The Human Apolipoprotein AV Gene Is Regulated by Peroxisome Proliferator-activated Receptor-
and Contains a Novel Farnesoid X-activated Receptor Response Element*
Xavier Prieur,
Hervé Coste and
Joan C. Rodríguez
From the
GlaxoSmithKline, 25 Avenue du Québec, 91951 Les Ulis cedex,
France
Received for publication, February 6, 2003
, and in revised form, March 21, 2003.
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ABSTRACT
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The newly identified apolipoprotein AV (apoAV) gene is a key player in
determining plasma triglyceride concentrations. Because hypertriglyceridemia
is a major independent risk factor in coronary artery disease, the
understanding of the regulation of the expression of this gene is of
considerable importance. We presently characterize the structure, the
transcription start site, and the promoter of the human apoAV gene. Since the
peroxisome proliferator-activated receptor-
(PPAR
) and the
farnesoid X-activated receptor (FXR) have been shown to modulate the
expression of genes involved in triglyceride metabolism, we evaluated the
potential role of these nuclear receptors in the regulation of apoAV
transcription. Bile acids and FXR induced the apoAV gene promoter activity.
5'-Deletion, mutagenesis, and gel shift analysis identified a heretofore
unknown element at positions 103/84 consisting of an inverted
repeat of two consensus receptor-binding hexads separated by 8 nucleotides
(IR8), which was required for the response to bile acid-activated FXR. The
isolated IR8 element conferred FXR responsiveness on a heterologous promoter.
On the other hand, in apoAV-expressing human hepatic Hep3B cells, transfection
of PPAR
specifically enhanced apoAV promoter activity. By deletion,
site-directed mutagenesis, and binding analysis, a PPAR
response
element located 271 bp upstream of the transcription start site was
identified. Finally, treatment with a specific PPAR
activator led to a
significant induction of apoAV mRNA expression in hepatocytes. The
identification of apoAV as a PPAR
target gene has major implications
with respect to mechanisms whereby pharmacological PPAR
agonists may
exert their beneficial hypotriglyceridemic actions.
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INTRODUCTION
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Recent epidemiological studies have shown that hypertriglyceridemia is a
major independent risk factor for coronary heart disease, which remains as a
major cause of mortality in the Western world
(16).
Understanding the factors that control plasma triglyceride levels and the
genes that primarily reflect circulating concentrations of triglyceride-rich
lipoproteins is thus of major importance and may provide new opportunities for
therapeutic intervention in atherogenic dyslipidemia.
Pharmacological activation of peroxisome proliferator-activated
receptor-
(PPAR
1; NR1C1)
lowers plasma levels of triglyceride-rich lipoproteins markedly
(7,
8). PPAR
is a fatty
acid-activated nuclear transcription factor that regulates the expression of
genes involved in lipid and energy metabolism
(9,
10). PPAR
heterodimerizes with the retinoid X receptor
(RXR
; NR2B1) and
binds to specific DNA sequence elements, designated PPREs, which consist of a
direct repeat of the hexanucleotide core motif PuGGTCA separated by 1 or 2
nucleotides (DR1 or DR2). PPAR
mediates the hypotriglyceridemic effect
of fibrates by regulating the transcription of key genes associated with
different intra- and extracellular metabolic pathways. Fibrates increase
cellular fatty acid transport and uptake, conversion to acyl-CoA derivatives,
and catabolism by
-oxidation, which results in decreased substrate
availability for triglyceride synthesis and very low density lipoprotein
production by the liver. In addition, activated PPAR
promotes lipolysis
and clearance of triglyceride-rich lipoproteins, thus decreasing the plasma
levels of triglycerides (11).
These extracellular effects have been attributed to an increase in LPL
activity via induction of its expression in the liver and reduction of hepatic
expression (and subsequently serum levels) of apolipoprotein CIII (apoCIII),
an established inhibitor of LPL activity and remnant particle catabolism
(7,
1215).
The farnesoid X-activated receptor (FXR, NR1H4) is another member of the
nuclear receptor superfamily that has been described as a key regulator in the
control of plasma triglyceride levels
(16). The expression of FXR is
restricted to the liver, intestine, kidney, and adrenal cortex
(17,
18). FXR is a bile
acid-activated receptor that alters transcription by binding as a heterodimer
with RXR to response elements (FXREs) within the regulatory regions of target
genes
(1621).
With two exceptions, FXREs consist of an inverted repeat of the canonical
hexanucleotide core motif PuGGTCA spaced by one base pair (IR1)
(16). Recently, an inverted
repeat with no spacer (IR0) in the dehydroepiandrosterone sulfotransferase
gene (22) and an everted
repeat of the core motif separated by 8 nucleotides (ER8) in the multidrug
resistance-associated protein 2 (MRP2) gene
(23) have also been reported
as elements required for FXR-dependent transcriptional activation. FXR acts as
a bile acid sensor in the liver, where it mediates the negative feedback of
bile acid biosynthesis via induction of small heterodimer partner (NR0B2) and
subsequent repression of cholesterol 7
-hydroxylase gene
(CYP7A1) expression
(2427),
the rate-limiting enzyme in this pathway, and the sterol 12
-hydroxylase
gene (CYP8B1), the specific enzyme required for cholic acid synthesis
(28). FXR also controls the
transport of bile acids in the liver and intestine. It promotes the excretion
of bile acids from hepatocytes into the bile by induction of bile salt export
pump gene expression (29) and
stimulates the enterohepatic circulation of bile acids by activation of the
transcription of ileal-bile acid-binding protein (I-BABP)
(30). In addition, recent
findings have shown that FXR regulates lipoprotein metabolism
(16). Phospholipid transfer
protein and apoAI, two major players in plasma high density lipoprotein
metabolism, have been described as FXR target genes
(3133).
Studies in rodents have revealed the role of FXR in regulating triglyceride
levels. The administration of either a synthetic FXR ligand (GW4064)
(34) or a natural FXR ligand
(cholic acid) (35) to rodents
resulted in at least a 50% decrease in plasma triglyceride levels. Consistent
with these observations, FXR-deficient mice exhibited a 150% increase in
plasma triglyceride levels
(36). Importantly, Edwards and
co-workers (35) have reported
that activated FXR induces the expression of apoCII, an obligate cofactor for
LPL, which in turn would result in the hydrolysis of triglycerides in
chylomicrons and very low density lipoprotein. These data provide a mechanism
to explain the hypotriglyceridemic effects of the natural FXR activator
chenodeoxycholic acid (CDCA) when administered to patients with
cholesterol-rich gallstones
(37).
Recently, a novel apolipoprotein, designated apoAV, was suggested to play a
significant role on plasma triglyceride metabolism. Pennacchio et al.
(38) described the discovery
of apoAV when 200 kb of orthologous sequences spanning the apoAI/CIII/AIV gene
cluster were compared in humans and mice. At the same time, van der Vliet
et al. (39)
identified apoAV as a protein associated with an early phase of liver
regeneration. Interestingly, the predicted amino acid sequence showed
appreciable homology with apoAI and apoAIV. Evidence for the involvement of
apoAV in triglyceride metabolism was obtained in mice, in which serum
triglyceride concentrations were decreased to one-third when a human apoAV
transgene was overexpressed
(38) or adenoviral vectors
expressing mouse apoAV were injected
(40). Conversely, plasma
triglyceride levels were 4-fold elevated in knockout mice lacking apoAV
compared with their wild-type littermates
(38). Furthermore, single
nucleotide polymorphisms (SNPs) across the locus of the apoAV gene were found
to be significantly associated with plasma triglyceride and very low density
lipoprotein mass levels in several ethnic groups
(38,
4145).
Notwithstanding, the specific biological functions of apoAV in triglyceride
metabolism or liver regeneration are still unknown. Pennacchio et al.
(38) reported a genomic
organization of human apoAV with four exons. In contrast, van der Vliet et
al. (39) reported three
exons. Both groups showed that the expression of apoAV mRNA was restricted to
the liver.
