Received for publication, June 8, 2000, and in revised form, October 16, 2000
Triglyceride-rich remnant lipoproteins are
considered as major risk factors contributing to the pathogenesis of
atherosclerosis. Because apolipoprotein (apo) C-III is a major
determinant of plasma triglyceride and remnant lipoprotein metabolism,
it is important to understand how the expression of this gene is
regulated. In the present study, we identified the orphan nuclear
receptor ROR
1 as a regulator of human and mouse apo C-III gene
expression. Plasma triglyceride and apo C-III protein concentrations in
staggerer (sg/sg) mice, homozygous
for a deletion in the ROR
gene, were significantly lower than in
wild type littermates. The lowered plasma apo C-III levels were
associated with reduced apo C-III mRNA levels in liver and
intestine of sg/sg mice. Transient transfection experiments
in human hepatoma HepG2, human colonic CaCO2, and rabbit kidney RK13
cells demonstrated that overexpression of the human ROR
1 isoform
specifically increases human apo C-III promoter activity, indicating
that ROR
1 enhances human apo C-III gene transcription. ROR
1
response elements were mapped by promoter deletion analysis and gel
shift experiments to two AGGTCA half-sites located at positions
83/
78 (within the C3P site) and
23/
18 (downstream of the TATA
box) in the human apo C-III promoter, with the
23/
18 site
exhibiting the highest binding affinity. Transfection of site-directed
mutated constructs in HepG2 cells indicated that the ROR
1 effect is
predominantly mediated by the
23/
18 site. This site is conserved in
the mouse apo C-III gene promoter. Moreover, ROR
binds to the
equivalent mouse site and activates constructs containing three copies
of the mouse site cloned in front of an heterologous promoter. Taken
together, our data identify ROR
as a transcriptional regulator of
apo C-III gene expression, providing a novel, physiological role for
ROR
1 in the regulation of genes controlling triglyceride metabolism.
 |
INTRODUCTION |
Several epidemiological studies support the idea that, in addition
to elevated low density lipoprotein and reduced high density lipoprotein cholesterol, elevated triglycerides constitute an independent risk factor for coronary heart disease (1-4). More specifically, triglyceride-rich lipoprotein remnants are positively correlated to the progression of atherosclerosis (5, 6). Identifying
the factors or genes controlling triglyceride metabolism is therefore
of major importance and may provide means for pharmacological intervention in dyslipidemic patients.
Apolipoprotein (apo)1 C-III
is a 79-amino acid glycoprotein synthesized in the liver and, to a
lesser extent, in the intestine, that plays a key role in plasma
triglyceride metabolism as evidenced by pharmacological (7-9),
clinical (10, 11), genetic (12), and experimental data in transgenic
animal models (13). Apo C-III concentrations in plasma are positively
correlated with plasma triglyceride levels, both in the normal
population as well as in hypertriglyceridemic patients (10, 11, 14) or
in transgenic animals (15). Moreover, apo C-III deficiency in humans
(16) or apo C-III gene disruption in transgenic mice (17) results in
increased catabolism of very low density lipoprotein particles, whereas
increased apo C-III synthesis occurs in hypertriglyceridemic patients
(18). Results from both in vivo (19-22) and in
vitro (23-25) studies indicate that apo C-III delays the
catabolism of triglyceride-rich particles. Several potential mechanisms
may participate in the inhibitory effect of apo C-III on triglyceride catabolism. These include inhibition of lipolysis by lipoprotein (16,
26) or hepatic lipase (27), inhibition of triglyceride-rich particle
binding to glycosaminoglycans (22), as well as interference with apo
E-mediated receptor clearance of remnant particles from plasma (19, 20,
22, 25).
Apo C-III gene expression is tightly regulated, being down-regulated by
hormones such as insulin (28, 29) or thyroid hormones (30), cytokines
such as interleukin-1 (31) or tumor necrosis factor
(32), as well
as hypolipidemic drugs such as fibrates (7, 33) or
-blocked fatty
acids (8, 34). By contrast, its expression is increased by retinoids
(9). Regulatory sequences determining the tissue-specific expression
pattern of apo C-III have been delineated in its gene (35-38). The C3P
site located at position
87/
67 relative to the transcription start
site is a major determinant of apo C-III promoter activity (35, 36). It
contains a direct repeat of two AGGTCA half-sites separated by one
nucleotide (DR-1) to which the nuclear hormone receptors HNF-4, PPAR,
RXR, Ear2, COUPTF-I, and COUPTF-II are binding (8, 9, 37, 39, 40).
