(Received for publication, August 19, 1994; and in revised form, September 30, 1994)
From the
Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes the conjugation of long chain fatty acid and cholesterol to form cholesteryl esters. It is an integral membrane protein located in the endoplasmic reticulum. Experiments performed in intact mammalian cells have shown that the rate of cholesteryl ester synthesis in intact cells, as well as the ACAT activity from cell extracts, are greatly activated by the addition of low density lipoprotein (LDL) or oxygenated sterols such as 25-hydroxycholesterol to the growth medium. However, the molecular mechanism(s) by which sterol(s) stimulate the ACAT activity remains to be elucidated. Recently, our laboratory reported the expression cloning of human ACAT cDNA (Chang, C. C. Y., Huh, H. Y., Cadigan, K. M., and Chang, T. Y. 1993) J. Biol. Chem. 268, 20747-20755). In the current study, we report the expression of human ACAT cDNA in insect Sf9 cells. Uninfected Sf9 cells do not express detectable ACAT-like activity. Infecting these cells with recombinant virus containing ACAT cDNA caused these cells to express high levels of ACAT protein and high levels of ACAT activity when assayed in vitro. The catalytic properties of ACAT expressed in these cells were found to be similar to those found in human tissue culture cells. The combination of high level of ACAT protein expression and the low level of cellular cholesterol content in the infected cells have provided us a novel opportunity to establish a simple cell-free system, whereby stimulation of ACAT by sterols can be readily demonstrated. Using this system, we have shown that cholesterol itself can serve as an ACAT activator in vitro, in addition to its role as an ACAT substrate. The current work provides the experimental basis to hypothesize that, inside mammalian cells, cholesterol itself may serve as a physiological regulator of ACAT.
Acyl-coenzyme A:cholesterol acyltransferase (ACAT) ()is an intracellular enzyme that catalyzes the conjugation
of long chain fatty acids and cholesterol to form cholesteryl esters.
ACAT is believed to play important roles in lipoprotein assembly, in
dietary cholesterol absorption, and in cholesterol homeostasis (for
reviews see Suckling and Stange, 1985; Chang et al., 1994b).
Under pathological conditions, accumulation of ACAT reaction products
as foamy lipid droplets in the cytoplasm of macrophages and smooth
muscle cells is a characteristic feature of early lesions of
atherosclerotic plaques (for reviews see Brown and Goldstein, 1983;
Ross, 1986). Cholesterol ester synthesis and ACAT activity in the
arteries of experimental animals are increased upon feeding of
cholesterol to induce experimental atherosclerosis (for early
references see Chang and Doolittle, 1983). Experiments performed in
cultured human fibroblasts and CHO cells have shown that the rates of
cholesteryl ester synthesis in intact cells, as well as the ACAT
activity from cell extracts, are greatly activated by the addition of
low density lipoprotein (LDL) or oxygenated sterols such as
25-hydroxycholesterol to the growth medium (Goldstein et al.,
1974; Brown et al., 1975; Doolittle and Chang, 1982a). Results
from studies employing mammalian cell mutants defective in
intracellular cholesterol trafficking (Kruth et al., 1986;
Liscum et al., 1989; Cadigan et al., 1990; Dahl et al., 1992) suggest that the release of LDL-derived free
cholesterol from the lysosomal compartment may be required for ACAT
activation. Other evidence suggests that most of the newly liberated
LDL-bound cholesterol released from the lysosome may not move directly
to ACAT, but first move to the plasma membrane (Tabas et al.,
1988; Lange et al., 1993; for a review, see Liscum and Faust,
1994).
The process of activation of ACAT by LDL or by 25-hydroxycholesterol is insensitive to the protein synthesis inhibitor cycloheximide (Goldstein and Brown, 1977; Doolittle and Chang, 1982b), implying that the elevation of ACAT activity is not due to a change in enzyme content. A reconstituted vesicle assay for measuring ACAT activity in vitro (Cadigan and Chang, 1988; Doolittle and Chang, 1982a) has been developed. This method allowed ACAT activity to be measured independently of the enzyme's original lipid environment. Cell extracts prepared from CHO cells or human fibroblasts incubated in sterol-containing medium or in sterol-depleted medium had ACAT activities that vary up to 20-fold. The differences in the activity were essentially abolished in the reconstituted vesicle assay, supporting the notion that modulating the amount of ACAT enzyme may not be an important mechanism for regulating ACAT activity by sterols in mammalian cells. However, the molecular mechanism(s) responsible for sterol regulation of ACAT remains to be elucidated.
ACAT is an integral membrane protein located in the endoplasmic reticulum (ER). This protein exists only in small quantities and little progress has been made to purify the protein in an active form from crude cell homogenates (Doolittle and Chang, 1982a). Recently, our laboratory reported the cloning of human ACAT cDNA K1 by a somatic cell genetic and molecular biological approach (Chang et al., 1993, reviewed in Chang et al., 1994b). This gene has been localized to human chromosome 1 q25 (Chang et al., 1994a). Transfecting of the coding region of cDNA K1 into an ACAT-deficient mutant CHO cell line resulted in the expression of ACAT activity. Heat inactivation studies indicated that the expressed ACAT activities were of human origin. This result, along with protein sequence homology analysis, indicated that the open reading frame (ORF) of cDNA K1 encodes a polypeptide essential for ACAT catalysis (Chang et al., 1993).
