© 1997 American Heart Association, Inc.
Articles |
Cholesterol-Mediated Changes of Neutral Cholesterol Esterase Activity in Macrophages
Mechanism for Mobilization of Cholesteryl Esters in Lipid Droplets by HDL
From the School of Pharmaceutical Sciences (S.M., T.C., H.N., K.N., I.T.) and Graduate School of Health Sciences (N.M., M.I., T.T.), University of Shizuoka, Japan.
Correspondence to Dr Takako Tomita, Graduate School of Health Sciences, University of Shizuoka, 52-1 Yada, Shizuoka, Japan 422. E-mail tomitat{at}sea.u-shizuoka-ken.ac.jp
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Abstract |
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Abstract Cholesteryl esters (CE) in lipid droplets undergo a continual cycle of hydrolysis and reesterification by neutral cholesterol esterase (N-CEase) and acyl CoA:cholesterol acyltransferase (ACAT), respectively. The mechanism by which HDL mobilizes CE from lipid droplets in J774 A.1 cells was investigated, focusing on N-CEase activity. We asked whether HDL enhances the activity and, if so, what signals induce the change of the activity. An incubation of cells with HDL enhanced the decline of cholesteryl-[1-14C]-oleate in foam cells and increased N-CEase activity in the supernatant of cell homogenate in a concentration-dependent manner, whereas incubation with LDL decreased the activity. In addition, N-CEase activity was fivefold higher when cells were cultured in 10% lipoprotein-deficient serum (LPDS) medium (2 µg cholesterol/mL) than when cultured in 10% fetal calf serum medium (31 µg cholesterol/mL), suggesting that changes in N-CEase activity are mediated by cholesterol. An addition of cholesterol (0 to 30 µg/mL) in LPDS medium markedly inhibited N-CEase activity with a concomitant increase in cellular cholesterol concentration. This inhibitory effect of cholesterol was also observed in mouse peritoneal macrophages. In vitro addition of cholesterol did not affect N-CEase activity. Treatment of cells with HMG-CoA reductase inhibitors enhanced N-CEase activity, whereas ACAT inhibitor decreased the activity. Northern blot analysis of N-CEase mRNA showed that the expression was not altered by the presence of cholesterol in LPDS medium. These results suggest that cholesterol downregulates N-CEase activity, probably through cholesterol-dependent appearance of some factors.
Key Words: high-density lipoprotein (HDL) • neutral cholesterol esterase • J774 A.1 cells • cholesterol efflux
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The presence of foam cells that accumulate CE in the subendothelial space is one of the initial events in atheroma formation, and these foam cells are believed to be derived mainly from macrophages.1 2 3 4 5 Modified LDL and ß-VLDL are taken up into macrophages through the respective receptors, and CE in these lipoproteins undergo hydrolytic conversion to free cholesterol by acid cholesterol esterase in lysosomes. Then, free cholesterol released into cytosolic space is partly excreted from the cells in the presence of its acceptors, whereas the excess cholesterol is reesterified by ACAT, leading to CE accumulation in cytoplasm as lipid droplets. CE stored in the cytoplasm undergoes a continual cycle of hydrolysis and reesterification by the action of N-CEase and ACAT, respectively. Whether a foam cell will increase or decrease its stores of CE depends on the relative activity of N-CEase and ACAT.6
Epidemiological studies have generally shown a strong inverse correlation between serum HDL concentration and the development of atherosclerosis.7 In seeking an explanation of this inverse relationship, most investigators have focused on the role of HDL in reverse cholesterol transport.8 It has also become clear that HDL has the potential to limit oxidative modification of LDL.9 10 11 HDL is believed to play a primary role in reverse cholesterol transport by accepting cholesterol in plasma membranes, resulting in a marked CE reduction in foam cells.8 Cholesterol efflux by HDL occurs through physicochemical cholesterol exchange between cell membrane and HDL surfaces,12 an unknown signal transduction, or both. HDL-binding protein on the cell surface is postulated as playing a specific role by being linked to intracellular signal transduction. This transduction results in translocation of intracellular cholesterol to the cellular surface.13 14 Circumstantial evidence indicates that HDL not only removes cholesterol from the surface of cell membranes but also mobilizes CE from lipid droplets of foam cells, provided there is an adequate concentration of cholesterol acceptors available.15 16 Because cells are unable to excrete CE and excrete only free cholesterol, hydrolysis of CE in droplets should precede the mobilization.
