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J. Biol. Chem., Vol. 275, Issue 36, 28240-28245, September 8, 2000
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From the Division of Molecular Medicine, Department of Medicine, Columbia University, New York, New York 10032
Received for publication, April 19, 2000, and in revised form, May 28, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Tangier disease, a condition characterized by low
levels of high density lipoprotein and cholesterol accumulation
in macrophages, is caused by mutations in the ATP-binding cassette
transporter ABC1. In cultured macrophages, ABC1 mRNA was induced in
an additive fashion by 22(R)-hydroxycholesterol and
9-cis-retinoic acid (9CRA), suggesting induction by nuclear
hormone receptors of the liver X receptor (LXR) and retinoid X receptor
(RXR) family. We cloned the 5'-end of the human ABC1 transcript from
cholesterol-loaded THP1 macrophages. When transfected into
RAW macrophages, the upstream promoter was induced 7-fold by
22(R)-hydroxycholesterol, 8-fold by 9CRA, and 37-fold by
9CRA and 22(R)-hydroxycholesterol. Furthermore, promoter
activity was increased in a sterol-responsive fashion when
cotransfected with LXR Plasma
HDL1-cholesterol
levels are inversely related to the incidence of coronary artery
disease (1). Two genetic diseases illustrate this phenomenon, the rare
Tangier disease and the more common familial HDL deficiency. Tangier
disease is characterized by an extremely low concentration of
circulating HDL and the accumulation of cholesteryl esters in tonsils,
liver, spleen, and intestinal mucosa, mostly in macrophage foam cells
(2). Patients with familial HDL deficiency exhibit a low concentration
of HDL particles and an increased risk of coronary artery disease (3).
A common explanation for the cardioprotective effect of HDL is the
major role it plays in reverse cholesterol transport (4). It is
commonly accepted that the efflux of cholesterol from cells is caused
by two different pathways: the first is passive and promotes efflux from the cell membrane to HDL and the second is
energy-dependent and apolipoprotein-mediated (5). The
latter was characterized in fibroblasts and macrophages and involves
lipid-poor or -free apolipoproteins such as apoA-I, apoA-II, and apo-E
(5-7). This active pathway has been reported to be defective in both
Tangier disease and familial HDL deficiency (8-10). It was recently
demonstrated that ABC1 is a key gene in this process
(11) and that mutations of ABC1 are the major cause of both
Tangier disease and familial HDL deficiency (3, 12-17).
ABC1 (ABCA1) belongs to the large ATP-binding cassette transporter
family. These transmembrane proteins transport many diverse substrates
across membranes because of their channel-like topology (18, 19). The
human ABC1 gene was assigned to chromosome 9q31, spanning a minimum of 70 kilobases and containing at least 49 exons
(14, 16, 20). Whereas its expression is ubiquitous, the highest levels
of human or murine mRNAs were found in placenta, fetal tissues,
liver, lung, and adrenal glands (21, 22). The predicted human protein
contains 2201 amino acids (220-kDa protein) (21).
The expression of hABC1 is induced by cholesterol loading of human
macrophages. Both the protein and the mRNA are up-regulated in the
presence of acetylated LDL, and down-regulated by cholesterol unloading
via HDL3 (21). Whereas the cholesterol-mediated regulation of genes
involved in cholesterol uptake or biosynthesis via sterol regulatory element binding protein (SREBP) pathways is well understood (23), much less is known about direct mechanisms of sterol-mediated up-regulation of gene expression. Two families of nuclear receptors are
known to be activated by oxysterols and to mediate a positive response
by binding to specific DNA elements, the liver X receptor (LXR) and
steroidogenic factor 1 (SF1) (24-27). SF1 acts as a monomer and has
been implicated in the regulation of steroidogenic acute regulatory
protein gene expression (StAR) activity (26). Recently, two different
genes involved in the reverse cholesterol transport pathways,
cholesterol 7 Here we report the sequence of the hABC1 promoter and show
that this promoter is active in macrophages and that its
sterol-mediated activation depends on the binding of LXR/RXR 5'-RACE PCR--
5'-RACE PCR was performed with the SMART RACE
cDNA kit (CLONTECH, Palo Alto, CA) using 1 µg
of poly(A)+ mRNA from HepG2 and THP-1 cells that
were differentiated into macrophage with phorbol 12-myristate
13-acetate and exposed to acetylated LDL (25 µg/ml) for 48 h.
