From the
The hypolipidemic effect exerted by
Aryloxyalkanoic fibrates (e.g. clofibrate
(1) and bezafibrate
(2) ), substituted long chain
dicarboxylic acids (e.g. Medica 16
(3, 4) ), and
other amphipathic carboxylates lower plasma triglycerides and
cholesterol levels, and some are extensively used in humans as drugs of
choice for treating hypertriglyceridemia or combined
hypertriglyceridemia/hypercholesterolemia
(5) . The hypolipidemic
effect induced by these drugs may essentially be ascribed to enhanced
catabolism of plasma triglyceride-rich lipoproteins (VLDL
The
hypolipidemic effect exerted by hypolipidemic amphipathic carboxylates
is accompanied in rodents and some other species by an increase in
liver size and liver peroxisomes with a concomitant induction of
specific peroxisomal and other genes (e.g. peroxisomal
acyl-CoA oxidase, cytochrome P-4504A1) (reviewed in Refs. 19 and 20).
Induction of peroxisomal genes as well as of cytochrome P-4504A1 by
hypolipidemic drugs/peroxisome proliferators (HD/PP) is due to
transcriptional activation
(21) mediated by binding of the
peroxisome proliferator activated receptor (PPAR) homodimer or the
PPAR-retinoic acid-X-receptor (RXR) heterodimer to sequence specific
response elements in the 5`-flanking promoters of the concerned genes
(22-26). The apparent linkage observed between the hypolipidemic
and the peroxisomal effects exerted by HD/PP was claimed to indicate a
causal-sequential relationship between peroxisome
proliferation/induction of peroxisomal
The mode
of action of HD/PP in suppressing plasma apoC-III levels will be shown
here to result from transcriptional suppression of the apoC-III gene
mediated by PPAR-RXR. Transcriptional suppression of the apoC-III gene
along with transcriptional activation of the respective peroxisomal
genes by PPAR-RXR may rationalize the linkage between the hypolipidemic
and the peroxisome proliferative effects exerted by HD/PP in rodents.
The hypolipidemic effect of Medica 16 in rats is accounted
for by a 3-5-fold decrease in plasma apoC-III with a concomitant
5-10-fold activation of VLDL and chylomicron uptake into the
liver
(8, 9) . Plasma apoC-III clearance remained
unaffected by Medica 16
(9) , thus implying a decrease in the
production of liver apoC-III induced by Medica 16 treatment. As shown
in Fig. 1, A and B, the decrease in plasma
apoC-III induced by HD/PP treatment in rats may be accounted for by a
respective decrease in liver apoC-III mRNA induced either by an
aryloxyalkanoic drug (bezafibrate) or substituted long chain
dicarboxylic acid (Medica 16). Bezafibrate was somewhat more effective
than Medica 16, resulting in 5-fold decrease in liver apoC-III mRNA
following 3 days of treatment as compared with a 2-fold decrease
induced by Medica 16 treatment. Similarly, a decrease in apoC-III mRNA
was observed in human transformed liver cells (HepG2) incubated in the
presence of either Medica 16 (Fig. 1C) or bezafibrate,
thus pointing to a direct effect exerted by HD/PP on cells expressing
apoC-III. Medica 16 was significantly more effective than bezafibrate
in the human cells (2.5-3.0- versus 1.3-2.0-fold
decrease following 3 days of treatment). Suppression of liver apoC-III
mRNA was accompanied by inhibition of apoC-III transcription rate as
verified by run-on transcription assays in liver nuclei derived from
nontreated, Medica 16- or bezafibrate-treated rats (Fig. 2).
Hence, the hypolipidemic effect exerted by HD/PP may be ascribed to
suppression of transcription of the liver apoC-III gene by HD/PP.