Khoo et al17 demonstrated the presence of N-CEase activated by cAMP-dependent protein kinase in the cytosol of macrophages as well as in adipose tissue.18 Endogenously formed CE is degraded by this cytosolic enzyme, N-CEase, which is distinct from the lysosomal acid cholesterol esterase, which degrades CE in lipoproteins taken up into cells.6 Lesion regression demonstrated in experimental19 20 and human21 atherosclerosis might result from the increased rate of CE hydrolysis to reesterification of cholesterol. The N-CEase found in macrophages, which is presumably responsible for the hydrolysis of CE,22 23 is assumed to be the same enzyme in HSL in the adipose tissue from immunoblotting24 and Northern blot analysis.25 Khoo et al26 recently presented data that strongly suggested that the macrophage N-CEase and HSL of the adipose tissue are both products of a single gene.
Although several investigators suggest that mobilization of CE in foam cells by HDL is mediated by N-CEase in kinetic studies,27 28 no evidence has been presented so far on the change of N-CEase activity itself. We therefore asked whether HDL enhances the activity of N-CEase through some signals and, if so, what signals mediate the change. The results showed that cytosolic N-CEase activity is regulated by cellular cholesterol concentration.
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Methods |
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Materials
J774 A.1 cells (ATCC No. TIB67) were obtained from American Type Culture Collection; FCS was obtained from CSL Ltd and was heat-inactivated before use; DMEM, L-glutamine, and PBS(-) were obtained from Nissui Pharmaceutical Co, LTD; streptomycin and penicillin G potassium salt were obtained from Meiji Seika Co, LTD; sodium taurocholate, cholesteryloleate, cholesterol, 3-(p-hydroxyphenyl) propionic acid, and salmon sperm DNA were purchased from Sigma Chemical Co; bovine serum albumin (fatty acid free) was purchased from Miles; 3',5'-cAMP, ATP 2Na, cholesterol oxidase, cholesterol esterase, and horseradish peroxidase were obtained from Boehringer Mannheim GmbH; [1-14C]-oleic acid (56.0 mCi/mmol) and cholesteryl-[1-14C]-oleate (59.5 mCi/mmol) were purchased from DuPont-New England Nuclear Research; and [
-32P]-dCTP (3000 Ci/mmol) was purchased from ICN
Pharmaceuticals, Inc. Tissue culture plastic wares were purchased from
Sumitomo Bakelite Co, LTD; Centricell ultrafilters (30 000 NMWL) were
obtained from Polyscience Inc; and syringe filters (0.22 and 0.45
µm) were purchased from Sartorius. TLC plates (Kieselgel 60, No.
13 894, 20x20 cm, 0.5 mm thick) were obtained from E. Merck;
ISOGEN and Denhardt's solution were purchased from Nippon gene; nylon
membrane (Hybond-N+) and Megaprime DNA labeling systems came from
Amersham; pGEM-3Zf(+) came from Promega; and Bioimaging
analyzers Fujix BAS IIIs, Fujix BAS 2000II, and MacBAS version
2.x were from Fuji Film. ML-236B (Compactin), simvastatin, and (R) N-2-(1,3-benzodioxol-4-yl) heptyl-N'-2,6-diisopropylphenylurea (EAB-309) were supplied by Sankyo Co., Banyu Pharmaceutical Co, and Mitsubishi Kagaku Co, respectively.
Cell Cultures
J774 A.1 cells were grown and maintained in 75-cm2
flasks with DMEM containing 10% (vol/vol) FCS, penicillin
G (100 U/mL), streptomycin (100 µg/mL), and
L-glutamine (0.3 mmL) (FCS-DMEM) in 5% CO2
and humidified air at 37°C.
Mouse peritoneal macrophages were collected from nonstimulated male ICR mice (weight, 35 to 40 g) and suspended at 4x106 cells/mL with FCS-DMEM. To each 25-cm2 flask was added 4 mL of the cell suspension, and the flasks were incubated for 2 hours. Cell monolayers formed were washed three times with 4 mL of PBS (-) and used for experiments.