After reverse transcription (M-MLV reverse transcriptase, Life
Technologies Inc.), a first PCR (hot start, 94 °C 30 s,
65 °C 30 s, 72 °C 3 min, 25 cycles, and then 72 °C 10 min) was performed using the reverse primer 5'-CCCCCTCCCTCGGGATGCCCGCAGACAA-3'. A second PCR (hot start, 94 °C
30 s, 55 °C 30 s, 72 °C 3 min, 25 cycles, and then
72 °C 10 min) was done on 2.5 µl of the 50×-diluted first PCR
sample with the nested primer, 5'-GCCTCCGAGCATCTGAGAACAGGC-3'. The
forward primers were provided by CLONTECH.
Cloning of the hABC1 Promoter and Introns 1 and 2--
The
screening of the human RPC.11 BAC clone library was performed
(Research Genetics, Inc., Huntsville, AL) with a 68-mer oligonucleotide
probe corresponding to nucleotides 11-79 of the published
hABC1 sequence. Two BAC clones were recovered that were positive by PCR for exon 1 (BAC553F19) and exon 3 (BAC 522C12). After
digestion by PstI, a Southern blot was performed using the 32P-radiolabeled probes generated by PCR with the
previously cited exons. Positive bands were cloned in pBluescript KS(+)
(Stratagene, La Jolla, California). A colony hybridization (probes used
for Southern blot) (33) allowed us to isolate positive clones for the
hABC1 promoter (5 kilobases) and intron 2. Sequencing
performed on both strands showed that we also cloned intron 2 from BAC
522C12. The sequences of these introns are contained in the sequence of human genomic clone RP11-1M10, which also contains exons 1, 2, and 3 (see Fig. 2a).
Cell Cultures and Transfection Experiments--
The cell lines
were purchased from ATCC (Manassas, VA). The murine RAW 264.7, African
green monkey CV-1, and human 293 or HepG2 cell lines were maintained in
Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin. THP-1 cells were
maintained in RPMI 1640 containing L-glutamine, 10%
heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin supplemented with 0.5 µM
Transfections were performed in 24-well plates with LipofectAMINE
reagent (transactivation experiments in CV-1 and 293 cells, see Figs. 4
and 5) or LipofectAMINE-Plus reagent (basal activation experiments in
RAW 264.7, see Figs. 4, 6, and 8) according to the manufacturer's
instructions (Life Technologies Inc.). For basal activation
experiments, a total of 0.15 µg of reporter DNA and 0.05 µg of
PRL-CMV (Renilla, Promega) per well were used. For
transactivation studies, we used 0.025 µg/well PRL-CMV, 0.2 µg of
reporter DNA, and 0.1 µg of each receptor (CMX-hRXR Northern Blot Analysis--
Total RNA was isolated with RNAzol B
reagent (TEL-TEST, Inc., Friendwood, TX). Northern blots were performed
as described previously (33). A human ABC1 probe
corresponding to exons 2-8 of the published sequence was synthesized
by reverse transcriptase-PCR using the forward primer,
5'-AGGTGGCCTGGCCTCTATTTATCTTC-3' and the reverse primer,
5'-GCCTCCGAGCATCTGAGAACAGGC-3'. LXR probes were synthesized from human
LXR Electrophoretic Mobility Shift Assays--
Electrophoretic
mobility shift assays were performed as described previously (25).
Nuclear extracts were prepared from 293 cells cotransfected with
LXR Increased ABC1 mRNA in Human Macrophages Treated with Sterols
and/or Retinoic Acid--
To investigate whether the endogenous
ABC1 gene can be activated by oxysterols and/or retinoic
acid in macrophages, we performed Northern blot analysis of total RNA
from human THP-1 macrophages. Fig. 1
shows a significant increase of ABC1 mRNA in cells treated with
22(R)-Hch (2-fold induction, p < 0.05) or
9CRA (2-fold, p < 0.05). An additive effect was
obtained with combined treatment (4-fold, p < 0.05 when compared with separate treatments). These responses suggest
possible activation of transcription by LXR/RXR (32).
