(Received for publication, December 18, 1996, and in revised form, March 18, 1997)
From Ligand Pharmaceuticals Inc., San Diego, California 92121
Using recombinant adenoviral vectors and a dominant negative mutant of HNF-4, we have examined the contribution of hepatocyte nuclear factor 4 (HNF-4) to endogenous apolipoprotein AI and CIII mRNA expression. Overexpression of HNF-4 leads to a 7.4-fold increase in apolipoprotein CIII expression, while infection with the dominant negative mutant of HNF-4 reduces the level of apolipoprotein CIII mRNA by 80%, demonstrating that endogenous HNF-4 is necessary for apolipoprotein CIII expression. Experiments using the hepatoma cell lines, HepG2 and Hep3B, indicate that HNF-4 is also involved in the regulation of apolipoprotein AI expression in these lines. However, the effect of HNF-4 on apolipoprotein AI expression is much more dramatic in cell lines derived from intestinal epithelium. Infection of the intestinal-derived cell line IEC-6 with the HNF-4 adenovirus resulted in a greater than 20-fold increase in the level of apolipoprotein AI mRNA. These results indicate that HNF-4 does regulate apolipoprotein AI and CIII mRNA expression and suggest that HNF-4 is critical for intestinal apolipoprotein AI expression.
The apolipoproteins are lipid-binding polypeptides involved in the transport and metabolism of cholesterol, triglycerides, and phospholipids (1). These proteins regulate the structural characteristics of lipoprotein particles as well as their metabolism and uptake by cell surface receptors. While it has been demonstrated that genetic defects in apolipoprotein structure or biosynthesis can lead to severe disorders of plasma lipid transport and the development of atherosclerotic disease, recent clinical studies indicate that even relatively small imbalances in lipoprotein concentrations can increase the risk of atherosclerosis (2-4). Apolipoprotein gene transcription is a regulated process, and there have been significant advances in our understanding of the DNA elements that control apolipoprotein expression. However, for the most part, the identity of the factors that recognize these sequences and the signal transduction pathways that control the level of apolipoprotein expression remains unclear.
Hepatocyte nuclear factor 4 (HNF-4)1 is a member of the steroid/thyroid superfamily of ligand-dependent transcription factors that was originally isolated from liver nuclear extracts (5). HNF-4 mRNA is present in the intestine, kidney, and pancreas as well as the liver (5, 6). At present no ligand for HNF-4 has been identified, therefore HNF-4 is referred to as an orphan member of the intracellular receptor superfamily (7-9). Whether an activity-modulating ligand for HNF-4 exists is not known, but HNF-4 is capable of activating transcription in the absence of exogenously added ligand (10-12). The structure of HNF-4 is very highly conserved; there is 96% sequence identity at the amino acid level between the human and rat HNF-4s (13).
Previous studies have identified several sequences capable of binding
HNF-4 that are located within the promoters of apolipoproteins, including apolipoprotein AI (apoAI) and apolipoprotein CIII (apoCIII) (5, 11, 14, 15). To examine the role of HNF-4 on apoAI and apoCIII
expression we have utilized recombinant adenoviral vectors expressing
wild-type and a dominant negative mutant of HNF-4. HNF-4 binds DNA as a
homodimer and does not appear to form heterodimers with several other
intracellular receptor family members, including retinoid X receptor
,
,
, retinoid acid receptor
, or thyroid hormone receptor
(16). We have taken advantage of this and created a mutant of HNF-4
which lacks DNA binding activity and inhibits transcriptional
activation by wild-type HNF-4 via the formation of heterodimers
consisting of wild-type HNF-4 and the non-DNA-binding HNF-4 mutant that
exhibit decreased DNA binding avidity. Since the apoAI and apoCIII
genes are closely linked and appear to share regulatory elements (17),
we have constructed recombinant adenoviral vectors for the dominant
negative mutants and wild-type HNF-4 that has allowed us to examine the effects of modulating HNF-4 transcriptional activity on the endogenous expression levels of the apoAI and apoCIII genes.
The 1.4-kilobase
pair BglII-EcoRI apoAI (1378 to +11) promoter
fragment was isolated from pGF1, which was a generous gift from Dr.