Preparation of Lipoproteins and LPDS
LPDS (d>1.225 g/mL) was prepared from
heat-inactivated FCS by discontinuous density gradient
ultracentrifugation according to the method of
Goldstein et al29 using an ultracentrifuge
(70P-72, Hitachi) and a vertical rotor (RPV50T, Hitachi). After
dialysis for 48 hours against Tris-HCl buffer (10 mmol/L,
pH 7.4) containing KCl (0.15 mol/L) at 4°C, LPDS was filtered
through syringe filters (pore size, 0.22 µm) and stored at
-20°C until use.
ß-VLDL (d=1.006 g/mL) was prepared from serum (cholesterol >1500 mg/dL) obtained from cholesterol (1%)–fed Japanese white rabbits by repeated discontinuous density gradient ultracentrifugation according to the method of Chung et al.30 LDL (d=1.019 to 1.063 g/mL) and HDL (d=1.063 to 1.21 g/mL) were prepared from fresh porcine serum by the discontinuous density gradient ultracentrifugation.30 LDL was purified by fast protein liquid chromatography by use of a Sephacryl S400HR column (600x16-mm ID., Pharmacia Co). They were eluted with 10 mmol/L Tris-HCl, pH 7.4, containing 150 mmol/L NaCl as the eluting solvents in the presence of 1 mmol/L EDTA. After dialysis against PBS (-) for 24 hours at 4°C, each lipoprotein was filtered through syringe filters (pore size, 0.45 µm) before use.
Loading Cholesteryl-[1-14C]-oleate in J774 A.1
Cells
J774 A.1 cells (4x104 cells) were plated in 22-mm
dishes in FCS-DMEM and cultured for 48 hours in 1 mL DMEM containing
10% (vol/vol) LPDS (LPDS-DMEM). After being washed with PBS
(-), cells were cultured for 36 hours in 1 mL LPDS-DMEM,
[1-14C]-oleic acid (0.2 mmol/L, 2
µCi/mL)-albumin (1.2 mg/mL) complex prepared as
described by Goldstein et al,29 and ß-VLDL (0.5 mg
cholesterol/mL).
Incubation of Cholesteryl-[1-14C]-oleate–loaded
Cells with HDL
Cholesteryl-[1-14C]-oleate loaded cells were
washed two times—first with PBS (-) with BSA (2 mg/mL) and
then with PBS (-) without BSA—and cultured for the indicated times in
fresh 1 mL LPDS-DMEM in the presence and absence of HDL (0 to 500 µg
protein/mL). Cells were washed three times each—with Tris-HCl buffer
(50 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.4)
containing BSA (2 mg/mL) and with Tris-HCl buffer only—and
lipids were extracted twice with 1 mL of a mixture of
n-hexane-isopropanol (3:2) containing cholesteryl oleate (50 µg).
Lipids were resolved by TLC (petroleum ether:ether:acetic
acid=70:30:1), and the radioactivity of cholesteryloleate was counted
in a liquid scintillation spectrometer (Aloka LSC-602).
Incubation of J774 A.1 Cells with Lipoproteins,
Cholesterol, and Various Inhibitors
J774 A.1 cells (2x106 cells) and mouse peritoneal
macrophages (1.6x107 cells) were plated in
25-cm2 flasks in FCS-DMEM and cultured for 24 and 2 hours,
respectively. Cells were washed three times with PBS (-) and cultured
for the indicated times in 4 mL LPDS-DMEM in the presence and the
absence of lipoproteins (0 to 100 µg protein/mL),
cholesterol (0 to 30 µg/mL), HMG-CoA reductase
inhibitors (ML-236B or simvastatin 0 to 30
ng/mL), or ACAT inhibitor (EAB-309, 0 to 1
µmol/L). Except for the experiments with lipoproteins, each
medium was adjusted to contain the same concentration of the vehicles:
0.3% ethanol in cholesterol experiment, 0.1% ethanol in
HMG-CoA reductase inhibitor experiment, and 0.1% dimethyl
sulfoxide in ACAT inhibitor experiment.
Cholesterol and HMG-CoA reductase inhibitors
were dissolved in ethanol, and ACAT inhibitor was dissolved
in dimethyl sulfoxide.