Characterization of the 5' Region of the hABC1 Gene--
To
identify the promoter of the human ABC1 gene, we performed
5'-RACE PCR using poly(A)+ mRNA from cholesterol-loaded
THP-1 macrophages and HepG2 cells (Fig.
2b). In macrophages this
revealed a single major transcript (transcript A) consisting of a first
exon of 217 bp followed by a second exon of 160 bp, 73% identical to
mouse exon 1 (GenBankTM/EBI accession number X75926). This exon
is then followed by the published human exons 2, 3, and 4 (21).
In HepG2 cells, 5'-RACE PCR revealed three different transcripts (Fig.
2b). Transcript B represents a truncated version of exon 2 found in THP-1 cells (only the last 29 bp) followed by the published
exons 2, 3, and 4 (21). Transcript C contains one exon of 372 bp
upstream of the published exon 2, which is different from the exons
found in THP1 cells. Transcript D has the same 5' structure as
transcript C but lacks the published exon 3.
A BLAST search of the GenBankTM/EBI Data Bank (htgs) revealed
100% homology of these exons (Fig. 2a, exons
1-3) with fragments of the human genomic clone RP11-1M10
(working draft sequence, GenBankTM/EBI accession number
AC012230). A comparison of the sequences from the published exon
2, the 5'-RACE PCR product, and RP11-1M10 revealed a C instead of a T
at position +15 and a G instead of an A at position +17.
Conceptual translation of the transcripts revealed two new start codons
in frame with the previously published ATG located in exon 5 (14) (Fig.
2, a and c). In the case of the transcript characteristic of THP1 cells, a new ATG located in exon 2 resulted in
an extra 60 amino acids at the amino terminus. In the case of HepG2
cells, a new start codon at the 3'-end of exon 3 may be functional in
transcript C and also transcript D, which lacks the previously
published start codon. This results in an extra 39-amino acid fragment
for transcript C.
A comparison of the putative amino-terminal amino acid sequences of
ABC1 (transcripts A, B) with nucleotide data bases revealed strong homology to the amino-terminal sequences of two members of the
ABC1 family (57% identity with ABCR and 45% identity with ABC3
(Fig. 1c). This strongly suggests that the amino-terminal sequence of
hABC1 is authentic.
Sequence of the hABC1 Promoter--
The promoter region upstream
of exon 1 was responsive to sterols when transfected into cells (see
below), whereas the 2.3-kilobase region upstream of transcript B was
not responsive (data not shown). Thus, we focused our attention on the
former region.
Fig. 3 presents a partial sequence of
genomic DNA with a fragment of exon 1 cloned from the human RPCI.11 BAC
library. A potential TATA box is present at hABC1 Promoter Is Functional and Sterol-responsive in
Macrophages--
To investigate the function of the potential
hABC1 promoter, we transfected the macrophage-like RAW 264.7 cell line with a promoter-luciferase construct (Fig.
4a). To test for activation by
sterols we used 22(R)-Hch, a potent activator of LXR·RXR
(36) but a poor activator of SF1 (27) and 9CRA, to activate endogenous RXR. Compared with basal conditions, transfected cells treated with
22(R)-Hch or 9CRA exhibited 7- and 8-fold higher
promoter activity, respectively (p < 0.001) (Fig.
4a). When both compounds were added together, there was a
synergistic 37-fold induction (p < 0.001). A similar
response was obtained with promoter fragments containing 928 bp (Fig.
4a) or 469 bp (data not shown) of upstream sequence.
Next we compared the response of the ABC1 promoter to
different sterols (Fig. 4b). We treated the transfected
cells with 25-Hch, which is a good activator of SF1 and a weak
activator of LXR (27). Cells were also treated with 7-Kch, which is
relatively abundant in human arterial foam cells (37). 25-Hch is a poor
inducer of the hABC1 promoter compared with
22(R)-Hch (1.5-fold activation, p < 0.05).