Whereas Ear2, COUPTF-I, and COUPTF-II repress apo C-III promoter
activity via these sites (37, 39), HNF-4 activates it (35-37, 39, 40).
PPAR/RXR heterodimers also enhance the activity of reporter construct
containing the C3P site cloned in front of an heterologous promoter
(9). In addition, the
592/
792 fragment of the apo C-III promoter
acts as an enhancer that potentiates the strength of the proximal apo
C-III promoter (38). Functional positive HNF-4 and Sp1 binding sites as
well as negative COUPTF-I and COUPTF-II binding sites have been mapped in this region (38, 40). In addition, a CCAAT/enhancer binding protein
binding site located in the proximal apo C-III promoter (
171/
137) seems to be involved in the negative regulation of apo
C-III expression by interleukin-1 (31). Finally, T3R
(41), ATF-2 (42), NF
B (43), and Jun (42) regulatory elements have
also been identified in the human apo C-III promoter.
The ROR (retinoic acid receptor related orphan
receptor; also termed RZR) orphan receptors (44-46) are a
subfamily of orphan nuclear receptors consisting of three different
genes ROR
,
, and
(44, 46, 47). RORs were initially reported
to bind as monomers to response elements consisting of a 6-base pair
AT-rich sequence preceding the half-core PuGGTCA motif (44, 48, 49), but more complex response elements have also been described (50, 51).
Because of alternative splicing and promoter usage, the ROR
gene
gives rise to four isoforms:
1,
2,
3, and RZR
(44-46), which differ in their N-terminal domains and display distinct DNA
recognition and transactivation properties (44). In contrast to ROR
,
the expression of which is restricted to brain, retina, and pineal
gland (52), both ROR
and ROR
are widely expressed in peripheral
tissues (44, 45, 47, 49). Based on the presence of putative response
elements in their promoter, several target genes for ROR subfamily
members were proposed and analyzed in vitro (53-56). A role
for ROR
1 has been proposed in muscle differentiation (57), whereas
ROR
expression is induced during adipose tissue differentiation
(58). Transgenic mice have been developed that carry a deleted ROR
gene (59, 60). Their phenotype is similar to the one of
staggerer mice, which carry a natural deletion in the ROR
gene that prevents the translation of its putative ligand-binding domain, thereby presumably disrupting the normal function of this transcription factor (61). These mice exhibit deficient intestinal apo
A-I expression (62), suggesting that the mouse apo A-I gene is an
in vivo target of ROR
. Moreover, when maintained on a
high fat atherogenic diet staggerer (sg/sg) mice
develop a severe hypo-alphalipoproteinemia and atherosclerosis,
suggesting an important role for ROR
in cardiovascular and metabolic
diseases (63).
In the present study, we investigated the regulation of apo C-III
expression and triglyceride metabolism by the orphan nuclear receptor
ROR
in vivo using the staggerer mouse model.
We observed a striking reduction in both triglyceride and apo C-III
plasma levels in mutant compared with wild type mice. Next, we studied in vitro the molecular mechanisms regulating apo C-III gene
transcription by ROR
. Our results indicate that ROR
enhances the
activity of the human
1415/+24 apo C-III promoter. Furthermore, a
ROR
response element was identified at position
23/
18 that
confers ROR
responsiveness to the human apo C-III promoter. This
response element is preserved in both the human and the mouse apo C-III gene promoters and confers ROR
responsiveness to a heterologous promoter. Taken together, our results identify ROR
as a positive regulator of apo C-III gene transcription and support a role of ROR
as a regulator of lipid and lipoprotein metabolism.
 |
MATERIALS AND METHODS |
Mice--
staggerer mutant mice were obtained by
crossing heterozygote (+/sg) mice maintained in a C57BL/6
genetic background and identifying homozygous offspring by polymerase
chain reaction genotyping and by their clinical ataxia. Mice were
maintained on chow diet purchased from UAR (France) as described
previously (62). Wild type littermates of the same age as the
homozygous mutants were used as control. 10-week-old mice fasted
overnight were killed by ether overdose. Blood, liver, and intestine
samples were taken and stored for further analysis.
RNA Analysis--
RNA extractions, Northern blot hybridizations,
and measurements of mRNA levels were performed as described
previously (33) using rat apo C-III (33) and 36B4 control probes (64).
Autoradiograms were analyzed by quantitative scanning densitometry
(Bio-Rad GS670 Densitometer) as described (33).
