Hormone-Sensitive Lipase Overexpression Increases Cholesteryl Ester Hydrolysis in Macrophage Foam Cells

From the Lipid Research Laboratory, West Los Angeles VA Medical Center, and the Department of Medicine, University of California, Los Angeles, Calif.

Correspondence to Michael C. Schotz, Lipid Research Laboratory, Building 113, Room 312, West Los Angeles VA Medical Center, Los Angeles, CA 90073. E-mail schotz1{at}ucla.edu

	Abstract

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Abstract—Atherosclerosis is a complex physiopathologic process initiated by the formation of cholesterol-rich lesions in the arterial wall. Macrophages play a crucial role in this process because they accumulate large amounts of cholesterol esters (CEs) to form the foam cells that initiate the formation of the lesion and participate actively in the development of the lesion. Therefore, prevention or reversal of CE accumulation in macrophage foam cells could result in protection from multiple pathological effects. In this report, we show that the CE hydrolysis catalyzed by neutral cholesterol ester hydrolase (nCEH) can be modulated by overexpression of hormone-sensitive lipase (HSL) in macrophage foam cells. For these studies, RAW 264.7 cells, a murine macrophage cell line, were found to be a suitable model of foam cell formation. HSL expression and nCEH activity in these cells and in peritoneal macrophages were comparable. In addition, antibody titration showed that essentially all nCEH activity in murine macrophages was accounted for by HSL. To examine the effect of HSL overexpression on foam cell formation, RAW 264.7 cells were stably transfected with a rat HSL cDNA. The resulting HSL overexpression increased hydrolysis of cellular CEs 2- to 3-fold in lipid-laden cells in the presence of an acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitor. Furthermore, addition of cAMP produced a 5-fold higher rate of CE hydrolysis in cholesterol-laden, HSL-overexpressing cells than in control cells and resulted in nearly complete hydrolysis of cellular CEs in only 9 hours, compared with <50% hydrolysis in control cells. Thus, HSL overexpression stimulated the net hydrolysis of CEs, leading to faster hydrolysis of lipid deposits in model foam cells. These data suggest that HSL overexpression in macrophages, alone or in combination with ACAT inhibitors, may constitute a useful therapeutic approach for impeding CE accumulation in macrophages in vivo.

Key Words: hormone-sensitive lipase • foam cells • cholesteryl ester hydrolysis

	Introduction

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Monocytes/macrophages play a central role in the development of atherosclerosis, specifically in the initial accumulation of cholesterol in the arterial wall intima as well as in the progression of lesions to advanced plaques.¹ The appearance of large amounts of cholesterol stored as CEs in cytoplasmic lipid droplets accounts for the description of these macrophages as foam cells.^{2 3} Foam cells can also affect the stability of a plaque, potentially leading to its rupture and the threat of coronary thrombosis.^{4 5} Thus, the ability to alter the formation and the development of macrophage foam cells could be beneficial in mitigating their multiple pathological effects.

The accumulation of exogenous cholesterol initially involves the nonregulated uptake of modified plasma lipoproteins by macrophage-specific SR.¹ The level of intracellular CE is subsequently governed by a balance between its synthesis and degradation. In mouse peritoneal macrophages, the free cholesterol derived from lysosomal catabolism of lipoproteins is trapped in a continual cycle of esterification catalyzed by the enzyme ACAT and its subsequent release catalyzed by an nCEH. In the absence of an extracellular acceptor, free cholesterol is readily reesterified, leading to cytoplasmic deposits of CEs.^{6 7 8} Mechanisms to decrease the accumulation of CEs in foam cells could include inhibition of CE synthesis or stimulation of CE hydrolysis. Although it has been shown that the rate of CE hydrolysis cannot be modified under a variety of conditions, inhibition of ACAT activity with progesterone was shown to promote the net hydrolysis of CEs from lipid-laden mouse peritoneal macrophages. Moreover, the use of an extracellular acceptor of free cholesterol, eg, HDL, also enhanced the net hydrolysis of CEs by stimulating the efflux of free cholesterol from these cells.⁸

Investigations by other groups^{9 10} have attributed the CE cycle hydrolase activity in mouse macrophage cell lines to HSL. In mammals, HSL is a key enzyme in lipid metabolism, catalyzing the rate-limiting step in the hydrolysis of stored TGs in adipose tissue.¹¹ In addition, HSL can hydrolyze CEs to the same extent as TGs,¹¹ which may account for HSL's role in the metabolism of cholesterol in steroidogenic tissue as well.^{12 13 14 15 16 17} Furthermore, HSL expression^{18 19} and its regulation²⁰ by cholesterol accumulation in J774.2 mouse macrophages has been reported. Finally, our laboratory has isolated and characterized the cDNA²¹ and the gene for HSL²² and documented HSL expression in primary macrophages from both mice²³ and humans.²⁴

The current studies were conducted to investigate the role of HSL in cholesterol metabolism of macrophage foam cells. We used the RAW 264.7 murine macrophage cell line, which expresses SR^{25 26} and can be loaded with CEs by incubation with modified lipoproteins (Reference 27²⁷ and vide infra). In addition, RAW 264.7 cells are unusual among macrophage cell lines in that stable transfection with recombinant DNA molecules can be achieved and utilized to alter expression levels of key proteins involved in cholesterol metabolism (References 27 and 28^{27 28} and vide infra). We have noted that CE turnover in RAW 264.7 cells is similar to that previously observed in lipid-laden peritoneal macrophages.⁸ In this study, we also show that HSL expression and nCEH activity levels in both types of cells are similar. Furthermore, antibody titration experiments indicate that essentially all of the nCEH activity in both cell types is accounted for by HSL. Finally, we demonstrate that increased expression levels of HSL in macrophage foam cells produced by transfection of RAW 264.7 cells with an HSL cDNA result in increased nCEH activity and net hydrolysis of cellular CEs.

