From the Institute of Biochemistry and Cell Biology,
Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, Shanghai 200031, China and the § Department of
Biochemistry, Dartmouth Medical School, Hanover, New Hampshire
03755
Received for publication, December 20, 2000, and in revised form, February 23, 2001
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ABSTRACT |
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Acyl-coenzyme A:cholesterol acyltransferase
(ACAT) is an intracellular enzyme involved in cellular cholesterol
homeostasis and in atherosclerotic foam cell formation. Human
ACAT-1 gene contains two promoters (P1 and P7), each
located in a different chromosome (1 and 7) (Li, B. L., Li,
X. L., Duan, Z. J., Lee, O., Lin, S., Ma, Z. M., Chang,
C. C., Yang, X. Y., Park, J. P., Mohandas, T. K.,
Noll, W., Chan, L., and Chang, T. Y. (1999) J. Biol
Chem. 274, 11060-11071). Interferon- ACAT1 is an
intracellular enzyme responsible for catalyzing the intracellular
formation of cholesteryl esters from cholesterol and long-chain fatty
acyl-coenzyme A (1). In mammals, two ACAT genes have been
identified (2-5). In adult human tissues, ACAT-1 is the major enzyme
present in various tissues, including macrophages, liver (hepatocytes
and Kupffer cells), and adrenal gland (6, 7). ACAT-1 is also present in
the intestine; however, the major enzyme involved in the intestinal
cholesterol absorption may be ACAT-2, which is mainly located in the
apical region of the intestinal villi (7). The relative tissue
distributions of ACAT-1 and ACAT-2 in mice and monkeys are not entirely
consistent with those found in humans (8, 9) raising the possibility
that the distribution of the two ACATs in various tissues may be
species dependent. In macrophages and other cell types, a dynamic
cholesterol-cholesteryl ester cycle exist; the formation of
intracellular cholesteryl esters is catalyzed by ACAT-1, while the
hydrolysis of cholesteryl esters is catalyzed by the enzyme neutral
cholesteryl ester hydrolase (10, 11). The net accumulation of
intracellular cholesteryl esters is affected at the substrate level, as
well as at the levels of the enzymes ACAT and neutral cholesteryl ester
hydrolase (12-14). The main mode of sterol-specific regulation of
ACAT-1 has been identified at the post-translational level, involving
allosteric regulation by its substrate cholesterol (1, 15). On the
other hand, the cellular and molecular nature of non-sterol-mediated ACAT-1 regulation remains largely unknown. Recently, using mouse macrophage-derived foam cells, Panousis and Zuckerman (12) reported that IFN- Cell Culture and Treatments--
Human monocytes were isolated
according to a published procedure (21) with slight modification: human
leukocyte packs were obtained from Shanghai Blood Service Center and
used within 1 day. The cells were diluted (2:1, v/v) with cold
phosphate-buffered saline (PBS), layered on an equal volume of
Ficoll-Paque (Amersham Pharmacia Biotech), and centrifuged for 20 min
at 2,500 rpm at room temperature. Mononuclear cells were collected and
washed three times at 4 °C (to remove platelets) by adding 100 ml of PBS followed by centrifugation at 1,000 rpm for 10 min. The remaining red blood cells in the pellet were lysed by treatment with 10 ml of
0.2% NaCl for 45 s, followed by sequential additions of 10 ml of
1.6% NaCl and 30 ml of cold PBS. The pelleted cells were suspended in
cold RPMI 1640 with 7% human type AB serum to a density of 5 × 106/ml, plated onto 60-mm tissue culture dishes that were
precoated with 2 ml/dish of FBS, and incubated for 90 min at 37 °C.
Next, the dishes were washed three times with warm RPMI 1640 (37 °C) to remove unadhered cells. The adhered cells were judged to be more
than 95% monocytes by Reagents--
Human type AB serum was from Sigma. Fetal
bovine serum was obtained from Life Technologies, Inc. (Life
Technologies, Grand Island, NY). Purified recombinant human IFN- Chimeric Plasmid Construction--
The 632-bp DNA fragment
containing the human ACAT-1 P1 promoter ( Deletion and Site-directed Mutagenesis of the Sp1 and GAS
Elements in ACAT-1 P1 Promoter in pGL2-E--
Deletions were achieved
by PCR-mediated mutagenesis using the corresponding set of mutant
primers that included a 6-bp KpnI or NheI linker
flanking the primer sequences on the vector. The primer sequences on
the vector are: GLP1, 5'-TGTATCTTATGGTACTGTAACTG-3'; GLP2,
5'-CTTTATGTTTTTGGCGTCTTCCA-3'. The primers used to make 5'- and
3'-deletion mutations (to generate the STAT1 Mutant Expression Plasmid--
The human
STAT1 cDNA (encoding the 750 amino acids of STAT1
protein) in pRC/CMV (pRC/CMV-STAT1) was a gift from Dr. Darnell (Rockefeller University). The mutant STAT1 expression
plasmid was created using the site-directed mutagenesis procedure of Ho et al. (27). Two sets of primers
5'-GGAGAGAAGCTTCTTGGT-3'/5'-CTGAAGTCTAGAAGGGTG-3' and
5'-AAGGAACTGGATTTATCAAGACTGA-3'/5'-TCAGTCTTGATAAATCCAGTTCCTT-3' were
used to mutate STAT1 at amino acid 701 (the Jak1/2 phosphorylation site) from tyrosine to phenylalanine (28). The expression plasmid pRC/CMV-STAT1-Y701Fm was constructed by inserting the mutant PCR product digested with HindIII/XbaI into the same
sites of pRC/CMV.
Transfection and Luciferase Assay--
A series of
ACAT-1 P1 promoter/luciferase reporter (Luc) constructs were
transfected into THP-1 or U937 cells using the DEAE-dextran method (29,
30). After washing twice with PBS, 1 × 106
cells were transfected with 1.5 µg of ACAT-1 promoter/Luc
plasmid and 0.75 µg pCH110 as internal control in 1 ml of STBE (25 mM Tris-HCl, pH 7.4, 5 mM KCl, 0.7 mM CaCl2, 137 mM NaCl, 0.6 mM Na2HPO4, 0.5 mM
MgCl2) containing 150 µg of DEAE-dextran. The cells were
incubated for 20 min at 37 °C, washed once with RPMI 1640 without
FBS, then resuspended in 5 ml of fresh RPMI 1640 with 10% FBS, and
plated at 2 × 105 cells/ml/well in a 24-well plate
for 40 h. HepG2, CACO-2, and HEK293 cells were transfected by the
methods of calcium phosphate co-precipitation essentially as described
by Liu et al. (31). Briefly, cells were plated at 1 × 105 cells/well in 1 ml of medium in 24-well tissue culture
plates 1 day before transfection. One h before transfection, cells were replaced with fresh medium. Calcium phosphate precipitates containing (per well) 0.3 µg of ACAT-1 promoter/Luc and 0.15 µg of
pCH110 were prepared. The DNA/calcium phosphate precipitates were
incubated with the cells at 37 °C for 8 h, after which time the
cells were washed once with PBS, and replaced with Dulbecco's modified
Eagle's medium or minimal essential medium containing 10% FBS. After
incubation for 7 h, cells were treated with or without IFN- Reverse Transcription-Polymerase Chain Rreaction--
The total
RNA (4 µg) prepared according to the single step acid guanidinium
thiocyanate phenol chloroform method (Trizol Regent, Life Technologies,
Inc.) was annealed with 1 µg of oligo(dT) (12-18 in length) in a
total volume of 20 µl and reverse transcribed with 5 units of avian
myeloblastosis virus reverse transcriptase (Life Technologies, Inc.) at
42 °C for 50 min, and then diluted to a volume of 80 µl as the
ss-cDNA product. The 4 µl of diluted ss-cDNA product was
added to a reaction mixture in a final volume of 20 µl containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.5 mM dNTP, 0.5 mM each pair of primers, and 1 unit of Taq DNA
polymerase (Life Technologies, Inc.). To serve as controls, GAPDH gene
expression was assessed to ascertain that equal amounts of cDNA
were added to each PCR. The PCR products (10 µl), taken at several
different cycles (from 26 to 32), were separated in agarose gel and
quantified by using the UVP Labwork Software (UVP Inc.). The sets of
primers used are 5'-AAAGGAGTCCCTAGAG-3'/5'-GGATGAGAACTCTTGC-3' for ACAT-1 P1 product (hACAT-1 cDNA K1 1486-2043,
amplifying a 558-bp fragment),
5'-ACCCACCATTATCTAA-3'/5'-ACCCACCATTATCTAA-3' for human
ACAT-1 P7 product (hACAT-1 cDNA K1 982-1670,
amplifying a 689-bp fragment) (2, 26),
5'-GCCCGACCCTATTACAAAAA-3'/5'-CTGCCAACTCAACACCTCTG-3' for STAT1 coding
sequence (amplifying a 646-bp fragment) (32) and
5'-GAGTCAACGGATTTGGTCG-3'/5'-GAAGTGGTGGTACCTCTTCC-3' for GAPDH (amplifying a 291-bp fragment) (32).