Lipid and Lipoprotein Analysis--
Plasma triglyceride
concentrations were determined by enzymatic assays using commercially
available reagents (Roche Molecular Biochemicals), whereas plasma
levels of apo C-III were measured by an immunonephelometric assay using
a specific polyclonal antibody as described previously (65).
Cloning of Recombinant Plasmids--
The plasmid containing the
1415/+24 sequence of the human apo C-III gene promoter cloned in
front of the chloramphenicol acetyltransferase (CAT) reporter gene
(
1415/+24WT-CAT) has been described previously (9). The luciferase
gene from the plasmid pGL3 (Promega, Madison, WI)
(SacI-BamHI) was subcloned between the
corresponding sites of the vector pBKCMV (Stratagene, La Jolla, CA)
(pBKCMV-Luc+). The CAT reporter gene of the plasmid
1415/+24WT-CAT was then excised by digestion with KpnI and BamHI
and replaced by the luciferase-containing
KpnI-BglII fragment of the plasmid pBKCMV-Luc+ plasmid. The
1415/+24 fragment of the apo C-III promoter was excised from the resulting construct by HindIII
digestion and cloned in the corresponding site of the vectors pGL3
(construct
1415/+24WTpGL3) and pSL301 (Amersham Pharmacia Biotech)
(construct PSL301
1415/+24hCIII). The pSL301
1415/+24hCIII construct
was partially digested with EcoO109I and religated. The resulting construct was digested with XbaI and HindIII. The
insert was cloned in the corresponding sites of pGL3 to create the
construct
108/+24WTpGL3. The plasmid
1415/+24WTpGL3 was then used
as template to polymerase chain reaction amplify fragments of different
length of the human apo C-III promoter using forward primers annealing
to specific parts of the promoter sequence and containing a
NheI restriction site and a reverse primer annealing
downstream of the pGL3 polylinker. The polymerase chain reaction
products were cut with NheI and HindIII and
cloned in the corresponding sites of the pGL3 vector. Site-directed
mutagenesis of the construct
1415/+24WTpGL3 was performed using the
Quick Change site-directed mutagenesis kit (Stratagene) following the
manufacturer's instructions. The constructs (
58/
27)8sTkpGL3 and (
47/
79)1sTkpGL3
were obtained following the described previously strategy (66) based on
intermediary cloning in the BamHI and BglII sites
of the vector pic20H using double-stranded oligonucleotides with
sequences corresponding to the indicated fragment of the apo C-III
promoter flanked by protruding ends compatible with BamHI
and BglII sites. The oligonucleotide multimers were excised
from pic20H with SalI and XhoI and cloned in the
XhoI site of the described previously vector TkpGL3 (34). Alternatively, these oligonucleotide multimers were directly cloned into the BglII site of the TkpGL3 vector after disruption of
the BamHI site by Klenow blunting. The construct
pCDNA3-hROR
1 containing the hROR
1 cDNA cloned in the
KpnI and XbaI sites of the pCDNA3 vector was
a gift of Dr. A. Shevelev. The Renilla luciferase gene of the pRLnull
construct (Promega) was excised by the enzymes NheI and
XbaI and cloned in the XbaI site of the plasmid
pBKCMV. The resulting construct was cut by HindIII and
XbaI, and the insert was cloned in the corresponding sites
of the pGL3 control vector (Promega) to yield the pRenConT+ construct
used to evaluate transfection efficiency.
Cell Culture and Transient Transfection Assays--
Human
hepatoma HepG2, CaCO2 and RK13 cells were obtained from European
Collection of Animal Cell Cultures (Porton Down, Salisbury, UK). Cell
lines were maintained in standard culture conditions (Dulbecco's
modified Eagle's minimal essential medium supplemented with 10% fetal
calf serum at 37 °C in a humidified atmosphere of 5%
CO2/95% air). Medium was changed every 2 days.
Cells were seeded in 24-well plates at a density of 5 × 104, 6 × 104, or 105
cells/well for RK13, HepG2, or CaCO2, respectively, and incubated at
37 °C for 16 h prior to transfection. Cells were transfected using the cationic lipid RPR 120535B as described previously (34) with
reporter plasmids (at 50 ng/well), expression vectors (pCDNA3 or
pCDNA3-hROR
1 at 100 ng/well), and the control plasmids
(pRenCont+ at 1 ng/well or pSV-
-gal at 50 ng/well). At the end of
the experiment, the cells were washed once with ice-cold 0.15 M NaCl, 0.01 M sodium phosphate buffer, pH 7.2, and the luciferase activity was measured with the
Dual-LuciferaseTM Reporter Assay System (Promega) according
to the manufacturer's instructions. All transfection experiments were
performed at least three times. The
-galactosidase activity was
measured as described previously (66). Protein content of the extract
was evaluated by the Bradford assay using the kit from Bio-Rad.