Assay of N-CEase Activity
Assay of N-CEase activity was carried out principally as
described in our previous paper.31 32 The enzyme solution
was as follows. Cultured J774 A.1 cells and mouse peritoneal
macrophages were placed in a 25-cm2 flask that was
rinsed twice with 3 mL of PBS (-) and scraped with a rubber spatula in
4 mL of PBS (-). The cell suspension was centrifuged at
100g for 5 minutes at 10°C. Cells in the pellets were
homogenized in Tris-HCl buffer (10 mmol/L, pH
7.0) containing EDTA (0.1 mmol/L) and sucrose (0.25
mol/L) by freezing and thawing and centrifuged for 30
minutes at 43 000g at 4°C. The supernatant was used as
N-CEase enzyme solution. The substrate solution was as follows. Both
labeled and unlabeled cholesteryloleate was purified by TLC (developing
solvent, petroleum ether:ether:acetic acid, 70:30:1) before use. A
micellar substrate was prepared as follows: cholesteryloleate (400
nmol) containing 4.76 mCi cholesteryl-[1-14C]-oleate, and
a mixture (1.6 µmol) of egg phosphatidylcholine and
phosphatidylethanolamine (1:1) were lyophilized for 4 hours, and then
sonicated (50 W, 10 minutes; Ohtake) at 46°C in 2 mL of potassium
phosphate buffer (100 mmol/L, pH 7.0) containing sodium
taurocholate (1.8 µmol). For the assay, enzyme solution (
1 mg
protein/mL, 100 µL), was incubated for 60 minutes at 37°C with 50
µL substrate solution (cholesteryl-[1-14C]-oleate,
2x105 dpm, 2 nmol) in potassium phosphate buffer
(100 mmol/L, pH 7.0) (total, 200 µL). When indicated,
ATP · 2Na (0.5 mmol/L), Mg acetate (5
mmol/L), and cAMP (1 µmol/L) were added to assay
mixture. The reaction was terminated by the addition of 0.3 N NaOH (0.6
mL) and a mixture (3 mL) of benzene:CHCl3:MeOH (1:0.5:1.2).
The reaction tubes were shaken vigorously for 2 minutes and
centrifuged at 1500g for 10 minutes at room
temperature. Radioactivity of the upper layer was counted.
RNA Preparation
Total RNA from J774 A.1 cells was isolated by a single-step acid
guanidium thiocyanate-phenol-chloroform extraction
method33 using ISOGEN. All glassware and plastic ware were
autoclaved before use. RNA was purified via a series of ethanol
precipitations. RNA pellets were dissolved in diethyl
pyrocarbonate–treated sterile water, and samples of whole-cell RNA
were quantified by optical density at 260 nm.
Cloning of HSL cDNA From J774 A.1 Cell RNA
Total RNA from J774 A.1 cells was used as a template for cDNA
synthesis and amplified by polymerase chain reaction according to the
method of Khoo et al.26 The primers used were as follows:
5'-GCTGGTGCAGAGAGACAC-3' (nucleotides 1515 through 1532 of
mouse HSL coding sequence) as a forward primer and
5'-GAAAGCAGCGCGCACGCG-3' (nucleotides 1906 through 1923 of
mouse HSL coding sequence) as a reverse primer. The resulting 408
nucleotide product was purified by electrophoresis and
ligated into the SalI and BamHI sites of the
plasmid pGEM-3Zf(+). This partial mouse HSL cDNA was used as a probe
for Northern hybridization.
Northern Blot Analysis
Total RNA was electrophoresed for 15 hours at 30 V through 1.2%
(wt/vol) agarose containing 1% (vol/vol) formaldehyde and 1x
Goldberg buffer (40 mmol/L 3-morpholinopropanesulfonic
acid, 5 mmol/L sodium citrate, and 0.5 mmol/L
EDTA, pH 7.2). RNA was transferred to nylon membrane (Hybond-N+) in
0.05 N NaOH, and the nylon membrane was washed with 2x SSPE (0.36
mol/L NaCl, 0.02 mol/L sodium phosphate, 2 mmol/L
EDTA, pH 7.7). RNA was prehybridized for 4 hours in 5x SSPE, 50%
(vol/vol) formamide, 5x Denhardt's solution (0.1% BSA, 0.1%
polyvinylpyrrolidone, 0.1% Ficoll type 400), and 0.5% (wt/vol) SDS
containing 0.1 mg/mL heat-denatured salmon sperm DNA at 42°C.