	Methods

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nCEH Assay
Whole-cell extracts were prepared from cells washed three times in cold PBS and suspended at 2.0x10⁷ cells per mL in cold buffer containing 25 mmol/L Tris HCl, pH 7.4, 1 mmol/L EDTA, and 20% glycerol, followed by sonication on ice twice for 10 seconds with a Branson Sonifier microtip at the lowest power setting. Protein content was determined with the bicinchoninic acid reaction in the presence of 2% SDS,²⁹ with BSA as the standard. The 0.2-mL nCEH reaction contained 60 mmol/L potassium phosphate, pH 7.0; 0.5 mmol/L EDTA; 0.5 mmol/L DTT; 25 mg/mL fatty acid–free BSA (Bayer); 70 nmol cholesteryl [1-¹⁴C]oleate (6000 counts per minute/nmol); 175 µg/mL phosphatidylcholine/phosphatidylinositol (3:1, wt/wt); and freshly sonicated cell extract. After 20 minutes at 37°C, the [¹⁴C]oleate released was extracted and assayed by liquid scintillation counting.³⁰ One unit of enzyme hydrolyzes 1 µmol of CE in 1 minute. The amount of sonicate used (5 µg protein) was in the linear range of the assay and introduced very little (<5%) unlabeled substrate from cholesterol-laden cells.

Antibody Titration of nCEH Activity
Mouse peritoneal macrophages were elicited with sterile thioglycollate (28.9 g/dL, Sigma) and harvested from three C57BL/6J adult females, and resuspended in the medium described in the "Cell Culture and Transfection" paragraph plus amphotericin B (0.25 µg/dL). Cultures were seeded at 2x10⁷ cells per 60-mm plate and incubated for 24 hours at 37°C in 5% CO₂.

Enzyme extracts containing 8 to 40 µg protein prepared from wild-type RAW 264.7 cells (40 µg), 5X-HSL transfectants (8 µg), and mouse C57BL/6J peritoneal macrophages (20 µg) were preincubated in 0.1 mL with 0 to 160 µg chicken anti-rat HSL or nonimmune IgG at 37°C for 1 hour. To assay for the remaining nCEH activity, the reaction volume was brought to 0.2 mL.

Plasmid Construction
The pAL1 vector was kindly supplied by Dr C.K. Glass (University of California, San Diego). pAL1 contains 697 bp of the human SR promoter sequence linked to a 4.4-kb upstream enhancer fragment,³¹ a 48-bp polylinker, and 961 bp of part of exon 3 through exon 5 and the poly(A) tail of the human growth hormone gene, cloned into the Bluescript II SK(-) vector sites, XhoI and NotI. To produce a macrophage-specific HSL expression plasmid, a fragment containing the entire rat HSL cDNA plus 100 bp of upstream and 342 bp of 3'-untranslated sequences was isolated by digesting full-length rat HSL cDNA (American Type Culture Collection, Rockville, Md; 87191)³² with AvrII and EcoRV. Ends were filled with the Klenow fragment of DNA polymerase I (Promega), and the cDNA was subcloned into the EcoRV site of pAL1. The resulting plasmid, pAL1-HSL, contained the rat HSL cDNA in a sense orientation downstream from the SR promoter/enhancer (see Figure 2).

View larger version (18K): [in this window] [in a new window]   Figure 2. Macrophage-specific rat HSL expression construct. a, pAL1 XhoI/NotI fragment used as a control for RAW 264.7 cell transfection (see "Methods"). Arrow indicates site of transcription initiation of the hSR gene. b, pAL1-HSL XhoI/NotI fragment used for stable overexpression of HSL in RAW 264.7 cells. Initiation (ATG) and termination (TGA) codons in the cDNA and cloning sites (A2, AvrII; E5, EcoRV) are indicated. Nucleotide numbers shown are based on human SR gene31 and rat HSL cDNA.32 hSR indicates human SR; enh, enhancer; MCS, multiple cloning site; and hGH, human growth hormone.

Cell Culture and Transfection
The RAW 264.7 murine macrophage cell line (ATCC; TIB-71) was grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 1 mmol/L sodium pyruvate, 0.1 mmol/L nonessential amino acids, 2 mmol/L L-glutamine, 50 U/mL penicillin, and 50 µg/mL streptomycin. For transfection, the Bluescript II SK(-) vector sequences of pAL1-HSL were removed by digestion with XhoI and NotI (pAL1-HSL XhoI/NotI fragment; see Figure 2). Cotransfection of 5x10⁶ RAW cells was performed by electroporation (Bio-Rad Gene Pulser) in cell culture medium with 6 µg of the pAL1-HSL XhoI/NotI fragment plus 5 µg of pcDNA3 carrying the neomycin resistance gene (Invitrogen) linearized with SacII, at 300 V and 960 µF (=60 ms). Control transfection used the same molar equivalent (4.3 µg) of pAL1 XhoI/NotI (see Figure 2) plus 5 µg of pcDNA3/SacII. After electroporation, the cells were left on ice for 10 minutes before they were plated at 3x10⁵ cells/100-mm tissue culture dish and incubated at 37°C in 5% CO₂. After 60 hours, G418 (GIBCO-BRL) selection (400 µg/mL) was applied for 12 days.

PCR Detection of Transgenic Clones
Stable clones were expanded and analyzed by PCR. To detect integration of the control pAL1 fragment, the forward (5'-AGGGGAATCAAGGACCATCCTGAC-3') and reverse (5'-TGAATTCGATAATTCCTGCAGCCC-3') primers were derived from the SR promoter sequence. To detect integration of the pAL1-HSL fragment, the same forward primer was used in combination with a reverse primer corresponding to the rat HSL cDNA upstream untranslated sequence (nucleotides 589 to 566; 5'-GAAGATGGGAAGGTCTGGGATTAC-3'). PCR amplification was carried out in a PTC-100 thermal cycler (MJ Research) and a hot-start "touchdown" protocol.³³ The annealing temperature was decreased from 65°C by 0.5°C at each cycle for 20 cycles and maintained at 55°C for the final 10 cycles. PCR reactions were analyzed by electrophoresis on 1% agarose gels.