Electrophoretic Mobility Shift Assays (EMSAs)--
Nuclear
extract was prepared as described (33). THP-1 cells were harvested and
washed twice with cold PBS at 4 °C, and resuspended gently in 400 µl of buffer A (10 mM HEPES, pH 7.9, 10 mM
KCl, 1.5 mM MgCl2, 0.5 mM
dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride), and
stored on ice for 10 min, then vortexed for 10 s. Nuclei were
pelleted (10,000 × g, 10 s), resuspended with
ice-cold buffer C (20 mM HEPES, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride), and incubated on ice for
20 min. The mixture was subjected to centrifugation (10,000 × g) for 2 min at 4 °C, and the supernatant as nuclear
extract was stored in aliquots at RNA Preparation and Northern Blot
Analysis--
THP-1 cells were cultured at 2 × 105/ml in 60-mm dishes. Human blood monocytes were cultured
at 1.5 × 106/60-mm dishes. Cells were treated with
IFN- Western Blot Analysis--
Cells were harvested with 10% SDS in
50 mM Tris, 1 mM EDTA (pH 7.5) with 25 mM dithiothreitol, and incubated at 37 °C for 20 min,
then sheared with a syringe fitted with an 18-gauge needle. Protein
concentration of the cell extract was determined by a modified Lowry
method (34). The affinity purified anti-ACAT-1 IgGs (designated as
DM10) was used as the primary antibodies against ACAT-1 (35). Western
blots, using freshly prepared cell extracts in SDS, were conducted
according to a previously described procedure (35).
ACAT Activity Assay--
The assay was performed essentially as
described previously (15, 35). AC29, 25RA, and THP-1 cells were
cultured at 2 × 105/ml in 60-mm dishes and then
treated in various manners for 40 h as indicated. The
ACAT-1-deficient mutant cell line AC29, and its parental cell 25RA
derived from Chinese hamster ovary cells (2, 15, 35) were used to
ensure that the ACAT activity assayed in vitro work
properly. For THP-1 cells, the suspended and adherent cells were
collected by direct centrifugation and scrapping, respectively, at room
temperature. The two groups of cells collected from the same dish were
pooled together, washed with PBS once, and centrifuged to collect the
cell pellets. Cold 1 mM Tris, 1 mM EDTA
(pH 7.8), at 100 µl/sample, was added to cell pellet chilled on ice.
The mixtures were left on ice for 5 min. Brief but vigorous vortexing
(30 s to 1 min) was used to cause extensive cell lysis. The protein
concentration of the cell homogenates was kept at 2-4 mg/ml in buffer
A (50 mM Tris, 1 mM EDTA at pH 7.8 with
protease inhibitors). The enzyme was solubilized and assayed in mixed
micelle condition as previously described (35).
A 159-bp Core Region with 4 Sp1 Elements Is Responsible for Human
ACAT-1 P1 Promoter Activity--
Human ACAT-1 gene is
located in two different chromosomes (1 and 7), each chromosome
containing a separate ACAT-1 promoter (P1 and P7). Northern
analyses have revealed the presence of four ACAT-1 mRNAs (7.0, 4.3, 3.6, and 2.8-knt) in all the human tissues and cell lines examined (3).
The 2.8 and 3.6-knt messages are produced from the P1 promoter, while
the 4.3-knt mRNA is produced from two different chromosomes by a
novel RNA recombination event that presumably involves trans-splicing
(26). The P1 promoter is contiguous with the coding sequence and spans
from
To demonstrate the functional importance of the 4 Sp1 elements involved
in basal transcription, we next performed EMSA. We used nuclear
extracts of THP-1 cells and the 159-bp DNA fragments containing
mutations in each of all the 4 Sp1 elements as labeled probes. The
bindings of labeled probes were tested by competing with unlabeled
wild-type or mutated probes in 100-fold molar excess. The results (Fig.
2C) illustrated that the wild-type DNA fragment formed
several DNA-protein complexes (lanes 2, 4, and
5); the bindings were eliminated upon incubation with excess
unlabeled probe (lane 3) and supershifted by incubation with
the anti-Sp1 antibodies (lane 5). Additional control
experiments showed that the fragment containing mutations in all 4 Sp1
elements had no specific binding (lanes 7-10).
Interferon- The Combination of IFN-
To ascertain that the activating effect of ATRA/IFN- IFN-
To further examine the functional significance of the GAS element, we
isolated nuclear extracts from THP-1 cells co-treated with IFN- ATRA Induces STAT1 Expression, While IFN- ACAT-1 mRNAs and protein contents are significantly increased
during the human monocyte-macrophage differentiation process in
vitro (21, 41). Its protein content is amply present in macrophage-derived foam cells localized in the human atherosclerotic lesion, implying that up-regulation of the ACAT-1 gene plays
important roles in macrophage foam cell formation in atherosclerosis
(42). In mouse macrophages, ACAT-1 message was found to be
up-regulated by cells with IFN- In atherosclerosis, the infiltration of T-cells and monocyte-derived
macrophages into the intimal layer of the artery is believed to lead to
foam cell formation. Activated T cells found in human atheroma secrete
high levels of IFN- IFN- (IFN-
), a cytokine that exerts many pro-atherosclerotic effects in vivo,
causes up-regulation of ACAT-1 mRNA in human blood
monocyte-derived macrophages and macrophage-like cells but not in other
cell types. To examine the molecular nature of this observation, we
identified within the ACAT-1 P1 promoter a 159-base pair
core region. This region contains 4 Sp1 elements and an IFN-
activated sequence (GAS) that overlaps with the second Sp1 element. In
the monocytic cell line THP-1 cell, the combination of IFN-
and
all-trans-retinoic acid (a known differentiation
agent) enhances the ACAT-1 P1 promoter but not the P7
promoter. Additional experiments showed that
all-trans-retinoic acid causes large induction of the
transcription factor STAT1, while IFN-
causes activation of STAT1
such that it binds to the GAS/Sp1 site in the ACAT-1 P1
promoter. Our work provides a molecular mechanism to account for the
effect of IFN-
in causing transcriptional activation of
ACAT-1 in macrophage-like cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
increased the cellular cholesteryl ester content and reduced high density lipoprotein-mediated cholesterol efflux; its cellular effects were attributed to its ability to increase ACAT-1 message (12) and to induce down-regulation of the
Tangier Disease gene (the ABC1 transporter) (16). In the current work, we showed that IFN-
increased ACAT-1 message and protein
content in human monocyte-derived macrophages. To examine the molecular mechanism of IFN-
action on ACAT-1 gene regulation in
macrophages, we identified the important cis-acting elements in the
human ACAT-1 P1 promoter. In order to perform transient
transfection experiments, we used THP-1 cell, a monocytic human cell
line as the cell model. Upon treatment with retinoids, including
all-trans-retinoic acid (ATRA), THP-1 cells differentiate
into macrophage-like cells (17-20). Our results show that ATRA and
IFN-
synergistically caused up-regulation of ACAT-1 gene
expression. Additional experiments revealed that ATRA causes increased
gene expression of the transcription factor STAT1, while IFN-
is
essential to cause STAT1 to undergo phosphorylation dependent
dimerization and to bind to the GAS site present in the
ACAT-1 P1 promoter.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-naphthylacetate esterase staining. The cells
were cultured for up to 16 days in RPMI 1640 medium supplemented with
7% human type AB serum, with a medium change every other day. Other
cell lines were from ATCC. Cells were incubated 60-mm dishes in a
37 °C incubator with 5% atmospheric CO2. All media were
supplemented with 100 µg/ml kanamycin, 50 units/ml streptomycin, 2 g/liters sodium bicarbonate, plus 10% fetal bovine serum (FBS). THP-1
and U937 cells were grown in RPMI 1640 medium. HepG2 and Caco-2 cells
were grown in Dulbecco's modified Eagle's medium. HEK293 cells were
grown in minimal essential medium medium. Chinese hamster ovary cell
lines AC29 and 25RA (22, 23) were grown in F12 medium.