Cloned cDNAs were radiolabeled with [-32P]-dCTP using
a Megaprime DNA labeling system. Northern hybridization was performed
for 16 hours with denatured 32P-labeled HSL or GAPDH cDNA
probes (5x106 cpm/mL) at 42°C in the same buffer system
as above. Sequential 30-minute washes were performed with 2x SSC
buffer, 1% (wt/vol) SDS, and 0.2% (wt/vol)
Na4P2O7 10H2O at
65°C, and two 10-minute washes were done with 0.2x SSC and 0.1%
(wt/vol) SDS at room temperature. Washed membranes were exposed to
Imaging plate Fujix BAS IIIs. Densitometry of bands was performed with
a Fujix BAS 2000II and a MacBAS version 2.x.
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Results |
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Enhanced Hydrolysis of CE in Foam Cells by HDL
Purified ß-VLDL prepared from cholesterol-fed rabbits showed only apolipoprotein B-100 and apolipoprotein E spots by SDS-PAGE and contained total cholesterol 7 to 11 mg/mg protein, depending on preparations. J774 A.1 cells preincubated in LPDS-DMEM for 24 hours were loaded with cholesteryl-[1-14C]-oleate by incubating for 36 hours with ß-VLDL (0.5 mg cholesterol/mL) and [1-14C]-oleic acid–albumin complex in LPDS-DMEM. The foam cells contained 135 nmol cholesteryl-[1-14C]-oleate (
1x105 dpm)/mg
cell protein on the basis of specific activity of
[1-14C]-oleic acid in [1-14C]-oleic
acid–albumin complex. The mass content of CE in foam cells was
233 nmol cholesterol/mg cell protein. When these
foam cells were incubated in LPDS-DMEM for varying times, both
cholesteryl-[1-14C]-oleate and mass CE progressively
decreased with increasing time at analogous rates; the remaining CE as
a percent of the initial amounts was 90% at 6 hours, 61% at 12 hours,
38% at 24 hours, and 38% at 36 hours in
cholesteryl-[1-14C]-oleate, and it was 86% at 6 hours,
43% at 12 hours, 31% at 24 hours, and 38% at 36 hours in mass
CE.
To confirm that HDL enhances the decrease of CE in lipid droplets,
cholesteryl-[1-14C]-oleate–loaded foam cells were
incubated for 36 hours with varying concentrations of HDL (125, 250,
and 500 µg protein/mL) in LPDS-DMEM, and the remaining cellular
cholesteryl-[1-14C]-oleate was quantified by TLC (Fig 1
). Forty-four percent of
cholesteryl-[1-14C]-oleate was hydrolyzed after 36 hours
of incubation in control cells. HDL significantly enhanced the
hydrolysis of cholesteryl-[1-14C]-oleate in a
concentration-dependent manner; the hydrolysis rates were higher by
10% (125 µg protein/mL), 23% (250 µg protein/mL), and 61% (500
µg protein/mL) than the control value (in the absence of HDL) at 36
hours.
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Changes of N-CEase Activity by Incubation With
Lipoproteins
To examine whether the enhanced decline of CE by HDL results from
the activation of N-CEase, which specifically hydrolyzes
endogenously formed CE in lipid droplets, cells were
incubated for 24 hours in LPDS-DMEM in the presence and absence of HDL
(0 to 100 µg protein/mL). Then, N-CEase activity in
43 000g supernatant of cell homogenate was
assayed using a micellar substrate (Fig 2A). HDL significantly raised the
activity in a concentration-dependent manner. The activation was also
time dependent; HDL (100 µg protein/mL) raised the activity by 11%
at 12 hours and by 58% at 24 hours compared with that of the control.
This effect of HDL was observed regardless of the presence and absence
of cofactors (0.5 mmol/L ATP · 2Na, 5
mmol/L Mg acetate, and 1 µmol/L cAMP). In
contrast, incubation with LDL reduced the activity dose dependently at
smaller concentrations than those of HDL (Fig 2B). A significant LDL
effect was seen even at 3 µg protein/mL. Supernatant enzyme
preparations of cell homogenate used for N-CEase assay
contained similar amounts of CE <1 nmol/100 µg protein per
assay) in HDL- and LDL-treated and control cells. The substrate amount
was 10 nmol/100 µg protein per assay.