RT-PCR
cDNA was synthesized from 10 µg total RNA isolated with TRIzol (GIBCO-BRL) and AMV reverse transcriptase and an oligo(dT) primer (cDNA cycle kit, Invitrogen). PCR amplification was performed as described above by using 1/20th of the cDNA. Primers for HSL (forward, 5'-CTCCTCATGGCTCAACTCCTTCC-3'; reverse, 5'-AGGGGTTCTTGACTATGGGTG-3') and ß-actin (forward, 5'-AGAGATGGCCACGGCTGCTT-3'; reverse, 5'-ATTTGCGGTGGACGATGAG-3') were designed to span an intron so that products resulting from amplification of cDNA sequences (433 bp for HSL, 426 bp for ß-actin) could be distinguished from products that would arise from potential genomic DNA contamination.²⁴ ß-Actin amplification served as a control for equivalent cDNA synthesis in both control and 5X-HSL cells. PCR products from 31- (HSL) or 36- (ß-actin) cycle amplifications were analyzed by agarose gel electrophoresis.

Western Blotting
Sonicated control or 5X-HSL whole-cell extracts containing 100 µg or less total protein were centrifuged at 16 000g for 15 minutes at 4°C. Supernatants were denatured with SDS and ß-mercaptoethanol (2% final concentration each), boiled for 2 minutes, and electrophoresed on 9% polyacrylamide gels containing 0.1% SDS. Fifty nanograms of pure recombinant rat HSL (kindly supplied by Cecilia Holm, Lund University, Lund, Sweden) and Rainbow protein molecular weight markers (Amersham) served as standards. Proteins were electrotransferred onto Hybond polyvinylidine difluoride membranes (Amersham) with a buffer consisting of 10 mmol/L 3-cyclohexylamino-1-propanesulfonic acid (Sigma Chemical Co) in 10% (vol/vol) methanol. Blots were blocked in 20 mmol/L Tris HCl, pH 7.5; 500 mmol/L NaCl; and 3% BSA (fraction V, Sigma) for 4 to 5 hours at room temperature with gentle shaking. Antibody probing was performed in fresh blocking buffer with rabbit anti-HSL IgG (1:3000 dilution) as the primary antibody (antiserum kindly provided by F.B. Kraemer, Stanford University, Stanford, Calif; also see Reference 15¹⁵ ), or rabbit nonimmune IgG (1:1000 dilution) and biotin-conjugated goat anti-rabbit IgG (Pierce Chemical Co, 1:5000 dilution) as the secondary antibody. Membrane washes were performed in 100 mmol/L Tris HCl, pH 7.5; 10 mmol/L EDTA, pH 8.0; 5% Triton X-100; 5% N-lauroylsarcosine; and 0.5% SDS. The blots were developed with enhanced chemiluminescence (Pierce) and exposed to Hyperfilm-ECL film (Amersham). Only the anti-HSL IgG recognized 84-kDa HSL in cell extracts as well as recombinant HSL (open arrow in Figure 3b). Proteins other than HSL were also exhibited with nonimmune IgG.

View larger version (35K): [in this window] [in a new window]   Figure 3. nCEH activity and HSL protein expression in 5X-HSL cells. a, nCEH activity in transfected RAW 264.7 cells and titration with HSL antibody. Specific activities for control cells and the HSL-overexpressing clone (5X-HSL) are shown. Control average corresponds to 0.5 mU/mg, the same as the level in wild-type RAW 264.7 cells and C57BL/6J peritoneal macrophages. nCEH activities in control (data not shown) and 5X-HSL extracts were titrated with anti-HSL IgG, giving similar results. 5X-HSL activity was measured after incubation with anti-HSL antibody for 1 hour (100% activity represented 700 cpm above background). Incubation with equal amounts of nonimmune IgG (x) showed no significant changes in activity. b, Western blot analysis of 100 µg total protein from control (Con) and 5X-HSL cell extracts was done as described in "Methods." HSL protein (open arrow) overexpression in 5X-HSL cells was estimated by diluting the 5X-HSL extract 1:5, 1:10, and 1:20. Fifty nanograms of pure rat HSL (84 kDa) and protein molecular weight markers served as standards. As described in "Methods," bands above and below HSL were not recognized by anti-HSL IgG specifically, as they were also observed with nonimmune IgG. Blot is representative of six different experiments.

Cholesterol Accumulation and Turnover Studies
Foam cells were produced by incubation of control or 5X-HSL cells at 1.2x10⁶ per well in 12-well tissue culture plates with 0.5 mL of 150 µg/mL AcLDL (as prepared according to the method of Basu et al³⁴ ), 0.2 mmol/L oleate-BSA and 2 mg/mL BSA in Dulbecco's modified Eagle's medium at 37°C. At 24 or 48 hours, duplicate cultures were washed three times with Dulbecco's modified Eagle's medium and then PBS, followed by lipid (for colorimetric assay of cholesterol) extraction, protein extraction, or whole-cell extraction for nCEH assays. Cells loaded for 48 hours received fresh medium at 24 hours.

CE turnover studies were performed as described by Brown et al.⁸ The rate of CE hydrolysis was measured by a pulse/chase of cholesteryl oleate. Cells were loaded with cholesterol for 24 hours as described above, except that [³H]oleate-BSA (180 000 disintegrations per minute/nmol) was used. To make accurate turnover measurements, steady-state levels of CE were necessary. Thus, cholesterol and oleic acid pools were equilibrated by further incubation for 24 hours without lipoprotein and with 1 mg/mL BSA and 0.2 mmol/L [³H]oleate-BSA. At the end of the equilibration (0 hours), [³H]oleate-BSA was removed, and chase measurements were begun with sampling of the cultures by washing as described above, followed by lipid (for colorimetric and chromatographic assays) and protein extraction of each. An ACAT inhibitor, 20 µg/mL progesterone (Sigma) or 10 µmol/L CI-976 (provided by Dr Roger Newton at Parke-Davis; also see Reference 35³⁵ ) and where indicated, 1 mmol/L dibutyryl cAMP (Sigma), were added in the equilibration medium to the remaining cultures at 0 hours. At subsequent indicated times, the cultures were washed with PBS and extracted for both lipid and protein. The rate of CE resynthesis was measured in the absence of the ACAT inhibitor by labeling with [¹⁴C]oleate-BSA (3000 dpm/nmol) added at 0 hours. Radiolabeled cholesteryl oleate was isolated from lipid extracts and quantified as described below.