Michael Saunders. This construct was sequenced and the apoAI promoter
region was identical to that reported by Karathanasis and colleagues
(18) This was ligated into BamHI and EcoRI
digested Bluescript KS
(Stratagene) and digested with
HindIII and SpeI. The apoAI promoter fragment was
gel-purified and ligated into HindIII and NheI
digested pGL2 (Promega). This construct (p1400 apoAI-luc) was digested
with SmaI, the 5.9-kilobase pair fragment containing the
apoAI (
256 to +11) and pGL2 sequences was isolated and religated to
create p256A1-luc.
The construct p3XA was made by ligating together oligonucletides
corresponding to 210 to
188 of the apoAI promoter that had
BamHI overhangs (5
-GATCACTGAACCCTTGACCCCTGCCCT-3
) and then ligating the multimers into the BamHI site of pBL-tk-LUC
(19).
The apoCIII reporter vector was created using
5-ACGAGAGAATCAGTCCTGGT-3
and 5
-TGCCTCTAGGGATGAACT-3
as primers for
polymerase chain reaction from human genomic DNA to create a fragment
containing from
810 to +23 of the apoCIII promoter. The product was
then ligated into Bluescript KS
via the BamHI and
SpeI sites. The apoCIII promoter was sequenced and then the
BamHI-SpeI fragment was ligated into pGL2
digested with BglII and SmaI to create
p810CIII-luc.
To construct the HNF-4 expression vectors, the
BamHI-HindIII HNF41 cDNA fragment from
pLEN4 (5) was first ligated into Bluescript KS
. This was ligated into
Bluescript KS
to create pK-HNF4
1. Polymerase chain reaction
mutagenesis was also used to add the additional sequences of the
2
splice form (pK-HNF4
2) (20, 21). The HNF-4 expression vectors
pC-HNF4
1 and pC-HNF4
2 were created by ligating the
BamHI-HindIII fragments from the pK-HNF4
constructs into pCDNA3 (Invitrogen).
The 111HNF4 mutant was created using
GATGGTACCGCCGCCACCATGGACTTCCGGGCTGGCATGAAGAAAGAAGCC and T3 primer
(Stratagene) for polymerase chain reaction from pK-HNF4
1. The
resulting truncated HNF-4 was also ligated into pCDNA3 to create
pC-
111HNF4
1. All polymerase chain reaction mutants were
sequenced. Transient transfections of HepG2 and Caco2 cells were
performed as described previously (17, 19). Each transfection included
Rous sarcoma virus-
-galactosidase, and all luciferase values were
normalized to
-galactosidase values. Each result is representative
of at least three independent transfections.
Full-length HNF-41 and
111HNF-4
1 were cloned into pACCMVpLpA (22) using the
BamHI and HindIII sites to create pAC-HNF4
1 and pAC-
111HNF4
1, respectively. Recombinant adenovirus were prepared, purified, and titered as described previously (22). Multiplicity of infection (m.o.i.) that resulted in the highest percentage of cells being infected was determined by infection with
Ad
gal followed by staining for
-galactosidase activity.
Peptides corresponding to amino acids 37-53 and 126-148 of rat HNF-4 were synthesized along with a cysteine at the amino terminus for coupling. The peptides were then conjugated with keyhole limpet hemocyanin. New Zealand White rabbits were injected subcutaneously with 0.5 mg of linked peptide using standard procedures and subsequently boosted with 0.3-mg subcutaneous injections.
Gel Mobility Shift AssayNuclear extracts were prepared
from adenovirus-infected HepG2 as described previously (23). Protein
concentrations were determined using the Bradford method (24).
DNA-protein binding assays were done by incubating nuclear extracts at
4 °C in reaction buffer containing 10 mM Hepes (pH 7.8),
40 mM KCl, 0.5 mM MgCl2,1 mM dithiothreitol, 10% glycerol, 5 µg of bovine serum
albumin; 1 µg of poly(dI-dC) was added as a nonspecific competitor,
and 0.5 ng of 32P-labeled (5,000-20,000 cpm) apoAI
promoter probe (210 to
188). The final volume was 10 µl.
Complexes were separated on 6% polyacrylamide gels with 22.5 mM Tris borate (pH 8.0) and 1 mM EDTA
buffer.