(1 × 107 units/mg of protein) was a generous gift
from Professor Xin-yuan Liu (24) at the Shanghai Institute of
Biochemistry and Cell Biology. ATRA was from Sigma. Rabbit
anti-Sp1 (PEP2, catalog number sc-59-G, 200 µg/ml) and anti-STAT1
(C-111, catalog number sc-417, 200 µg/ml) polyclonal antibodies were
from Santa Cruz Biotechnology. [
-32P]- and
[
-32P]dATP (6000 Ci/mmol) were from Amersham Pharmacia
Biotech. CHAPS, taurocholate, oleoyl-coenzyme A, egg
phosphatidycholine, cholesteryl oleate, cholesterol, and
fatty acid-free bovine serum albumin were all from Sigma.
Reagent-grade solvents were from Fisher. [3H]Oleoyl-coenzyme A was chemically synthesized as
described (25). Radioactive reagents were from Amersham Pharmacia
Biotech.
598/+34) (26) was
inserted into the multiple cloning sites of the luciferase reporter
gene vector pGL2-E (Promega). This fragment was stepwise deleted from
both ends by various suitable restriction endonucleases to create
plasmids that contained the
324/+34,
188/+34,
125/+34,
598/
126, and the
598/
189 fragments, respectively.
110/+34,
100/+34,
100/
7,
100/
17, and the
100/
27 fragments) were
5'-aaaggtaccGGTGGGCGGAAC-3', 5'-aaaggtaccACTGGCAACCTG-3',
5'-aaagctagCCGGCCCCTACGC-3', 5'-aaagctagCGCCCCCTGCCTC-3', and
5'-aaagctagCTCCGAGCACCGC-3', respectively. The PCR products were then
digested with KpnI and NheI and subcloned into an
empty pGL2-E vector. The fidelity of all these constructs was verified by sequencing. Site-directed mutagenesis was undertaken using a
modification of the procedure described by Ho et al. (27). Briefly, two overlapping fragments of the promoter subcloned into the
pGL2-E vector were amplified separately. The first reaction used a
flanking primer that hybridized with the vector at the 5'-end of the
inserted sequence, and an internal primer that hybridized at the site
of the desired mutation and contained the mismatched base. The second
reaction included one flanking primer that hybridized with the vector
at the 3'-end of the inserted sequence, and an internal primer that
overlapped with the site of the desired mutation and also contained
the mismatched base. The two overlapping fragments generated by PCR are
"fused" by denaturing and annealing in a subsequent primer
extension reaction. Finally the "fusion" product was amplified by
PCR using the primers GLP1 and GLP2. The product of the final PCR was
digested with KpnI and NheI and subcloned into
the pGL2-E vector. To guard against PCR-associated nucleotide incorporation errors, the integrity of all the constructs generated was sequenced using an automated ABI Prism 377 DNA sequencer
(Perkin-Elmer Applied Biosystems, Canada Inc., Mississaug, ON). To
generate the fragments Sp1-1m, Sp1-2m, Sp1-3m, Sp1-4m, and GASm,
respectively, the following sets of primer pairs were used:
5'-CCTCCCCGTTCCGGTACCTCCCC-3'/5'-GGGGAGGTACCGGAACGGGGAGG-3', 5'-CAGTTCCGTTCACCTCCCCGCC-3'/5'-GGCGGGGAGGTGAACGGAACTG-3',
5'-GAGGCAGGAAGCGTAGGGGCCG-3'/5'-CGGCCCCTACGCTTCCTGCCTC-3', 5'-GGGCGTAGAAGCCGGGCTGTCC-3'/5'-GGACAGCCCGGCTTCTACGCCC-3',
5'-GTTGCCAGCCCCGCCCACCTCC-3'/5'-GGAGGTGGGCGGGGCTGGCAAC-3'. For
additional mutagenesis work, we used Sp1-1m as the template and
5'-CAGTTCCGTTCACCTCCCCGTT-3'/5'-AACGGGGAGGTGAACGGAACTG-3' as the primer
pair to generate Sp1-12m, used Sp1-12m as the template and
5'-GAGGCAGGAAGCGTAGGGGCCG-3'/5'-CGGCCCCTACGCTTCCTGCCTC-3' as the primer
pair to generate Sp1-123m, used Sp1-123m as the template and
5'-AAGCGTAGAAGCCGGGCTGTCC-3'/5'-GGACAGCCCGGCTTCTACGCTT-3' as the primer
pair to generate Sp1-1234m.
(100 units/ml), or ATRA (10
6 M), or IFN-
(100 units/ml) plus ATRA (10
6 M). 40 h
later, the cells were harvested and the cell pellets were lysed in 200 µl of lysis buffer (Reporter lysis buffer, Promega, catalog number
E397A), vortexed for 5 s, and spun at 2000 × g for 5 min at room temperature. 60 µl of the cell lysate was mixed with 60 µl of luciferase assay buffer (Promega) for luciferase activity measurement (Promega Instruction Bulletin Part number TB101)
in an Auto Lumat BG-P luminometer (MGM Instrument Inc.). For
-galactosidase activity assay, the luminescent
-galactosidase detection Kit II was used (CLONTECH User Manual
PT2106-1).
80 °C. For EMSA, 10 µg of
protein of nuclear extract was incubated for 10 min on ice in 10 µl
of binding buffer (10 mM Tris-HCl, pH 7.5, 1 mM
MgCl2, 4% glycerol, 50 mM NaCl, 0.5 mM dithiothreitol, 0.5 mM EDTA, and 3 µg of
poly(dI)-(dC) from Amersham Pharmacia Biotech Inc.). DNA probes were
labeled using T4 polynucleotide kinase (Promega) and [
-32P]dATP. 1 ng of labeled probe (~1 × 104 dpm) was added to the binding reaction mixture and
incubated at 25 °C for 30 min. For "supershift" analyses, 1 µl
of each antibody as indicated was added and incubated 30 min at
25 °C before adding the probe. Binding reactions were size
fractionated on a nondenaturing 4.5% acrylamide gel (29:1, mass:mass,
acrylamide:N,N'-methylenebisacrylamide), ran at 200 V for
3 h in 0.5 × TBE buffer. The gel was dried and autoradiographed with PhosphorImager scanning system.
as indicated for 40 h before harvest. The preparation of
total RNA was according to the single step acid guanidinium thiocyanate
phenol chloroform method (Trizol Regent, Life Technologies, Inc.).