No significant effect of 7-Kch was detected. However, when added in
combination with 9CRA, a significant additive effect was detected for
7-Kch (2-fold when compared with 9CRA alone, p < 0.01). This pattern of sterol responsiveness is consistent with a
transcriptional mechanism involving LXR.
LXR LXR/RXR Transactivates the hABC1 Promoter in 293 and CV-1
Cells--
To define the involvement of hRXR
As a positive control for these experiments we used a construct
containing three copies of the LXR·hRXR
In CV-1 cells a significant sterol-activation of the ABC1
promoter was detected without transfected receptors (4-fold,
p < 0.01, Fig. 5d). Cotransfection with
LXR Deletional Analysis of hABC1 Promoter--
To define the region of
the hABC1 promoter involved in its sterol-mediated
activation we carried out further deletional analysis (Fig.
6). Similar results to Fig. 4a
were obtained with fragments of the promoter from Mutational Analysis of the DR4 Element--
To test the hypothesis
that the DR4 element is responsible for sterol activation, we
introduced a mutated version of this element in the longer version of
the hABC1 promoter (from The DR4 Element Binds LXR/RXR--
To determine whether the DR4
element binds LXR/RXR, we used oligonucleotides containing the
wild-type DR4 or the mutated version (as in the functional assay, Fig.
7) to perform electrophoretic mobility shift assays using nuclear
extracts from 293 cells cotransfected with LXR In this study we have identified a region of the human
ABC1 promoter, which is active in macrophages and is induced
by 22(R)-hydroxycholesterol and 9-cis-retinoic
acid. Our characterization of the major transcript in
cholesterol-loaded THP1 macrophages led to the identification of this
promoter and also showed that most of the potential upstream initiation
codons (ATG) in the previously published cDNA (21) are unlikely to
represent the authentic translation initiation site. LXR A unique DR4 element mediates the sterol up-regulation of the
hABC1 gene through LXR/RXR, and mutational analysis suggests that this is the only site involved. Unlike previously identified DR4
elements (25, 36), the element in the ABC1 promoter is found
in an inverse orientation on the non-coding strand (Fig. 6). The almost
canonical sequence of this element might explain its high efficiency.
Mutations in the DR4 result in a decrease of the basal expression of
the promoter, but significant activity is still detectable. A weak
activation of the mutated promoter by 9CRA (Fig. 7) suggests the
presence of another binding site for RXR. However, the DR4 element we
identified is responsible for the entire sterol-mediated activation of
the 1-kilobase hABC1 promoter (Fig. 3).
The pattern of activation of the ABC1 promoter by sterols
suggests that its expression may be suboptimal in atherosclerotic lesions. Thus, 7-KCh is relatively abundant in oxidized-LDL and in
atheroma foam cells (37) and is a poor activator of the
hABC1 promoter (Fig. 4b). 27-Hch, also abundant
in foam cells, is a relatively poor activator of LXR (32, 36). Thus,
the accumulation of oxysterols in atherosclerotic lesions probably does
not result in optimal activation of hABC1. This suggests that small
molecules that are optimal LXR activators might be effective drugs at
reversing foam cell formation and that they might be useful as a
treatment for atherosclerosis. The activation of the hABC1
promoter by 9CRA is increased 2-fold when given with 7-Kch (Fig.
4b). This further suggests that with regard to the induction
of ABC1 by oxysterols, an unfavourable foam cell environment could also
be switched to a more favorable one by delivery of ligands for RXR.
The inability of LXR To conclude, we have shown that hABC1 is up-regulated at the
transcriptional level by oxysterols and 9CRA acting though LXR/RXR which binds a proximal DR4 element located on the non-coding strand. These results provide strong support for the idea that LXRs act directly to coordinate the activities of molecules mediating reverse cholesterol transport (25). This may lead to a functional coordination of different steps of reverse cholesterol transport. For example, CETP
activity results in the remodeling of HDL into small particles and
liberates free apoA-I from HDL (38). Free apoA-I appears to be the
optimal substrate for ABC1 (39, 40). Thus, coordinate induction of CETP
and ABC1 by LXR/RXR might act synergistically to enhance cholesterol
efflux from macrophage foam cells.