Nuclear extracts derived from adenovirus-infected HepG2 cells were separated on 10% SDS-polyacrylamide gels followed by electrophoretic transfer to polyvinylidene difluoride membrane (Bio-Rad). The membranes were blocked using 20 mM Tris (pH 8.0), 150 mM NaCl, 0.5% Tween 20 (TBST), and 5% non-fat dry milk for 1 h at room temperature and then incubated for 1 h with the rabbit anti-HNF4 126-148 antisera. The blots were washed three times for 5 min with TBST and then incubated for 30 min with a horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin antisera (Bio-Rad). After three washes the specific HNF-4 bands were detected using chemiluminescence (Pierce).
Northern Blot AnalysisRNA was extracted from the tissue culture cells using RNAzol (Tel-Test, Inc.). The RNA samples were electrophoresed (20 µg/lane) and transferred to Hybond N (Amersham Corp.) as described in the protocol supplied by the manufacturer. The indicated mRNAs were detected using random primed probes generated from the 440-base pair PstI fragment of human apoCIII, the HindIII-PstI fragment of human apoAI, the 1,500-base pair BamHI-HindIII fragment from rat HNF-4, and the 350-base pair EcoRI-XhoI fragment fragment from human glyceraldehyde phosphate dehydrogenase. The hybridization conditions and washes were done using the Rapid-Hyb system (Amersham Life Sciences) as described by the manufacturer. To quantify differences in mRNA expression, results were analyzed by PhosphorImager (Molecular Dynamics) and normalized to the level of glyceraldehyde phosphate dehydrogenase signal.
To examine the role of
HNF-4 on apolipoprotein gene transcription, we constructed expression
vectors coding for full-length cDNAs of the HNF-41 and HNF-4
2
splice forms as well as a truncated, dominant negative form of
HNF-4
1 (Fig. 1A).
111HNF-4
1 contains a deletion of the amino-terminal 111 amino acids, which include the
DNA-binding domain.
111HNF4
1 fails to display any detectable DNA
binding activity (data not shown) and should act as a dominant negative
of HNF-4-induced transcription due to dimerization with wild-type
HNF-4. The subsequent reduction in DNA binding avidity has previously
been demonstrated for DNA-binding domain mutants of several other
members of the intracellular receptor superfamily (25-28).
Cotransfection of HepG2 with a reporter construct containing three
copies of the 210 to
188 portion of the apoAI promoter (p3XA-luc)
along with expression vectors for either HNF-4
1 or HNF-4
2
resulted in 40-50-fold increases in luciferase activity (Fig.
1B). Although there was some experimental variation, the two
splice forms appeared to activate the p3XA-luc vector to aproximately the same degree. To determine whether the DNA binding domain mutant,
111HNF-4
1, inhibits HNF-4-induced transcription, submaximal levels of HNF-4
1 or HNF-4
2 were cotransfected into HepG2 cells along with p3XA-luc and increasing amounts of pC-
111HNF4
1. The
HNF-4
1 deletion mutant blocked the transfection-induced promoter activity of either HNF-4
1 or HNF-4
2 (Fig. 1C). These
results indicate that this DNA-binding domain mutant of HNF-4
1
behaves as a dominant negative and that HNF-4
1 and HNF-4
2 can
dimerize with each other. This result appeared to be specific for
HNF-4-induced activity as cotransfection of
111HNF-4
1 had no
effect on peroxisome proliferator-activated receptor
or retinoid X
receptor
-induced promoter activity (data not shown).
While these and previous studies have
demonstrated that the 210 to
188 region of the apoAI promoter can
bind HNF-4 and that artificial constructs containing multiple copies of
this region express increased levels of reporter gene activity in
response to cotransfected HNF-4, there is little evidence that
increasing the level of HNF-4 leads to significant increases in apoAI
promoter activity in hepatic-derived cells. One possible explanation
for this result is that the hepatoma cell lines used for these studies contain sufficient endogenous HNF-4 for maximal apoAI expression. To
address this we cotransfected HepG2 cells with p256A1-luc, a construct
that contains from
256 to +11 of the apoAI promoter linked to firefly
luciferase, and the individual HNF-4 expression vectors.
Cotransfection of either pCHNF-4
1 or pCHNF-4
2 resulted in
only small changes in apoAI promoter activity (Fig. 2,
A and B). However, cotransfection of the dominant
negative pC
111HNF4
1 reduced apoAI promoter expression by
51%, suggesting that HNF-4 does contribute substantially to apoAI gene
expression in this cell line (Fig. 2C). In contrast to HepG2
cells, cotransfection of the intestinal cell line Caco2 with pCHNF4
1
resulted in a 4-fold activation of apoAI-luc (Fig. 2D).