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Cellular cholesterol contents in cells incubated either
with HDL or LDL (100 µg protein/mL) under the same condition as above
are shown in Table 1.
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Influence of Culture Media on N-CEase Activity
Table 2 shows that N-CEase activity
in J774 A.1 cells varies, depending on culture media. The N-CEase
activity in J774 A.1 cells cultured for 24 hours in LPDS-DMEM was
5-fold higher than that of cells cultured in FCS-DMEM. Conversely,
total cholesterol concentration in FCS-DMEM was 31 µg
cholesterol/mL, 16-fold higher than that found in
LPDS-DMEM.
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Inhibition of N-CEase Activity in Macrophages by
Cholesterol
The results in Fig 2
and Table 2 suggest that the change of
N-CEase activity is mediated by cholesterol. To show this
possibility more directly, J774 A.1 cells were incubated for various
times in LPDS-DMEM with and without the supplement of
cholesterol (30 µg/mL), a comparable amount as
found in FCS-DMEM (Fig 3), or for 24
hours with varying concentrations of cholesterol (Fig 4). N-CEase activity of cells cultured in
LPDS-DMEM was enhanced with increasing time; the activity was 57%
higher at 6 hours and reached a plateau at 12 hours (2.8-fold) compared
with that of time zero. A supplement of cholesterol (30
µg/mL), however, completely blocked the enhancement of N-CEase
activity (Fig 3). When cells were cultured for 24 hours in LPDS-DMEM
containing varying amounts of cholesterol (0 to 30
µg/mL), N-CEase activity was greatly inhibited with increasing
concentrations of cholesterol. The presence of 10 µg
cholesterol/mL reduced the activity by 74% (Fig 4A). Addition of oleic acid as an albumin complex to the medium
did not influence N-CEase activity up to 1 mmol/L
concentration. In vitro addition of cholesterol 1 to 100
ng/per tube (5 to 500 ng/mL) to the assay mixture of
N-CEase did not alter the activity (Table 3). Free cholesterol
concentration in assay mixture is usually nondetectable.
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and absence (
) of cholesterol (30 µg/mL). N-CEase
activity in the supernatant (43 000g, 30 minutes) of cell
homogenate was assayed as described in "Methods."
N-CEase activity at time zero was 1.04 nmol CE hydrolyzed per hour per
1 mg protein. Data represent mean±SD of triplicate
determinations. *P<.05, **P<.01 vs time
zero.
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Lipids were extracted from whole cells with n-hexane-isopropanol, and
cholesterol was measured by an enzyme fluorescent
method. The contents of cellular free and esterified
cholesterol were concomitantly elevated with increasing
concentration of cholesterol in the medium (Fig 4B).
To see whether this regulation of N-CEase by cholesterol is
specific to J774 A.1 cells, resident mouse peritoneal
macrophages were incubated with either cholesterol
or 25-hydroxycholesterol under the conditions used for J774
A.1 cells. Table 4 shows that both
cholesterol (1 to 10 µg/mL) and
25-hydroxycholesterol (3 µg/mL) significantly
inhibited N-CEase activity.
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Effects of Inhibitors of HMG-CoA Reductase and ACAT on
N-CEase Activity
HMG-CoA reductase inhibitors reduce cellular
cholesterol concentration by inhibiting
cholesterol synthesis,34 while ACAT
inhibitors elevate the concentration by inhibiting
esterification of cellular free cholesterol.35
Cells were cultured for 24 hours with HMG-CoA reductase
inhibitors ML-236B or simvastatin or with the
ACAT inhibitor EAB-309 in LPDS-DMEM to manipulate cellular
cholesterol concentration, and N-CEase activity was assayed
(Fig 5). ML-236B is a cell permeable
derivative of Pravastatin.36 37 Fig 5A
illustrates the effects of ML-236B and simvastatin (0 to 30
ng/mL). ML-236B increased N-CEase activity in a
concentration-dependent manner; the lowest concentration of ML-236B 0.3
ng/mL raised the activity by 26%, and the highest concentration
(30 ng/mL) raised it by 54% compared with control.