Total RNA, 20 µg/sample, were electrophoresed in a 1% agarose gel
containing 2.2 M formaldehyde and transferred to a Nytran
membrane (Schleicher and Schuell, Dassel, Germany) with 3.0 M sodium chloride, 0.3 M sodium citrate
(20 × SSC) as the transfer buffer. The membrane was cross-linked
by UV irradiation and incubated for 10 min at 65 °C in 0.5 M sodium phosphate buffer (pH 7.2), 7% SDS, 1 mM EDTA (prehybridization buffer). The polymerase chain
reaction products of human ACAT-1 cDNA (1486-2686, 1.2-kilobases)
and human GAPDH cDNA (291-bp) were used as templates for labeling
probes. Labeled probes were made with [
-32P]dATP by
the random primer method using a Random-labeling Kit (Promega). Blots
were prehybridized and hybridized with labeled probes and washed under
high stringency conditions. Hybridization was carried out at 65 °C
in the same solution as prehybridization, except for the addition of
labeled probe. The membrane was washed with 40 mM sodium
phosphate buffer (pH 7.2), 0.1% SDS at room temperature for 5 min
three times, and at 65 °C for 20 min. After washing, the membrane
was exposed and the intensity of the bands was quantified by
densitometric analysis using the UVP Labwork software (UVP Inc.). To
serve as control, rehybridization of the same blot with the human GAPDH
probe was carried out. The sample mRNA expression levels were
normalized by the intensity of the human GAPDH mRNA bands.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
598 to +65 of the ACAT-1 genomic DNA (26). To
determine the minimal region of the P1 promoter, we transfected THP-1
cells with constructs containing various deleted fragments fused
upstream to a luciferase reporter gene of the pGL2-enhancer vector
(pGL2-E), and measured luciferase activities. As shown in the
right panel of Fig.
1A, the results indicated that
the maximal transcriptional activity is located within the 159-base
pair from
125 to +34 (Fig. 1A). Sequence analysis by
computer revealed 4 Sp1 elements are located in this core region (Fig.
1B). We next performed various specific deletion analyses to
test the relative importance of these 4 Sp1 elements. The results (Fig.
2, A and B) showed
that the most important basal transcription activity is present in the
first two Sp1 elements from the 5'-end of the 159-bp core region.
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Fig. 1.
A 159-bp core region comprises the basal
transcriptional activity of ACAT-1 P1 promoter.
A, Luc constructs (bars shown on the left
panel) containing serial 5' and 3' deletions as indicated between
598 and +34 of the ACAT-1 P1 promoter were co-transfected
with pCH110 into THP-1 cells. The cells were harvested 48 h after
transfection for activity assays. The luciferase activity per each cell
extract was normalized by using the
-galactosidase value found in
the same cell extract. The reporter construct activities shown on the
right panel were expressed as relative luciferase
activities, using the value of the reporter activity driven by the SV40
promoter as one. Values were means of triplicate determinations. Sizes
of error bars indicated 1 S.E. B, nucleotide
sequence analysis of ACAT-1 P1 promoter core region. The
four Sp1 elements were boxed. Asterisks indicate the three
major transcriptional initiation sites (Li et al.
(26)). Sequence of Exon 1 was underlined.
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Fig. 2.
Four Sp1 elements are functionally present in
ACAT-1 P1 promoter. A, Luc
constructs containing serial 5'- and 3'-deletion (bars shown
on the left) of the 159-bp core region were transfected into
THP-1 cells. The luciferase activities (shown on right) were
determined in the same manner as described in the legend to Fig.
1A. The promoterless plasmid pGL2-E was used as a negative
control. B, Luc constructs containing single or multiple Sp1
mutations (marked by the ×) of the 159-bp core region were transfected
into THP-1 cells. The luciferase activities shown on right
panel were determined as described in the legend to Fig. 1A.
C, EMSAs using nuclear extracts of THP-1 cells. The wild-type and
Sp1-1234-mutant DNA fragments of ACAT-1 P1 promoter
(depicted at the left panel) were, respectively, labeled and
1 × 104 dpm of labeled probe was used for each
binding reaction. Lane 1, 32P-labeled wild-type
DNA as probe alone. Lane 2, binding reaction between labeled
wild-type DNA probe and nuclear extracts. Lane 3,
competition by adding 100-fold molar excess of cold probe to the
binding reaction described for lane 2. Lane 4, competition
by adding 100-fold molar excess of nonspecific DNA to the binding
reaction described for lane 2. Lane 5, supershift reaction
by adding 1 µl of anti-Sp1 antibody to the binding reaction described
for lane 2. Lanes 6-10, the same conditions as described
for lanes 1-5, except using the Sp1-1234-mutant DNA as the
labeled probe.
Causes up-regulation of Human ACAT-1 Expression in
Blood Monocyte-derived Macrophages--
The human blood monocytes were
incubated in culture for up to 16 days. This procedure causes monocytic
cells to differentiate into mature macrophages within several days.
Cells incubated for various time points were treated with or without
IFN-
for 40 h. The total RNAs and proteins of treated and
untreated cells were extracted for RT-PCR and Western blot. The results
show that both the ACAT-1 P1 promoter transcript and the
ACAT-1 protein level increased during the monocyte differentiation
process; and these increases were further augmented in cells treated
with IFN-
(Fig. 3, A and
C). In contrast, the level of the human ACAT-1 P7
promoter transcript was not significantly altered throughout the time
course of the experiment (Fig. 3B).
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Fig. 3.
Effect of IFN- on
the ACAT-1 gene expression during the human blood
monocyte-macrophage differentiation process. Human blood monocytes
were cultured at 1.5 × 106/60-mm dish for various
days as indicated and then treated with or without IFN-
(100 units/ml) for 40 h before harvested for preparation of total RNA
and protein extract. A and B, quantitation by
RT-PCR (26 to 32 cycles). Appropriate primers described under
"Experimental Procedures" were used to obtain the ACAT-1
P1 promoter transcript (designated as the P1 product), the
ACAT-1 P7 promoter transcript (designated as P7 product),
and the transcript for the control gene (indicated as GAPDH) by RT-PCR.
Control experiments indicated that between cycles 25 and 35, the
ACAT-1 P1 transcript and the ACAT-1 P7 transcript
could be estimated semiquantitatively by RT-PCR (data not shown). The
ratio of DNA contents (shown at the bottom panels) was
determined by using the UVP Labwork software (UVP Inc.). C,
immunoblotting of ACAT-1 protein from extracts of blood
monocyte-derived macrophages treated with or without IFN-
(100 units/ml) for 40 h. Protein extracts were prepared and
immunoblotting were conducted as described under "Experimental
Procedures." Samples used (40 µg of protein/lane) were freshly
prepared with SDS. The membrane was incubated with DM10 (0.5 µg/ml)
as the primary antibody. The immunoreactive proteins were visualized
using the ECL detection system and autoradiography. The intensities of
bands were determined by using the UVP Labwork software (UVP Inc.). The
data are expressed as relative ACAT-1 protein level using the value in
untreated cells as 1.0. The ratios of the ACAT-1 protein from treated
or untreated cells were shown at the bottom panel.
and ATRA Is Needed to Enhance ACAT-1 P1
Promoter Activity in THP-1 Cell--
Using THP-1 cell, we tested the
functional responses of the ACAT-1 P1 promoter toward
IFN-
, ATRA, or a combination of both. As shown in Fig.
4A, treating cells with
IFN-
and ATRA, but not with IFN-
or with ATRA alone,
synergistically enhanced the luciferase expression driven by the
ACAT-1 P1 promoter. To investigate the cell type and
promoter specificity of this effect, we tested the human
ACAT-1 P1, P7, and SV40 promoters in THP-1 cells, using the
luciferase reporter activity assays. The results (Fig. 4A) showed that neither IFN-
, ATRA, nor their combination, had any detectable effect on the ACAT-1 P7 or the SV40 promoter. We
also tested the potential effect of ATRA and/or IFN-
on
ACAT-1 P1 promoter in other human cell lines including
HEK293, HepG2, CaCO-2, and U937. The results (Fig. 4B)
demonstrated that the synergistic effect of ATRA and IFN-
occurred
only in the monocytic cell lines (e.g. U937, THP-1), but not
in other cell types (e.g. HEK293, HepG2, or CACO-2). Using
THP-1 cells, we next investigated the time and dose requirements. For
IFN-
, the results showed that the enhancement exhibited a saturable
process, with maximal enhancement seen when IFN-
reached 500 units/ml (Fig. 5A). For ATRA,
a non-saturable, linear relationship between concentration and effect
was observed (Fig. 5B). The effect of IFN-
and ATRA
continued to increase within the time frame examined (60 h, Fig.
5C).
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Fig. 4.
The synergistic effect of
IFN- and ATRA on ACAT-1 P1
promoter is promoter specific and cell type specific.