Cholesterol Determination
The cholesterol content of control and 5X-HSL foam cells was analyzed by extracting cellular lipids⁸ in the culture well with 0.5 mL hexane/isopropanol (3:2, vol/vol) at room temperature for 30 minutes, followed by a wash with another 0.5 mL of solvent. Lipid extracts were dried under N₂ and resuspended in 0.2 mL isopropanol. Protein in the lipid-free wells was extracted with 0.5 mL of 0.2N NaOH for 15 minutes at room temperature and measured as described above in the nCEH assay. Cholesterol was measured by using an adaptation of an enzymatic colorimetric assay.³⁶ The 1-mL reaction contained 50 mmol/L PIPES, pH 6.9, 200 mmol/L KCl, 3 mmol/L sodium cholate, 0.1% Triton X-100, 5 mmol/L of 4-aminoantipyrine, 3 mmol/L 2-hydroxy-3,5-dichlorobenzenesulfonic acid, 0.2 U cholesterol oxidase, and 10 U horseradish peroxidase with or without 0.2 U cholesterol ester hydrolase to measure total or free cholesterol levels, respectively, and CEs by subtraction. Absorbance at 515 nm was read after incubation at 37°C for 15 minutes.

The rates of cholesteryl oleate hydrolysis and resynthesis in cholesterol-laden control and 5X-HSL cells were determined by radioisotopic labeling in the cholesteryl oleate turnover protocol described above. Aliquots of isopropanol suspensions of extracted lipid along with 13 µg unlabeled cholesteryl oleate carrier (Sigma) were applied to silica thin-layer chromatography plates (Sigma) with CE and TG standards and resolved with hexane/ethyl ether/acetic acid (80:20:1, vol/vol/vol). TG and CEs were well separated and were visualized by iodine staining. The CEs were collected and measured by liquid scintillation counting. The analysis procedure accounted for sample recovery and isotope crossover in double-label counting.

	Results

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HSL Activity in Mouse Peritoneal Macrophages and the RAW 264.7 Murine Macrophage Cell Line
It has been established that primary macrophages isolated from mouse peritoneum exhibit nCEH activity and express HSL mRNA.²³ We have now determined that the RAW mouse macrophage cell line exhibits levels of nCEH activity similar to those seen in peritoneal macrophages measured in parallel (0.5 mU/mg protein). Furthermore, titration of the nCEH activity with HSL-specific IgG indicated that nCEH activity was nearly completely inhibited in both peritoneal macrophages isolated from C57BL/6 mice and RAW 264.7 cells (Figure 1). Analysis of Western blots prepared with peritoneal macrophage and RAW 264.7 cell homogenates also revealed comparable levels of HSL protein (data not shown). These results suggest that HSL accounts for essentially all of the nCEH activity in both primary mouse macrophages and in the RAW 264.7 cell line.

View larger version (18K): [in this window] [in a new window]   Figure 1. nCEH activity in murine macrophages and titration with HSL antibody. Titration of nCEH activity in wild-type RAW 264.7 cell and C57BL/6J peritoneal macrophage extracts. Both extracts had specific activities of 0.5 mU/mg protein and were essentially completely inhibited by incubation with anti-HSL IgG for 1 hour. Intact (100%) nCEH activity for RAW 264.7 extract was the release of 763 cpm above background of [14C]oleate, whereas 100% for C57BL/6J peritoneal macrophages was 442 cpm. Nonimmune chicken IgG had little effect on either extract; each retained 90% activity with 160 µg IgG.

Increased HSL Expression and nCEH Activity in Stably Transfected RAW 264.7 Cells and Inhibition by HSL Antibody
To determine the role of HSL in foam cell development, we transfected the mouse macrophage cell line RAW 264.7 with a rat HSL cDNA. Expression was placed under the control of human SR promoter and enhancer sequences (Figure 2), which have previously been shown to direct gene expression in macrophages both in vitro and in atherosclerotic lesions in vivo.³⁷ Construction of the macrophage-specific HSL expression plasmid pAL1-HSL is described in "Methods." The pAL1-HSL XhoI/NotI fragment (Figure 2b) was coelectroporated with the neomycin resistance vector pcDNA3 into RAW 264.7 cells. In parallel, control transfections were carried out with the pAL1 XhoI/NotI fragment (Figure 2a) and pcDNA3. After 12 days of selection in the presence of G418, 28 pAL1-HSL and 10 pAL1 control colonies were picked, expanded, and screened by PCR. All pAL1 and 18 pAL1-HSL clones were positive for the respective transgenes.

Cell extracts of seven pAL1-HSL–positive clones exhibited 2 to 5 times more nCEH activity than did pAL1 control clones, which had the same specific activity as wild-type RAW cells. Expression of transgenic HSL mRNA by these positive clones was verified by RT-PCR (data not shown). The elevated nCEH activity in the pAL1-HSL clone (referred to as 5X-HSL) exhibiting a 5-fold increase was titrated with HSL-specific IgG. As shown in Figure 3a, essentially all of the activity could be attributed to HSL. The 5X-HSL clone expressed 10- to 20-fold more HSL protein than did pAL1 control clones (Figure 3b), which contained the same level of HSL as did wild-type RAW 264.7 cells and mouse peritoneal macrophages (data not shown). In addition identical patterns and amounts of other proteins were detected by the antibody on Western blots of all four extracts (data not shown). However, as described in "Methods," these other proteins were also recognized by nonimmune IgG and did not exhibit nCEH activity (Figure 3b).

Conversion of RAW 264.7 Cells and 5X-HSL Transfectants Into Foam Cells and Effect on HSL Activity
The control and 5X-HSL clones formed foam cells when continuously cultured with AcLDL in lipoprotein-deficient serum without an extracellular acceptor to mediate cholesterol efflux as described in "Methods." Figure 4 shows the total accumulation of cholesterol in control and 5X-HSL cells that resulted from the sum of endogenous cholesterol, uptake of AcLDL, CE hydrolysis, and cholesterol reesterification. After 24 hours, >50% of the cholesterol was in the form of CE, thereby making these cells a suitable model for foam cell formation. Cells incubated without AcLDL contained no CE and 50 nmol free cholesterol per mg protein. Interestingly, although they were challenged with the continuous uptake of AcLDL and lack of extracellular cholesterol acceptors, the HSL-overexpressing cells managed to accumulate 38.4% (33.9% to 42.9%; average and range from two experiments) less CEs and 28.0% (27.7% to 28.3%) less total cholesterol but had the same level of free cholesterol as control cells (Figure 4).