In the current study, we report the expression of ACAT human cDNA in insect Sf9 cells. Uninfected Sf9 cells do not express detectable ACAT-like activity (see ``Result''). Infecting these cells with recombinant virus containing ACAT cDNA K1 caused these cells to express high levels of ACAT protein and high levels of ACAT activity assayed in vitro. The catalytic properties of ACAT expressed in these cells were found to be similar to those found in human tissue culture cells. We also observed that the cellular cholesterol content of Sf9 cells grown in medium with 10% fetal bovine serum (FBS) is much lower (by approximately 10-20-fold) than the values typically found in various mammalian cells grown under the same condition. The combination of high level ACAT protein expression and the low level cellular cholesterol content in the infected cells has provided us a novel opportunity to establish a simple cell-free system, whereby stimulation of ACAT by sterols can be readily demonstrated. Using this cell-free system, we have shown that cholesterol itself can serve as an ACAT activator, in addition to its role as an ACAT substrate.
An antiserum was raised in rabbit against a synthetic decapeptide (EKNNHRAKDL) located at amino acid residues 103-112 of the predicted human ACAT protein sequence (Chang et al., 1993). The specific IgGs of this antiserum were purified by affinity column chromatography using the peptide EKNNHRAKDL as the ligand. The affinity column was synthesized and utilized according to the instructions of Pierce ImmunoPure Ag/Ab Immobilization Kit. Western blots were prepared essentially according to the method of Towbin et al.(1979) and briefly described as follows: after the samples were separated by SDS-PAGE on 10% acrylamide slab gels, the proteins were transferred to Immobilon-P (Millipore, Bedford, MA) at 300 mA for 4 h. The transfer buffer consisted of 25 mM Tris, 190 mM glycine, 20% methanol, and 0.005% SDS. Proteins were immunoblotted as follows: the blots were blocked in 5% Carnation nonfat milk and probed with 2.5 µg/ml of the affinity-purified specific antibody in 1% Carnation milk. The horseradish peroxidase-conjugated enzyme immunoassay grade affinity-purified goat anti-rabbit IgG (from Bio-Rad) at 1/3000 dilution in 1% milk was used as the secondary antibody to probe the immunoabsorbed proteins on the blot. The buffer used for blotting and washing was 20 mM Tris-HCl, 150 mM NaCl, and 0.3% Tween-20 at pH 7.6. The ECL Western blotting detection reagent was used to for detection.
The
[S]methionine metabolic labeling technique was
utilized to analyze the newly synthesized proteins after the Sf9 cells
were infected with the recombinant virus. It is known that baculovirus
infection results in shut-off of host gene expression and host protein
synthesis such that, by 24-36 h post-infection, gene expression
is primarily viral-specific (O'Reilly et al., 1992). As
shown in Fig. 1A, at 48-h post-infection, two prominent
metabolically labeled protein bands, with apparent sizes at 50 and 56
kDa, were detected after SDS-PAGE (lane 4). In contrast, cells
infected with the control recombinant virus containing
-galactosidase (lane 8) or with the wild-type virus (lane 12) produced the expected 110-kDa
-galactosidase
protein band or the 32-kDa viral polyhedrin protein band, respectively.
We next examined proteins of the infected Sf9 cells by Western blot
analysis. An affinity purified, anti-peptide antibody raised in rabbit
against the synthetic decapeptide EKNNHRAKDL, located at amino acid
103-112 of the predicted human ACAT protein sequence, was
employed to probe the Western blot. This antibody specifically
recognized proteins produced in Sf9 cells infected with the
ACAT-recombinant virus (lanes 2-5, Fig. 1B). In contrast, no signal was detectable by this
antibody in cell extracts prepared from Sf9 cells infected with the
control recombinant virus or with the wild-type virus (lanes 6 and 7). Before the 36 h post-infection time point, the
antibody mainly recognized the 50 kDa protein band only. However, after
48 h, an additional 56 kDa band, as seen in Fig. 1A, lane 4, became apparent. The same results were obtained
irrespective of whether the following methods were used to prepare the
protein samples: 1) lysis buffer (8 M urea and 2% SDS) with or
without 1%
-mercaptoethanol, with or without various protease
inhibitors; 2) lysis buffer with different SDS concentrations (from 1
to 5%); 3) boiling urea-SDS buffer to solubilize the cells, or long
periods (30-60 min) of low heating (50 °C) to treat protein
samples prior to electrophoresis; 4) treatment of intact cells with the N-glycosylation inhibitor tunicamycin at 20 µg/ml in the
growth medium for 6 h before cell lysis. In addition, the same results
were seen using three independently isolated and purified recombinant
viral particles. Another unusual, and perhaps related observation is
that, the apparent size of the 56-kDa protein and the 50-kDa protein in
SDS-PAGE deviate significantly from the estimated size of the
hypothetical ACAT protein (63.8 kDa) deduced from the ORF analysis of
human ACAT cDNA K1 1.7 kb (Chang et al., 1993). We noticed
from the protein sequence analysis that the hypothetical ACAT protein
K1 is a hydrophobic, membrane-spanning protein with a calculated
isoelectric point (PI) of 9.78. In the literature, there is ample
precedent for a discrepancy in molecular weight estimation among
membrane proteins (for example, Kartner et al., 1991, and
earlier references cited therein). Perhaps the combination of the
hydrophobic and basic nature of the ACAT protein causes a higher amount
of SDS associated with it and makes the ACAT protein move faster in
SDS-PAGE. The reason(s) for the appearance of the 50/56 kDa doublet
protein bands at 48 h post-infection time remains obscure. It is
possible that, due to the abnormal protein structure, the 56-kDa
protein may represent a minor portion of the ACAT protein that
maintains a certain non-random configuration under various denaturing
conditions. This hypothesis can explain the existence of the 100 and
200 kDa bands seen in the immunoblots, which may be the dimeric and
tetrameric forms of the ACAT protein; these aggregates can not be
completely dissociated into monomers even under the harsh conditions
described above. However, other possibilities cannot be ruled out. More
experiments are needed to clarify these observations.