The current studies were undertaken to provide a comprehensive analysis of
the regulation of human apoAV gene expression. We presently describe the
genomic structure, the transcription start site, and the promoter of the human
apoAV gene. Inasmuch as PPAR
and FXR play key roles in the regulation
of genes involved in triglyceride metabolism, we sought to evaluate their
potential effects on human apoAV gene expression. We show that a novel FXRE is
present in the promoter of human apoAV and demonstrate that human apoAV is a
bona fide PPAR
target gene. Our findings provide new insights
into the mechanisms whereby PPAR
and its ligands lower plasma
triglyceride levels.
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EXPERIMENTAL PROCEDURES
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RNA Ligase-mediated Rapid Amplification of cDNA Ends
(RLM-RACE)The start of transcription of human apoAV was identified
by using GeneRacerTM (Invitrogen). This kit provides a modification of
the RACE method that ensures the capture of only full-length 5' ends of
specific transcripts via elimination of truncated messages from the
amplification process. Human liver total RNA (Clontech) was treated with calf
intestinal phosphatase and tobacco acid pyrophosphatase according to the
instructions of the kit. After the selective ligation of an RNA
oligonucleotide to the 5'-ends of decapped mRNA, first strand cDNA
synthesis was carried out with the ThermoScriptTM RNase
H reverse transcriptase (Invitrogen; not supplied in the
GeneRacerTM kit) at 50 °C for 50 min and a reverse human
apoAV-specific primer (JCR81; see Table
I). The cDNA 5'-end was amplified by using PlatinumTM
TaqDNA Polymerase High Fidelity (Invitrogen; not supplied in the
GeneRacerTM kit) and primers GeneRacerTM 5' and JCR80
(Table I). The PCR conditions
were as follows: 94 °C for 2 min followed by 30 cycles of 94 °C for 30
s, 65 °C for 30 s, and 68 °C for 1 min. The sample was then diluted
20-fold, and 1 µl was used in a second PCR with nested primers
(GeneRacerTM 5' Nested and JCR79). DNA products were cloned into
pCR4-TOPOTM vector (Invitrogen) and then sequenced.
PlasmidsSeveral constructs containing the 5'-flanking
region of human apoAV (2455/+18, 617/+18, 617/+529,
535/+18, 437/+18, 242/+18, and 82/+18, relative to
the transcription start site reported in this paper) were obtained by PCR from
human bacterial artificial chromosome-RPCI-11442E11 DNA (BACPAC
Resources, Children's Hospital Oakland Research Institute) with
PlatinumTM TaqDNA Polymerase High Fidelity (Invitrogen). Forward
primers were tailed with a KpnI restriction site. Reverse primers
were tailed with an NheI site. The PCR products were digested with
KpnI and NheI and cloned into the corresponding sites of the
promoterless firefly (Photinus pyralis) luciferase reporter plasmid
pGL3-basic (Promega), generating p2455/+18hAvLUC,
p617/+18hAvLUC, p617/+529hAvLUC, p535/+18hAvLUC,
p437/+18hAvLUC, p242/+18hAvLUC, and p82/+18hAvLUC.
Site-directed mutagenesis of the constructs p2455/+18hAvLUC,
p617/+18hAvLUC, and p242/+18hAvLUC were accomplished using the
QuikChangeTM site-directed mutagenesis kit (Stratagene) according to the
recommendations of the manufacturer. The vector pGL3-TK contains a fragment
corresponding to nt 109 to +20 of the thymidine kinase (TK) gene
promoter of herpes simplex virus
(46) subcloned into the
BglII/HindIII sites of the pGL3-basic vector. The reporter
plasmids p(AvIR8)n-TK (n = 14) were
generated by insertion of 14 copies of a double-stranded
oligonucleotide (obtained by annealing JCR139 and JCR140; see
Table I) containing the
sequences spanning nt 109 to 80 into the BglII site of
pGL3-TK. Similarly, the four-copy mutant IR8 constructs
p(mt1AvIR8)4-TK, p(mt2AvIR8)4-TK, and
p(mt3AvIR8)4-TK were generated by annealing oligonucleotides JCR159
with JCR160, JCR161 with JCR162, and JCR163 with JCR164, respectively, before
ligation into BglII-digested pGL3-TK. The luciferase reporter plasmid
p(ER8)4-TK contains four copies of the rat MRP2 ER8 element
(23) in front of TK promoter
and was provided by L. Moore (GlaxoSmithKline, Research Triangle Park, NC).
Plasmids expressing human cDNAs for PPAR
, RXR
, and FXR were
provided by J. A. Holt and J. M. Maglich (GlaxoSmithKline, Research Triangle
Park, NC). The backbone of those plasmids was the mammalian expression vector
pSG5 with a modified polylinker. Plasmid DNA was prepared using the Qiagen
endotoxin-free maxipreparation method and quantitated spectrophotometrically.
The integrities of all plasmids were verified by DNA sequencing.
Cell Transfection and Reporter AssaysHuman hepatoblastoma
Hep3B and monkey kidney CV-1 cell lines were cultured in Eagle's basal medium
supplemented with nonessential amino acids, 2 mM L-glutamine, 100
units/ml penicillin, and 100 µg/ml streptomycin sulfate (medium A) and 10%
(v/v) fetal calf serum (FCS). On day 0, cells were seeded on 24-well plates at
a density of 3 x 105 or 5 x 104 cells/well
for Hep3B or CV-1, respectively. On day 1, cells were transfected with FuGENE
6 reagent (Roche Applied Science) according to the manufacturer's
instructions. Typically, each well of a 24-well plate received 100 ng of
firefly luciferase reporter plasmid and, when indicated, 200 ng of plasmids
expressing human PPAR
or FXR. Effector plasmid dosage was kept constant
by the addition of appropriate amounts of the empty expression vector pSG5.
100 ng/well of a sea pansy (Renilla reniformis) luciferase plasmid
pRL-TK (Promega) was included in all transfections as an internal control for
transfection efficiency. After 5 h, cells were switched to medium A
supplemented with 10% delipidated calf serum (Sigma) in the presence, when
indicated, of either 1 µM GW9003 (GlaxoSmithKline), 50 or 100
µM CDCA, 1 µM GW4064 (GlaxoSmithKline), or the
vehicle (Me2SO). On day 3, cell lysates were prepared by shaking
the cells in 200 µlof1x Promega lysis buffer for 15 min at room
temperature. Firefly and Renilla luciferase activities were measured
using a Dual-Luciferase® Reporter Assay System (Promega) and a Lumistar
luminometer (BMG Lab Technologies). Firefly luciferase activity values were
divided by Renilla luciferase activity values to obtain normalized
luciferase activities. To facilitate comparisons within a given experiment,
activity data were presented either as relative luciferase activities or as
-fold induction over the normalized activity of the reporter plasmid in the
absence of nuclear receptor cotransfection and agonist supplementation. All
transfection experiments were performed at least three times and with each
experimental point done in triplicate. The data are expressed as the means
± S.D. Statistic significance analysis were done with Student's
t test.
Treatment of HepG2 CellsOn day 0, human hepatoblastoma
HepG2 cells were plated on 24-well plates at 3.5 x 105
cells/well in medium A supplemented with 10% (v/v) FCS. On day 1, the medium
was replaced with fresh medium A supplemented with 1% (v/v) FCS and 1
µM GW9003, 1 µM GW4064, or vehicle
(Me2SO). On day 3, the cells were washed twice with
phosphate-buffered saline and harvested for isolation of RNA.
Treatment of Primary Cultured Cynomolgus Monkey Hepatocytes
Cynomolgus monkey hepatocytes (batch L-1811; Biopredict) were seeded on day 0
at a density of 6 x 105 cells/well on 12-well plates in
Williams' medium E supplemented with 2 mM L-glutamine, 100 units/ml
penicillin, 100 µg/ml streptomycin sulfate, 4 µg/ml insulin (medium B),
and 10% (v/v) FCS. On day 1, cells were switched to medium B containing 100
nM dexamethasone, 1% (v/v) FCS and 1 µM GW9003, 1
µM GW4064, 30 µM lithocholic acid (LCA), or
vehicle (Me2SO). After 24 h, a fresh experimental medium was
provided. On day 3, the cells were washed twice with PBS and harvested for
isolation of RNA.
