esterification activity we observed is currently unknown. The most well understood cellular cholesterol-esterifying enzyme is ACAT, which uses cholesterol and acyl-CoA as substrates. Two main isoforms have been described, ACAT1 and ACAT2 (34, 48). In addition, a novel splice variant of human ACAT1 has recently been described whose biochemical characteristics have not yet been reported (49). However, several lines of evidence support the conclusion that the cholesteryl [3H]oleate synthesis we observed under the above-described conditions was not catalyzed by canonical ACAT. (i) In living cells, synthesis continued in ACAT-deficient cells and in the presence of an ACAT inhibitor (Figs. 6 and 8); (ii) the pH optimum for cholesteryl [3H]oleate synthesis by endosomal fractions in vitro was 4.5 (Fig. 5), whereas the pH optimum for ACATs is above neutral (50); (iii) cholesteryl [3H]oleate produced in vivo co-fractionated with endosomal/lysosomal vesicles (Figs. 6 and 8), whereas ACAT resides predominantly in the ER and its products are released either into the cytoplasm or the ER lumen (44, 51-53); (iv) unlike ACAT (25, 34-36), the acidic esterification activity was not stimulated by 25-hydroxy-cholesterol (data not shown).
The only acidic cholesterol-esterifying enzyme that has been described is aCEH, which in reversal of its well known hydrolytic activity can synthesize cholesteryl ester from cholesterol and fatty acids (14). Previous studies with aCEH-deficient cell extracts have indicated that aCEH is the only acidic cholesterol-esterifying enzyme in human fibroblasts (54). However, based on the current data, the existence of another acidic cholesterol-esterifying enzyme in macrophages or other cell types cannot be ruled out.
Effectively, the dependence of endosomal cholesteryl ester hydrolysis on the cellular cholesterol content can be regarded as another safeguard protecting cells against the potentially toxic effects of excess lipoprotein-derived cholesterol (47). In the sequence of feedback responses that follows the cellular uptake of cholesteryl ester, endosomal cholesteryl ester retention is an early effect that may diminish over time as other mechanisms of cholesterol reduction take over (10, 55, 56). However, cholesteryl ester does persist in lysosomes under certain conditions such as in atherosclerotic foam cells containing large quantities of cholesterol. Based on the current results, we propose that atheromatous cells accumulate cholesteryl ester in lysosomes due to local re-esterification of cholesterol.
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FOOTNOTES |
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Supported by a fellowship from the China Scholarship Council.
These authors contributed equally.
¶ Supported by a Harvard Graduate School of Arts and Sciences Prize fellowship, the Ford Foundation, and through a Merck-Wiley award.
|| To whom correspondence should be addressed: The Biological Laboratories, Dept. of Molecular and Cellular Biology, Harvard University, 16 Divinity Ave., Cambridge, MA 02138. Tel.: 617-384-5846; Fax: 617-384-7423; E-mail: axno{at}mcb.harvard.edu.
1 The abbreviations used are: ER, endoplasmic reticulum; ACAT, acyl-coenzyme A:cholesterol acyltransferase; aCEH, acidic cholesteryl ester hydrolase; LDL, low density lipoprotein; AcLDL, acetylated LDL; LPDS, lipoprotein-deficient serum; FBS, fetal bovine serum; PBS, phosphate-buffered saline; PVT, polyvinyltoluene; -hex, -hexosaminidase; CCR, cytochrome c reductase.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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