A, the Lus constructs containing
ACAT-1 P1, or P7, or the SV40 promoters ligated to the
luciferase reporter vector (pGL2-C) were transfected into THP-1 cells.
7 h after transfection, cells were treated with or without IFN-
(100 units/ml), or with ATRA (10
6 M) or
IFN-
(100 units/ml) plus ATRA (10
6 M),
respectively. The luciferase activity was determined in lysates of
THP-1 cells 40 h later, and normalized by using values of
-galactosidase. The data were expressed as luciferase activities
relative to the value from untreated cells as one. Values represented
the means from triplicate determinations. Sizes of error
bars represented 1 S.E. B, the Luc construct containing
ACAT-1 P1 promoter was transfected into THP-1, U937, HepG2,
Caco-2, or HEK293 cells as indicated by methods described under
"Experimental Procedures." The cells were then treated and assayed
as described in A.
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Fig. 5.
The dose and time dependence of the
IFN- and ATRA effects. The Luc constructs
containing ACAT-1 P1 promoter was transfected into THP-1
cells. 7 h after transfection, A, cells were treated
with or without ATRA (10
6 M) alone, or with
ATRA plus the indicated concentrations of IFN-
, respectively, for
40 h; B, cells were treated with or without IFN-
(100 units/ml) alone, or with IFN-
plus indicated concentrations of
ATRA for 40 h; C, cells were treated with or without
ATRA (10
6 M) plus IFN-
(100 units/ml) for
the indicated lengths of time, respectively. Afterward, the luciferase
activity was determined as described under "Experimental
Procedures."
on
ACAT-1 promoter bears biological relevance, we treated THP-1
cells for 40 h with either IFN-
or ATRA, or a combination of
both, then examined the ACAT-1 at the levels of mRNAs, protein, and enzyme activities. Semiquantitative RT-PCR analysis showed that the
level of human ACAT-1 P1 promoter transcripts in treated
cells increased by about 3-fold (Fig.
6A), while the level of human ACAT-1 P7 promoter transcripts were not significantly
altered (Fig. 6B). Consistent with these results, Northern
blotting (Fig. 6C) showed that the amounts of 3.6- and
2.8-knt mRNAs (26) were significantly enhanced by the combination
treatment of IFN-
and ATRA. In addition, the ACAT-1 protein as
analyzed by Western blotting (Fig.
7A), as well as by the ACAT
enzyme activity, measured in cholesterol-independent manner, was all
significantly and proportionally increased (Fig. 7B).
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Fig. 6.
IFN- and ATRA
synergistically increase ACAT-1 mRNA. Total RNAs were prepared
from THP-1 cells treated for 40 h with or without IFN-
(100 units/ml), or with ATRA (10
6 M) or with
IFN-
(100 units/ml) plus ATRA (10
6 M),
respectively. A and B, quantitation by RT-PCR (26 to 32 cycles). Appropriate primers described under "Experimental
Procedures" were used to obtain the ACAT-1 P1 promoter
transcript (designated as the P1 product), the ACAT-1 P7
promoter transcript (designated as P7 product), and the transcript for
the control gene (indicated as GAPDH) by RT-PCR. Control experiments
indicated that between cycles 25 and 35, the ACAT-1 P1
transcript and the ACAT-1 P7 transcript could be estimated
semiquantitatively by RT-PCR (data not shown). The ratio of DNA
contents (shown at the bottom panels) was determined using
the UVP Labwork software (UVP Inc.). C, quantitation by
Northern analysis. 20 µg of total RNAs per lane from cells treated in
various manners as indicated was employed, using a
32P-labeled ACAT-1 cDNA probe; the same filter was
rehybridized with a 32P-labeled human GAPDH cDNA probe.
After exposing with PhosphorImager, the intensities of the 2.8- and
3.6-knt ACAT-1 were normalized to that of the GAPDH mRNA levels;
the intensities of bands were determined by using the UVP Labwork
software (UVP Inc.). The ratios of the 2.8- and 3.6-knt message from
cells treated in various manners as indicated were shown on the
right panel.
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Fig. 7.
IFN- and ATRA
synergistically increase ACAT-1 protein and enzyme activity.
A, immunoblotting of ACAT-1 protein from extracts
of THP-1 cells were treated in various manners as indicated. Cells were
seeded at 2 × 105/ml in 60-mm dishes with 7.5 ml of
medium, and treated for 40 h with or without IFN-
(100 units/ml), or with ATRA (10
6 M) or IFN-
(100 units/ml) plus ATRA (10
6 M) as
indicated. Cell extracts were prepared and immunoblotting were
conducted as described under "Experimental Procedures." Samples (40 µg of protein/lane) were freshly prepared with SDS. The membrane was
incubated with DM10 (0.5 µg/ml) as the primary antibody. The
immunoreactive proteins were visualized using the ECL detection system
and autoradiography. The intensities of bands were determined by using
the UVP Labwork software (UVP Inc.). The data are expressed as relative
protein using the value in untreated THP-1 cells as 1.0. The ratios of
the protein from cells treated in various manners as indicated were
shown at the bottom panel. B, ACAT activity assayed in
vitro. AC29, 25RA, and THP-1 cells were cultured at 2 × 105/ml in 60-mm dishes with 7.5 ml of medium and treated
for 40 h with or without IFN-
(100 units/ml), or with ATRA
(10
6 M), or with IFN-
(100 units/ml) plus
ATRA (10
6 M) as indicated. ACAT activities
were assayed as described under "Experimental Procedures." The data
are expressed as relative ACAT activity using the value in untreated
THP-1 cells as 1.0. The ACAT specific activity in untreated cell
extracts was 74 pmol/mg/min.
Activated Sequence (GAS) Is Required for the Synergistic
Effect by IFN-
and ATRA--
Sequence analysis by computer showed
that the core region of human ACAT-1 P1 promoter contained
an GAS that overlaps exactly with the second Sp1 element from 5'-end
(Fig. 8A). To test its functional significance, a series of P1 promoter deletion and point
mutation constructs were made, linked to a luciferase reporter gene,
and used in transient transfection studies in THP-1 cells. The results
indicated that the two constructs containing the GAS element (at the
top of Fig. 8A) responded to IFN-
and ATRA,
while the shorter promoter lacking the GAS element and first two Sp1 elements (at the bottom of Fig. 8A) did not.
Specific mutations in the GAS element, but not mutations in the Sp1
elements, abrogated the synergistic effect by IFN-
and ATRA (Fig.
8B). Therefore, the GAS element, rather than the 4 Sp1 sites
plays an important role in mediating the regulatory response to IFN-
and ATRA.
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Fig. 8.
Identification of a functional GAS element in
ACAT-1 P1 promoter. A,
individual Luc constructs containing the 159-bp core region (shown on
the left panel; with wild-type at the top, two different
deletions as indicated in the middle or the
bottom) were transfected into THP-1 cells. 7 h after
transfection, cells were treated with or without IFN- (100 units/ml), or with ATRA (10
6 M), or with
IFN-
(100 units/ml) plus ATRA (10
6 M) as
indicated for 40 h. Results of luciferase activities are shown on
the right panel. B, individual Luc constructs with or
without various mutations in Sp1 or in GAS (as indicated on the
left panel) were transfected into THP-1 cells. Cells were
treated and assayed in the same way as described in A. C and
D, EMSAs using nuclear extracts of THP-1 cells treated for
40 h with IFN-
(100 units/ml) plus ATRA (10
6
M). The wild-type and mutant GAS DNA fragments as indicated
were labeled; 1 × 104 dpm of labeled probe was used
in each binding reaction. C, lane 1, 32P-labeled
wild-type fragment as the probe alone serving as negative control.