Real Time PCR Quantification of mRNAsTotal RNA was prepared
from primary cynomolgus hepatocytes, human THP-1 monocytes differentiated to
macrophages, HepG2, Hep3B, Huh-7, and Caco-2 cells with the RNeasyTM Mini
kit, the QIAshredderTM homogenizers, and the RNase-Free DNase set
(Qiagen) according to the manufacturer's instructions. A 1-µg aliquot was
used as a template for cDNA synthesis, employing the TaqManTM Reverse
Transcription Reagents kit (Applied Biosystems). Specific primers for each
gene were designed with Primer Express Software (PerkinElmer Life Sciences).
The sequences of forward and reverse primers are shown in
Table I. The specificity of the
primers was verified by showing that the real time reverse transcriptase
(RT)-PCR reaction product generated a single band after agarose gel
electrophoresis. In addition, each couple of primers was tested in successive
dilutions of cDNA to analyze and validate its efficiency. The reactions
contained, in a final volume of 25 µl, 5 µl of diluted (1:10) cDNA, a
300 nM concentration of the forward and reverse primers, and
2x SYBRTM Green PCR Master Mix (Applied Biosystems). Real time PCRs
were carried out in 96-well plates by using the ABI PRISMTM 7700 sequence
detection system (Applied Biosystems). Since there is a high level of sequence
identity for apoAV between humans and monkeys, the primers were valid for RNA
from both primates. Levels of apoAV were normalized to 18 S to compensate for
variations in input RNA amounts (18 S levels were unaffected by the
treatments). All points were performed in triplicate during two independent
experiments, and the RT-PCRs were carried out in duplicate for each sample.
The relative amounts of mRNAs were calculated using the Comparative
CT method as described in Ref.
55.
In Vitro Transcription/Translation and Electrophoretic
Mobility Shift Assay (EMSA)Human PPAR
, RXR
, and FXR
proteins were synthesized in vitro from the corresponding expression
plasmids in rabbit reticulocyte lysate by using the TNT® Quick Coupled
transcription/translation system (Promega) according to the instructions of
the manufacturer. In order to obtain an unprogrammed lysate as a negative
control for EMSA, a reaction was performed with the empty vector pSG5.
Double-stranded oligonucleotides were radiolabeled by fill in with the Klenow
fragment of DNA polymerase I and used as probes. For competition experiments,
increasing -fold molar excesses of unlabeled probes were included during a
15-min preincubation on ice. Protein-DNA binding assays were performed as
described (47). AvDR1 is a
double-stranded oligonucleotide corresponding to nt 275 to 247
of human apoAV promoter. mtAvDR1 is a modified version of AvDR1 that contains
mutations corresponding to nt 263(G
C)/262(G
A)/261(T
A). The control probe used in EMSA with
PPAR
-RXR
contains the PPRE of the rat mitochondrial
3-hydroxy-3-methylglutaryl-CoA synthase (mitHMGS) gene promoter
(48). The probe AvIR8 contains
the sequence spanning nt 109 to 80 of the human apoAV promoter.
The mutations present in the modified versions of AvIR8 (mt1AvIR8, mt2AvIR8,
and mt3AvIR8) are shown in Fig.
10B. The probe used as a control for competition
experiments with FXR-RXR
contains the FXRE of the human I-BABP gene
promoter (30). Samples were
electrophoresed at 4 °C on a 4.5% polyacrylamide gel in 0.5x TBE
buffer (45 mM Tris, 45 mM boric acid, 1 mM
EDTA, pH 8.0). Gels were dried and analyzed using an Amersham Biosciences
PhosphorImager STORM 860 and ImageQuant software (Amersham Biosciences).

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FIG. 10. The IR8 element present in the promoter of the human apoAV gene (AvIR8)
confers FXR responsiveness to heterologous promoters. A, the
apoAV IR8 element confers transactivation by bile acid-activated FXR in a copy
number-dependent manner. Hep3B cells were transiently transfected with
plasmids expressing human FXR or the empty pSG5 vector as control, together
with reporter constructs containing 14 copies of the sequence
corresponding to nt 109 to 80 (AvIR8) of the human
apoAV gene promoter or four copies of the ER8 element present in the MRP2 gene
promoter (ER8) cloned in front of a heterologous TK promoter-driven
luciferase. The empty pGL3-TK reporter vector was used as negative control.
Cells were treated with vehicle or 100 µM CDCA for 48 h, and
luciferase activities were measured as described under "Experimental
Procedures." Results are expressed as -fold induction over control.
B, human apoAV gene promoter sequence surrounding the IR8 element and
mutated versions used in C. The hexameric consensus sites are in
boldface type, and their orientations are indicated by
arrows. Mutations in half-sites are shown in lowercase type
and within gray squares. C, functional effects of mutations on
hexameric consensus sites. Experiments were performed as in A with
reporter constructs containing four copies of the wild-type (wt)
(wtAvIR8) or mutant (mt1AvIR8, mt2AvIR8, mt3AvIR8) sequences
corresponding to nt 109 to 80 of the human apoAV gene promoter
cloned in front of TK promoter-driven luciferase.
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RESULTS
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Characterization of the Structure of the Human ApoAV Gene,
Identification of an Alternatively 5'-Untranslated Region, and
Determination of the Transcription Start SiteTwo previous reports
coincided to show by Northern blot analysis that apoAV has transcripts of
1.3 and 1.9 kb in the human liver, which are likely to result from
alternative polyadenylation
(38,
39). However, the number of
exons and the details of the exon/intron organization in the 5' region
of the human apoAV gene have been contradictory. In order to delineate the
structure of the gene, we performed RLM-RACE. This powerful technique is a
modification of the RACE method that ensures the capture of only full-length
specific transcripts via elimination of truncated messages from the
amplification process. Thus, using antisense primers from the 3' region
of mRNA, it is possible to amplify all of the upstream exons and hence to
detect the presence of alternatively spliced exons. A primer was designed at
the last exon of human apoAV, and a specific reverse transcription was carried
out from human liver RNA. In order to avoid secondary structures of mRNA, we
made the first strand cDNA synthesis at 50 °C with a reverse transcriptase
having a high thermal stability (see "Experimental Procedures").
Subsequently, specific PCRs were performed using additional apoAV nested
primers (Table I). The PCR
products were cloned and sequenced. Based on the genomic sequence spanning
part of human chromosome 11q23 present in the GenBankTM data bank
(accession number AC007707
[GenBank]
), we obtained an exon/intron organization of apoAV
with two distinct 5'-untranslated regions
(Fig. 1A). As shown in
Fig. 1B, the four-exon
structure of apoAV is also present in apoAI and apoCIII genes (as deduced from
the analysis of sequences in NM_000039
[GenBank]
, NM_000040
[GenBank]
, X01038
[GenBank]
, X03120
[GenBank]
, and J00098
[GenBank]
)
but not in apoAIV (structure taken from Ref.
50). The most important
feature of the RLM-RACE method is that it permits the determination of the
transcription start site with more precision than the primer extension and the
classic 5'-RACE procedures. As shown in
Fig. 1C, a single
human apoAV transcription start site was identified. The newly identified
first exon, which was named exon 1, has only 12 untranslated nt and contains a
stop codon in frame with the first ATG, which is located in the following exon
(Fig. 1C).
Interestingly, a sequence of 24 nt was observed just after exon 1 in 4 of 20
clones. This novel sequence, which we named exon 2a, was flanked on the
5' side by an ag consensus splice acceptor site on the first intron and
on the 3' side by the exon previously described in the literature as
exon 1, which we renamed exon 2b (Fig.
1C). Therefore, the second exon has either 57 nt (exon
2b) or 81 nt (exon 2a plus exon 2b), depending on the utilization of an
internal acceptor site (Fig.
1A). No novel exons were observed, so the coding region
is distributed in three exons, in agreement with earlier results
(39). Considered together, the
human apoAV gene consists of four exons, the second exon with two alternative
acceptor sites and the last exon with two alternative polyadenylation sites.
On the other hand, we noticed the presence of an SNP (A
G) 3 nt upstream
from the predicted translation start codon
(Fig. 1C).