View larger version (32K): [in this window] [in a new window]   Figure 4. Cholesterol accumulation by control and 5X-HSL cells. Cells were loaded with cholesterol by incubation with 150 µg/mL AcLDL for 24 hours. Levels of intracellular free cholesterol, CEs, and total cholesterol were determined enzymatically as described in "Methods." Means and ranges of duplicate cultures from a representative experiment are presented.

Decreases in HSL protein and enzyme activity have been associated with sterol accumulation in murine macrophage J774.2 cells.²⁰ In two different experiments, cholesterol accumulation in control RAW 264.7 cells for 24 or 48 hours also resulted in decreases in endogenous nCEH activity of 28% (23% to 32%; average and range) and 46% (38% to 53%), respectively. In contrast, the decrease in 5X-HSL cells was only 18% (17% to 19%) at 24 and 16% (15% to 17%) at 48 hours, indicating that expression of the endogenous HSL gene, but not the transfected heterologous HSL, was diminished as a result of cholesterol accumulation. It has been previously demonstrated that expression driven by SR gene regulatory elements utilized in the pAL1-HSL construct is not downregulated by cellular cholesterol accumulation.³⁷

Increased CE Hydrolysis in 5X-HSL Cells
CE turnover in RAW 264.7 cells was investigated by following the procedure previously applied to peritoneal macrophages using double-labeled oleic acid.⁸ In brief, foam cells were obtained by incubating control RAW 264.7 cells and 5X-HSL transfectants with AcLDL and [³H]oleate for 24 hours, followed by a 24-hour equilibration period without lipoprotein. At 0 hours [³H]oleate was removed, further CE synthesis was prevented by an ACAT inhibitor, and hydrolysis was determined by assaying the [³H]cholesteryl oleate remaining at subsequent times. The rate of CE resynthesis was measured in the absence of the ACAT inhibitor by adding [¹⁴C]oleate at 0 hours and assaying the [¹⁴C]cholesteryl oleate at subsequent times. In control cells (data not shown), rates of cholesteryl oleate hydrolysis and resynthesis were similar to those reported for peritoneal macrophages.⁸ In the 5X-HSL cells the rate of cholesteryl oleate resynthesis was increased 2-fold during the initial period of measurement (data not shown). However, inhibition of ACAT with progesterone⁸ revealed a significantly higher absolute rate of cholesteryl oleate hydrolysis in the 5X-HSL cells than in the control cells; logarithmic transformation of the data gave a slope 2.4 times higher for the 5X-HSL cells (Figure 5). Similar results were obtained when another ACAT inhibitor, CI-976, was used: in three independent experiments, 5X-HSL cells had a rate of hydrolysis 2.0±0.1 times (mean±SEM) higher than did control cells. Addition of human HDL (250 µg/mL at 0 hours) had little effect (<50% increase), which is similar to a finding reported for mouse peritoneal macrophages.⁸ Thus, when cholesterol esterification is prevented, increased HSL expression produces a 2- to 3-fold increase in CE hydrolysis in lipid-laden cells. In addition, HSL overexpression increased the net hydrolysis of cholesteryl oleate, resulting in almost complete hydrolysis (<10% remaining) by 5X-HSL cells in 24 hours, whereas >30% of the ester present at 0 hours remained in control cells (Figure 5).

View larger version (13K): [in this window] [in a new window]   Figure 5. CE hydrolysis in control and 5X-HSL foam cells. Cholesteryl oleate hydrolysis was analyzed as described in "Methods." In brief, foam cells were obtained by incubating RAW 264.7 cells with AcLDL and [3H]oleate for 24 hours, followed by a 24-hour equilibration period without lipoprotein. At 0 hours, [3H]oleate was removed, and duplicate cultures were washed and extracted for lipid and protein. CE synthesis was inhibited with 20 µg/mL progesterone added at 0 hours, and hydrolysis was determined by assaying [3H]cholesteryl oleate remaining at indicated times. CEs were isolated by silica thin-layer chromatography and measured by liquid scintillation counting. 5X-HSL cells had a 2.4-fold higher rate of hydrolysis than did control cells. Semilogarithmic transformation of data with means and SEs of three independent experiments is presented. At 0 hours, 100% (log=2.0) for control cells was 97.8±8.3 nmol [3H]cholesteryl oleate per mg protein and 75.4±0.5 for 5X-HSL cells. At 24 hours, nearly complete hydrolysis of cholesteryl oleate was achieved in 5X-HSL cells (6.6±2.1 nmol [3H]cholesteryl oleate per mg protein), whereas control cells still contained 32% (31.3±3.8 nmol [3H]cholesteryl oleate per mg protein) of the ester present at 0 hours.

cAMP Stimulation of CE Hydrolysis in 5X-HSL Cells
HSL activity is stimulated by phosphorylation of a specific serine residue catalyzed by cAMP-dependent protein kinase.¹¹ To determine whether the hydrolysis of CEs could be stimulated by cAMP, dibutyryl cAMP was added along with the ACAT inhibitor CI-976 to lipid-laden control RAW 264.7 and 5X-HSL cells at 0 hours after cholesterol accumulation and labeling with [³H]oleate. The rate of hydrolysis in 5X-HSL cells was 5-fold higher than in control cells (Figure 6a). Thus, the rate of hydrolysis in HSL-overexpressing cells was stimulated 2- to 3-fold by incubation with cAMP (compare Figures 5 and 6a), whereas hydrolysis in control cells was increased by only 1.3-fold with cAMP treatment. In addition, cAMP further stimulated the net hydrolysis of CEs in 5X-HSL cells in the presence of an ACAT inhibitor. Whereas almost complete hydrolysis of CEs by 5X-HSL cells occurred after 24 hours without cAMP (Figure 5), hydrolysis required only 9 hours in the presence of cAMP (Figure 6b). In contrast, at 9 hours control cells treated with cAMP still contained 55% of the cholesteryl oleate present at 0 hours (Figure 6b).