Surprisingly, cotransfection with the dominant negative
pC
111HNF4
1 failed to significantly affect apoAI promoter activity
in Caco2 cells (Fig. 2E).
The affects of HNF-4 cotransfection on apoAI
promoter activity depends on the cell phenotype. A, HepG2
cells were transiently transfected with p256AI-luc and the indicated
dose of pC-HNF41. B, HepG2 cells were transiently
transfected with p256AI-luc and the indicated dose (in nanograms per 1.5 × 106
cells) of pC-HNF4
2. C, HepG2 cells were transiently
transfected with p256AI-luc and the indicated dose of
pC-
111HNF4
1. D, Caco2 cells were transiently
transfected with p256AI-luc and the indicated dose of pC-HNF4
1.
E, Caco2 cells were transiently transfected with p256A1-luc
and the indicated dose of pC-
111HNF4
1. All transfections received
5 µg of the p256AI-luc reporter vector and the indicated quantities
of expression vector per 1.5 × 106 cells. The
error bars represent the standard deviation; each condition
was performed in triplicate.
ApoCIII Expression
Cotransfection of either pCHNF41 or
pCHNF4
2 along with a promoter construct containing from
810 to +23
of the apoCIII promoter linked to firefly luciferase (p810CIII-luc)
resulted in 4-5-fold increases in luciferase activity (Fig.
3, A and B). This indicates that,
similar to the response observed using the construct consisting of
multiple copies of the apoAI A site, both of these forms of HNF-4 can
recognize the apoCIII HNF-4 response element(s) and that both have
roughly the same activation potential for this promoter. To address
whether or not endogenous HNF-4 is involved in the basal expression of
the apoCIII promoter by HepG2, cells were cotransfected with
p810CIII-luc and increasing amounts of pC
111HNF4
1. Cotransfection
of the dominant negative resulted in a 70-80% decrease in
p810CIII-luc promoter activity (Fig. 3C).
Generation of Recombinant Adenovirus Vectors for the Expression of HNF-4
It can be difficult to recapitulate the bona
fide regulation of endogenous genes from recombinant promoter
constructs that may lack critical regulatory sequences. To address this
limitation, we generated recombinant adenovirus vectors to express
HNF-41 and the dominant negative deletion mutant
111HNF-4
1. In
many cell lines, adenovirus vectors can achieve nearly 100% infection, thus allowing us to examine the effect of expressing either wild-type HNF-4
1 or the dominant negative mutant on endogenous gene
expression. The ability of the recombinant adenoviral vectors to
express HNF-4
1 was tested by infection of HepG2 cells at titers of
adenovirus that resulted in 80-95% of the cells being infected (data
not shown) followed by extract preparation and immunoblotting using a
polyclonal antiserum specific for HNF-4. Infection of HepG2 cells with
AdHNF4
1 resulted in greatly increased levels of a 55-kDa polypeptide
recognized by this antiserum compared with cells infected with a
recombinant adenovirus that expresses
-galactosidase (Fig.
4A, lanes 1 and 4). Coinfection of
HepG2 with AdHNF4
1 and Ad
111HNF4
1 resulted in the expression
of an additional immunoreactive polypeptide with an apparent molecular
mass of 42 kDa (Fig. 4A, lanes 2 and 3).
Coinfection with the dominant negative mutant of HNF-4 did not have any
detectable effect on the level of HNF-4
1 expression (Fig.
4A).
Nuclear extracts prepared from HepG2 cells 2 days after infection with
AdHNF41 show a large increase in binding to double-stranded oligonucleotides corresponding to the apoAI (
210 to
188) (Fig. 4B). The major complex formed in the nuclear extracts from
AdHNF4
1-infected cells contains HNF-4 as it is recognized by
anti-HNF-4 antiserum (Fig. 4B, lane 3). Coinfection of HepG2
cells with the dominant negative truncation mutant of HNF-4 and
wild-type HNF4
1 resulted in substantially decreased HNF-4 DNA
binding compared with extracts obtained from cells infected with
AdHNF4
1 alone (Fig. 4C).