Treatment of rats with either bezafibrate or Medica 16 resulted
indeed in a decrease in liver HNF-4 mRNA with a pronounced
time-dependent decrease in HNF-4 protein levels (Fig. 3). The 50%
decrease in HNF-4 mRNA induced by either Medica 16 or bezafibrate could
be accounted for by transcriptional suppression of the HNF-4 gene as
verified in liver nuclei derived from Medica 16- or bezafibrate-treated
rats or in HepG2 cells incubated in the presence of the respective
HD/PP (Fig. 3A). Transcriptional suppression of HNF-4 in
HepG2 cells was significantly more pronounced in the presence of Medica
16 as compared with bezafibrate and in accordance with apoC-III mRNA
levels in liver cells treated with the two respective effectors. The
reduced availability of HNF-4 for binding and transactivating apoC-III
transcription in nuclear extracts derived from HD/PP-treated rats was
further verified by analyzing the binding of respective nuclear
extracts to the rat apoC-III C3P element. Bound HNF-4 in the respective
extracts was identified by the extent of HNF-4 supershift in the
presence of added anti HNF-4 antibodies. As shown in Fig. 3D (lanes 1, 5, and 9), nuclear extract
binding to the rat apoC-III C3P element was significantly reduced in
HD/PP-treated rats. Reduced binding was accounted for by the lower
availability of HNF-4 in nuclear extracts derived from HD/PP-treated
rats (lanes 6 and 10 as compared with 2,
lanes 7 and 11 as compared with 3, and
lanes 8 and 12 as compared with 4). Hence,
HNF-4 suppression may account for apoC-III suppression by HD/PP.
Decrease in plasma apoC-III induced in rats by treatment with
two structurally different hypolipidemic amphipathic carboxylates was
shown here to result from transcriptional suppression of the liver
apoC-III gene as verified by run-on transcription assays in liver
nuclei derived from rats treated in vivo. The extent of
decrease in transcription rate induced by HD/PP essentially correlates
with the decrease observed in apoC-III mRNA and plasma apoC-III content
induced by treatment with HD/PP. In line with the previously reported
inhibition of clearance of plasma triglyceride-rich lipoproteins by
apoC-III
(10, 11, 12, 13, 14) ,
the hypolipoproteinemia induced in humans inflicted by apoC-III
deficiency
(17) , the hyperlipoproteinemia induced in h-apoC-III
transgenic mice
(16, 17) , as well as hypotriglyceridemia
and protection from postprandial hypertriglyceridemia in animals
lacking apoC-III
(18) , transcriptional suppression of the
apoC-III gene by hypolipidemic amphipathic carboxylates with a
concomitant increase in plasma triglyceride-rich lipoproteins clearance
may account for the hypolipidemic effect exerted by these drugs.
Transcriptional suppression of the apoC-III gene by HD/PP was found
here to be related to HNF-4-enhanced transcription of the apoC-III gene
and ascribed to direct and indirect complementary modes of action,
namely, displacement of HNF-4 from the apoC-III promoter as well as
suppressing HNF-4 levels by HD/PP. HNF-4 displacement by PPAR-RXR was
implied in light of the inhibition of HNF-4-enhanced transcription by
PPAR-RXR in cells transfected with a reporter plasmid promoted by the
homologous apoC-III promoter. Inhibition of HNF-4-enhanced
transcription by PPAR-RXR could be accounted for by binding of PPAR-RXR
to the apoC-III HNF-4 element as verified by using gel shift binding
assays. Since transcription of the apoC-III gene is activated by HNF-4
(44, 45, Fig. 4) while remaining unaffected by PPAR-RXR, the
extent of inhibition of HNF-4 enhanced transcription by PPAR-RXR may be
expected to reflect the prevailing content of the concerned
transcription factors and their respective binding affinities to the
apoC-III C3P element. In this respect, the PPAR-RXR heterodimer behaves
similarly to other previously reported transcription factors which may
compete with HNF-4 for binding to the apoC-III C3P promoter element,
e.g. ARP-1, EAR-3/coup, or EAR-2
(44, 45) . It
should be pointed out, however, that generalizing the direct mode of
action of HD/PP as verified here in transiently transfected cells for
the endogenous apoC-III promoter still remains to be complemented by
studying the role played by additional regulatory sequences of the
apoC-III promoter not present in the transiently transfected promoter
as well as by the chromatin context of the endogenous gene. The
difference between the endogenous and the transiently transfected
promoter is indeed reflected in the requirement for the respective
transcription factors (e.g. HNF-4, PPAR) for suppression of
the transfected apoC-III gene by HD/PP. Suppression of the transiently
transfected promoter by HD/PP requires cotransfection of expression
vectors for the respective transcription factors (Fig. 4),
whereas the endogenous promoter may be directly suppressed by HD/PP in
the absence of added transcription factors ( Fig. 1and
Fig. 2
). This difference is similar to that previously reported
for other members of the steroid/thyroid hormone receptors superfamily
(57) where endogenous transcription factors sequestered within
the chromatin may become nonaccessible to the transiently transfected
promoter but still available to the endogenous promoter.