/RXR or LXR
/RXR. Further experiments identified a direct repeat spaced by four nucleotides (from 
70 to
55 base pairs) as a binding site for LXR/RXR or LXR/RXR. Mutations in this element abolished the sterol-mediated activation of
the promoter. The results show sterol-dependent
transactivation of the ABC1 promoter by LXR/RXR and suggest
that small molecule agonists of LXR could be useful drugs to reverse
foam cell formation and atherogenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-hydoxylase (24) and cholesterol ester transfer protein
(CETP) (25), have been shown to be up-regulated by the
heterodimer LXR·RXR. This suggests the hypothesis that LXRs
might coordinate different steps of reverse cholesterol transport (25).
LXR (NR1H3) and LXR (NR1H2) heterodimerize with their partner
RXR. The resulting complex up-regulates genes through binding sites
typically composed of direct repeats (DR) of the motif AGGTCA, spaced
by 4 nucleotides (LXR and LXR) or 1 nucleotide (LXR) (28-30).
The dimer can be activated by both the ligands of RXR (retinoids) and
LXR (oxysterols) separately or together (29, 31, 32).
to a
DR4 element.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol. Confluent cells were differentiated with 0.2 µM phorbol 12-myristate 13-acetate (Sigma) in ethanol over 72 h. Thioglycolate-elicited peritoneal macrophages were isolated from C57 Bl/6 mice as described previously (34).
, CMX-hLXR, CMV-mLXR). pcDNA3.1 plasmid was included to obtain a final
quantity of 0.45 µg of total DNA per well. The transfected cells were
cultured in lipoprotein-deficient serum medium in the presence of 4 µg/ml (see Figs. 4, 6, and 8) or 2 µg/ml (transactivation
experiments, see Fig. 5) of 22(R)-hydroxycholesterol
(22(R)-Hch), 25-hydroxycholesterol (25-Hch), or
7-ketocholesterol (7-Kch), 10 µM
9-cis-retinoic acid (9CRA, Sigma) or ethanol alone for
24 h. The luciferase activities were measured using the Promega
dual luciferase assay system. A reporter plasmid used to analyze the
activity of the hABC1 promoter was constructed by subcloning
a 1029-bp PCR fragment of the hABC1 promoter (from 928 to
+101 bp) into the pGL3-Luc basic vector (Promega). A shorter promoter
(from 469 to +101 bp) was generated by digestion of this plasmid with
SacI. Deletions (see Fig. 7) were performed by enzymatic
digestion (ApaI, from 156 to +101 bp) or PCR. The sequence
of the PCR fragments were verified. Where shown, error bars
represent S.D. Non-parametric Mann Whitney tests were performed to
obtain p values.
and mouse LXR sequences (28, 35). A
mouse glycerol-3-phosphate dehydrogenase probe was used as an internal
standard (reverse transcriptase-PCR synthesized fragment: forward
primer, 5'-ACCACAGTCCATGCCATCAC-3' and reverse primer,
5'-TCCACCACCCTGTTGCTGTA-3'). Signals were quantitated with phosphor
imaging. Non-parametric Mann Whitney tests were performed to obtain
p values.
and hRXR or LXR and hRXR. Double-stranded oligonucleotides
containing the DR4 element or its mutated version (see Fig. 7) were
synthesized with overhangs and used at a final concentration of 0.1 pM (hABC1DR4) or 0.5 µM (competitors). An oligonucleotide corresponding to a canonical half-site sequence (AGGTCA) was added to each sample to reduce the background (1 µM). Polyclonal antibodies against peptides from LXR
(P20, sc-1202X), LXR/ (C19, sc-2101X), RXR (D20, sc-553X), and
ROR (K-20, sc-6063X) were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of hABC1 in THP1 macrophages.
THP-1 cells were exposed for 72 h to phorbol 12-myristate
13-acetate to induce differentiation in macrophages. On day 4, cells
were treated for 24 h with vehicle (ethanol), 22(R)-Hch
(10 µM), and/or 9CRA (10 µM)
(n = 4 per treatment). Top, Northern blot
with 40 µg of total RNA from each sample. The membrane was hybridized
with hABC1 probe and mouse glycerol-3-phosphate dehydrogenase
(G3PDH) as an internal standard. Bottom,
quantitation of blot, error bars represent S.D. Significance
of treatment versus ethanol is indicated by *,
p < 0.05.