Gel Retardation Assays--
ROR
was in vitro
transcribed from the pCDNA3-hROR
plasmid using T7 polymerase and
subsequently translated using the TNT-coupled transcription/translation
system (Promega) following the manufacturer's instructions.
DNA-protein binding assays were conducted as described (68) using the
following binding buffer: 10 mM Hepes, 50 mM KCl, 1% glycerol, 2.5 mM MgCl2, 1.25 mM dithiothreitol, 0.1 µg/µl poly(dI-dC), 50 ng/µl
herring sperm DNA, 1 µg/µl bovine serum albumin containing 10% of
programmed or unprogrammed reticulocyte lysate. Double-stranded
oligonucleotides were end-labeled using T4 polynucleotide kinase and
[
-32P]ATP and used as probe. For competition
experiments, the indicated amounts of cold oligonucleotide were
included 15 min before adding labeled oligonucleotides.
 |
RESULTS |
Plasma Triglyceride and apo C-III Concentrations as Well as Hepatic
and Intestinal apo C-III mRNA Levels Are Decreased in staggerer
Mice--
To determine whether staggerer mice display
altered triglyceride metabolism, plasma triglycerides were measured in
overnight fasted female staggerer mice and compared with
age-matched wild type C57BL/6 littermates (Fig.
1). Interestingly, staggerer
mice exhibited 50% lower blood triglyceride levels compared with wild type littermates (Fig. 1A). Because apo C-III is a major
determinant of plasma triglyceride levels (13), its plasma
concentrations were measured next. A 70% decrease of plasma apo C-III
concentration was observed in mutant mice (Fig. 1B). To
determine whether this reduction was associated with a decreased
expression of the apo C-III gene, hepatic and intestinal apo C-III
mRNA levels were analyzed by Northern blotting. Both intestinal and
liver apo C-III mRNA levels were reduced in mutant mice compared
with wild type mice (Fig. 1C). Hepatic and intestinal 36B4
mRNA levels measured as control were similar in both groups.

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Fig. 1.
staggerer mice have decreased
plasma triglyceride and apo C-III levels associated with decreased
hepatic and intestinal apo C-III mRNA levels compared with wild
type littermates. Overnight fasted 11-week-old homozygous female
staggerer mice carrying a nonfunctional ROR 1 gene and
their wild type littermates were killed by ether overdose. Plasma
triglyceride (A) and apo C-III levels (B) were
measured as described under "Materials and Methods" (four
animals/group). Total RNA was extracted from liver and intestinal
tissues and analyzed by Northern blotting as described under
"Materials and Methods." C shows the relative apo CIII
mRNA levels (three animals/group) as evaluated by quantitative
scanning densitometry (Bio-Rad GS670 Densitometer) of the Northern blot
presented. The data are expressed as the mean values ± S.D.
(Mann-Whitney nonparametric test). *, p < 0.05.
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Overexpression of hROR
1 Enhances the Activity of the Human apo
C-III Gene Promoter--
Because staggerer
(sg/sg) mice carry a nonfunctional ROR
gene (61), the
above data suggest that ROR
is a positive regulator of apo C-III
transcription. Transient transfection assays were performed to
determine whether human ROR
controls the transcription of the human
apo C-III gene. In hepatoma HepG2 cells, which produce apo C-III,
cotransfection of a human nuclear receptor ROR
1 expression plasmid
resulted in an increased activity of the luciferase reporter gene
driven by the
1415/+24 fragment of the human apo C-III gene promoter
(Fig. 2A). An activation was
also observed in rabbit kidney RK13 cells that do not express apo C-III
(Fig. 2B) and in human intestinal CaCO2 cells (Fig.
2C) that produce apo C-III. The effect of hROR
1
overexpression was promoter-dependent because the
promotor-less vector pGL3 was unaffected in all cell lines studied.

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Fig. 2.
hROR 1 enhances the
activity of the human apo C-III gene promoter. HepG2
(A), RK13 (B), or CaCO2 (C) cells were
transiently cotransfected with the 1415/+24wtpGL3 reporter plasmid
(50 ng) containing the 1415/+24 fragment of the human apo C-III
promoter cloned in front of the luciferase reporter gene or the empty
pGL3 vector as control (50 ng) and the expression plasmid (100 ng)
pCDNA3-hROR 1 (hROR 1) or the empty
pCDNA3 vector as control (Cont.). Cells were transfected
and luciferase activity measured and expressed as described under
"Materials and Methods." The fold induction above control level is
indicated for the 1415/+24wtpGL3 construct.