Simvastatin elevated the activity even more at 0.3 and 1
ng/mL, but N-CEase activity was lower at concentrations higher
than 3 ng/mL in cells treated with simvastatin than
in cells treated with ML-236B. This enhancing effect was not observed
when cells were cultured in FCS-DMEM. Fig 5B shows the
inhibitory effect of EAB-309 (0 to 1 µmol/L)
on N-CEase activity in LPDS-DMEM. It reduced the activity in a
concentration-dependent manner. These results strongly indicate that
cholesterol is one of the potent endogenous
modulators for N-CEase activity.
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Effects of Cholesterol on Expression of N-CEase
mRNA
Northern blot analysis was performed to determine whether
these cholesterol-mediated changes in N-CEase activity are
associated with the expression of N-CEase mRNA. Based on evidence
showing that N-CEase from macrophages and HSL from mouse
adipose tissue are products from a single gene,26 a
cDNA probe corresponding to nucleotides 1515 through 1923
of mouse HSL was used. Expression of N-CEase mRNA in cells incubated
for 3, 6, 9, and 12 hours with cholesterol (30
µg/mL) in LPDS-DMEM was unaltered compared with those in cells
incubated without cholesterol (Fig 6).
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Discussion |
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In the course of our investigation of the mechanism by which HDL mobilizes CE in foam cells specifically focusing on N-CEase, we have found that cholesterol downregulates N-CEase activity.
Cholesterol efflux rates from foam cells incubated in LPDS-DMEM or HDL were slower in our experiments with J774 A.1 cells compared with previous results in mouse peritoneal macrophages.15 38 The slow rate of cholesterol efflux in our study is probably due to the lack of apolipoprotein E synthesis in J774 A.1 cells39 and partly a result of the use of whole HDL fraction in place of HDL3 fraction used in other experiments.15 38 Human monocyte-derived macrophages, mouse peritoneal macrophages, and the human macrophage cell line THP-1 express apolipoprotein E gene and secrete apolipoprotein E,40 41 42 whereas the J774 A.1 cell does not express an apolipoprotein E gene.39 HDL3-mediated efflux is very active in cells secreting with apolipoprotein E but not in J774 A.1 cells. J774 A.1 cells transfected with a human apolipoprotein E cDNA expression vector, however, enhanced cholesterol efflux to HDL3.43
cAMP-dependent N-CEase activity, which has neutral pH optima, is present in adipose tissues,18 the heart,44 the adrenal cortex,45 the testis,46 arterial smooth muscle cells,47 and a variety of other cells, including macrophages.17 This enzyme serves to supply cholesterol for steroid hormone synthesis in the endocrine glands.48 49 There is a similarity between HSL and N-CEase. Small et al.50 51 have shown that an antibody against HSL from adipose tissue completely inhibits the N-CEase activity in both mouse peritoneal macrophages and the WEHI macrophage cell line. This antibody recognized a protein band with a molecular mass of 84 kD, identical to that of HSL.50 51 Recently, Khoo et al.26 have reported that macrophage N-CEase and HSL from the adipose tissue are both products of a single gene.
We observed in this study that incubation of J774 A.1 cells with
either HDL or LDL altered N-CEase activity in opposite directions: HDL
progressively enhanced the activity, whereas LDL decreased it with
increasing concentration. Furthermore, we found that N-CEase activity
in cells changes to a great degree, depending on the incubation media;
incubation in FCS-DMEM with a much higher cholesterol
concentration (31 µg/mL) gave significantly lower N-CEase
activity than incubation in LPDS-DMEM with a lower
cholesterol concentration (1.9 µg/mL). These
results suggested that HDL-induced mobilization of CE is presumably
mediated by N-CEase and N-CEase activity changes, depending on cellular
cholesterol concentration. We have shown that cellular
cholesterol is a potent regulator for N-CEase by the
following experiments: (1) complete blocking of the enhancement of
N-CEase in LPDS-DMEM by an addition of cholesterol to the
medium, (2) progressive inhibition of N-CEase by increasing
cholesterol concentration in the medium, and (3) opposite
changes of N-CEase activity in cells treated with HMG-CoA reductase
inhibitors and an ACAT inhibitor. This
downregulation of N-CEase by cholesterol was observed in
mouse peritoneal macrophages (Table 4) and in adipocytes
transformed from 3T3-L1 cells (data not shown).