The
indirect mode of action of HD/PP in suppressing apoC-III gene
transcription was verified here by analyzing HNF-4 transcription rates
and transcript levels as well as measuring HNF-4 protein levels in
nuclear extracts of HD/PP-treated animals. Both the overall content of
nuclear extract HNF-4 and that available for binding to the apoC-III
C3P promoter element were found to be significantly reduced by
treatment with HD/PP. Since HNF-4 expression is positively modulated by
HNF-4 itself
(41) , suppression of HNF-4 gene expression by HD/PP
may perhaps result as well from displacement of HNF-4 from its putative
C3P element in the HNF-4 promoter. Hence, the direct and indirect
complementary effects of HD/PP in suppressing apoC-III gene expression
may actually reflect a unified mode of action where displacement of
HNF-4 from its C3P element in the apoC-III or HNF-4 gene promoters is
exerted by PPAR-RXR binding.
The mode of action of HD/PP in
initiating binding of the PPAR-RXR heterodimer to C3P elements of
peroxisomal genes, apoC-III, and other promoters has still to be
investigated, since, in contrast to other members of the
steroid/thyroid hormone receptors superfamily, the putative binding of
HD/PP to PPAR still remains unclear
(22) . Thus, PPAR binding to
its C3P element may be directly initiated by binding of the free HD/PP
to a putative ligand binding site of PPAR, albeit with low binding
affinity
(22) , or HD/PP could indirectly affect targeting or
affinity of PPAR to its C3P response elements. The extreme structural
diversity of HD/PP
(19, 20, 27, 40, 46) is indeed in contrast with the strict structural
specificity characteristically required for ligand binding by members
of the superfamily.
This proposed mode of action of amphipathic
carboxylic peroxisome proliferators as hypolipidemic drugs may indeed
rationalize the enigmatic linkage previously observed between the
hypolipidemic and peroxisome proliferative effects exerted by
HD/PP
(27) . Rather than pointing to a causal-sequential linkage
between peroxisome proliferation and
hypolipidemia
(27, 28) , the observed linkage may reflect
transcriptional modulation of genes involved in the hypolipidemic and
peroxisome proliferative effects by a common transduction pathway
consisting of binding PPAR to C3P response elements of the respective
concerned promoters. The net effect exerted by PPAR binding which may
result either in transcriptional activation or inhibition of the
concerned genes will depend, however, on the interplay between the
various transcription factors which may bind to a particular C3P
element, their prevailing nuclear concentrations, and their respective
binding affinities as well as the specific promoter context of the
concerned gene. Thus, binding of PPAR-RXR to a C3P element of
peroxisomal acyl-CoA oxidase in the context of the h-acyl-CoA oxidase
promoter, results in transcriptional activation of that gene while
binding the heterodimer to a C3P element in the context of the apoC-III
promoter does not modulate apoC-III transcription in the absence of
added HNF-4.
Transcriptional suppression of the apoC-III gene by
HD/PP mediated by modulating HNF-4 expression and its extent of binding
to the apoC-III gene promoter may offer a rationale for predicting and
realizing additional inhibitory effects exerted by amphipathic
carboxylates acting as HD/PP. Thus, other genes reported to be
transcriptionally transactivated by HNF-4
(41) should be
considered as candidates for transcriptional suppression by HD/PP
similar to that reported here for apoC-III. Transthyrethin is
transactivated by HNF-4 and has indeed been recently reported to be
inhibited by HD/PP
(47) . A particular candidate of interest
consists of the apoB gene recently reported to be transcriptionally
activated upon HNF-4 binding to its AF-1 promoter site
(48) .