Figure 1:
Functional expression of ACAT cDNA K1
in Sf9 cells. A, time course of protein synthesis in
baculovirus-infected Sf9 cells. Sf9 cells were infected with
recombinant baculovirus containing ACAT cDNA K1 1.7 kb (lanes
1-4) or -galactosidase (lanes 5-8) or
with wild-type baculovirus (lanes 9-12). The cells were
metabolically labeled with [
S]methionine and
were lysed at 12 (lanes 1, 5, and 9), 24 (lanes 2, 6, and 10), 36 (lanes 3, 7, and 11), or 48 (lanes 4, 8, and 12) h post-infection as described under ``Experimental
Procedures.'' Each lane was loaded with
50 µg of protein
and was analyzed by 10% SDS-PAGE. The gel was fixed and
autoradiographed at -80 °C for 1 h. B, immunoblot
analysis of proteins from Sf9 cells uninfected or infected with
recombinant virus containing ACAT cDNA K1 or control viruses. Cell
lysates of uninfected Sf9 cells (lane 1), Sf9 cells infected
with ACAT-recombinant virus (lanes 2-5), with
-galactosidase-recombinant virus (lane 6), or with
wild-type virus (lane 7) were prepared after 12 h (lane
2), 24 h (lane 3), 36 h (lane 4), or 48 h (lanes 5-7) of infection. Infections were performed in a
staggered manner such that at the end, cells were harvested at the same
time. Approximately 50 µg of protein was loaded in each lane. After
10% SDS-PAGE, proteins were transferred onto a filter, and Western blot
analysis was performed as described under ``Experimental
Procedures.'' C, analysis of ACAT activity in Sf9 cells
infected with recombinant viruses at various points post infection. Sf9
cells were infected with ACAT-recombinant virus (
),
-galactosidase-recombinant virus (
), or wild-type virus
(
). At each time point post-infection, the cell lysates were
prepared, and the ACAT activities were measured by the reconstituted
vesicle assay as described under ``Experimental Procedures.''
Similar results as reported in A-C were obtained in three
independent experiments. The inset of C gives the
result of a separate experiment, which showed dependence of ACAT
activity on cholesterol concentration present in the reconstituted
vesicles. ACAT activities were measured by the reconstituted vesicle
assay; vesicles containing 10 mg/ml egg phosphatidylcholine (PC) and various indicated concentrations of cholesterol,
prepared according to the procedure described previously (Ventimiglia et al., 1986). The values are the averages of duplicate assays
from the same detergent-treated extracts and varied within 10% of the
mean.
To test whether these virally produced proteins possess ACAT enzyme activity, we measured ACAT activity in Sf9 cells in vitro at various post-infection time points (Fig. 1C). To ensure that the measured ACAT activity would be independent of cellular lipid composition, we employed a solubilization and reconstitution into vesicle procedure (Cadigan and Chang, 1988). Uninfected Sf9 cells or cells infected with the control recombinant virus or with the wild-type virus did not exhibit any ACAT activity throughout the time course. In contrast, Sf9 cells infected with the ACAT recombinant virus began to express increasing amount of ACAT activity with time. At 48 h post-infection, the specific activity of ACAT was at least 10-fold higher than those detected in various human cell lines, including human A431 cells and human fibroblast cells (Cadigan and Chang, 1988). In the reconstituted vesicle assay, the ACAT enzyme residing in the vesicle utilizes cholesterol present in the same vesicle as its substrate. This assay avoids the step of cholesterol transfer between vesicles. Thus, a sensitive activity-cholesterol concentration curve could be demonstrated. In the inset of Fig. 1C, the shape of the ACAT activity-cholesterol concentration curve was shown to be sigmoidal (instead of hyperbolic), suggesting cooperative interaction between cholesterol and the ACAT protein during catalysis. A similar observation was made when extracts of human fibroblasts, or CHO cells, or pig liver microsome were used as the ACAT enzyme source (Doolittle and Chang, 1982a, 1982b; Cadigan and Chang, 1988).