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FIG. 1. Characterization of the structure, the transcription start site, and the
promoter of the human apoAV gene. A, schematic representation of
the exon-intron structure of the human apoAV gene. Exons are shown in
boxes with translated regions shaded in black. The
predicted initiation codon ATG is located in the second exon. The
relative positions of the gene-specific primers used in the RLM-RACE are
indicated by horizontal arrows. The last exon has two alternative
polyadenylation sites (39),
and the second exon has two alternative acceptor sites. A bent arrow
depicts the transcription start site. The relative position of an SNP (A
G) is shown. B, the structure of the human apoAV gene is compared
with that of the other components of the human apolipoprotein gene cluster.
The structures of human apoAI and apoCIII genes are deduced from sequences in
NM_000039
[GenBank]
, NM_000040
[GenBank]
, X01038
[GenBank]
, X03120
[GenBank]
, and J00098
[GenBank]
. The structure of the apoAIV
gene is taken from Ref. 50.
The exon lengths in nt are indicated by numbers above the bars.
Numbers below the lines depict intron lengths. C,
nucleotide sequence corresponding to the 5' region of the human apoAV
gene. Exons are in boldface. Intron sequences are shown in
lowercase. The predicted initiation codon is double
underlined. Numbers are relative to the transcription start site (+1). A
putative TATA box and the DR1 and IR8 elements described in the current study
are boxed. The consensus receptor-binding hexads are indicated by
horizontal arrows.
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Expression of ApoAV in Human Cell LinesPreviously published
Northern blots with RNA from several different human tissues have shown that
apoAV expression is restricted to the liver
(38,
39). We carried out real time
PCR quantification assays with RNA from several human cell lines with
different origins. We found that apoAV is expressed in HepG2 and Hep3B
hepatoma cells with levels comparable with the liver, but not in hepatic Huh-7
cells. Consistent with the hepatic specific expression, intestinal Caco-2
cells and THP-1 monocytes differentiated to macrophages showed no detectable
expression of apoAV (data not shown).
Cloning and Characterization of the Human ApoAV Gene
PromoterAnalysis of the 5'-flanking region of the human
apoAV gene identified a TATA box 30 nt upstream of position +1
(Fig. 1C), further
confirming the mapped transcription start site. No consensus Sp1 binding sites
were found within the first 3 kb, although several putative nuclear receptor
response elements containing imperfect repeats of the half-site PuGGTCA were
detected (see below). To investigate whether this 5'-flanking region
could drive transcription of a reporter gene, we subcloned
0.6 kb of the
human apoAV sequence upstream of the transcription start site into the firefly
luciferase pGL3-basic vector. The resulting construct, p617/+18hAvLUC,
was transiently transfected into HepG2 and Hep3B cells. The activity of this
construct was about 5-fold the activity of the parental promoterless vector in
both cell lines (Fig. 2). Next,
we observed that neither the addition of a more upstream region, in construct
p2455/+18hAvLUC, nor the inclusion of the first intron, in
p617/+529hAvLUC, substantially altered promoter activity (data not
shown).
Overexpression of the Nuclear Receptor PPAR
Enhances
the Activity of the Human ApoAV Gene PromoterApoAV is an important
determinant of plasma triglyceride levels
(38,
4045).
Inasmuch as PPAR
regulates the expression of genes involved in
triglyceride metabolism (10),
we evaluated whether this nuclear receptor modulates human apoAV gene
expression. For this purpose, transient transfection studies were performed in
Hep3B cells. Cotransfection of a human PPAR
expression plasmid resulted
in a significant increase of the activity of the firefly luciferase reporter
gene driven by the 617/+18 sequence of the human apoAV promoter
(Fig. 2). The effect of
PPAR
overexpression was promoter-dependent, because it was not observed
with the promoterless pGL3-basic vector. In line with previous studies in
hepatic cells (48,
49), transiently transfected
PPAR
showed substantial constitutive activity, possibly due to
activation by endogenous ligands. Nevertheless, we observed that it could be
further activated upon the exogenous addition of the specific PPAR
ligand GW9003 (Fig. 2).
Mapping of the Human ApoAV Promoter Site Conferring Responsiveness to
PPAR
In order to localize the region required for
PPAR
activation, a series of progressively larger 5'-deletion
human apoAV promoter constructs from nt 2455 to +18 were transiently
transfected into Hep3B cells together with a human PPAR
expression
plasmid in the presence or absence of GW9003. As shown in
Fig. 3, the sequence upstream
to position 437 can be deleted without loss of the response to
PPAR
. However, further deletion up to nt 242 eliminated the
PPAR
responsiveness. Analysis of the sequence in the
437/242 fragment revealed two potential hexamer binding sites
separated by a single nucleotide between nt 271 and 259
(Fig. 1C), thereby
conforming to the DR1 response element for PPAR
(PPRE).
To unequivocally characterize the DR1 as a PPRE, Hep3B cells were
cotransfected with a human PPAR
expression vector and an apoAV
promoter-luciferase reporter plasmid in which the DR1 sequence was mutated in
the 3'-hexamer (Fig.
4A). In contrast to the native promoter construct,
PPAR
and GW9003 had only a modest residual effect on the luciferase
gene expression of the construct bearing the mutated DR1
(Fig. 4B). These
results demonstrate that this DR1 indeed confers PPAR
response to the
apoAV promoter.
Binding Analysis of PPAR
-RXR
Heterodimers to the apoAV DR1 ElementPPAR
binds to DNA
as a heterodimer with RXR
(10). To investigate whether
PPAR
-RXR heterodimers directly bind to the human apoAV gene promoter,
radiolabeled double-stranded oligonucleotides corresponding to portions of the
617/+18 fragment of the apoAV promoter were used as probes in EMSA
experiments (Fig. 5). As
expected, the addition of in vitro translated PPAR
and
RXR
proteins resulted in the appearance of a robust retarded complex
when using a 275/247 fragment containing the apoAV DR1
(Fig. 5A, lane
4), but not when 617/593, 169/147,
144/117, and 52/31 probes
(Fig. 5A, lanes 2,
6, 8, and 10, respectively) or 484/462,
399/374, 298/263, and 109/80
fragments were employed (data not shown). Furthermore, the specificity of the
RXR
-PPAR
heterodimerapoAV DR1 interaction was demonstrated by
competition analysis. The formation of the retarded complex was inhibited by
the addition of increasing concentrations of either the unlabeled apoAV DR1
probe (Fig. 5B,
lanes 57) or a cold probe containing the PPRE of the mitHMGS
gene (Fig. 5B,
lanes 1113). In contrast, a cold double-stranded
oligonucleotide that is equivalent to the apoAV DR1 probe but harbors point
mutations in the 3' half-site (nt 263G
C/262G
A/261T
A in Fig.
4A) could not displace the labeled wild-type element
(Fig. 5B, lanes
810). In addition, it is worth noting that a more retarded band of
the labeled apoAV DR1 containing probe was visible upon the addition of
RXR
alone (Fig.
5B). Further investigations will be done to elucidate the
nature of this band. Taken together, these results show that this apoAV DR1 is
a genuine PPRE.

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FIG. 5. PPAR -RXR heterodimers can specifically bind to the DR1
element of the human apoAV promoter. A, EMSAs were performed
using in vitro transcribed/translated human PPAR (2.5 µl),
human RXR (2.5 µl), or unprogrammed reticulocyte lysate (),
when indicated, and labeled double-stranded oligonucleotides corresponding to
the indicated fragments of the human apoAV gene promoter as described under
"Experimental Procedures." The lysate volumes were kept constant
by the addition of unprogrammed lysate. CONTROL, a labeled probe that
contains the PPRE of the mitHMGS gene (3-fold less was used). B,
competition experiments for binding of PPAR -RXR heterodimers to
the labeled double-stranded oligonucleotides corresponding to nt 275 to
247 (AvDR1) were performed by adding a 10-, 50-, and 250-fold
molar excess of the indicated unlabeled probes. mtAvDR1 is a modified
version of wtAvDR1 with mutations corresponding to nt
263G C/262G A/261T A. The specific
PPAR -RXR -PPRE complex is indicated by an arrow. CONT,
PPRE of mitHMGS gene (48);
wt, wild type; mt, mutated.
|
|
PPAR
Agonists Induce ApoAV mRNA Levels in Human
Hepatoma HepG2 Cells and Primary Cultures of Cynomolgus
HepatocytesTo analyze whether apoAV mRNA expression is regulated
by PPAR
agonists, primary hepatocytes isolated from cynomolgus liver
and human hepatoma HepG2 cells were incubated for 48 h in medium containing
GW9003 or vehicle. As determined by quantitative real time RT-PCR, treatment
with the PPAR
agonist markedly increased apoAV mRNA levels
(Fig. 6).