Lane 2, binding reaction between labeled probe and the
nuclear extracts. Lane 3, competition by adding 100-fold
molar excess of cold wild-type probe; lanes 4-6, supershift
reactions by adding 1 µl of anti-Sp1 antibody, 1 µl of anti-STAT1
antibody, or 1 µl of anti-Sp1 antibody and 1 µl of anti-STAT1
antibody as indicated to the binding reaction; lanes 7-10,
competition by adding 100-fold molar excess of probe containing
mutation within the first, or second, or the first two, or all four Sp1
elements as indicated to the binding reaction; lanes 11 and
12, supershift reactions by adding 1 µl of anti-Sp1
antibody or 1 µl of anti-STAT1 antibody as indicated to the binding
reaction described for lane 10. D, lanes 1-3,
the same conditions as described for Fig. 7C, lane 1-3,
were employed; lane 4, competition by adding 100-fold molar
excess of probe containing the mutant GAS element; lanes 5 and 6, supershift reactions by adding 1 µl of anti-Sp1
antibody or anti-STAT1 antibody as indicated to the binding reaction
described for lane 4; lanes 7-9, the same conditions as
described for lanes 1-3 were employed, except the mutant
GAS DNA fragment was used as the labeled probe; lanes 10 and
11, supershift reactions by adding 1 µl of anti-STAT1
antibody or anti-Sp1 antibody as indicated to the binding reactions
described in lane 8.
and
ATRA, and performed EMSA using the wild-type P1 promoter (the 159-bp
DNA) as the labeled probe. As shown in Fig. 8, C and
D, two specific bands, one migrating slower than the other,
were detectable (lane 2). These two bands were abolished by
preincubation with unlabeled competitors containing all the Sp1 and GAS
elements (lane 3). As shown in Fig. 8C, gel
supershift assays using either anti-Sp1 antibodies (lane 4)
or anti-STAT1 antibodies (lane 5), or both antibodies
(lane 6), indicated that these two bands were specific
complexes formed between STAT1 and Sp1. When excess unlabeled probes
containing either the first or the second Sp1 element were used as
competitors, the two bands were also competed out (lanes 7 and 8). When unlabeled probe containing only the first two
mutant or all four mutant Sp1 elements were used as competitors, both
bands moved faster (lanes 9 and 10) than those in
the control lane (lane 2). These two bands were supershifted
by using the anti-Sp1 antibody (lane 11) but not by using
the anti-STAT1 antibody (lane 12). Additional experiments (Fig. 8D) showed that when labeled wild-type DNA fragments
were used as probe and unlabeled DNA fragment containing all four
wild-type Sp1 elements and the mutant GAS (GASm) as competitors, one
band was found to move faster than the control lane (comparing
lanes 4-6 with lane 2). This band was
supershifted by adding anti-STAT1 antibody, but not by adding anti-Sp1
antibody (comparing lanes 4-6). When labeled probe
containing mutant GAS was used, two bands moved faster (lanes
8); they were supershifted by anti-Sp1 antibody but not by
anti-STAT1 antibody (comparing lanes 10 and 11).
Together, these results demonstrate that the first two Sp1 sites and
the GAS site are functionally important, and that the second Sp1 site overlaps with a GAS site to form a novel overlapping GAS/Sp1 element. This GAS/Sp1 element is recognized by both STAT1 and Sp1 present in the
nuclear extracts of treated THP-1 cells.
Causes the STAT1 to
Dimerize and Bind to GAS Element in the ACAT-1 Promoter--
STAT1 is
a key component of the IFN-
-dependent transcriptional
activation complex (36, 37). We examined the transcript level of STAT1
in control and treated THP-1 cells by RT-PCR. As shown in Fig.
9A, STAT1
transcript was not detectable in control THP-1 cells (lane
1). Treating cells with ATRA gave rise to a remarkable increase
(lane 3), while treating cells with IFN-
caused only a
modest increase in the STAT1 transcript (lane 2). Treating cells with ATRA with or without IFN-
caused large increases in similar fashion (lane 4). These results indicated that
treating THP-1 cells with ATRA, with or without IFN-
, increased
significant gene expression of STAT1. It has been shown that STAT1 can
be activated as a homodimer that moves into the nucleus and acts as a
mature transcription factor by binding to the GAS element (39). The
dimerization of STAT1 requires tyrosine phosphorylation of STAT1 in a
manner triggered by IFN-
(38). Mutant STAT1 (STAT1-Y701Fm, replacing
tyrosine 701 with phenylalanine) is unable to undergo the tyrosine
phosphorylation dependent dimerization process (40). To test the
possibility that IFN-
may be involved in activating STAT1 to
up-regulate the ACAT-1 gene, we prepared wild-type STAT1 cDNA (STAT1-Y701) and the mutant STAT1 cDNA (STAT1-Y701Fm) in pRC/CMV vector. We then transfected these constructs individually into
the IFN-
and/or ATRA-treated THP-1 cells, and measured
ACAT-1 P1 promoter activity. As shown in Fig. 9B,
when cells were treated with IFN-
alone, a significant enhancement
of the P1 promoter was seen when these cells were transfected with
wild-type STAT1 cDNA. The enhancement was not seen when mutant
STAT1 cDNA was used (comparing the sizes of the second
bar in the STAT1-Y701 panel with the second
bar in the STAT1-Y701Fm panel). These results imply
that IFN-
is involved in stimulating the phosphorylation of STAT1,
causing the dimeric form of STAT1 to bind to the GAS element. To
further test this interpretation, we treated THP-1 cells with IFN-
alone, and transfected with or without the wild-type or the mutant
STAT1 cDNAs, then prepared the nuclear extracts of these
cells and performed EMSAs. As shown in Fig. 9C, the two GAS-specific bands were detectable in the nuclear extracts of cells
transfected with wild-type STAT1 cDNA (comparing
lane 6 with lane 2). These two bands were not
detectable in extracts of cells transfected with the mutant
STAT1 cDNA (comparing lane 10 with lane
2). Results of the gel supershift assays using anti-STAT1 and
anti-Sp1 antibodies confirmed that these two bands were complexes resulting from specific interactions of STAT1 and Sp1 with the ACAT-1 P1 promoter (lanes 8 and 9).
Additional EMSAs, using the nuclear extracts from control
(untransfected) THP-1 cells or from mutant STAT1
cDNA-transfected cells showed that the two bands described above
migrated faster. These bands were not supershifted with anti-STAT1
antibodies, but were supershifted with anti-Sp1 antibodies (comparing
lane 4 and 5 with 12 and
13). These results showed that the GAS site, not the Sp1
sites, formed specific complexes with the wild-type STAT1 after
activation by IFN-
through the tyrosine-phosphorylation dependent
mechanism.
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Fig. 9.
STAT1 is involved in the synergistic effect
of IFN- and ATRA. Total RNAs were
prepared from THP-1 cells treated for 40 h with or without IFN-
(100 units/ml), or ATRA (10
6 M), or IFN-
(100 units/ml) plus ATRA (10
6 M) as
indicated. A, quantitation by RT-PCR (30 cycles). Primers
were used for STAT1 and the GAPDH cDNAs as described under
"Experimental Procedures." Control experiments indicated that
between cycles 25 and 35, the IFN-
receptor transcripts and the
STAT1 transcript could be estimated semiquantitatively by RT-PCR (data
not shown). B, THP-1 cells were co-transfected with the Luc
construct containing the wild-type ACAT-1 P1 promoter core
region, and the wild-type STAT1 (indicated as STAT1-Y701), or the
mutant STAT1 (indicated as STAT1-Y701Fm), or the empty vector
(indicated as control). 7 h after transfection, the cells were
treated in various manners as indicated for 40 h. The luciferase
activities of treated cell extracts were then determined in the same
way as described in the legend to Fig. 1A. C,
EMSAs using nuclear extracts from the transfected THP-1 cells treated
for 40 h with IFN-
(100 units/ml). The wild-type 159-bp core
region DNA was labeled as probe; 1 × 104 dpm of
labeled probe was used for each binding reaction. Lane 1,
32P-labeled probe alone. Lane 2, binding
reaction between labeled probe and the nuclear extracts. Lane
3, competition by adding 100-fold molar excess of cold probe.