Transcriptional suppression of apoB by HD/PP similar to that reported
here for apoC-III may result in decreased liver production of apoB with
a concomitant decrease in liver VLDL production as previously reported
for Medica 16
(49) . Transcriptional suppression of apoB may thus
complement plasma VLDL lowering effect of HD/PP induced by apoC-III
suppression. It should be pointed out, however, that additional factors
and constraints other than having a C3P element, and, in particular,
the specific promoter context concerned, may determine HNF-4 binding
and transactivation of a particular promoter, as well as suppression of
HNF-4-enhanced transcription by HD/PP.
Transcriptional suppression
of HNF-4-enhanced genes together with transcriptional activation of a
variety of other genes (e.g. peroxisomal
We thank T. Leff, T. Hashimoto, F. M. Sladek, S.
Green, R. M. Evans, and T. Grange for their kind gifts of the
respective plasmids and T. Leff for critical review of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
,
`-tetramethylhexadecanedioic acid (Medica 16) is accounted
for by enhanced catabolism of plasma triglyceride-rich lipoproteins due
to a decrease in plasma apolipoprotein C-III (Frenkel, B., Mayorek, N.,
Hertz, R., and Bar-Tana, J.(1988) J. Biol. Chem. 263,
8491-8497; Frenkel, B., Bishara-Shieban, J., and Bar-Tana,
J.(1994) Biochem. J. 298, 409-414). Decrease in
apolipoprotein C-III exerted by peroxisome proliferators/hypolipidemic
amphipathic carboxylates (e.g. Medica 16, fibrate drugs) is
shown here to result from suppression of apolipoprotein C-III gene
expression. Transcriptional suppression of apolipoprotein C-III is due
to transcriptional suppression of hepatic nuclear factor (HNF)-4 as
well as displacement of HNF-4 from the apolipoprotein C-III promoter.
HNF-4 displacement exerted by peroxisome proliferators/hypolipidemic
amphipathic carboxylates is mediated by the peroxisome proliferators
activated receptor (PPAR). Transcriptional suppression of
HNF-4-enhanced genes (e.g. apolipoprotein C-III) along with
transcriptional activation of peroxisomal and other genes by
hypolipidemic drugs may account for their broad spectrum
pharmacological effect.
(
)
and chylomicrons) with a concomitant increase in their
hepatic uptake
(6, 7, 8, 9) . Since
apolipoprotein (apo) C-III potently inhibits plasma triglyceride-rich
lipoprotein catabolism due to inhibition of their intravascular
lipolysis by lipoprotein lipase as well as their liver
receptor-mediated
uptake
(10, 11, 12, 13, 14) , and
in light of the hyperlipoproteinemia induced in h-apoC-III transgenic
mice (15, 16), or the hypotriglyceridemia induced in human
(17) or animals (18) lacking apoC-III, a decrease in plasma
apoC-III could account for the hypolipidemic effect exerted by these
drugs. Medica 16 has indeed been found to dramatically decrease plasma
apoC-III by 3-5-fold with a concomitant 5-10-fold increase
in chylomicrons
(8) and VLDL
(9) plasma clearance and
hepatic uptake. Hypolipidemic fibrate drugs have been similarly found
to decrease plasma apoC-III.
(
)
The mode of action
of hypolipidemic amphipathic carboxylates in suppressing plasma
apo-C-III levels remained, however, to be investigated.
-oxidation and
hypolipidemia (27, 28). This claim appeared, however, to be refuted in
humans where the hypolipidemic effect exerted by hypolipidemic fibrates
is unaccompanied by an increase in liver peroxisomes and peroxisomal
-oxidation (29 and references therein), thus leaving open the
question concerning the enigmatic linkage observed between the
hypolipidemic and the peroxisomal effects exerted by HD/PP.
Animals and Cultures
Male albino rats weighing
150-200 g were fed with laboratory chow diet. 0.25% (w/w) Medica
16 or 0.2% (w/w) bezafibrate was added to their diet where indicated.
HepG2, HeLa, and COS-7 cells were cultured in Dulbecco's modified
Eagle's media supplemented with 10% fetal calf serum with either
dimethyl sulfoxide as vehicle, Medica 16, or bezafibrate added to the
culture medium as indicated.
