© 1998 American Heart Association, Inc.
Original Contributions |
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
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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
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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 groups9 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 expression18 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 SR25 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 2827 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.
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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
).
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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.
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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.
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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.
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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).
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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).
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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).
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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.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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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.
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