This laboratory previously reported that the ACAT activity in CHO cells is more thermolabile than the ACAT activity in human cells (Cadigan et al., 1989; Chang et al., 1993). This result may reflect the structural difference between the CHO cell ACAT protein and the human cell ACAT protein. In order to examine the heat stability of ACAT activity expressed in the Sf9 cells, a heat inactivation experiment was carried out. As shown in Fig. 2, ACAT activity expressed in Sf9 insect cells exhibits heat stability comparable to that of human ACAT; its heat stability is significantly greater than that of hamster ACAT.
Figure 2:
Heat
inactivation of ACAT activities in CHO 25-RA cells (), T2-8
cells (CHO cell line transfected with human ACAT) (
), human A431
cells (+), and Sf9 cells infected with the human ACAT-recombinant
virus (
). Sf9 cells infected with ACAT recombinant virus at 5
pfu/cell were harvested at 48 h post-infection time point. 25-RA and
T2-8 cells were grown in F-12 medium + 10% fetal bovine
serum. A431 cells were grown in Dulbecco's modified Eagle's
medium + 10% fetal bovine serum. They were harvested, and the cell
extracts were prepared at
2 mg protein/ml, treated with 1%
deoxycholate, and reconstituted into vesicles according the previously
described method (Cadigan and Chang, 1988). The reconstituted vesicles
were incubated at 45 °C for various indicated times, then placed on
ice until assayed for enzyme activity. The control activity (without
heat treatment) for 25-RA, T2-8, A431, and infected Sf9 cells
were 97.9, 41.8, 18.9, 479.8 pmol/min/mg protein, respectively. The
values are the averages of duplicate assays from the same
detergent-treated extracts and varied within 10% of the
mean.
Three active site-directed ACAT inhibitors (CI 976, octimibate, and
Dup 128), each with distinct structural features, were employed to
probe the sensitivity of active site(s) of ACAT expressed in Sf9 cells.
The ACAT inhibition curves (Fig. 3) indicate that insect
cell-expressed ACAT activity shows similar sensitivity to inhibition as
the CHO cell ACAT activity and the human cell ACAT activity. For all
the ACAT activities examined, the IC values for CI 976,
octimibate, and Dup 128 were approximately 5, 50, and 0.5
µM, respectively. These values are similar to those
reported by Harte et al.(1993) using rat liver ACAT as the
enzyme source.
Figure 3:
Dose-response curves of ACAT inhibitors on
ACAT activities from various sources. Sf9 cells infected with ACAT
recombinant virus at 5 pfu/cell for 48 h were used. Cell extracts were
prepared from infected Sf9 cells (), 25-RA cells (
),
T2-8 cells (
), A431 cells (
), or wild-type CHO cells
(
). The reconstituted cell lysates, prepared according to the
previously described method (Cadigan and Chang, 1988), were incubated
with CI 976 (A), octimibate (B), or Dup 128 (C) at various final concentrations for 20 min on ice.
Me
SO (DMSO) was used as the solvent of the ACAT
inhibitors. Final concentration of Me
SO in all the assay
tubes was 2.5% (v/v). The ACAT assays were performed as described
(Cadigan and Chang, 1988). The curves were drawn according to the
values obtained from Sf9 cell-expressed ACAT activity (
). The
control ACAT activities (2.5% Me
SO treated) for 25-RA,
T2-8, A431, wild-type CHO, and infected Sf9 cells were 93.6,
51.0, 19.3, 67.8, and 550.9 for pmol/min/mg protein, respectively. The
values are the averages of duplicate assays from the same detergent
extracts and varied within 10% of the mean.
To find out whether the human ACAT enzyme expressed in Sf9
cells can be activated by sterols added in growth medium, we used the
[H]oleate pulse method to measure the cholesterol
esterification rate in intact cells. Sf9 cells infected with the
ACAT-expressing virus grown in 10% FBS medium exhibited basal
esterification rate, at approximately 0.03-0.1 pmol/min/mg, which
was at least 300-fold less than the values found in mammalian cells
grown under similar condition (30-100 pmol/min/mg; Goldstein et al., 1974; Chang et al., 1986). Adding
25-hydroxycholesterol to the growth medium caused a very large increase
in cholesterol esterification rate; the fold increase varied between
20-60-fold in several different experiments. The activation by
25-hydroxycholesterol was both time- (Fig. 4A) and
concentration-dependent (Fig. 4B). At a concentration
of 5 µg/ml, the time required to cause maximal activation was
approximately 60 min. The concentration to cause half-maximal
activation was approximately 1-2 µg/ml when the cells were
treated with 25-hydroxycholesterol for 60 min. In control experiments,
we found that in uninfected or mock infected Sf9 cells, treated or
untreated with 25-hydroxycholesterol, the cholesterol esterification
rate was undetectable (result not shown). To examine if sterols other
than 25-hydroxycholesterol possess a similar capacity to activate
cholesterol esterification, we tested 7-ketocholesterol,
6-ketocholestanol, 7
-hydroxycholesterol,
-sitosterol,
cholate, deoxycholate, and cholesterol. The result (Fig. 5)
showed that none of these sterols were nearly as effective as
25-hydroxycholesterol in activating cholesterol esterification in
intact cells.