The N-CEase assay was not significantly influenced by the presence of the endogenous substrate in enzyme preparations because we used the supernatant of cell homogenate (43 000g) as the enzyme source. The CE content was similar in the various supernatant preparations. These supernatant contained <1/10 CE in an assay tube compared with the amount of the added substrate.
Cellular cholesterol homeostasis is maintained by a balance between the supply through the exogenous and endogenous pathways, and the removal through the reesterification and the efflux. When cells accumulate excess sterol, they suppress uptake of exogenous sterol through repression of the gene for the LDL receptor.52 Synthesis of endogenous cholesterol is reduced through repression of the genes for several enzymes in the cholesterol biosynthetic pathway, including HMG-CoA reductase and HMG-CoA synthase.52 Studies using cloned genes for these three proteins showed that this negative feedback regulation is mediated at the transcriptional level and is determined by sequences (sterol regulatory elements) in the 5' flanking regions of the genes.52 It was reported recently that sterols block the proteolytic maturation of sterol regulatory element binding proteins to its active form, which binds to sterol regulatory element-1 and controls the transcription activity.53 Sterol-regulated proteolysis appears to mediate the first step of the cleavage of sterol regulatory element binding proteins followed by a sterol-independent cleavage.54
ACAT is known to be upregulated by cholesterol.34 ACAT esterifies cholesterol to CE when a certain threshold level of cholesterol is reached in cells. The mechanism for the upregulation is reported to be posttranscriptional,55 but the nature of the critical posttranscriptional event has not yet been clearly elucidated. Recently, Schissel et al56 reported that the treatment of macrophage and CHO cells with cysteine protease inhibitors leads to the inhibition of cholesterol esterification, and they speculated that an endogenous protease plays an important role in cholesterol esterification, perhaps by cleaving an endogenous inhibitor of the interaction of the expanded cholesterol pools with ACAT.
In our experiment, Northern blot analysis indicated that the expression of N-CEase mRNA was not altered by an addition of cholesterol in the incubation medium. Although a more sensitive assay such as a competitive reverse-transcriptase polymerase chain reaction might be applied to differentiate a rather small change in N-CEase mRNA level in the future studies, our result suggests the possibility that altered transcription of a gene encoding N-CEase other than HSL could account for the difference in the N-CEase activity observed. Contreras and Lasunción27 reported that HSL mRNA was not detected in human monocyte-derived macrophages by means of reverse-transcriptase polymerase chain reaction technique and suggested that a different and as-yet-unidentified enzyme must be present to mobilize CE from foam cells in human macrophages. Another possibility is the cholesterol-dependent appearance of factors modifying N-CEase activity. Overloading macrophages with cholesterol stimulates the cells to synthesize apolipoprotein E,57 58 lipoprotein lipase,59 60 protease,61 and various cytokines. The expression of apolipoprotein E by macrophages is enhanced by increasing cellular cholesterol concentration.57 58 This is achieved primarily at the level of apolipoprotein E gene transcription.41
Shand et al62 63 have reported that a number of
cells, including hepatocytes62 and
macrophages,63 contain a cytosolic protein
inhibitor of N-CEase, and the inhibitor
activity can be increased under conditions characterized by enhanced
accumulation of CE.63 Bottalico et al64 found
that a marked increase in an isoform of -enolase is associated with
macrophage foam cell formation. Shand and West65
later reported that enolases have the inhibitory activity
of N-CEase and suggested that glycolytic enzyme enolase play a
secondary function as a negative regulator of the hydrolytic phase in
the CE cycle. Besides this inhibitor protein expression
associated with foam cell formation, the appearance of specific
phosphatases to dephosphorylate phospholylated
N-CEase,66 the active form of N-CEase, and the expression
of proteases to degrade the enzyme might be involved in
cholesterol-mediated change of N-CEase activity.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by a grant for basic research on human sciences from Human Science Foundation of Japan (1995) and a research promotion fund from Ono Pharmaceutical Co Ltd in Japan.
Received April 22, 1996; accepted August 26, 1996.
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