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FIG. 6. PPAR agonists enhance apoAV mRNA levels in human HepG2 cells and
primary cynomolgus hepatocytes. Hepatocytes isolated from cynomolgus liver
(Cyno) and human hepatoma HepG2 cells were treated for 48h with
vehicle (Control) or 1 µM GW9003. Total RNA was
extracted for analysis by real time RT-PCR as described under
"Experimental Procedures." Specific apoAV mRNA levels normalized
to 18 S content are expressed relative to untreated cells set as 100 (mean
± S.D.). *, p < 0.01 versus control.
|
|
Bile Acids Induce Human apoAV Gene Promoter Activity via the Nuclear
Receptor FXRFXR regulates lipoprotein metabolism
(16), and FXR agonists have
hypotriglyceridemic effects on rodents and humans
(34,
35,
37). To determine whether FXR
and bile acids are able to modulate the apoAV gene promoter activity,
transient transfection assays were performed with a firefly luciferase
reporter gene expression vector driven by the 617/+18 sequence of the
human apoAV promoter. In Hep3B, the bile acid CDCA alone induced apoAV
promoter activity to some extent (Fig.
7A). Cotransfection of a human FXR expression plasmid
significantly enhanced CDCA-induced promoter activity. To further confirm the
role of FXR in the bile acid-induction of the apoAV promoter, monkey kidney
CV-1 cells, a cellular model frequently used in order to avoid the background
of endogenous FXR
(1921,
32), were also employed. As
shown in Fig. 7B,
promoter activity was unaffected by CDCA in the absence of FXR supplied
exogenously. By contrast, cotransfection of FXR led to a more than 10-fold
induction of apoAV promoter activity in the presence of 100 µM
CDCA. In both cell lines, the transactivation of the apoAV promoter-luciferase
reporter plasmid by CDCA via FXR was dose-dependent
(Fig. 7, A and
B). Moreover, the effects of CDCA and FXR were
promoter-dependent, because they were not observed with the promoterless
pGL3-basic vector. Since bile acids may also exert their actions through
FXR-independent pathways, we also tested the synthetic nonsteroidal specific
FXR ligand GW4064 (34). As
expected, treatment of Hep3B cells with GW4064 further enhanced the
transactivation of the apoAV promoter by FXR
(Fig. 7C). These data
clearly demonstrated the existence of an FXR response element in the promoter
of the human apoAV gene.

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FIG. 7. Bile acids induce human apoAV promoter activity via FXR. Hep3B
(A and C) and CV-1 (B) cells were transfected with
a plasmid containing a luciferase reporter gene driven by the
5'-flanking region (617/+18) of the human apoAV gene or the empty
pGL3-basic vector along with a plasmid expressing human FXR or the empty
vector pSG5 as control. Cells were treated for 48 h with vehicle alone, with
increasing concentrations of CDCA (A and B), or with 1
µM GW4064 (C), and luciferase activities were measured
and expressed as described under "Experimental Procedures."
Significant differences compared with the corresponding pSG5 control are as
follows: *, p < 0.005; **, p < 0.001; #, p
< 0.0005.
|
|
A Novel Element in the Human ApoAV Promoter Is Required for
Transcriptional Activation by FXRTo localize the region within the
human apoAV promoter that confers transcriptional responsiveness to FXR and
bile acids, a series of constructs containing sequential 5'-deletions
from nt 2455 to +18 of the apoAV promoter in front of the firefly
luciferase reporter gene were transiently transfected into CV-1 cells together
with a human FXR expression plasmid in the presence or the absence of 100
µM CDCA (Fig. 8).
The results showed that the nucleotide sequence between 2455 and
242 could be removed without preventing strong activation of the
reporter gene by bile acid-activated FXR. In contrast, deletion of the
fragment between nt 242 and 82 completely abolished the
induction of apoAV promoter activity by CDCA-activated FXR, indicating that
this region mediates the effects of bile acids.

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FIG. 8. The region between nt 242 and 82 of the human apoAV
promoter mediates the response to bile acid-activated FXR. CV-1 cells were
cotransfected with reporter plasmids containing the firefly luciferase gene
driven by progressively 5'-shortened fragments of the apoAV promoter
together with the empty vector pSG5 or a plasmid expressing human FXR. Cells
were treated with vehicle or 100 µM CDCA for 48 h, and
luciferase activities were measured as described under "Experimental
Procedures." Results are expressed as -fold induction over control.
LUC, luciferase.
|
|
Surprisingly, analysis of the sequence in the 242/82 fragment
did not reveal any of the previously described FXR response element (IR0, IR1,
or ER8) motifs (16,
22,
23). The only repeat of the
hexanucleotide core motif PuGGTCA with a low degree of degeneration present
within this region corresponds to an inverted repeat separated by 8
nucleotides (IR8) between nt 103 and 84
(Fig. 1C). Although a
response element with this kind of organization has never been reported
before, we mutated both halves of the IR8 site
(Fig. 9A) in the
context of the apoAV promoter on both the longest plasmid
(Fig. 9B) and the
shortest construct that still responds to FXR
(Fig. 9C) and
performed transient transfections in Hep3B cells. Whereas cotransfection of an
FXR expression plasmid significantly enhanced wild-type apoAV promoter
activity, FXR failed to induce the activity of the mutated constructs. These
results unequivocally show that this IR8 element is required for the response
to FXR and that there is no other FXRE in the promoter of human apoAV.

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FIG. 9. Mutation of the IR8 element on the human apoAV promoter eliminates the
response to FXR. A, human apoAV gene promoter sequence
surrounding the IR8 element. The gray box denotes the IR8 sequence.
The AGGTCA half-sites are indicated by horizontal arrows. The
wild-type nucleotides that were modified by site-directed mutagenesis are
underlined. The corresponding mutated nucleotides are shown
below the vertical arrows and within the gray squares.
B and C, Hep3B cells were transfected with a plasmid expressing
human FXR or the empty pSG5 vector as control together with reporter
constructs containing the wild-type or site-directed mutated 5'-flanking
regions of the human apoAV gene or the empty pGL3-basic vector as negative
control. Cells were treated for 48 h with vehicle, 100 µM CDCA,
or 1 µM GW4064, and luciferase activities were measured and
expressed as described under "Experimental Procedures."
Significant differences compared with the corresponding pSG5 control are as
follows: *, p < 0.001; **, p < 0.0005. The
crosses depict the presence of site-directed mutations in the IR8
element. LUC, luciferase.
|
|
The ApoAV IR8 Element Confers FXR Responsiveness to Heterologous
PromotersTo evaluate whether this IR8 element could confer FXR
responsiveness to a heterologous promoter, we linked the apoAV IR8 site
upstream of the TK promoter and the luciferase reporter gene. Reporter
constructs containing 14 copies of the apoAV IR8 element were
transiently transfected into Hep3B cells along with a human FXR expression
plasmid in the presence or the absence of CDCA. As demonstrated in
Fig. 10A, bile
acid-activated FXR enhanced the activity of apoAV IR8-driven promoter
constructs, whereas the reporter construct with the TK promoter alone was not
stimulated at all. Indeed, an induction of about 50-fold was reached with four
copies, and the response was manifested in a copy number-dependent manner,
thereby confirming that the IR8 element functions as an FXRE.