Figure 4:
Activation of cholesterol esterification
rate by 25-hydroxycholesterol in intact Sf9 cells expressing human
ACAT. A, time dependence. B, concentration
dependence. Sf9 cells grown in 25-cm flasks were infected
with ACAT recombinant virus at 5 pfu/cell. After infection for 48 h,
cells were incubated with 5 µg/ml of 25-hydroxycholesterol at 37
°C for various time as indicated (A) or were incubated
with various indicated concentrations of 25-hydroxycholesterol at 37
°C for 60 min (B). Cholesterol esterification rate as
measured by the [
H]oleate pulse assay in intact
cells was performed according to the procedure described under
``Experimental Procedures.'' The final concentrations of
ethanol in the media were kept at 0.1% in all the flasks. The values
are the averages of duplicate flasks and varied within 10% of the mean.
Similar results were reproduced in three other independent
experiments.
Figure 5:
Sterol specificity in activating the
cholesterol esterification rate in intact Sf9 cells expressing ACAT.
The experiment was performed as described in Fig. 4. The symbols
were: , 25-hydroxycholesterol;
, cholesterol;
,
7-ketocholesterol;
, 6-ketocholestanol;
,
-sitosterol;
, 7
-hydroxycholesterol;
, cholate;
, deoxycholate. The values are the averages of duplicate flasks
and varied within 10% of the mean.
To examine if the activation by 25-hydroxycholesterol involves an increase in ACAT protein content in cells, we used immunoblotting to monitor the ACAT protein levels and found that neither the content nor the eletrophoretic mobility of ACAT protein underwent detectable changes, after the intact cells were treated with 25-hydroxycholesterol with concentrations ranging from 0.1 to 10 µg/ml in the medium (Fig. 6).
Figure 6: ACAT protein electrophoretic mobility and content after treating intact cells with varying concentrations of 25-hydroxycholesterol. The experiment was conducted as described in Fig. 4B. A parallel set of cell culture were used for infection and incubations, and whole-cell protein lysates were prepared by lysing the cells in 8 M urea, 2% SDS. Western blot analysis was performed as described under ``Experimental Procedures.'' Lane 1 represents lysate from the infected cells without any treatment. Lanes 2-7 represent lysates from infected cells treated with 0, 0.1, 0.5, 1, 5, or 10 µg/ml 25-hydroxycholesterol, respectively. 25-Hydroxycholesterol was delivered from ethanol stock solution. The final concentrations of ethanol in media during the incubation for lanes 2-7 were 0.1%.
To characterize the in vitro ACAT activation system, we next tested the effect of incubation temperature on the degree of activation. The result presented in lines 2 and 3 of Table 1showed that incubating 25-hydroxycholesterol with extracts at two different temperatures (4 or 37 °C) did not affect the degree of activation (6.7- versus 7.2-fold); we did observe that incubating cell extracts at 37 °C for 30 min caused approximately a 2-fold increase in basal ACAT activity (from 8.6 pmol/min/mg to 16.5 pmol/min/mg; similar observations were made in two separate experiments). In another experiment, we tested the possible effect of incubation time on the degree of activation and found that 25-hydroxycholesterol added at 10 µg/ml to the cell extracts at 37 °C reached its maximal activating effect within 10 min (result not shown). For the purpose of consistency, we chose a 30-min sterol incubation time at 37 °C to perform the remainder of the experiments reported in this article.
Using the in vitro condition described above, we examined
the effect of sterol concentration and found that the concentration of
25-hydroxycholesterol needed to cause half-maximal activation of ACAT
activity was 1-2 µg/ml (Fig. 7), a value similar to
the value found to cause half-maximal activation of cholesterol
esterification rate in intact cells (Fig. 4B). We next
compared the effect of various sterols (at 10 or 100 µg/ml) on ACAT
activity and found that two oxysterols, 7-ketocholesterol and
6-ketocholestanol, added at high concentration (100 µg/ml), caused
modest increase in ACAT activity, while 7-hydroxycholesterol
caused significant inhibition in ACAT activity.
-Sitosterol,
cholate, or deoxycholate did not cause detectable change in ACAT
activity. Among the various sterols tested, other than
25-hydroxycholesterol, the only sterol that caused significant increase
in ACAT activity was cholesterol (Fig. 8). Since cholesterol is
the substrate for the ACAT enzyme, we could not conclude from this
result whether the stimulation of ACAT by cholesterol was due to an
increase in substrate availability, or was due to a specific activating
effect in addition to the substrate effect.