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Mapping of the Human apo C-III Promoter Sites Conferring
Responsiveness to hROR
1--
To identify the response element(s)
required for hROR
1 activation of the apo C-III gene promoter, 5'
nested deletions of the apo C-III promoter were cotransfected with the
hROR
1 expression vector in HepG2 cells. Deletion of the promoter led
to a decrease in its basal activity (Fig.
3), corroborating previous observations that the
792/
592 fragment of the apo C-III promoter acts as a
strong hepatic enhancer (38). However, hROR
1 activation was still
observed with the shortest construct
108/+24WTpGL3, indicating that
the first 108 nucleotides of the human apo C-III promoter contain
sequence determinants sufficient to confer hROR
1 responsiveness (compare the 3.1-fold induction of the
1415/+24wtpGL3 construct with
the 2.6-fold induction of the
108/+24wtpGL3 construct). To verify
whether hROR
1 directly binds to the proximal apo C-III promoter,
radiolabeled overlapping oligonucleotides corresponding to portions of
the
108/+24 fragment of the apo C-III promoter were used as probes in
gel shift assays. Specific binding of in vitro translated
hROR
1 protein was observed only on the
33/
16 and
90/
64
fragments (Fig. 4A). Both
fragments contain an AGGTCA half-site preceded by a degenerated
A/T-rich region that could function as hROR
1 response element.
Binding of hROR
1 to the
33/
16 fragment of the apo C-III promoter
was lost after mutation of the AGGTCA half-site present in position
23/
18 (
33/
16mt:
22G
C,
21G
A) (Fig. 4B). The binding of
hROR
1 to the
33/
16 fragment of the apo C-III promoter was
displaced by increasing amounts of either the cold
33/
16
double-stranded oligonucleotide or a cold double-stranded
oligonucleotide that contains one copy of the hROR
1 consensus
binding site (Fig. 5A). This
binding was not displaced by the mutated cold
33/
16 double-stranded
oligonucleotide (Fig. 5A). Binding of hROR
1 to the
90/
64 fragment corresponding to the C3P site of the human apo C-III
gene promoter was specific because it could be displaced by increasing
amounts of either cold
90/
64 (Fig. 5B) or hROR
1
consensus binding site double-stranded oligonucleotides (data not
shown). By contrast, binding of hROR
1 to the
90/
64 fragment of
the human apo C-III gene promoter was abrogated when the AGGTCA
half-site located at position
82/
77 was mutated (
90/
64mt:
78G
C,
79G
A;
Fig. 4B). No significant binding was observed to other
fragments of the proximal human apo C-III promoter (Fig.
4A). Taken together, our results suggest the presence of two
binding site for hROR
1 on the proximal human apo C-III promoter, the
first downstream of the TATA box (
23/
18) and the second within the
C3P site (
82/
77).

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Fig. 3.
Identification of the human apo C-III
promoter elements conferring its responsiveness to
hROR 1. HepG2 cells were cotransfected
with pCDNA3-hROR 1 expression vector (100 ng;
hROR 1) or the empty pCDNA3 vector as
control (Cont.) and reporter constructs (50 ng) containing
the indicated nested fragments of the human apo C-III promoter cloned
in front of the luciferase reporter gene. The empty pGL3 vector (50 ng)
was used as control. Cells were transfected and luciferase activity
measured and expressed as described under "Materials and Methods."
The fold induction above control level is indicated for each
construct.
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Fig. 4.
hROR 1 binding to
labeled probes covering the 16/ 125 region of the human apo C-III
promoter. Double-stranded oligonucleotides corresponding to
overlapping fragments of the human apo C-III promoter comprised between
positions 16 and 125 were prepared and labeled as described under
"Materials and Methods." These probes were incubated as indicated
with in vitro translated hROR 1 protein or unprogrammed
lysate as control. DNA/protein complexes were resolved by
nondenaturating PAGE (A) as described under "Materials and
Methods." In B, double-stranded oligonucleotides
corresponding to the wild type or mutated 33/ 16
( 22G C, 21G A)
and 90/ 64 ( 78G C,
79G A) fragments of the human apo C-III
promoter were labeled and incubated with in vitro translated
hROR 1 protein or unprogrammed lysate as control. DNA/protein
complexes were resolved by nondenaturating PAGE as described under
"Materials and Methods." Specific complexes not observed with
unprogrammed lysate are indicated by arrows.