View larger version (18K): [in this window] [in a new window]   Figure 6. Effect of cAMP on rate and net hydrolysis of CEs in 5X-HSL cells. Cholesteryl oleate hydrolysis was analyzed as described in the legend for Figure 5. a, 5X-HSL cells had a 5-fold higher rate of hydrolysis than did control cells in the presence of 1 mmol/L dibutyryl cAMP plus 10 µmol/L CI-976 added at 0 hours. Semilogarithmic transformation of data is presented. At 0 hours, 100% (log=2.0) for control cells was 80 nmol [3H]cholesteryl oleate per mg protein, and 74 for 5X-HSL cells. b, cAMP further stimulated net hydrolysis of cholesteryl oleate in 5X-HSL cells compared with that in control cells. In fact, treatment with 1 mmol/L dibutyryl cAMP and 10 µmol/L CI-976 decreased the time required for complete cholesteryl oleate hydrolysis to only 9 hours (compare with Figure 5), whereas control cells still retained 55% of the cholesteryl oleate present at 0 hours. Total cholesteryl oleate is the sum of [3H] and [14C]cholesteryl oleate.

	Discussion

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Formation of foam cells by cholesterol accumulation in monocytes/macrophages is a primary step in the development of atherosclerotic lesions. Thus, the ability to inhibit cholesterol accumulation in macrophages or to remove CEs from mature foam cells in lesions would be of considerable therapeutic benefit. In macrophage foam cells, ACAT has been recognized to be responsible for CE synthesis. Furthermore, it has been proposed that whereas ACAT activity is regulated by the level of intracellular free cholesterol, the rate of CE hydrolysis remains unchanged under a variety of conditions.⁸ In addition, net hydrolysis of CEs obtained by inhibition of ACAT in peritoneal macrophages⁸ has provided a basis for designing therapeutic ACAT inhibitors to prevent cholesterol accumulation in macrophages.

It has been proposed that HSL is responsible for the nCEH activity in macrophage cell lines.^{9 10} In this report we present further evidence that HSL is present and accounts for essentially all of the nCEH activity in murine macrophages. Specifically, we have shown that nCEH activity in extracts of both RAW 264.7 and C57BL/6J peritoneal macrophages was neutralized by anti-HSL IgG (Figure 1). However, because HSL-specific antibody neutralized most but not all of the nCEH activity in macrophage extracts, it remains possible that other unidentified proteins along with HSL account for nCEH activity in murine macrophages.

HSL expression and nCEH activity were similar in wild-type RAW 264.7 cells and C57BL/6J peritoneal macrophages. In addition, control RAW 264.7 cells were converted to foam cells when incubated in the presence of AcLDL (Figure 4), and kinetic analysis revealed that the CE turnover cycle in these cells was comparable to that reported for peritoneal macrophages.⁸ Thus, we have used overexpression of HSL in cholesterol-laden RAW 264.7 cells to study the role of this lipase in CE hydrolysis in macrophage foam cells. SR gene regulatory elements were chosen to direct stable HSL expression in RAW 264.7 cells (Figure 2), because the SR gene is upregulated during monocyte-to-macrophage differentiation and not downregulated by increased intracellular levels of cholesterol.³⁷ It should be noted that monocytes/macrophages and macrophage cell lines have been reported to be resistant to both transient and stable transfection.²⁸ However, we have successfully obtained stable transfectants of RAW 264.7 cells overexpressing HSL (Figure 3) by observing the limiting amount of 2 µg DNA per 10⁶ cells that can be successfully incorporated by macrophages.²⁸ The modest levels of HSL overexpression observed in the transgenic clones could be attributed to the low concentration of DNA used to obtain viable transfected cells.

5X-HSL foam cells reproducibly contained 30% less total cholesterol than did control cells (Figure 4). Although this could be due to clonal variation, the properties of the 5X-HSL cells, especially their enhanced hydrolysis of CEs, support an explanation based on the expression of the HSL transgene. There could be less uptake of AcLDL caused by a decrease in SR expression at the surface of 5X-HSL cells. A decrease in SR expression could be caused by a titration of transcription factors needed for endogenous SR gene expression by competing SR regulatory sequences in copies of the pAL1-HSL transgene in 5X-HSL cells. On the other hand, although 5X-HSL foam cells contained less total cholesterol, the level of free cholesterol was not decreased in these cells compared with control cells (Figure 4). Therefore, cholesterol-laden, HSL-overexpressing cells contained proportionally more free cholesterol and less esterified cholesterol than did control cells. The 40% drop in CEs (Figure 4) is consistent with greater CE hydrolysis in 5X-HSL cells. In accordance with this observation, a 2- to 3-fold increase in hydrolysis of CEs was seen in cholesterol-laden 5X-HSL compared with control cells when ACAT was inhibited by progesterone (Figure 5) or CI-976.

cAMP plus CI-976 gave a 5-fold higher rate of CE hydrolysis in 5X-HSL than in control cells (Figure 6a), suggesting incomplete phosphorylation of the overexpressed transgenic rat HSL protein in 5X-HSL cells without cAMP stimulation. In the presence of an ACAT inhibitor, HSL overexpression enhanced the net hydrolysis of CEs, which was further stimulated by cAMP, presumably through additional phosphorylation of transgenic HSL (Figure 6b). Because cAMP also stimulated CE hydrolysis in overexpressing cells more than in control cells, these data are consistent with previous findings that HSL activity in murine macrophages is regulated by cAMP-dependent phosphorylation as in adipocytes.^{9 10 11 38 39 40} Although it is unclear how phosphorylation stimulates HSL, this modification may be necessary for access of HSL to lipid droplets in cells.⁴¹ Thus, without cAMP stimulation, only a portion of the overexpressed enzyme may be able to interact with the substrate. Incomplete phosphorylation of overexpressed HSL could also account for the lower nCEH activity in extracts than expected on the basis of protein levels (Figure 3a and 3b).

Endogenous HSL appeared to be downregulated by cholesterol accumulation in control cells, as previously reported for J774.2 macrophages.²⁰ However, the heterologous transfected enzyme was apparently not affected in cholesterol-laden 5X-HSL cells, perhaps due to a difference in stability between mouse and rat HSL proteins or the replacement of HSL gene regulatory sequences with SR sequences in the transgene.