Next, the effects of selective mutations of each half-site in the IR8
element (Fig. 10B)
were examined in Hep3B cells transfected with reporter constructs driven by
four copies of the apoAV IR8 in front of the TK promoter. The FXR-dependent
induction was abrogated when mutations were introduced in any half-site
(Fig. 10C). These
observations confirm the organization of this FXR response element as an
inverted repeat of the canonical hexanucleotide separated by 8 nt. Finally,
the ability of the apoAV IR8 to respond to FXR and bile acids was assessed by
comparison with the ER8 element described in the promoter of MRP2 gene
(23). The FXR-dependent
response of the MRP2 ER8 in front of TK was
2.5-fold higher than the
apoAV IR8 (Fig.
10A).
The FXR-RXR
Heterodimer Binds Specifically to the ApoAV
IR8 ElementDirect binding of FXR/RXR
heterodimers to the
human apoAV IR8 element was examined. For this purpose, gel shift assays were
performed using in vitro translated human FXR and RXR
and a
radiolabeled double-stranded oligonucleotide containing the apoAV IR8 element.
The addition of FXR resulted in the appearance of a weak protein-DNA complex
band (Fig. 11, lane
2), a phenomenon that we also perceived in previously published gel shift
analysis performed with other FXREs (Fig. 5B in Ref.
23, Fig. 3B in Ref.
29, and Fig. 5A in
Ref. 30). Nevertheless, this
faint band disappeared, and a robust and more shifted band emerged when both
FXR and RXR
were present (Fig.
11, lane 4). This RXR
-FXR-IR8 binding was
specific, as demonstrated by competition of an excess of either unlabeled
wild-type apoAV IR8 oligonucleotide (Fig.
11, lanes 57) or a cold probe containing the IR1
sequence of the human I-BABP FXRE (Fig.
11, lanes 1719). Furthermore, mutation of either
half-site (Fig. 10B)
abolished the ability of the probe to compete
(Fig. 11, lanes
816). These results demonstrate that FXR-RXR
heterodimers
are able to directly and specifically interact with the apoAV IR8 and not with
cryptic binding motifs embedded within this site. We conclude that this novel
IR8 is a bona fide FXR response element.

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FIG. 11. The FXR-RXR heterodimer binds specifically to the IR8 element of
the human apoAV promoter. EMSAs were performed using in vitro
transcribed/translated human FXR (2.5 µl), human RXR (2.5 µl), or
unprogrammed reticulocyte lysate (), when indicated, and a labeled
double-stranded oligonucleotide corresponding to nt 109 to 80
(AvIR8) of the human apoAV gene promoter as described under
"Experimental Procedures." The lysate volumes were kept constant
by the addition of unprogrammed lysate. The competition experiments for
binding of FXR-RXR heterodimers were performed by adding a 50-, 250-,
and 500-fold molar excess of the indicated unlabeled double-stranded probes.
The mutations present in the modified versions of wtAvIR8
(mt1AvIR8, mt2AvIR8, and mt3AvIR8) are shown in
Fig. 10B. The
specific FXR-RXR -FXRE complex is indicated by an arrow. CONT,
FXRE of human I-BABP gene
(30); wt, wild-type;
mt, mutated.
|
|
ApoAV mRNA Levels Are Not Induced by Treatments with a FXR Agonist or a
Bile AcidTo analyze whether apoAV mRNA expression is regulated by
FXR agonists and bile acids, primary hepatocytes isolated from cynomolgus
liver and human hepatoma HepG2 cells were incubated for 48 h in medium
containing GW4064, LCA, or vehicle. Whereas the treatment of hepatocytes with
the FXR agonist or LCA increased the expression of small heterodimer partner
and bile salt export pump genes, no significant induction of apoAV expression
was observed (Fig. 12).

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FIG. 12. apoAV mRNA levels are not induced by treatments with an FXR agonist or a
bile acid. Hepatocytes isolated from cynomolgus liver (Cyno) and
human hepatoma HepG2 cells were treated for 48 h with vehicle
(Control), 1 µM GW4064 or 30 µM LCA.
Total RNA was extracted for analysis by real time RT-PCR as described under
"Experimental Procedures." Specific mRNA levels normalized to 18 S
content are expressed relative to untreated cells set as 100 for each gene
(mean ± S.D.). Significant differences compared with the untreated
control are as follows: *, p < 0.005; #, p < 0.01; **,
p < 0.0001.
|
|
 |
DISCUSSION
|
---|
The present study aimed to comprehend the mechanisms controlling the human
apoAV gene expression and to identify factors capable of positively modulating
its transcription. Our data reveal that two nuclear receptors involved in the
regulation of triglyceride metabolism, namely PPAR
and FXR, may control
the human apoAV gene.
The apoAV gene resides
27 kb distal to apoAIV in the apoAI/CIII/AIV
gene cluster on human chromosome 11q23
(38,
39). Our results showed that
the human apoAV gene contains four exons, with the first intron within the
5'-untranslated regions (Fig.
1A). With the exception of apoAIV
(50), both features are also
present in the rest of the apolipoprotein genes
(Fig. 1B). This
similarity gives further insight into the understanding of the process of
evolution of the apolipoprotein genes from a common ancestor
(51). In addition to the
previously reported alternative 3'-untranslated regions
(39), we found that the second
exon may have alternative length depending on the use of two distinct acceptor
sites. Interestingly, alternative transcripts have not been reported for other
members within this gene cluster. However, although some variation in terms of
mRNA stability may be inferred, none of the alternative transcripts contains a
different coding sequence. Strikingly, we came across an SNP (A
G) 3 nt
upstream from the predicted translation start codon
(Fig. 1C). The
incidence of this SNP (called c.3A
G) and its association with
higher triglyceride levels were recently reported
(42). There is another ATG in
frame 9 nt downstream of the start codon
(Fig. 1C). The
nucleotide sequences surrounding both methionine codons conform to the Kozak
consensus sequence (RNNATGG), with a G at +4 and a purine (R), preferably A, 3
nucleotides upstream (52). It
remains as an interesting question for future research to determine whether
this c.3A
G polymorphism could modify the start of translation or
reduce the rate of apoAV protein synthesis. On the other hand, a T
C SNP
in the 5'-region of the human apoAV gene, called SNP3, has also been
associated with elevated plasma triglyceride levels
(38,
4145).
Luciferase reporter assays revealed that the change of the common allele T to
C, which is located at position 600 relative to the transcription start
site (Fig. 1C), has no
significant effect on the basal activity of the apoAV promoter. Similarly, the
induction by either PPAR
or FXR of the apoAV promoter activity was not
affected by this change of T to C (data not shown). Analysis of the sequence
surrounding SNP3 reveals no apparent transcription factor binding sites. In
addition, EMSA showed no binding of nuclear receptors to this region
(Fig. 5A and data not
shown). Considered together, these data suggest that this polymorphism may not
be functional but, as proposed by others
(43), may act as a marker for
another functional change elsewhere in the gene cluster.
The presence of a TATA box 30 nt upstream of the start of transcription
identified by RLM-RACE along with the results from reporter experiments
showing transcriptional activity and responsiveness to nuclear receptors
validate this proximal 5'-flanking region as the promoter of the human
apoAV gene. The expression of apoAI, apoCIII, and apoAIV is controlled by a
common enhancer sequence located at the distal regulatory region of the
apoCIII gene (53). Based on
the notion that apoAV and these apolipoprotein genes evolved from a common
ancestral gene, it is tempting to speculate that a distal enhancer also
regulates the apoAV gene. If this is the case, however, then the enhancer for
apoAV should be elsewhere within a 26-kb XhoI fragment that contains
only apoAV, which was able to induce specific expression of human apoAV when
injected into mouse eggs (38).
Further work will be required to fully assess this hypothesis.
Deletion, site-directed mutagenesis, and binding analysis demonstrated
unequivocally that the apoAV gene promoter responds to bile acids and FXR via
a novel response element (IR8). Furthermore, the IR8 element was capable of
conferring FXR responsiveness on a heterologous promoter in a copy
number-dependent manner, and selective mutation of each half-site in the IR8
abolished the nuclear receptor response. However, despite exhaustive efforts,
we have not been able to demonstrate a significant induction of apoAV mRNA
levels by either bile acids or the FXR ligand GW4064 in hepatocytes
(Fig. 12). This paradox
suggests that other factors might potentially interfere in the regulation by
FXR. Recent studies are beginning to establish the notion that the DNA
sequences of hormone response elements for several nuclear receptors, once
thought to be specific, can be quite varied. The MRP2 ER8, for example, has
been shown to function as a pregnane X receptor (NR1I2), constitutive
androstane receptor (NR1I3), and FXR response element
(23). It is possible that the
novel IR8 could also act as response element for the convergence of
alternative transcription factors present in the systems that we have
analyzed, and the response to bile acids depends on the ratio between FXR and
these factors. Alternatively, sequences in the 5'-flanking region of
human apoAV upstream to position 2455 might respond negatively to bile
acids and thereby counteract the effect through the proximal FXRE. Further
studies will be necessary to determine whether potential activation of the
apoAV gene by bile acids is dependent on a particular environmental or
physiological cue.