Infection of papoCIII-luc transfected HepG2 cells with the adenoviral
vectors encoding HNF-4 or the dominant negative mutant resulted in
similar effects on apoCIII promoter activity as those obtained in the
cotransfection experiments. Infection with AdHNF41 lead to a
3-4-fold increase in luciferase activity, while infection with
Ad
111HNF4
1 resulted in a 70% decrease in reporter expression (Fig. 5). Reporter activity in HepG2 cells transfected
with the p256A1-luc vector was unaffected by infection with AdHNF4
1
and decreased 27% after infection with the dominant negative
Ad
111HNF4
1 (data not shown).
To determine whether the observed effects of HNF-4 modulation on
recombinant promoter constructs was reflective of endogenous gene
expression as well as to determine whether the tight genetic linkage
between apoAI and apoCIII resulted in common HNF-4 regulatory elements,
the hepatoma lines HepG2 and Hep3B as well as the intestinal cell lines
Caco2 and IEC-6 cells were infected with AdHNF41, Ad
111HNF4
1,
or Ad
gal. Adenoviral infection levels of 80-95% were obtained for
the HepG2, Hep3B, and IEC-6 cell lines with no detectable toxicity.
However we were unable to achieve greater than 30% adenoviral
infection of the Caco2 cells without significant cell death, therefore
adenoviral studies with this line were discontinued. The level of
glyceraldehyde phosphate dehydrogenase mRNA was unaffected by
infection with any of the recombinant viruses, indicating that infection with the viral vectors does not have significant nonspecific affects on cellular gene expression (Fig. 6). As was
previously observed in the transient transfection assays with the apoAI
promoter construct in HepG2 cells, the level of apoAI mRNA was
relatively unaffected by expression of the recombinant HNF-4
1 (Fig.
6 and Table I). However, addition of AdHNF-4
1 did
result in a 3-fold increase in apoAI expression in Hep3B cells, and
infections with AdHNF-4
1 resulted in a dramatic induction of apoAI
mRNA expression in the rat intestinal cell line IEC-6 (Fig. 6 and
Table I). Infection with the dominant negative Ad
111HNF-4
1
caused a 33 and 54% decrease in HepG2 and Hep3B, respectively. The
basal level of apoAI mRNA expression was quite low in the IEC-6
cells and addition of the dominant negative did not detectably alter
apoAI mRNA levels in this cell line (Table I).
|
In contrast to the effect on apoAI expression in HepG2 cells,
expression of exogenous HNF-4 increased the level of apoCIII mRNA,
while infection with the dominant negative mutant Ad111HNF4
1 resulted in decreased message levels (Fig. 6). Quanitation of the
apoCIII mRNA levels revealed a 7-fold increase in apoCIII mRNA
after infection with AdHNF4
1 and an 80% decrease after addition of
the dominant negative mutant (Table I); confirming that HNF-4 is a
significant contributor to apoCIII gene expression. We were unable to
detect basal apoCIII expression in either the Hep3B or IEC-6 cells and
infection with the HNF-4 adenovirus failed to induce detectable apoCIII
mRNA (data not shown).
HNF-4 is an orphan member of the steroid/thyroid superfamily that
is expressed in the liver and small intestine, where the majority of
apolipoproteins are synthesized. HNF-4 is capable of binding to
sequences present in the apolipoprotein A1, apolipoprotein B,
apolipoprotein CIII, and apolipoprotein AIV promoters and has been
proposed to regulate the expression of these apolipoprotein genes (11,
12, 17, 29, 30). However, the evidence for HNF-4-regulated
apolipoprotein expression has been primarily based on the existence of
sequences within the apolipoprotein promoters to which HNF-4 binds in
gel shift experiments and transient transfections utilizing
overexpressed HNF-4 and recombinant promoter-reporter constructs. To
complement experiments utilizing overexpression of HNF-4, we have
constructed a mutant of HNF-41 that lacks the DNA-binding domain.
Since HNF-4 binds to DNA as a homodimer and the
111HNF-4
1 mutant
contains the sequences necessary for dimerization,
111HNF-4
1
should form dimers with full-length HNF-4 that have reduced DNA binding
avidity similar to that previously described for mutants of
c-erb-
, thyroid hormone receptor, and the estrogen receptor (25-28). HNF-4 fails to form heterodimers with several of the
other intracellular receptors (16), suggesting that the dominant
negative HNF-4 mutant should act as a specific inhibitor of
HNF-4-induced transcription. The
111HNF-4
1 mutant demonstrates no
detectable DNA binding activity and efficiently blocks both HNF-4
1
and
2-induced transcription, indicating that the two splice forms
can dimerize with each other despite the sequence changes within the
carboxyl-terminal domain. While we could not detect any affect of
111HNF-4
1 cotransfection on promoter activity induced by other
intracellular receptors, it is also possible that the carboxyl-terminal
domain of HNF-4 sequesters out coactivators that are necessary for
HNF-4-induced activity.