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Fig. 5.
Specificity of hROR 1
binding to 33/ 16 or the 90/ 64 fragments of the human apo C-III
promoter. Double-stranded oligonucleotides corresponding to the
33/ 16 (A) and 90/ 64 (B) fragments of the
human apo C-III promoter were labeled as described under "Materials
and Methods." In vitro translated hROR 1 protein or
unprogrammed lysate were incubated with 10-, 50-, and 100-fold excess
of the indicated unlabeled double-stranded oligonucleotides for 15 min
at 4 °C before labeled probes were added for 5 min at room
temperature. DNA/protein complexes were resolved by nondenaturating
PAGE as described under "Materials and Methods." Specific complexes
not observed with unprogrammed lysate are indicated by
arrows.
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Functional Characterization of hROR
1 Response Elements Present
in the Proximal Human and Mouse apo C-III Promoters--
To evaluate
whether these two putative response elements were functional in the
context of the proximal human apo C-III promoter, the half-sites
present downstream of the TATA box in position
23/
18
(
22G
C,
21G
A)
or in the C3P site in position
82/
77
(
78G
C,
79G
A)
of the human apo C-III promoter were mutated by site-directed mutagenesis in the
1415/+24WTpGL3 construct either alone
(
82/
77mt,
23/
18mt, respectively) or in combination (
82/
77mt +
23/
18mt). Mutation of the
82/
77 half-site reduced the basal
activity of the apo C-III promoter but did not prevent its activation
by hROR
1 (Fig. 6). Mutation of the
23/
18 half-site enhanced the basal activity of the apo C-III
promoter in HepG2 cells and abrogated hROR
1 responsiveness. The
activity of the promoter and its hROR
1 responsiveness were lost when
both half-sites were mutated simultaneously. These data suggest that
the
23/
18 half-site plays a major role in the hROR
1
responsiveness of the apo C-III promoter in HepG2 cells.

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Fig. 6.
Functional evaluation of the
hROR 1 response elements present in the
1415/+24 fragment of the human apo C-III promoter. HepG2 cells
were cotransfected with pCDNA3-hROR 1 expression vector
(hROR 1; 100 ng) or the empty pCDNA3 vector
as control (Cont.) and reporter constructs (50 ng)
containing the wild type or site-directed mutated 1415/+24 fragments
of the human apo C-III promoter cloned in front of the luciferase
reporter gene. The empty pGL3 vector was used as negative control. The
AGGTCA half-sites present in position 23/ 18
( 22G C, 21G A)
and 82/ 77 ( 78G C,
79G A) were mutated alone ( 23/ 18,
82/ 77mt, respectively) or in combination ( 23/ 18 + 82/ 77mt)
as indicated. Cells were transfected and luciferase activity measured
and expressed as described under "Materials and Methods." The fold
induction above control level is indicated for each constructs.
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|
To evaluate whether these two sites could confer ROR
responsiveness
to an heterologous promoter and to exclude that the
108/+24 fragment
of the apo C-III promoter contains other hROR
1-responsive elements,
overlapping fragments of the apo C-III promoter (covering the
100/
16 region of the apo C-III promoter) were cloned in front of a
thymidine kinase (Tk) promoter-driven luciferase reporter vector. These
constructs were cotransfected with a hROR
1 expression vector in
HepG2 cells. The (
33/
16)3STkpGL3 construct was strongly activated by hROR
1, whereas the (
83/
67)4STkpGL3
construct was weakly stimulated (Fig.
7A). The other constructs were
not activated by hROR
1 (Fig. 7A). Both the
33/
16 and
83/
67 fragments contain an AGGTCA half-site preceded by a
degenerated A/T-rich region. To evaluate the specificity of ROR
action, these half-sites were next mutated to create the constructs
(
33/
16mt)3STkpGL3 (
22G
C,
21G
A) and
(
87/
67mt)4STkpGL3 (
78G
C,
79G
A) that were cotransfected with a
hROR
1 expression vector in HepG2 cells. In contrast to the wild type
constructs, these mutated constructs were not activated by hROR
1
(Fig. 7B). These data suggest that the two AGGTCA half-sites
to which hROR
1 bind in the proximal human apo C-III promoter are
also functional in the context of a heterologous promoter.