Figure 7: Activation of ACAT activity in vitro by adding varying amounts of 25-hydroxycholesterol to the cell extracts. Sf9 cells were infected with ACAT-recombinant virus for 48 h at 5 pfu/cell. Cell extracts were prepared as described under ``Experimental Procedures.'' The cell extracts were incubated with various indicated concentrations of 25-hydroxycholesterol at 37 °C for 30 min. After incubation, extracts were assayed immediately for ACAT activity. The final ethanol concentration present in each assay tube was 1%. The values are the averages of duplicate assays from the same cell extracts and varied within 5% of the mean.
Figure 8: Sterol specificity in activating ACAT activity in vitro. Conditions for virus infection, cell extract preparation, and incubation with different sterols and ACAT assay in vitro were the same as described in Fig. 7, except cholate and deoxycholate were delivered from stock solutions in water at 1/100 volume of the cell extract. The final concentration of ethanol in each tube was adjusted to 1%. The control value indicated by the arrow is the value obtained after the cell extract was incubated with 1% ethanol alone. Solid bars represent the values obtained after cell extracts were incubated with 10 µg/ml of sterol; hatched bars represent the values obtained after the cell extracts were incubated with 100 µg/ml of sterol. The values are the averages of the duplicate assays from the same cell extracts and varied within 5% of the mean.
We tested the stimulation effect of 25-hydroxycholesterol on ACAT in vitro in the presence of varying amounts of cholesterol. Cell extracts from cells unexposed to 25-hydroxycholesterol were preincubated with increasing amounts of cholesterol, then incubated with or without 10 µg/ml 25-hydroxycholesterol, and assayed for ACAT activity. The result (Fig. 9A, replotted in Fig. 9B) showed that as the cholesterol concentration added to the cell extracts increased, the fold activation of ACAT activity by 25-hydroxycholesterol decreased (from 3.5- to 1.5-fold).
Figure 9:
A, cholesterol dose-response curve of ACAT
activity in vitro, with or without preincubating the cell
extracts with 25-hydroxycholesterol. Conditions for virus infection,
cell extract preparation, and ACAT assay were as described in Fig. 7. Cell extracts were preincubated with () or
without (
) 10 µg/ml 25-hydroxycholesterol at 37 °C for
30 min. Then various indicated amounts of cholesterol were added, and
an additional incubation at 37 °C for 30 min was performed before
the extracts were assayed for ACAT activity. Final concentration of
ethanol in each tube was at 2%. B, fold activation of ACAT
activity by 25-hydroxycholesterol diminishes as cholesterol
concentration in the cell extracts increases. This is a replot of A. Fold activation of ACAT activity is defined as
ACAT activity with 25-hydroxycholesterol preincubation divided by ACAT
activity without 25-hydroxycholesterol preincubation at a given
cholesterol concentration in cell extracts. Similar results were
reproduced in two other independent
experiments.
Figure 10:
Cholesterol activation of ACAT activity in vitro using 25-hydroxycholesterol as the substrate.
Conditions for Sf9 cell infection, preparation of cell extracts were as
described in Fig. 7.
[H]25-Hydroxycholesterol (2.5 pmol, 0.05 µCi)
in 1 µl of ethanol was delivered to each 60 µl of cell extracts
kept at 0 °C. After a brief vortex, either 10 µg/ml final
concentration of cholesterol was added (
) or vehicle ethanol
(
) was added (A); various amounts of cholesterol as
indicated were added (B). Final concentration of ethanol in
each tube was adjusted to 2%. The mixtures were incubated at 37 °C
30 min. Esterification of 25-hydroxycholesterol was initiated by adding
40 µl of assay mixture containing 12.5 mg/ml fatty acid-free bovine
serum albumin, 0.25 mM oleyl-CoA, 100 mM Tris-HCl, pH
7.8. The assay time was as indicated in A and was 5 min in B. Lipid extraction procedure and TLC analysis was as
described under ``Experimental Procedures.'' The values are
the averages of duplicate assays from the same cell extracts and varied
within 5% of the mean. The same results were seen in two independent
experiments.
Using
[H]25-hydroxycholesterol as the substrate, we
found that cholesterol added at 10 µg/ml activated the
25-hydroxycholesteryl ester formation by approximately 3-fold (Fig. 10A). If cholesterol was not an activator, but
served only as a substrate for ACAT, one would have seen that
cholesterol competed with the
[
H]25-hydroxycholesterol and reduced
[
H]25-hydroxycholesteryl ester formation. The
result shown in Fig. 10, therefore, is a demonstration that
cholesterol also serves as an activator for ACAT. The activation by
cholesterol was concentration-dependent; approximately 1-2
µg/ml of cholesterol was required to cause half-maximal activation (Fig. 10B).
In this study, we have shown that expression of human ACAT
cDNA K1 1.7 kb in Sf9 insect cells caused these cells to produce ACAT
activity. Sf9 cells do not contain endogenous ACAT-like activity.
Infecting these cells with the ACAT gene-containing recombinant
baculovirus for 48 h caused these cells to express ACAT, with specific
activities at least 10 times higher than those found in various human
cell lines. Time course experiments showed that the expression of ACAT
activities correlated well with the appearance and accumulation of new
polypeptides, which were detected by both the
[S]methionine metabolic labeling and Western
blotting. The heat stability characteristics of the Sf9 cell-expressed
ACAT activity were very similar to that of the human enzyme.