Lane 4, supershift reaction by adding 1 µl of anti-Sp1
antibody. Lane 5, supershift reaction by adding 1 µl of
anti-STAT1 antibody. For lanes 2-5, nuclear extracts were
from cells transfected with the empty vector. Lanes 6-9 and
10-13 are results using the same series of reaction
conditions as described in lanes 2-5, but using the nucleic
extracts from THP-1 cells transfected with wild-type STAT1 cDNA or
with mutant STAT1 cDNA, respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(12). The molecular basis of these
findings has not been pursued at the gene transcription level. In our
current work, we showed that IFN-
increased ACAT-1
mRNAs and protein contents during the human blood
monocyte-macrophage differentiation process. We then found that
treating the human monocyte-like THP-1 cells with ATRA and IFN-
caused up-regulation of ACAT-1 gene expression in cell-type
specific manner. To elucidate the molecular basis of this finding, we
identified a 159-bp core region with Sp1 elements that is responsible
for the P1 promoter activity. This region also contains an IFN-
activated sequence (GAS) that overlaps exactly with the second Sp1
element (TGGGCGGAA, with the Sp1 site
underlined). To our knowledge, this is the first example in literature
describing an overlapping Sp1/GAS site. Using luciferase constructs in
transient trasfection studies, we demonstrated that the combination of
IFN-
and ATRA is needed to enhance ACAT-1 P1 promoter
activity. Additional experiments using RT-PCR and EMSA showed that ATRA
caused large induction of the transcription factor STAT1, while IFN-
triggered the phosphorylation dependent activation of STAT1. The
activated STAT1 then acts by binding to the overlapping GAS/Sp1 site in
the ACAT-1 P1 promoter. Our work dissects the
non-sterol-mediated ACAT regulation at the transcriptional level, and
provides a molecular mechanism to account for part of the effects of
IFN-
in causing macrophage foam cell formation in
vitro.
(43, 44). IFN-
has been shown to exert
certain proatherosclerogenic actions in vitro. It induces
VCAM-1 on endothelial cells (45), decreases apoE secretion,
and increases uptake of hypertriblyderidemic very low density
lipoprotein on macrophages (46), induces myosin heavy chain-II on macrophages and smooth muscle cells
(47), and induces scavenger receptors on smooth muscle cells during
atherogenesis (48). On the other hand, IFN-
has also been shown to
exert protective action against atherosclerosis in certain in
vitro systems examined (49, 50). Recently, it has been shown that apoE knockout mice crossed with the IFN-
receptor knockout mice display reductions in lesion size, lipid accumulation, and cellularity (51). In addition, in mice, post-transplant graft arteriosclerosis is
associated with the presence of IFN-
; the serological neutralization or the genetic absence of IFN-
markedly reduces the extent of intimal expansion (52). These results support the notion that IFN-
is pro-atherogenic in vivo. If this concept holds true, then
our finding described here may explain some of the effects of IFN-
on foam cell formation in vivo.
exhibits antigrowth or antiproliferation effects in various
target cells. Its effects often occur synergistically with retinoids
(53). To cite a few examples, in various myelogenous leukemic cell
lines, Gianni et al. (54) showed that ATRA can bypass
IFN/IFN receptors and induce the expression of IFN-regulated genes
including STAT1; Matikainen et al. (55, 56)
showed that ATRA causes up-regulation of several IFN-specific
transcription factors and signal inducers including STAT,
and enhances their responsiveness toward IFNs. The molecular
mechanism(s) for the synergism observed between ATRA and IFNs in these
studies remain to be elucidated. Our work described here may serve to
explain some of the synergistic actions of ATRA and IFNs described in these studies.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Darnell for wild-type
STAT1 cDNA, and Dr. Xinyuan Liu for the gifts of
recombinant IFN-. We also acknowledge Deng-Hong Zhang for
participating in certain stages of this work, and thank Bao-Liang Song,
Nian-Yi Zhang, and Wei Qi for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by National Natural Scientific Foundation of China Grant 39425005 (to B. L. L.), Shanghai Science and Technology Commission Grant 97XD14022 (to B. L. L.), and National Institutes of Health Grant HL 36709 (to T. Y. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom corresponding may be addressed: Dept. of Biochemistry, Dartmouth Medical School, Hanover, NH. Tel.: 603-650-1622; Fax: 603-650-1483; E-mail: Ta.Yuan.Chang@Dartmouth.EDU.
To whom correspondence may be addressed: Institute of
Biochemistry and Cell Biology, 320 Yue-Yang Road, Shanghai 200031, China. Tel.: 86-21-6474-7035; Fax: 86-21-6433-8357;
E-mail:boliang@server.shcnc.ac.cn.
Published, JBC Papers in Press, February 28, 2001, DOI 10.1074/jbc.M011488200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
ACAT, acyl-coenzyme
A:cholesterol acyltransferase;
bp, base pair(s);
GAS, IFN- activated
sequence;
IFN-
, interferon-gamma;
ATRA, all-trans-retinoic acid;
CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate;
FBS, fetal bovine serum;
PBS, phosphate-buffered saline;
EMSA, electrophoretic mobility shift assays;
RT-PCR, reverse
transcriptase-polymerase chain reaction;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
knt, kelonucleotides.
![]() |
REFERENCES |
---|
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---|
1. | Chang, T. Y., Chang, C. C. Y., and Cheng, D. (1997) Annu. Rev. Biochem. 66, 613-638[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Chang, C. C. Y.,
Huh, H. Y.,
Cadigan, K. M.,
and Chang, T. Y.
(1993)
J. Biol. Chem.
268,
20747-20755 |
3. |
Anderson, R. A.,
Joyce, C.,
Davis, M.,
Reagan, J. W.,
Clark, M.,
Shelness, G. S.,
and Rudel, L. L.
(1998)
J. Biol. Chem.
273,
26747-26754 |
4. |
Cases, S.,
Novak, S.,
Zheng, Y. W.,
Myers, H. M.,
Lear, S. R.,
Sande, E.,
Welch, C. B.,
Lusis, A. J.,
Spencer, T. A.,
Krause, B. R.,
Erickson, S. K.,
and Farese, R. V., Jr.
(1998)
J. Biol. Chem.
273,
26755-26764 |
5. |
Oelkers, P.,
Behari, A.,
Cromley, D.,
Billheimer, J. T.,
and Sturley, S. L.
(1998)
J. Biol. Chem.
273,
26765-26771 |
6. |
Lee, O.,
Chang, C. C.,
Lee, W.,
and Chang, T. Y.
(1998)
J. Lipid Res.
39,
1722-1727 |
7. |
Chang, C. C.,
Sakashita, N.,
Ornvold, K.,
Lee, O.,
Chang, E. T.,
Dong, R.,
Lin, S.,
Lee, C. Y.,
Strom, S. C.,
Kashyap, R.,
Fung, J. J.,
Farese, R. V., Jr.,
Patoiseau, J. F.,
Delhon, A.,
and Chang, T. Y.
(2000)
J. Biol. Chem.
275,
28083-28092 |
8. | Buhman, K. K., Accad, M., Novak, S., Choi, R. S., Wong, J. S., Hamilton, R. L., Turley, S., and Farese, R. V., Jr. (2000) Nat. Med. 6, 1341-1347[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Lee, R. G.,
Willingham, M. C.,
Davis, M. A.,
Skinner, K. A.,
and Rudel, L. L.
(2000)
J. Lipid Res.
41,
1991-2001 |
10. | Brown, M. S., and Goldstein, J. L. (1983) Annu. Rev. Biochem. 52, 223-261[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Fielding, C. J.
(1992)
FASEB J.
6,
3162-3168 |
12. |
Panousis, C. G.,
and Zuckerman, S. H.
(2000)
J. Lipid Res.
41,
75-83 |
13. | Ross, R. (1993) Nature 362, 801-809[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Brown, M. S.,
Ho, Y. K.,
and Goldstein, J. L.
(1980)
J. Biol. Chem.
255,
9344-9352 |
15. |
Chang, C. C.,
Lee, C. Y.,
Chang, E. T.,
Cruz, J. C.,
Levesque, M. C.,
and Chang, T. Y.
(1998)
J. Biol. Chem.
273,
35132-35141 |
16. |
Panousis, C. G.,
and Zuckerman, S. H.
(2000)
Arterioscler. Thromb. Vasc. Biol.