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Fig. 2.
Analysis of hABC1 5'
sequence. a, partial gene structure; b, results
of 5'-RACE PCR; and c, amino-terminal sequence.
a, the hABC1 promoter was cloned from the human
library RPCI-11, and the structure of the 5'-end of the gene was
determined by sequence analysis. Positions of exons in RP11-M10
sequence are indicated in the inset. b, 5'-RACE
PCR was performed on cholesterol-loaded THP-1 macrophages. These cells
express exons 1 and 2 (transcript A). A 5'-RACE PCR was also
conducted on HepG2 cells, which express a truncated version of exon 2 and also exon 3 (transcripts B, C, D).
Two possible ATG start codons are present in exons 2 and 3. c, a comparison of the deduced amino-terminal sequence
(NH2-terminal sequence) of hABC1 with the
nucleotide data base (tBlastn) revealed similarities with two members
of the ABC1 family, ABCR and ABC3. |, identity; 
, conserved
substitution.
32 bp and an Sp1 site at
101 bp. An analysis of this sequence revealed several potential
transcription factor binding sites.
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Fig. 3.
hABC1 promoter sequence. This
sequence is identical to the working draft sequence of the genomic
clone RP11-1M10 (position 2335-3463 bp, GenBankTM/EBI accession
number AC012230) except for the italicized 5'-end which is new. The
fragment used in transfection covers from 928 to +101 bp. The
arrowhead at 469 bp shows the SacI site that
was used to generate a deleted promoter fragment. An analysis of the
sequence (Matinspector) revealed numerous putative transcription
binding sites (underlined). Dashed lines indicate
a transcription binding site on the complementary strand. The
bold type represents the 5'-end of exon 1.
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Fig. 4.
Activation of the hABC1 promoter by
oxysterols and retinoic acid in RAW 264.7 cell. a, a
fragment of the hABC1 promoter (from 828 to +101 bp) was
linked to the firefly luciferase reporter gene. The resulting plasmid
was cotransfected with a control reporter plasmid (Renilla
luciferase) in the mouse macrophage-like RAW 264.7 cells. Four
independent transfection experiments (each in triplicate) were
performed. The results are expressed as a ratio between the Firefly and
Renilla luciferase activities. Cells were treated with
vehicle (ethanol) or 22(R)-Hch (10 µM) or 9CRA
(10 µM) or 22(R)-Hch (10 µM) and
9CRA (10 µM) for 24 h in fetal bovine serum medium
complemented with 10% lipoprotein-deficient serum. b,
activation of the hABC1 promoter by various oxysterols
and/or 9CRA. Similar experiments to those performed in a
were done using 22(R)-Hch (10 µM), 25-Hch (10 µM), 7-Kch (10 µM), and 9CRA (10 µM). Three to four independent experiments in duplicate
or triplicate were performed. Bars indicate mean ± S.D. Significance of treatment versus ethanol is
indicated by ***, p < 0.001; *, p < 0.05.
and LXR Are Both Expressed in Macrophages--
To
further investigate the potential role of LXR and LXR in the
sterol-mediated up-regulation of hABC1, we verified that the mRNA
of these nuclear receptors was present in RAW264.7 cells (Fig.
5a). We also analyzed their
expression in vivo, using thioglycolate-elicited peritoneal
macrophages from mice. Both receptors were detected in macrophages by
Northern blot of total RNA, with a much stronger signal for LXR. We
also selected two cell lines for our transactivation experiments, CV-1
and 293 cells. As shown in Fig. 5, both LXR and LXR could be
detected in 293 cells but only LXR in CV-1 cells. Because the
quantities of mRNA for LXRs are relatively low in 293 cells and
CV-1 cells, we chose to perform transactivation experiments in these
cell lines.
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Fig. 5.
Expression of LXR
and LXR in various cell lines.