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Fig. 7.
hROR 1 response
elements present in the proximal 198/+24 fragment of the human apo
C-III promoter confer hROR 1 responsiveness to
heterologous promoters. A, HepG2 cells were
cotransfected with the pCDNA3-hROR 1 expression vector
(hROR 1; 100 ng) or the empty pCDNA3 vector
as control (Cont.) and reporter constructs (50 ng)
containing multiple copies of overlapping fragments ( 33/ 16,
58/ 24, 78/ 46, 83/ 67, and 100/ 80) of the human apo C-III
promoter inserted in front of the Herpes simplex thymidine kinase
promoter, cloned upstream of the luciferase reporter gene as described
under "Materials and Methods." The empty TkpGL3 reporter plasmid
was used as negative control. B, reporter constructs (50 ng)
containing multiple copies of mutated oligonucleotides corresponding to
the 33/ 16 or 83/ 67 fragments of the the human apo C-III
promoter cloned in front of the Herpes simplex thymidine kinase
promoter of the TkpGL3 reporter plasmid
(( 33/ 16mt)3STkpGL3 ( 22G C,
21G A) and
( 87/ 67mt)4STkpGL3 ( 78G C,
79G A)) and their wild type homologues were
cotransfected in HepG2 cells with pCDNA3-hROR 1 expression vector
(100 ng; hROR 1) or empty pCDNA3 vector as
control (Cont.) (B). Cells were transfected and
luciferase activity measured and expressed as described under
"Materials and Methods."
|
|
Because the sequence of the
33/
16 fragment of the human apo C-III
promoter is almost fully conserved in the mouse promoter (67), hROR
1
binding to the mouse sequence corresponding to this region was
analyzed. As shown in Fig. 8A,
hROR
1 bound with similar affinity to the
33/
14 fragment of the
mouse apo C-III gene promoter as to the
33/
16 fragment of the human
apo C-III gene promoter. To compare its activity with the corresponding human promoter sequence, three copies of the wild type
33/
14 fragment of the mouse apo C-III promoter were cloned in front of the
thymidine kinase promoter and tested in cotransfection assay. As shown
in Fig. 8B, the human (
33/
16)3asTkpGL3 and
the mouse (
33/
14)3asTkpGL3 were similarly activated by
hROR
overexpression in HepG2 cells. This indicates that the AGGTCA
half-sites located downstream of the TATA box both in the mouse and
human apo C-III promoters are equally functional in the context of an
heterologous promoter.

View larger version (40K):
[in this window]
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|
Fig. 8.
Functional comparison of putative
hROR 1 response elements present in the
proximal fragment of the human and mouse apo C-III promoters.
A, double-stranded oligonucleotides corresponding to the
33/ 16 fragment of the human apo C-III promoter or the 33/ 14
fragment of the mouse apo C-III promoter were prepared and labeled as
described under "Materials and Methods." These probes were
incubated as indicated with in vitro translated hROR 1
protein or unprogrammed lysate as control. In vitro
translated hROR 1 protein or unprogrammed lysate were incubated with
10-, 50-, and 100-fold excess of the indicated unlabeled
double-stranded oligonucleotides for 15 min at 4 °C before labeled
probes were added for 5 min at room temperature as indicated.
DNA/protein complexes were resolved by nondenaturating PAGE as
described under "Materials and Methods." Specific complexes not
observed with unprogrammed lysate are indicated by arrows.
B, HepG2 cells were cotransfected with the
pCDNA3-hROR 1 expression vector (hROR 1;
100 ng) or the empty pCDNA3 vector as control (Cont.)
and reporter constructs (50 ng) containing multiple copies of the wild
type or mutated 33/ 16 fragment of the human apo C-III promoter or
the wild type 33/ 14 fragment of the mouse apo C-III promoter
(( 33/ 16wt)3asTkpGL3, ( 33/ 16mt)3asTkpGL3
( 22G C, 21G A)
and ( 33/ 14wt)3asTkpGL3), respectively) inserted in
front of the Herpes simplex thymidine kinase promoter and cloned
upstream of the luciferase reporter gene as described under
"Materials and Methods." The empty TkpGL3 reporter plasmid was used
as negative control. Cells were transfected and luciferase activity
measured and expressed as described under "Materials and
Methods."
|
|
 |
DISCUSSION |
In the present study, we report that staggerer mice
lacking functional orphan nuclear receptor ROR
(61) have
significantly reduced plasma triglyceride levels compared with wild
type controls. These data indicate a physiological role of this
receptor in the regulation of plasma triglyceride metabolism in mice.