20,
1565-1571 |
17. | Banka, C. L., Black, A. S., Dyer, C. A., and Curtiss, L. K. (1991) J. Lipid Res. 32, 35-43[Abstract] |
18. |
Alessio, M.,
Monte, L. De.,
Scirea, A.,
Gruarin, P.,
Tandon, N. N.,
and Sitia, R.
(1996)
J. Biol. Chem.
271,
1770-1775 |
19. | Tontonoz, P. L., Nagy, J. G., Alvalez, A., Thomazy, V. A., and Evans, R. M. (1998) Cell 93, 241-252[Medline] [Order article via Infotrieve] |
20. |
Shiffman, D.,
Mikita, T.,
Tai, J. T.,
Wade, D. P.,
Porter, J. G.,
Seilhamer, J. J.,
Somogyi, R.,
Liang, S.,
and Lawn, R. M.
(2000)
J. Biol. Chem.
275,
37324-37332 |
21. |
Cheng, W.,
Kvilekval, K. V.,
and Abumrad, N. A.
(1995)
Am. J. Physiol.
269,
E642-648 |
22. | Cadigan, K. M., and Chang, T. Y. (1988) J. Lipid Res. 29, 1683-1692[Abstract] |
23. |
Chang, T. Y.,
and Limanek, J. S.
(1980)
J. Biol. Chem.
255,
7787-7795 |
24. | Jiang, C. L., Lu, C. L., Chen, Y. Z., and Liu, X. Y. (1999) Peptides 20, 1385-1388[CrossRef][Medline] [Order article via Infotrieve] |
25. | Bishop, J. E., and Hajra, A. K. (1980) Anal. Biochem. 106, 344-350[Medline] [Order article via Infotrieve] |
26. |
Li, B. L.,
Li, X. L.,
Duan, Z. J.,
Lee, O.,
Lin, S.,
Ma, Z. M.,
Chang, C. C.,
Yang, X. Y.,
Park, J. P.,
Mohandas, T. K.,
Noll, W.,
Chan, L.,
and Chang, T. Y.
(1999)
J. Biol. Chem.
274,
11060-11071 |
27. | Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 51-59[CrossRef][Medline] [Order article via Infotrieve] |
28. | Vinkemeier, U., Cohen, S. L., Moarefi, I., Chait, B. T., Kuriyan, J., and Darnell, J. E., Jr. (1996) EMBO J. 15, 5616-5626[Abstract] |
29. | Powell, J. T., Klaasse Bos, J. M., and van Mourik, J. A. (1992) FEBS Lett. 303, 173-177[CrossRef][Medline] [Order article via Infotrieve] |
30. | Mack, K. D., Wei, R., Elbagarri, A., Abbey, N., and McGrath, M. S. (1998) J. Immunol. Methods. 211, 79-86[CrossRef][Medline] [Order article via Infotrieve] |
31. | Liu, J., Streiff, R., Zhang, Y. L., Vestal, R. E., Spence, M. J., and Briggs, M. R. (1997) J. Lipid Res. 38, 2035-2048[Abstract] |
32. |
Sakamoto, S.,
Nie, J.,
and Taniguchi, T.
(1999)
J. Immunol.
162,
4381-4384 |
33. | Andrews, N. C., and Faller, D. V. (1991) Nucleic Acids Res. 19, 2499[Medline] [Order article via Infotrieve] |
34. | Oosta, G. M., Mathewson, N. S., and Catravas, G. N. (1978) Anal. Biochem. 89, 31-34[Medline] [Order article via Infotrieve] |
35. |
Chang, C. C.,
Chen, J.,
Thomas, M. A.,
Cheng, D.,
Del Priore, V. A.,
Newton, R. S.,
Pape, M. E.,
and Chang, T. Y.
(1995)
J. Biol. Chem.
270,
29532-29540 |
36. | Schindler, C., and Darnell, J. E., Jr. (1995) Annu. Rev. Biochem. 64, 621-651[CrossRef][Medline] [Order article via Infotrieve] |
37. | Darnell, J. E., Jr., Kerr, I. M., and Stark, G. R. (1994) Science 264, 1415-1421[Medline] [Order article via Infotrieve] |
38. | Aguet, M., Dembic, Z., and Merlin, G. (1988) Cell 55, 273-280[Medline] [Order article via Infotrieve] |
39. | Schindler, C., Shuai, K., Prezioso, V. R., and Darnell, J. E., Jr. (1992) Science 257, 809-813[Medline] [Order article via Infotrieve] |
40. |
Walter, M. J.,
Look, D. C.,
Tidwell, R. M.,
Roswit, W. T.,
and Holtzman, M. J.
(1997)
J. Biol. Chem.
272,
28582-28589 |
41. |
Wang, H.,
Germain, S. J.,
Benfield, P. P.,
and Gillies, P. J.
(1996)
Arterioscler. Thromb. Vasc. Biol.
16,
809-814 |
42. |
Miyazaki, A.,
Sakashita, N.,
Lee, O.,
Takahashi, K.,
Horiuchi, S.,
Hakamata, H.,
Morganelli, P. M.,
Chang, C. C. Y.,
and Chang, T. Y.
(1998)
Arterioscler. Thromb. Vasc. Bio.
18,
1568-1574 |
43. | Hansson, G. K., Holm, J., and Jonasson, L. (1989) Am. J. Pathol. 135, 169-175[Abstract] |
44. |
Geng, Y. J.,
Holm, J.,
Nygren, S.,
Bruzelius, M.,
Stemme, S.,
and Hansson, G. K.
(1995)
Arterioscler. Thromb. Vasc. Biol.
15,
1995-2002 |
45. | Li, H., Cybulsky, M. I., Gimbrone, M. A., Jr., and Libby, P. (1993) Arterioscler Thromb. 13, 197-204[Abstract] |
46. |
Whitman, S. C.,
Argmann, C. A.,
Sawyez, C. G.,
Miller, D. B.,
Hegele, R. A.,
and Huff, M. W.
(1999)
J. Lipid Res.
40,
1017-1028 |
47. | Jonasson, L., Holm, J., Skalli, O., Gabbiani, G., and Hansson, G. K. (1985) J. Clin. Invest. 76, 125-131[Medline] [Order article via Infotrieve] |
48. | Li, H., Freeman, M. W., and Libby, P. (1995) J. Clin. Invest. 95, 122-133[Medline] [Order article via Infotrieve] |
49. | Christen, S., Thomas, S. R., Garner, B., and Stocker, R. (1994) J. Clin. Invest 93, 2149-2158[Medline] [Order article via Infotrieve] |
50. | Geng, Y. J., and Hansson, G. K. (1992) J. Clin. Invest. 89, 1322-1330[Medline] [Order article via Infotrieve] |
51. |
Gupta, S.,
Pablo, A. M.,
Jiang, X. C.,
Wang, N.,
Tall, A. R.,
and Schindler, C.
(1997)
J. Clin. Invest.
99,
2752-2761 |
52. | Tellides, G., Tereb, D. A., Kirkiles-Smith, N. C., Kim, R. W., Wilson, J. H., Schechner, J. S., Lorber, M. I., and Pober, J. S. (2000) Nature 403, 207-211[CrossRef][Medline] [Order article via Infotrieve] |
53. | Mangelsdorf, D. J., Umesono, K., and Evans, R. M. (1994) in The Retinoids: Biology, Chemistry and Medicine (Sporn, M. B. , Roberts, A. B. , and Goodman, D. S., eds), 2nd Ed. , pp. 319-349, Raven Press, Ltd., New York |
54. |
Gianni, M.,
Terao, M.,
Fortino, I.,
LiCalzi, M.,
Viggiano, V.,
Barbui, T.,
Rambaldi, A.,
and Garattini, E.
(1997)
Blood
89,
1001-1012 |
55. | Matikainen, S., Lehtonen, A., Sareneva, T., and Julkunen, I. (1998) Leuk. & Lymphoma 30, 63-71[Medline] [Order article via Infotrieve] |
56. | Matikainen, S., Ronni, T., Lehtonen, A., Sareneva, T., Melen, K., Nordling, S., Levy, D. E., and Julkunen, I. (1997) Cell Growth Differ. 8, 687-698[Abstract] |