ACAT activity was also found to be stimulated 2-fold in cholesterol-laden 5X-HSL cells (data not shown). As previously established by others,⁸ in the absence of extracellular acceptors an increase of intracellular free cholesterol stimulates ACAT activity in macrophage foam cells. Therefore, the increase in ACAT activity could be due to the free cholesterol generated by the increased rate of CE hydrolysis in HSL-overexpressing cells. However, ACAT stimulation and even HSL downregulation by cholesterol may not occur to the same extent in macrophage foam cells of atherosclerotic lesions in vivo, where the presence of serum and extracellular cholesterol acceptors would significantly alter the level of free cholesterol in these cells.

In conclusion, the data reported here demonstrate for the first time that nCEH activity can be manipulated in macrophage foam cells by specifically overexpressing HSL. Without an ACAT inhibitor, HSL overexpression significantly prevented the accumulation of CEs in model foam cells. In the presence of an ACAT inhibitor, HSL overexpression significantly enhanced the rate and net hydrolysis of CEs, leading to faster depletion of CEs in these cells. Therefore, HSL overexpression in macrophages alone or in combination with ACAT inhibitors may constitute a useful therapeutic approach for impeding CE accumulation in foam cells in atherosclerotic lesions. We are currently examining this hypothesis with a transgenic mouse line that exhibits a 5-fold macrophage-specific overexpression of HSL.

	Selected Abbreviations and Acronyms

 

ACAT = acyl coenzyme A:cholesterol acyltransferase AcLDL = acetylated LDL CE = cholesteryl ester HSL = hormone-sensitive lipase nCEH = neutral cholesteryl ester hydrolase PCR = polymerase chain reaction RT = reverse transcription SR = scavenger receptor TG = triglyceride

	Acknowledgments

 
This study was supported by the Veterans Administration and Glaxo-Wellcome (to M.C.S.). The authors thank Alan Wagner at UCLA for providing human LDL; Qin Han for her assistance with cell culture; and Mark Doolittle, Osnat Ben-Zeev, and Véronique Briquet-Laugier for their advice on the Western blotting.