Sterol transactivation by LXR/RXR of the hABC1 promoter is
shown. a, cells were isolated and cultured as described
under "Material and Methods." A Northern blot was performed with 35 µg of total RNA for each cell line. Hybridizations were performed
using probes of similar specific activities for hLXR, mLXR, and
mouse glycerol-3-phosphate dehydrogenase (G3PDH) as an
internal standard. h, human; m, mouse.
b, 293 cells were transfected with the hABC1
promoter (from 469 to +101 bp) or a construct containing three copies
of a sterol-responsive element of the CETP promoter
(c) (25). These constructs were cotransfected with the
Renilla luciferase reporter gene and hLXR, mLXR,
and/or hRXR as designated. The cells were treated 24 h with
vehicle alone or 22(R)-Hch (5 µM) and/or 9CRA
(10 µM) in fetal bovine serum medium with 10%
lipoprotein-deficient serum. The results represent 2 independent
experiments in duplicate for the transfection using the
hABC1 promoter and 1-2 experiments in duplicates for the
transfection using the hCETP promoter. d, CV-1
cells were transfected and treated according to the protocols described
in b. 2-3 independent experiments in duplicates were
performed. Bars indicate mean ± S.D. Significance of
treatment versus ethanol is indicated by **,
p < 0.01; *, p < 0.05.
and/or LXR/ in
the sterol up-regulation of hABC1, we cotransfected 293 cells with the human ABC1 promoter and with these receptors (Fig.
5b). We used the shorter promoter (deletion 460 bp), which
was sterol-responsive in macrophages (data not shown). In 293 cells
without cotransfected LXR/hRXR we observed up-regulation of the
promoter by 22(R)-Hch (4.5-fold, p < 0.05)
and 9CRA (3.5-fold, p < 0.05) alone. The combination
of 9CRA and 22(R)-Hch resulted in an additive effect (10-fold, p < 0.05). Basal activity was slightly
increased (1.5-fold) when LXR/hRXR were cotransfected, as was the
sterol response, but there was no additional effect by 9CRA or 9CRA and
22(R)-Hch. However, cotransfection of LXR/RXR caused a
5-fold increase in basal expression and a synergistic effect of 9CRA
and 22(R)-Hch (19-fold induction compared with ethanol and
3-fold induction compared with 9CRA alone, p < 0.05).
binding site of the CETP promoter (25) (Fig. 5c). Even in the absence
of transfected receptors, this construct was highly sterol- and
retinoic acid-responsive in 293 cells (14- and 24-fold increase in
luciferase activity, respectively, p < 0.05), and an
additive or synergistic effect (58-fold) was obtained with cotreatment.
LXR transfection resulted in increased basal activity and increased
induction by sterols. However, 9CRA provided no further increase in
activity compared with non-transfected cells. These results suggest
that endogenous LXRs in 293 cells play a role in the response of both
ABC1 and CETP promoters with a further increase
in sterol-dependent promoter activity when LXR and RXR are cotransfected.
/hRXR or LXR/hRXR increased the basal activity of
the promoter (2- and 4-fold, respectively, both p < 0.01). Exposure to 22(R)-Hch resulted in increased
transactivation of the promoter (6- and 8-fold, respectively), compared
with the control with no receptor.
160 bp or 100 bp
to +101 bp. Deletion of exon sequences (from +3 bp to +101 bp) reduced
the basal activity, but the response to 22(R)-Hch and 9CRA
was maintained. Notably, deletion of sequences between 101 and 36
bp reduced the basal activity and abolished the response to sterol
and/or retinoic acid. Interestingly, the region covering 100 to +36
bp contains an almost perfect DR4 element located on the non-coding
strand between 70 and 55 bp (Fig. 6, inset).
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Fig. 6.
Deletional analysis of hABC1
promoter. Deletions were performed by enzymatic digestion or
PCR amplification of the hABC1 promoter. The results
represent two independent experiments of duplicates. Bars
indicate mean ± S.D. Significance of treatment versus
ethanol is indicated by ***, p < 0.001; *,
p < 0.05.
928 to +101 bp). Both half-sites
were mutated by changing nucleotides away from the nuclear hormone
binding consensus sequence, as shown in Fig.
7. This mutation reduced the basal
expression (2.5-fold), but the activity was still readily detectable.