Run-on Transcription
Rat liver nuclei were
prepared according to Refs. 30 and 31 or HepG2 nuclei were prepared
according to Ref. 32. Run-on transcription assays were carried out in
0.2 ml of buffer containing 25% glycerol, 50 mM Hepes (pH
7.5), 50 mM NaCl, 2.5 mM MgCl, 0.05
mM EDTA, 5 mM dithiothreitol, 1.25 mM ATP,
1.25 mM CTP, 1.25 mM GTP, 2 mM creatine
phosphate, 2 µg of creatine kinase, 0.1 mM
phenylmethylsulfonyl fluoride, and 250 µCi of
[
P]UTP, using nuclei samples amounting to 7.0
absorbance units (260 nm). Incubation was carried out at 26 °C for
30 min followed by addition of 20 units of RNase-free DNase I and then
30 µg of proteinase K. Newly synthesized
[
P]RNA was hybridized for 48 h at 42 °C with
the respective cDNA inserts affixed to GeneScreen. Hybridization was
carried out in 2 ml of 50% formamide containing 50 mM Tris-HCl
(pH 8.0), 10 mM EDTA, 6
SSC, 1% SDS, 0.12% Ficoll,
0.12% polyvinylpyrrolidone, 0.12% bovine serum albumin, and 300 µg
of salmon-sperm DNA. Washes were performed once in 5
SSC, 0.2%
SDS, once in 2
SSC, 0.1% SDS, and once in 0.5
SSC, 0.1%
SDS at 60 °C followed by a 30-min treatment with 10 µg/ml RNase
A.
Transfection Assays
Transcriptional activity was
measured in cells cultured in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum. Cells were transfected
for 6 h with the respective CsCl-purified plasmid DNA added by calcium
phosphate precipitation, washed, and further cultured for 42 h in the
absence or presence of 120 µM Medica 16 added as
1000 stock in dimethyl sulfoxide. The
-galactosidase
expression vector pRSGAL (2 µg) added to each precipitate served as
an internal control for transfection. When transfected with variable
amounts of expression vectors, total amount of DNA was kept constant
for each expression vector by supplementing with the parent pSG5
vector
(33) . Cell extracts were prepared by freeze-thawing and
assayed for
-galactosidase and CAT activities. Results are
expressed as -fold induction relative to CAT expression observed in
cells transfected with the parent pSG5 vector. Each point represents
the mean of duplicate cultures.
Gel Mobility Shift Assays
Gel mobility shift
assays were carried out using rat nuclear extracts
(30) , in
vitro-synthesized transcription factors, or whole Cos extracts
overexpressing the respectively transfected expression vectors. h-RXR
and r-HNF-4 cDNAs cloned in pSG5 were linearized by XbaI and
transcribed (Strategin) and translated in rabbit reticulocytes
(Promega). Cos extracts enriched with PPAR, RXR, or HNF-4 were prepared
from Cos-7 cells transfected for 5 h by calcium phosphate precipitation
with 10 µg of either pSG5, pSG5-PPAR, pSG5-RXR, pSG5-HNF-4, or
selected combinations of the above plasmids. Following transfection,
cells were glycerol-shocked, incubated for 48 h, harvested, and lysed
by three cycles of freezing-thawing in 100 µl/plate of lysis buffer
(600 mM KCl, 10 mM Tris-HCl, pH 7.5, 1 mM
dithiothreitol, 1 mM EDTA, 1 mM phenylmethylsulfonyl
fluoride, 1 µg/ml pepstatin, and 1 µg/ml leupeptin),
centrifuged at 20,000 g for 15 min, and the
supernatants were then aliquoted and stored at -70 °C. For
gel shift assays, programmed or unprogrammed reticulocytes lysates (2
µl) or whole Cos extracts (4 µg) as indicated were incubated
for 20 min on ice in 11 mM Hepes, pH 7.9, containing 50
mM KCl, 1 mM dithiothreitol, 2.5 mM
MgCl
, 10% glycerol, 1 µg of poly(dI-dC) in a final
volume of 20 µl. 0.1 ng of the respective
P-labeled
oligonucleotide was then added, and incubation was continued for an
additional 20 min at room temperature. Protein-DNA complexes were
resolved on a 5% nondenaturing polyacrylamide gel in 0.5
TBE.