Additionally, the inhibition constants of three structurally different
ACAT inhibitors against the Sf9 cell-expressed ACAT activity were very
similar to those against the ACAT activities produced by various
mammalian cell lines. These ACAT inhibitors contain different
functional groups (
)and are believed to be targeting at the
ACAT active site(s). Our results thus strongly suggest that the active
site(s) of Sf9 cell-expressed ACAT is very similar to the ACAT enzyme
present in human and hamster cell lines. Taken together, these data
demonstrate that the cDNA K1 1.7 kb encodes an essential catalytic
component of the ACAT enzyme. At present, we cannot unambiguously prove
that the protein encoded by cDNA K1 alone constitutes the entire ACAT
holoenzyme. This question can only be appropriately addressed by
examining the biological activities of the protein purified to
homogeneity. High level expression of the ACAT K1 protein reported here
will facilitate this investigation.
Using the intact Sf9 cells expressing ACAT to study ACAT regulation by sterols, we showed that the cholesterol esterification rate and the ACAT activity (using endogenous cholesterol as its substrate) in these cells was highly elevated by adding 25-hydroxycholesterol to the culture medium. Cholesterol added to the medium in the same manner failed to elicit the same response. The dependence in sterol concentration and the rapidity of the activation process (Fig. 4, A and B) are very similar to the earlier observations made in various mammalian cells (Goldstein et al., 1974; Doolittle and Chang, 1982a). Since the DNA elements of the ACAT gene other than the protein coding region were not included in constructing the recombinant virus, we can conclude that the ACAT activation process described in Fig. 4is strictly a post-translational event. Western blotting analysis showed that the increase in ACAT activity in oxysterol-treated cells did not involve any increase in the levels of the ACAT protein. However, our results do not exclude the possibility of transcriptional or translational mechanism in sterol-dependent regulation of ACAT in mammalian cells. The high specific activity of ACAT found in the extracts of ACAT-expressing Sf9 cells (higher than 1 nmol/min/mg; Fig. 1C) was measured by using in vitro assay A, utilizing optimal amounts of cholesterol present in reconstituted vesicle as the substrate. If in vitro assay B, utilizing endogenous cholesterol present in the Sf9 cell extract as the substrate was used, the specific activity of ACAT of these cells (5-20 pmol/min/mg) was actually lower than those found in various mammalian cells grown and assayed under the similar condition (30-50 pmol/min/mg in human fibroblasts or in CHO cells; Cadigan and Chang, 1988). We also noted that the cholesterol esterification rate in these cells grown in medium containing 10% FBS was much lower (0.03-0.1 pmol/min/mg, Fig. 4) than the rates found in various mammalian cells grown under similar conditions (versus 50-100 pmol/min/mg; Goldstein et al., 1974; Chang et al., 1986). Taken together, these results suggest that the ACAT enzyme residing within the cell interior rarely encounters cholesterol; when extracts were prepared from these cells, the ACAT activity remains largely dormant (based on the results using assay B). Using the cell extracts prepared from ACAT-expressing Sf9 cells as the enzyme source, we were able to demonstrate significant activation of ACAT by 25-hydroxycholesterol in vitro (Fig. 7). The capacity of 25-hydroxycholesterol to act as an ACAT activator was shown to be gradually diminished by adding increasing amounts of cholesterol to the cell extracts (Fig. 9). This result could explain why in earlier studies, when extracts of mammalian tissues or mammalian cells were used as the ACAT enzyme source, it was difficult to show a large increase in ACAT activity (no more than 2-fold) by adding oxysterol in vitro (Lichtenstein and Brecher, 1980; Erickson et al., 1980). During the cell homogenization step, the relatively high cholesterol content endogenously present in mammalian tissues might have adhered onto the ACAT protein and largely masked the effect of 25-hydroxycholesterol.
Previously, we and others (for early references, see Chang and Doolittle, 1983) had shown significant stimulation of ACAT catalyzed reaction by adding cholesterol in vitro. However, since cholesterol is a substrate of ACAT, one could not determine from these studies whether the stimulatory effect of cholesterol was due to its effect as a substrate, or as an activator, or as both. Using the in vitro system described here, and by exploiting the enzymatic characteristics of ACAT, which can use 25-hydroxycholesterol as an alternative substrate, we have now demonstrated that cholesterol itself served as an ACAT activator (Fig. 10). The unique features of the in vitro system described here (i.e. high enzyme expression in a low cholesterol environment) may be generally applicable for demonstrating specific effects of cholesterol on other enzymes or proteins involved in cholesterol or other metabolic pathways (Jingami et al., 1987; Skalnik et al., 1988; Wang et al., 1994).