The current study shows that treatment of hepatocytes with a PPAR
activator increased apoAV mRNA levels. Consistent with this observation,
induction of apoAV mRNA levels by fibrates has recently been reported
(54). Furthermore,
cotransfection of a PPAR
expression plasmid induced the activity of the
human apoAV promoter, suggesting that the increase by PPAR
activators
occurs, at least in part, via transcriptional mechanisms. Using
5'-deletion, mutagenesis, and gel shift analysis, a functional PPRE was
identified. A reporter construct bearing a mutated version of this PPRE still
showed a minimal transactivation by PPAR
. The basis for this residual
response is unclear, and the presence of an additional, weak response element
between nt 617 and 271 should not be excluded.
It is well established that PPAR
activators lower plasma
triglyceride levels by modulating the transcription of several lipid-related
genes (10). PPAR
induces the expression of enzymes involved in the uptake, transport, and
metabolism of fatty acids. As a result, the substrate availability for
triglyceride synthesis and very low density lipoprotein production is
diminished, which will ultimately decrease plasma triglyceride-rich
lipoprotein concentrations
(11). In addition, activated
PPAR
may increase LPL-mediated lipolysis and clearance of
triglyceride-rich lipoprotein particles by stimulating LPL expression in the
liver and by reducing hepatic production of apoCIII
(1215).
Our results demonstrate that apoAV is de facto a PPAR
target
gene, which is consistent with the triglyceride-lowering role proposed for
apoAV. These findings shed new light on the mechanisms whereby PPAR
agonists lower plasma triglyceride levels.
 |
FOOTNOTES
|
---|
* This work was supported by a Marie Curie Fellowship of the European
Community program Quality of Life under contract number QLK5-CT-2000-60009.
The costs of publication of this article were defrayed in part by the payment
of page charges. This 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. Tel.: 33-1-69-29-61-22; Fax:
33-1-69-07-48-92; E-mail:
jcrodrig{at}freesurf.fr.
1 The abbreviations and trivial name used are: PPAR
, peroxisome
proliferator-activated receptor
; apoAI, apoAIV, apoAV, apoCII, and
apoCIII, apolipoprotein AI, AIV, AV, CII, and CIII, respectively; CDCA,
chenodeoxycholic acid; DR, direct repeat; FXR, farnesoid X-activated receptor;
ER, everted repeat; FXRE, farnesoid X-activated receptor response element;
I-BABP, ileal-bile acid-binding protein; IR, inverted repeat; LCA, lithocholic
acid; LPL, lipoprotein lipase; mitHMGS, mitochondrial
3-hydroxy-3-methylglutaryl-CoA synthase; MRP2, multidrug resistance-associated
protein 2; nt, nucleotide(s); PPRE, peroxisome proliferator-activated receptor
response element; RACE, rapid amplification of cDNA ends; RLM-RACE, RNA
ligase-mediated RACE; RT-PCR, reverse transcriptase-PCR; RXR
, retinoid
X receptor
; SNP, single nucleotide polymorphism; TK, thymidine kinase;
FCS, fetal calf serum; GW9003, propanoic acid,
2-[4-[[[[2-(4-chlorophenyl)-4-methyl-5-thiazolyl]carbonyl]
amino]methyl]phenoxy]-2-methyl-(9Cl). 
 |
ACKNOWLEDGMENTS
|
---|
We thank J. Kirilovsky and M. Walker for encouragement of this work; D.
Grillot and P. Martres for scientific discussions; J. Pilot, V. Baudet, and G.
Krysa for technical assistance; P. A. Wilson for bioinformatics analysis; E.
Nicodeme for help with cynomolgus hepatocytes; J. Chapman (U551 INSERM,
France) for critical reading of the manuscript; and A. Brewster for manuscript
corrections.
 |
REFERENCES
|
---|
- Cullen, P. (2000) Am. J.
Cardiol. 86,
943949[CrossRef][Medline]
[Order article via Infotrieve]
- Jeppesen, J., Hein, H. O., Suadicani, P., and Gyntelberg, F.
(1998) Circulation
97,
10291036[Abstract/Free Full Text]
- Miller, M., Seidler, A., Moalemi, A., and Pearson, T. A.
(1998) J. Am. Coll. Cardiol.
31,
12521257[CrossRef][Medline]
[Order article via Infotrieve]
- Assmann, G., Schulte, H., and von Eckardstein, A.
(1996) Am. J. Cardiol.
77,
11791184[CrossRef][Medline]
[Order article via Infotrieve]
- Stampfer, M. J., Krauss, R. M., Ma, J., Blanche, P. J., Holl, L.
G., Sacks, F. M., and Hennekens, C. H. (1996) JAMA (J.
Am. Med. Assoc.) 276,
882888[Abstract]
- Fruchart, J. C., and Duriez, P. (2002)
Curr. Opin. Lipidol. 13,
605616[CrossRef][Medline]
[Order article via Infotrieve]
- Staels, B., Dallongeville, J., Auwerx, J., Schoonjans, K.,
Leitersdorf, E., and Fruchart, J. C. (1998)
Circulation 98,
20882093[Abstract/Free Full Text]
- Kersten, S., Desvergne, B., and Wahli, W. (2000)
Nature 405,
421424[CrossRef][Medline]
[Order article via Infotrieve]
- Issemann, I., and Green, S. (1990)
Nature 347,
645650[CrossRef][Medline]
[Order article via Infotrieve]
- Desvergne, B., and Wahli, W. (1999) Endocr.
Rev. 20,
649688[Abstract/Free Full Text]
- Barbier, O., Torra, I. P., Duguay, Y., Blanquart, C., Fruchart, J.
C., Glineur, C., and Staels, B. (2002) Arterioscler.
Thromb. Vasc. Biol. 22,
717726[Abstract/Free Full Text]
- Winegar, D. A., Brown, P. J., Wilkison, W. O., Lewis, M. C., Ott,
R. J., Tong, W. Q., Brown, H. R., Lehmann, J. M., Kliewer, S. A., Plunket, K.
D., Way, J. M., Bodkin, N. L., and Hansen, B. C. (2001)
J. Lipid Res. 42,
15431551[Abstract/Free Full Text]
- Hertz, R., Bishara-Shieban, J., and Bar-Tana, J.
(1995) J. Biol. Chem.
270,
1347013475[Abstract/Free Full Text]
- Staels, B., Vu-Dac, N., Kosykh, V. A., Saladin, R., Fruchart, J.
C., Dallongeville, J., and Auwerx, J. (1995) J. Clin.
Invest. 95,
705712[Medline]
[Order article via Infotrieve]
- Schoonjans, K., Peinado-Onsurbe, J., Lefebvre, A. M., Heyman, R.
A., Briggs, M., Deeb, S., Staels, B., and Auwerx, J. (1996)
EMBO J. 15,
53365348[Abstract]
- Edwards, P. A., Kast, H. R., and Anisfeld, A. M.
(2002) J. Lipid Res.
43,
212[Abstract/Free Full Text]
- Forman, B. M., Goode, E., Chen, J., Oro, A. E., Bradley, D. J.,
Perlmann, T., Noonan, D. J., Burka, L. T., McMorris, T., Lamph, W. W., Evans,
R. M., and Weinberger, C. (1995) Cell
81,
687693[Medline]
[Order article via Infotrieve]
- Lu, T. T., Repa, J. J., and Mangelsdorf, D. J. (2001)
J. Biol. Chem. 276,
3773537738[Free Full Text]
- Makishima, M., Okamoto, A. Y., Repa, J. J., Tu, H., Learned, R. M.,
Luk, A., Hull, M. V., Lustig, K. D., Mangelsdorf, D. J., and Shan, B.