In agreement with previous studies (11, 12, 30), cotransfection of
HepG2 cells with HNF-41 or
2 had fairly modest affects on apoAI
promoter expression. However, expression of the dominant negative in
HepG2 cells does lead to a 40-50% decrease in apoAI
promoter-activated expression, suggesting that endogenous HNF-4
contributes to apoAI expression in this cell model. Utilizing the
recombinant adenoviral vectors, we have extended these findings to
examine endogenous apoAI gene expression. The effect of increasing HNF-4 expression on endogenous apoAI mRNA levels in HepG2 cells is
simlar to the results of the recombinant promoter assays in that
increasing HNF-4 levels did not change apoAI expression, whereas
expression of the dominant negative mutant decreases expression of
apoAI mRNA. Overexpression of HNF-4 leads to a 3-fold increase in
apoAI mRNA levels in the Hep3B cell line, while expression of the
dominant negative results in a 54% reduction in apoAI mRNA. This
result is consistent with the notion that comparatively high levels of
HNF-4 expression in HepG2 cells blunt the effect of additional HNF-4,
since the Hep3B cell line appears to have lower levels of HNF-4
expression than HepG2 (data not shown).
HNF-4 overexpression appears to have a much more pronounced effect on apoAI expression in cell lines derived from intestinal epithelium. Transient transfection of HNF-4 up-regulated the promoter activity of apoAI-luciferase constructs by 3-5-fold in the enterocyte-like cell line Caco2 as has recently been described by Bisaha et al. (17). Infection of the IEC-6 cell line with the adenoviral vector encoding wild-type HNF-4 dramatically increased apoAI mRNA expression by greater than 20-fold. Both of these cell lines appear to have less HNF-4 than do the hepatic derived HepG2 or Hep3B, therefore magnifying the affect of adding exogenous HNF-4. But even so, the large induction using the adenovirus on the endogenous apoAI gene suggests a more critical role for HNF-4 on intestinal expression of apoAI. It is tempting to speculate that these differences are related to the previously described intestinal control region contained within the closely linked apoCIII gene, as this region does contain HNF-4 response elements that could contribute to apoAI gene expression by intestinal cells (17, 30, 31).
Unlike the apoAI promoter assays in the HepG2 cells, cotransfection of
either the 1 or
2 splice forms of HNF-4 leads to increased
reporter expression from an apoCIII reporter construct. The strong
induction of apoCIII expression by HNF-4 is also observed at the level
of apoCIII mRNA following infection with the adenovirus encoding
wild-type HNF-4. More importantly, infection with the dominant negative
mutant of HNF-4 results in an 80% decrease in apoCIII mRNA levels.
This indicates that endogenous HNF-4 is required for apoCIII promoter
activity and suggests that modulation of HNF-4 transcriptional activity
would lead to significant changes in the level of apoCIII expression.
High levels of apoCIII correlate with increased fasting triglycerides
in both clinical hypertriglyceridemic patients and murine model systems
(32-34). Recent clinical studies suggest that high serum triglycerides
are directly atherogenic as well as being inversely correlated with
high density lipoprotein cholesterol levels, a well documented risk
factor for atherosclerotic disease (35, 36). Therefore modulation of
apoCIII expression via HNF-4 might represent a potential therapeutic
target. A primary question is whether HNF-4 activity is regulated or
not. HNF-4 is capable of activating transcription in the absence of
exogenously added ligand. Therefore, if HNF-4 requires a ligand for
transcriptional activation, it must be present under normal tissue
culture conditions as has been found to be the case for nuclear
receptors such as peroxisome proliferator-activated receptor and
farnesoid X-activated receptor (37, 38). The question of whether or not
an unidentified ligand actually regulates HNF-4-induced transcription
awaits future studies as does elucidation of HNF-4s role in lipid
metabolism and cardiovascular disease.
We thank Dr. Robert Gerard for his assistance with the recombinant adenovirus. We thank Dr. Michael Saunders for his generous gift of plasmid pGF1. We thank Dr. Glenn Croston and Dr. James Paternitti for their helpful comments on this manuscript.