Importantly, the mutation abolished the response to
22(R)-Hch alone or in combination with 9CRA.
View larger version (29K):
[in a new window]
Fig. 7.
Mutational analysis of hABC1
promoter. Raw 264.7 cells were transfected with wild-type
(WT) hABC1 promoter (from 928 to +101 bp) or
its mutated version. The mutations are presented in Fig. 7. The results
represent two independent experiments of duplicates. Bars
indicate mean ± S.D. Significance of treatment versus
ethanol is indicated by ***, p < 0.001; *,
p < 0.05.
/hRXR (Fig.
8, top) or LXR/hRXR
(Fig. 8, bottom). When the hABC1 wild-type DR4
element is used alone, a single major shift in activity is detected for
both types of nuclear extracts (Fig. 8, lane 1), which
disappears with excess of cold competitor indicating specificity (Fig.
8, lane 2). Because the mutated version is unable to compete
with the intact oligonucleotide (Fig. 8, lane 3), the
integrity of the DR4 itself is necessary for transcription factor
binding. In cells cotransfected with LXR/RXR, antibodies recognizing
the LXR or LXR/ common region and anti-RXR antibodies markedly
reduced binding activity and produced supershifted bands (Fig.
8, top). In cells cotransfected with LXR/RXR, antibodies to LXR, LXR /, and RXR showed similar effects except that the LXR antibody did not produce a supershifted complex (25). Antibodies specific for ROR had no (or a minimal) effect as
expected. These results show that LXR/RXR or LXR/RXR bind
this DR4 element.
View larger version (59K):
[in a new window]
Fig. 8.
Electrophoretic mobility shift assay of human
ABC1 promoter fragment. Oligonucleotides
containing the DR4 element (see Fig. 6) that was identified as a
potential binding site for LXR/RXR or a mutated version (mut.
DR4) were 32P-radiolabeled. Competitors (lanes
2 and 3) correspond to cold wild-type or mutant
oligonucleotides. After incubation with these oligonucleotides and
nuclear extracts from 293 cells transfected with LXR /hRXR
(B) or LXR/hRXR (A), some samples were
incubated with polyclonal antibodies for LXR/, LXR, RXR, or
ROR (negative control) or with a monoclonal antibody targeting
LXR. Three different experiments were performed with similar
results. The arrow indicates the position of the retarded
complex.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/RXR or
LXR/RXR binds to a DR4 element in the hABC1 promoter
and mediates its activation by oxysterol and retinoic acid. Thus, LXR
and/or RXR agonists could be useful drugs to reverse foam cell
formation and atherogenesis.
to compensate for the lack of LXR in
LXR
/ mice (24) suggests that these receptors have
different targets. In vitro, both LXR and LXR are able
to up-regulate hABC1 (Fig. 5, b-d) or CETP (25), but
LXR is clearly more effective than LXR in mediating the sterol
response of hABC1 (Fig. 5b). This is also consistent with
the fact that LXR appears to be more highly expressed than LXR in
macrophages (Fig. 5a).
| |
FOOTNOTES |
|---|
* This work was supported by National Institutes of Health Grants HL54591 and HL56984.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 correspondence should be addressed: Division of Molecular
Medicine, Dept. of Medicine, Columbia University, New York, NY 10032. Tel.: 212-305-4899; Fax: 212-305-5052; E-mail:
art1@columbia.edu.
Published, JBC Papers in Press, June 16, 2000, DOI 10.1074/jbc.M003337200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: HDL, high density lipoprotein; 9CRA, 9-cis-retinoic acid; 22(R)-Hch, 22(R)-hydroxycholesterol; 25-Hch, 25-hydroxycholesterol; 7-Kch, 7-ketocholesterol; LXR, liver X receptor; RXR, retinoid X receptor; LXR/RXR, cotransfected mixture of two receptors; LXR·RXR, putative heterodimer complex; SF1, steroidogenic factor 1; CETP, cholesterol ester transfer protein; DR, direct repeat; RACE, rapid amplification of cDNA ends; bp, base pair(s); PCR, polymerase chain reaction; LDL, low density lipoprotein.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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