Molecular Probes and Plasmid Constructs
A
270-nucleotide fragment from the 3`-end of m-apoC-III gene cloned into
pGem-1 (pGmCIII 270)
(34) , h-apoC-III cDNA (pCIII 607) and
(-854/+22)h-apoC-III-CAT (35) were from T. Leff (Ann Arbor,
MI). Peroxisomal acyl-CoA oxidase pMJ125
(36) was from T.
Hashimoto (Nagano, Japan). HNF-4 recombinant DNA pLEN4S
(37) was
from F. M. Sladek (Riverside, CA). h-PABP cDNA
(38) cloned into
the EcoRI site of pGEM1 (Promega) was from T. Grange (Paris,
France). pSG5-HNF-4 expression plasmid was constructed by inserting the
BamHI fragment of pLEN4S encoding the entire 3-kilobase HNF-4
cDNA into the BamHI site of pSG5. pSG5-m-PPAR expression
plasmid
(22) and anti m-PPAR antiserum were from S. Green
(Zeneca, Cheshire, UK). RS-h-RXR
(39) was from R. Evans (La
Jolla, CA). pSG5-RXR expression plasmid was constructed by inserting
the EcoRI fragment of RS-h-RXR
into the EcoRI
site of pSG5.
HNF-4 Western Blot Analysis
Nuclear extracts of
pooled (n = 3) rat liver nuclei were prepared according
to Ref. 30, and 10 µg of protein were resolved by 10% SDS-PAGE and
electroblotted onto nitrocellulose filters. Filters were blocked in
0.5% gelatin for 2 h at room temperature and then incubated with anti
HNF-4 antisera (raised in rabbit to a synthesized peptide that
corresponded to amino acids 445-455 of the rat HNF-4
protein
(37) ) for 6 h at room temperature in 0.1 M
sodium phosphate buffer, pH 7.4, containing 0.25% gelatin and 0.25%
Tween 20. Following washes with the same buffer, filters were incubated
for 1 h with goat alkaline phosphatase-conjugated anti-rabbit IgG
antibodies (Promega), and the immune complexes were detected using the
nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate
(Sigma). Equal loading of protein in each lane was verified by
Coomassie blue staining.
Figure 1:
ApoC-III mRNA levels in
response to Medica 16 and bezafibrate. A, apoC-III mRNA in
rats. Total RNA from liver of male rats treated for 6 days with either
Medica 16 or bezafibrate as described under ``Experimental
Procedures'' was prepared according to Ref. 54, and 25 µg were
subjected to Northern blot analysis using GeneScreen membranes (DuPont
NEN). Rat apoC-III mRNA was determined using the 270-base pair
BamHI/EcoRI restriction fragment of pGmCIII 270
plasmid P-labeled by the random priming method (55).
B, kinetics of Medica 16 (
) and bezafibrate (
)
treatment in rats. Mean ± S.D. (n = 3).
C, apoC-III mRNA in HepG2 cells. HepG2 cells were cultured for
72 h as described under ``Experimental Procedures'' with
either vehicle (dimethyl sulfoxide) or 120 µM Medica 16.
Total RNA was extracted according to Ref. 56 and subjected to Northern
blot analysis. HepG2 apoC-III mRNA was probed using the 500-base pair
PstI restriction fragment of h-apoC-III
cDNA.
Figure 2:
Rat
apoC-III transcriptional activity in response to Medica 16 and
bezafibrate. Male rats were treated for 5 days with either Medica 16 or
bezafibrate as described under ``Experimental Procedures.''
Run-on transcription assays were carried out as described under
``Experimental Procedures.'' Newly synthesized
[P]RNA was hybridized with 0.5 µg of cDNA
BamHI/EcoRI 270-base pair insert of m-apoC-III and
the extent of hybridization was normalized to the signal obtained with
-actin cDNA. Numbers indicate relative densitometric units.
Representative experiment out of 4 independent
experiments.