Received November 5, 1997; accepted January 21, 1998.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Beisiegel U, St Clair RW. An emerging understanding of the interactions of plasma lipoproteins with the arterial wall that leads to the development of atherosclerosis. Curr Opin Lipidol. 1996;7:265–268.[Medline] [Order article via Infotrieve]
2. Bulkley BH, Buja LM, Ferrans VJ, Bulkley GB, Roberts WC. Tuberous xanthoma in homozygous type II hyperlipoproteinemia: a histologic, histochemical, and electron microscopical study. Arch Pathol. 1975;99:293–300.[Medline] [Order article via Infotrieve]
3. Buja LM, Kovanen PT, Bilheimer DW. Cellular pathology of homozygous familial hypercholesterolemia. Am J Pathol. 1979;97:327–357.[Abstract]
4. Libby P, Geng YJ, Aikawa M, Schoenbeck U, Mach F, Clinton SK, Sukhova GK, Lee RT. Macrophages and atherosclerotic plaque stability. Curr Opin Lipidol. 1996;7:330–335.[Medline] [Order article via Infotrieve]
5. Henney AM, Wakeley PR, Davies MJ, Foster K, Hembry R, Murphy G, Humphries S. Localization of stromelysin gene expression in atherosclerotic plaques by in situ hybridization. Proc Natl Acad Sci U S A. 1991;88:8154–8158.[Abstract/Free Full Text]
6. Brown MS, Goldstein JL, Krieger M, Ho YK, Anderson RGW. Reversible accumulation of cholesteryl esters in macrophages incubated with acetylated lipoproteins. J Cell Biol. 1979;82:597–613.[Abstract/Free Full Text]
7. Ho YK, Brown MS, Goldstein JL. Hydrolysis and excretion of cytoplasmic cholesteryl esters by macrophages: stimulation by high density lipoprotein and other agents. J Lipid Res. 1980;21:391–398.[Abstract]
8. Brown MS, Ho YK, Goldstein JL. The cholesteryl ester cycle in macrophage foam cells: continual hydrolysis and re-esterification of cytoplasmic cholesteryl esters. J Biol Chem. 1980;255:9344–9352.[Free Full Text]
9. Small CA, Goodacre JA, Yeaman SJ. Hormone-sensitive lipase is responsible for the neutral cholesterol ester hydrolase activity in macrophages. FEBS Lett. 1989;247:205–208.[Medline] [Order article via Infotrieve]
10. Small CA, Rogers MP, Goodacre JA, Yeaman SJ. Phosphorylation and activation of hormone-sensitive lipase in isolated macrophages. FEBS Lett. 1991;279:323–326.[Medline] [Order article via Infotrieve]
11. Strålfors P, Björgell P, Belfrage P. Hormonal regulation of hormone-sensitive lipase in intact adipocytes: identification of phosphorylated sites and effects on the phosphorylation by lipolytic hormones and insulin. Proc Natl Acad Sci U S A. 1984;81:3317–3321.[Abstract/Free Full Text]
12. Fredrikson G, Strålfors P, Nilsson NÖ, Belfrage P. Hormone-sensitive lipase of rat adipose tissue: purification and some properties. J Biol Chem. 1981;256:6311–6320.[Free Full Text]
13. Holm C, Belfrage P, Fredrikson G. Immunological evidence for the presence of hormone-sensitive lipase in rat tissues other than adipose tissue. Biochem Biophys Res Commun. 1987;148:99–105.[Medline] [Order article via Infotrieve]
14. Yeaman SJ. Hormone-sensitive lipase–a multipurpose enzyme in lipid metabolism. Biochim Biophys Acta. 1990;1052:128–132.[Medline] [Order article via Infotrieve]
15. Kraemer FB, Patel S, Saedi MS, Sztalryd C. Detection of hormone-sensitive lipase in various tissues: I: Expression of an HSL/bacterial fusion protein and generation of anti-HSL antibodies. J Lipid Res. 1993;34:663–671.[Abstract]
16. Kraemer FB, Patel S, Singh-Bist A, Gholami SS, Saedi MS, Sztalryd C. Detection of hormone-sensitive lipase in various tissues: II: Regulation in the rat testis by human chorionic gonadotropin. J Lipid Res. 1993;34:609–616.[Abstract]
17. Holst LS, Langin D, Mulder H, Laurell H, Grober J, Bergh A, Mohrenweiser HW, Edgren G, Holm C. Molecular cloning, genomic organization, and expression of a testicular isoform of hormone-sensitive lipase. Genomics. 1996;35:441–447.[Medline] [Order article via Infotrieve]
18. Contreras JA, Martin A, Lasunción MA, Holm C, Gaspar ML. Presence of hormone-sensitive lipase mRNA in J774 macrophages. Isr J Med Sci. 1994;30:778–781.[Medline] [Order article via Infotrieve]
19. Contreras JA, Lasunción MA. Essential differences in cholesteryl ester metabolism between human monocyte-derived and J774 macrophages: evidence against the presence of hormone-sensitive lipase in human macrophages. Arterioscler Thromb. 1994;14:443–452.[Abstract/Free Full Text]
20. Jepson CA, Harrison JA, Kraemer FB, Yeaman SJ. Down-regulation of hormone-sensitive lipase in sterol ester-laden J774.2 macrophages. Biochem J. 1996;318:173–177.
21. Holm C, Kirchgessner TG, Svenson KL, Fredrikson G, Nilsson S, Miller CG, Shively JE, Heinzmann C, Sparkes RS, Mohandas T, Lusis AJ, Belfrage P, Schotz MC. Hormone-sensitive lipase: sequence, expression, and chromosomal localization to 19cent-q13.3. Science. 1988;241:1503–1506.[Abstract/Free Full Text]
22. Li Z, Sumida M, Birchbauer A, Schotz MC, Reue K. Isolation and characterization of the gene for mouse hormone-sensitive lipase. Genomics. 1994;24:259–265.[Medline] [Order article via Infotrieve]
23. Khoo JC, Reue K, Steinberg D, Schotz MC. Expression of hormone-sensitive lipase mRNA in macrophages. J Lipid Res. 1993;34:1969–1974.[Abstract]
24. Reue K, Cohen RD, Schotz MC. Evidence for hormone-sensitive lipase mRNA expression in human macrophages. Arterioscler Thromb Vasc Biol. 1997;17:3428–3432.[Abstract/Free Full Text]
25. Berg KA, Petty HR. Cholesteryl ester handling by RAW264 macrophages: response to native and acetylated low density lipoprotein. Mol Cell Biochem. 1988;84:29–40.[Medline] [Order article via Infotrieve]
26. Via DP, Plant AL, Craig IF, Gotto AM Jr, Smith LC. Metabolism of normal and modified low-density lipoproteins by macrophage cell lines of murine and human origin. Biochim Biophys Acta. 1985;833:417–428.[Medline] [Order article via Infotrieve]
27. Smith JD, Miyata M, Ginsberg M, Grigaux C, Shmookler E, Plump AS. Cyclic AMP induces apolipoprotein E binding activity and promotes cholesterol efflux from a macrophage cell line to apolipoprotein acceptors. J Biol Chem. 1996;271:30647–30655.[Abstract/Free Full Text]
28. Stacey KJ, Ross IL, Hume DA. Electroporation, and DNA-dependent cell death in murine macrophages. Immunol Cell Biol. 1993;71:75–85.
29. Morton RE, Evans TA. Modification of the bicinchoninic acid protein assay to eliminate lipid interference in determining lipoprotein protein content. Anal Biochem. 1992;204:332–334.[Medline] [Order article via Infotrieve]
30. Belfrage P, Vaughan M. Simple liquid-liquid partition system for isolation of labeled oleic acid from mixtures with glycerides. J Lipid Res. 1969;10:341–344.[Abstract]
31. Moulton KS, Wu H, Barnett J, Parthasarathy S, Glass CK. Regulated expression of the human acetylated low density lipoprotein receptor gene and isolation of promoter sequences. Proc Natl Acad Sci U S A. 1992;89:8102–8106.[Abstract/Free Full Text]
32. Holm C, Kirchgessner TG, Svenson KL, Lusis AJ, Belfrage P, Schotz MC. Nucleotide sequence of rat adipose hormone sensitive lipase cDNA. Nucleic Acids Res. 1988;16:9879.[Free Full Text]
33. Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS. `Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 1991;19:4008.[Free Full Text]
34. Basu SK, Goldstein JL, Anderson RGW, Brown MS. Degradation of cationized low density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts. Proc Natl Acad Sci U S A. 1976;73:3178–3182.[Abstract/Free Full Text]
35. Krause BR, Anderson M, Bisgaier CL, Bocan T, Bousley R, DeHart P, Essenburg A, Hamelehle K, Homan R, Kieft K, McNally W, Stanfield R, Newton RS. In vivo evidence that the lipid-regulating activity of the ACAT inhibitor CI-976 in rats is due to inhibition of both intestinal and liver ACAT. J Lipid Res. 1993;34:279–294.[Abstract]
36. Allain CC, Poon LS, Chan CSG, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem. 1974;20:470–475.[Abstract]
37. Horvai A, Palinski W, Wu H, Moulton KS, Kalla K, Glass CK. Scavenger receptor A gene regulatory elements target gene expression to macrophages and to foam cells of atherosclerotic lesions. Proc Natl Acad Sci U S A. 1995;92:5391–5395.[Abstract/Free Full Text]
38. Khoo JC, Mahoney EM, Steinberg D. Neutral cholesterol esterase activity in macrophages and its enhancement by cAMP-dependent protein kinase. J Biol Chem. 1981;256:12659–12661.[Abstract/Free Full Text]
39. Goldberg DI, Khoo JC. Stimulation of a neutral cholesteryl ester hydrolase by cAMP system in P388D1 macrophages. Biochim Biophys Acta. 1990;1042:132–137.[Medline] [Order article via Infotrieve]
40. Bernard DW, Rodriguez A, Rothblat GH, Glick JM. cAMP stimulates cholesteryl ester clearance to high density lipoproteins in J774 macrophages. J Biol Chem. 1991;266:710–716.[Abstract/Free Full Text]
41. Egan JJ, Greenberg AS, Chang MK, Wek SA, Moos MC Jr, Londos C. Mechanism of hormone-stimulated lipolysis in adipocytes: translocation of hormone-sensitive lipase to the lipid storage droplet. Proc Natl Acad Sci U S A. 1992;89:8537–8541.[Abstract/Free Full Text]