It has long been speculated that inside the mammalian cells the true activator of ACAT may be an oxysterol, such as 25-hydroxycholesterol, rather than cholesterol itself. This hypothesis was based on the finding that in intact cells certain oxygenated sterols, when added from ethanol stock solutions to the culture medium, greatly stimulated cholesterol esterification rate and increased ACAT activity; when cholesterol was tested, it failed to elicit the same response (reproduced in the current study (Fig. 5)). To explain the negative result when cholesterol was tested as the stimulating agent in this type of experiment, an alternative interpretation is that the highly compartmentalized distribution of cholesterol in intact cells (DeGrella and Simoni, 1982; Lange and Ramos, 1983; reviewed by Dawidowicz, 1987) has caused the spatial separation of the incoming cholesterol from ACAT located at the cell interior. Once broken cell homogenates are used cholesterol added to the cell extracts is readily available to ACAT. If cholesterol itself can activate ACAT, then under carefully chosen conditions its activating effect should be readily demonstrable. The result of our current work has provided direct evidence in support of the latter interpretation. While not negating the possibility that oxysterol(s) may be physiological regulator(s) of ACAT under certain situations, our current work provides the experimental basis to hypothesize that, inside the mammalian cells, cholesterol itself may serve as a physiological regulator of ACAT.
The molecular mechanism by which cholesterol activates ACAT in vitro is not clear at present. In principle, cholesterol could activate ACAT activity by one of at least three mechanisms: first, it is possible that binding of cholesterol by ACAT causes a ligand induced structural/configurational change in the ACAT protein, which converts the enzyme from an inactive form to an active form (Monod et al., 1963; Koshl and and Neet, 1968). The ligand-induced configurational change may occur through interactions among multiple subunits in the ACAT holoenzyme, since the molecular mass of the functional ACAT enzyme has been reported to be approximately 220 kDa (Erickson et al., 1994), which is about four times the value as estimated by SDS-PAGE. This model explains the recurring finding that, the ACAT activity-cholesterol concentration curve is sigmoidal, once ACAT is reconstituted in vesicles with a defined lipid composition (inset of Fig. 1C; Doolittle and Chang 1982a, 1982b; Cadigan and Chang, 1988). If this model turns out to be correct, it would be interesting to find out, within the same ACAT protein, whether the cholesterol-binding site that causes activation to occur is the same as or distinct from the catalytic site for cholesterol esterification. It would also be important to determine, if 25-hydroxycholesterol shares the same site(s) with cholesterol. Since ACAT is believed to span the phospholipid bilayer (Chang et al., 1993), it is possible that the interaction between sterol and ACAT protein may involve phospholipid as another lipid participant. The second possibility is that there may be a cholesterol-induced specific covalent modification of ACAT protein, which in turn activates the enzyme. It is difficult to rationalize why such a mechanism would exist in the extracts of Sf9 cells which normally do not contain endogenous ACAT-like activity. However, the ACAT protein expressed in these cells may contain intrinsic activity for covalently modifying and activating itself upon encountering cholesterol. This model can be rigorously tested once the ACAT protein is purified to homogeneity. The third possibility is that cholesterol activates the ACAT catalysis not by acting on the ACAT protein, but by facilitating the translocation/delivery of substrate to the ACAT catalytic site(s). While this remains as a viable mechanism to explain some of the activation effects of oxysterols on ACAT activity seen in intact mammalian cells, it is not compatible with the result presented in Fig. 10, in which 25-hydroxycholesterol at 0.1 µg/ml was used as the substrate to measure ACAT activity. At this concentration, 25-hydroxycholesterol is 100% soluble in water (Sinensky(1981) reported that the solubility of 25-hydroxycholesterol in water is approximately 0.4 µg/ml). It is therefore difficult to envision a specific mechanism by which cholesterol added to the extracts could facilitate the delivery of a water-soluble substrate to ACAT. In any event, utilizing the cell-free system reported in the current study, further biochemical analyses will shed light on the molecular mechanism(s) by which cholesterol/oxysterol activates the ACAT activity.
During the preparation of this article, Becker et al.(1994) reported that expression of the full-length cDNA encoding the human macrophage carboxylesterase enzyme in wild-type CHO cells led to an approximately 3-fold increase in cellular ACAT activity. This gene was previously cloned and sequenced by Munger et al.(1991) who showed that it encodes the major serine esterase contained within and secreted by alveolar macrophages. This enzyme is also present in abundance in liver microsomes (Ozols, 1989). Shown by various investigators, this enzyme is capable of catalyzing the hydrolysis of esters, amides, and thioesters and is believed to function as a detoxication enzyme (Heymann, 1980). In vitro experiments have shown that long chain fatty-acyl CoAs are poor substrates for this enzyme (Kroetz et al., 1993). Ozols provided evidence(1989) that this enzyme resides within the lumen of the ER. This result suggests that physiologically this enzyme would not be able to use long chain fatty-acyl CoAs as its normal substrates, since the ER membranes are not permeable to long chain fatty-acyl CoAs (for a review, see Coleman and Bell, 1983). Since the human carboxylesterase gene has been expressed in Sf9 cells (Kroetz et al., 1993) it would be interesting to see if it constitutes ACAT-like enzyme activity with catalytic and regulatory properties consistent with what would be expected from the literature.