Because apo C-III plays an important role in intravascular triglyceride
metabolism (13), we subsequently evaluated the role of ROR
in the
control of apo C-III expression. Our observation that the decrease in
plasma triglyceride levels observed in staggerer mice is
associated with a strong decrease in apo C-III plasma concentrations
and hepatic as well as intestinal gene expression provides a possible
mechanistic explanation of the phenotype and suggests that apo C-III is
a ROR
target in mice. Despite the severe phenotype of
staggerer mice, few ROR
target genes have been identified
to date. To the best of our knowledge, beside rat apo A-I, apo C-III is
the second mouse ROR
target gene identified in peripheral tissues
using both molecular and physiological approaches. To evaluate whether
our results obtained in mice could be extended to humans, we measured
the effects of hROR
1 overexpression on human apo C-III promoter
activity by cotransfection assays in HepG2 cells. The observed
activation of human apo C-III promoter activity suggests that human apo
C-III could be a ROR
target gene in humans as well.
The demonstration that the
108/+24 apo C-III promoter fragment
remained sensitive to the action of hROR
1, although slightly less
than the
1415/+24 fragment indicated the presence of at least one
response element to hROR
1 in this region. Two potential response
elements located respectively in position
23/
18 and
82/
77 have
been identified. The
23/
18 site consists of a perfect AGGTCA
half-site preceded by an A/T rich region that deviates from the optimal
consensus only by a C in position
1 (44). However, this sequence was
shown to bind hRZR
in vitro, although more weakly than
the optimal consensus site (46). Our results confirm these binding data
on a natural response element with hROR
and demonstrate that this
sequence is transcriptionally active in the context of a natural
promoter. The
82/
77 site also consists of a perfect AGGTCA
half-site preceded by an A in position
1 and two Gs in positions
3
and
4. This larger divergence from the consensus sequence likely
explains the weaker binding of hROR
to this sequence and the weaker
transactivation of a construct containing three copies of this sequence
cloned in front of a heterologous promoter. Site-directed mutagenesis
of both sites confirmed that the
23/
18 half-site plays a major role in hROR
1 responsiveness of the apo C-III promoter in HepG2 cells. The better sensitivity of the
1415/+24 fragment of the apo C-III promoter, however, suggests either the presence of additional hROR
response elements in the upstream region of the promoter or the
cooperation with other nuclear factors binding to upstream sites of the
promoter that remain to be identified.
The sequence of the
33/
16 fragment of the human apo C-III promoter
is almost fully conserved in the mouse promoter (67). We observed
hROR
1 binding to the
33/
14 fragment of the mouse apo C-III
promoter and activation of a reporter construct containing three copies
of this site cloned in front of an heterologous promoter. This suggests
that ROR
could also act via this site on the mouse promoter.
Although elevated triglycerides likely affect atherosclerosis in
humans, the effect of hypertriglyceridemia in mice is modest (69). This
might explain why, despite their decreased hepatic apo C-III gene
expression (63), staggerer mice fed an atherogenic diet
develop more severe atherosclerosis than wild type mice. In humans, a
different picture may be expected because elevated serum triglycerides
are considered as an independent risk factor for coronary heart disease
and because the human apo A-I promoter is unaffected by hROR
1 (data
not shown). The enhancement of human apo C-III gene promoter activity
by overexpressing hROR
1 in HepG2 cells suggests that it acts at the
transcriptional level. Hence, hROR
1 could be a valuable target for
the development of hypotriglyceridemic agents.
In conclusion, the strong decrease in plasma triglyceride and apo C-III
levels associated with lowered hepatic apo C-III expression observed in
staggerer mice lacking functional ROR
identifies ROR
as a modulator of triglyceride levels in mice. Furthermore, the
observation that human apo C-III promoter activity was also enhanced by
hROR
1 extends the mice data to humans and suggests that hROR
1 is
another modulator of human apo C-III promoter activity and, hence, a
valuable target for the development of hypotriglyceridemic agents.
We thank B. Derudas, Y. Delplace, E. Baugé, O. Vidal, and C. Faure for excellent technical assistance
and J. Dallongeville for critical reading of the manuscript. We also
thank A. Shevelev for providing ROR expression vectors and G. Byk
(Rhône-Poulenc-Rorer, Paris, France) for the cationic lipid RPR
120535B (WO patent 97/18185).
Published, JBC Papers in Press, October 26, 2000, DOI 10.1074/jbc.M004982200
The abbreviations used are:
apo, apolipoprotein;
CAT, chloramphenicol acetyltransferase;
PAGE, polyacrylamide gel
electrophoresis.
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2071-2074[Medline]
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