(1999) Science
284,
13621365[Abstract/Free Full Text]
- Parks, D. J., Blanchard, S. G., Bledsoe, R. K., Chandra, G.,
Consler, T. G., Kliewer, S. A., Stimmel, J. B., Willson, T. M., Zavacki, A.
M., Moore, D. D., and Lehmann, J. M. (1999)
Science 284,
13651368[Abstract/Free Full Text]
- Wang, H., Chen, J., Hollister, K., Sowers, L. C., and Forman, B. M.
(1999) Mol. Cell
3,
543553[Medline]
[Order article via Infotrieve]
- Song, C. S., Echchgadda, I., Baek, B. S., Ahn, S. C., Oh, T., Roy,
A. K., and Chatterjee, B. (2001) J. Biol.
Chem. 276,
4254942556[Abstract/Free Full Text]
- Kast, H. R., Goodwin, B., Tarr, P. T., Jones, S. A., Anisfeld, A.
M., Stoltz, C. M., Tontonoz, P., Kliewer, S., Willson, T. M., and Edwards, P.
A. (2002) J. Biol. Chem.
277,
29082915[Abstract/Free Full Text]
- Russell, D. W. (1999) Cell
97,
539542[Medline]
[Order article via Infotrieve]
- Chawla, A., Saez, E., and Evans, R. M. (2000)
Cell 103,
14[Medline]
[Order article via Infotrieve]
- Chiang, J. Y., Kimmel, R., Weinberger, C., and Stroup, D.
(2000) J. Biol. Chem.
275,
1091810924[Abstract/Free Full Text]
- Davis, R. A., Miyake, J. H., Hui, T. Y., and Spann, N. J.
(2002) J. Lipid Res.
43,
533543[Abstract/Free Full Text]
- del Castillo-Olivares, A., and Gil, G. (2001)
Nucleic Acids Res. 29,
40354042[Abstract/Free Full Text]
- Ananthanarayanan, M., Balasubramanian, N., Makishima, M.,
Mangelsdorf, D. J., and Suchy, F. J. (2001) J. Biol.
Chem. 276,
2885728865[Abstract/Free Full Text]
- Grober, J., Zaghini, I., Fujii, H., Jones, S. A., Kliewer, S. A.,
Willson, T. M., Ono, T., and Besnard, P. (1999) J.
Biol. Chem. 274,
2974929754[Abstract/Free Full Text]
- Laffitte, B. A., Kast, H. R., Nguyen, C. M., Zavacki, A. M., Moore,
D. D., and Edwards, P. A. (2000) J. Biol.
Chem. 275,
1063810647[Abstract/Free Full Text]
- Urizar, N. L., Dowhan, D. H., and Moore, D. D. (2000)
J. Biol. Chem. 275,
3931339317[Abstract/Free Full Text]
- Claudel, T., Sturm, E., Duez, H., Torra, I. P., Sirvent, A.,
Kosykh, V., Fruchart, J. C., Dallongeville, J., Hum D. W., Kuipers, F., and
Staels, B. (2002) J. Clin. Invest.
109,
961971[Abstract/Free Full Text]
- Maloney, P. R., Parks, D. J., Haffner, C. D., Fivush, A. M.,
Chandra, G., Plunket, K. D., Creech, K. L., Moore, L. B., Wilson, J. G.,
Lewis, M. C., Jones, S. A., and Willson, T. M. (2000)
J. Med. Chem. 43,
29712974[CrossRef][Medline]
[Order article via Infotrieve]
- Kast, H. R., Nguyen, C. M., Sinal, C. J., Jones, S. A., Laffitte,
B. A., Reue, K., Gonzalez, F. J., Willson, T. M., and Edwards, P. A.
(2001) Mol. Endocrinol.
15,
17201728[Abstract/Free Full Text]
- Sinal, C. J., Tohkin, M., Miyata, M., Ward, J. M., Lambert, G., and
Gonzalez, F. J. (2000) Cell
102,
731744[Medline]
[Order article via Infotrieve]
- Iser, J. H., and Sali, A. (1981)
Drugs 21,
90119[Medline]
[Order article via Infotrieve]
- Pennacchio, L. A., Olivier, M., Hubacek, J. A., Cohen, J. C., Cox,
D. R., Fruchart, J. C., Krauss, R. M., and Rubin, E. M. (2001)
Science 294,
169173[Abstract/Free Full Text]
- van der Vliet, H. N., Sammels, M. G., Leegwater, A. C., Levels, J.
H., Reitsma, P. H., Boers, W., and Chamuleau, R. A. (2001)
J. Biol. Chem. 276,
4451244520[Abstract/Free Full Text]
- van der Vliet, H. N., Schaap, F. G., Levels, J. H., Ottenhoff, R.,
Looije, N., Wesseling, J. G., Groen, A. K., and Chamuleau, R. A.
(2002) Biochem. Biophys. Res. Commun.
295,
11561159[CrossRef][Medline]
[Order article via Infotrieve]
- Ribalta, J., Figuera, L., Fernandez-Ballart, J., Vilella, E.,
Castro Cabezas, M., Masana, L., and Joven, J. (2002)
Clin. Chem. 48,
15971600[Free Full Text]
- Pennacchio, L. A., Olivier, M., Hubacek, J. A., Krauss, R. M.,
Rubin, E. M., and Cohen, J. C. (2002) Hum. Mol.
Genet. 11,
30313038[Abstract/Free Full Text]
- Talmud, P. J., Hawe, E., Martin, S., Olivier, M., Miller, G. J.,
Rubin, E. M., Pennacchio, L. A., and Humphries, S. E. (2002)
Hum. Mol. Genet. 11,
30393046[Abstract/Free Full Text]
- Endo, K., Yanagi, H., Araki, J., Hirano, C., Yamakawa-Kobayashi,
K., and Tomura, S. (2002) Hum. Genet.
111,
570572[CrossRef][Medline]
[Order article via Infotrieve]
- Nabika, T., Nasreen, S., Kobayashi, S., and Masuda, J.
(2002) Atherosclerosis
165,
201204[CrossRef][Medline]
[Order article via Infotrieve]
- McKnight, S. L. (1982) Cell
31,
355365[Medline]
[Order article via Infotrieve]
- Coste, H., and Rodríguez, J. C. (2002)
J. Biol. Chem. 277,
2712027129[Abstract/Free Full Text]
- Rodríguez, J. C., Gil-Gómez, G., Hegardt, F. G., and
Haro, D. (1994) J. Biol. Chem.
269,
1876718772[Abstract/Free Full Text]
- Vu-Dac, N., Chopin-Delannoy, S., Gervois, P., Bonnelye, E., Martin,
G., Fruchart, J. C., Laudet, V., and Staels, B. (1998)
J. Biol. Chem. 273,
2571325720[Abstract/Free Full Text]
- Elshourbagy, N. A., Walker, D. W., Paik, Y. K., Boguski, M. S.,
Freeman, M., Gordon, J. I., and Taylor, J. M. (1987)
J. Biol. Chem. 262,
79737981[Abstract/Free Full Text]
- Haddad, I. A., Ordovas, J. M., Fitzpatrick, T., and Karathanasis,
S. K. (1986) J. Biol. Chem.
261,
1326813277[Abstract/Free Full Text]
- Kozak, M. (1996) Mamm. Genome
7,
563574[CrossRef][Medline]
[Order article via Infotrieve]
- Zannis, V. I., Kan, H. Y., Kritis, A., Zanni, E. E., and Kardassis,
D. (2001) Curr. Opin. Lipidol.
12,
181207[CrossRef][Medline]
[Order article via Infotrieve]
- Gervois, P., Vu-Dac, N., Jackel, H., Nowak, M., Staels, B.,
Pennacchio, L. A., Rubin, E. M., Fruchart, J., and Fruchart, J. C.
(2002) Circulation
106,
11301[Free Full Text]
- Applied Biosystems (2001) User Bulletin
2, Foster City, CA