Transcription of the apoC-III gene has previously been shown to be
affected by hepatic nuclear factor (HNF)-4, a liver-abundant orphan
member of the steroid/thyroid hormone superfamily receptors
(41) which may bind to the C3P 5`-flanking sequence of the
apoC-III gene promoter, resulting in activation of apoC-III
transcription
(42, 43, 44, 45) . Since
liver apoC-III transcription is dependent on HNF-4
levels
(44, 45) , transcriptional suppression of the
apoC-III gene by HD/PP could result from suppression of HNF-4 levels
and/or interference with HNF-4 binding to the apoC-III promoter.
Figure 3:
HNF-4 gene expression in response to
Medica 16 and bezafibrate. A, HNF-4 transcription rate. Male
rats were treated for 5 days with either Medica 16 or bezafibrate as
described under ``Experimental Procedures.'' HepG2 cells were
cultured for 72 h with either dimethyl sulfoxide or 120 µM
Medica 16. Run-on transcription assays for HNF-4 were carried out as
described under ``Experimental Procedures'' using HepG2 or
pooled (n = 3) rat liver nuclei. Newly synthesized
[P]RNA was hybridized with the HNF-4 recombinant
cDNA pLEN4S. The extent of hybridization was normalized to the signal
obtained with
-actin cDNA or poly(A)-binding protein (PABP).
Numbers indicate relative densitometric units. Transcriptional
activation of peroxisomal
-oxidation genes is exemplified by
hybridization with the peroxisomal acyl-CoA oxidase (AOX)
pMJ125 plasmid. B, HNF-4 mRNA. HNF-4 mRNA was determined by
Northern blot analysis of total RNA (20 µg) as in Fig. 1A and probed with the 3.5-kilobase EcoRI restriction
fragment of the recombinant cDNA pLEN4S. C, HNF-4 protein.
Male rats were treated for 1-3 days with either Medica 16 or
bezafibrate as described under ``Experimental Procedures.''
HNF-4 protein in rat liver nuclear extracts was determined by Western
blot analysis as described under ``Experimental Procedures.''
Western blot results were confirmed in 4 independent preparations of
nuclear extracts. D, nuclear extract and HNF-4 binding to the
r-apoC-III C3P element. Nuclear extracts prepared as described under
``Experimental Procedures'' from rats treated for 3 days with
either Medica 16 or bezafibrate were analyzed by gel shift as described
under ``Experimental Procedures'' for binding to the
r-apoC-III
P-labeled C3P element (filled
arrowhead). HNF-4 specific binding was determined by incubating
the respective extracts with anti rat HNF-4 antibodies, thus producing
the HNF-4 supershifted bands (filled arrow). Lanes 1,
5, and 9, 0.05 µg of the respective nuclear
extracts incubated with 1 µl of preimmune serum. Lanes 2,
6, and 10, 0.05 µg of the respective extracts
incubated with 1 µl of anti-HNF-4 immune serum. Lanes 3,
7, and 11, 0.1 µg of the respective extracts
incubated with 1 µl of anti-HNF-4 immune serum. Lanes 4,
8, and 12, 0.2 µg of the respective extracts
incubated with 1 µl of anti-HNF-4 immune
serum.
In
addition to indirectly affecting apoC-III transcription by modulating
HNF-4 gene expression and protein content, HD/PP exert a direct effect
on apoC-III promoter as verified in cells cotransfected with a CAT
reporter plasmid promoted by the 854 5`-flanking base pairs of the
human apoC-III gene
(42, 43) together with expression
vectors for HNF-4, PPAR, and RXR. Basal expression of the
(-854/+22)h-apoC-III CAT construct remained essentially
unaffected by Medica 16 or bezafibrate. However, the HNF-4-enhanced
transcription of this construct was repressed by PPAR and further
repressed by PPAR in the presence of added Medica 16
(Fig. 4A) or bezafibrate and as a function of the
relative proportions between transfected HNF-4 and PPAR. Suppression of
HNF-4-activated transcription by PPAR was further affected by
cotransfection with an expression vector for RXR
(Fig. 4B). Thus, the 20-fold activation of apoC-III
expression induced by HNF-4 could be essentially eliminated in the
presence of PPAR, RXR, and Medica 16. No effect of PPAR, RXR, or both
was observed in the absence of added HNF-4 (not shown), thus indicating
that PPAR and RXR specifically counteracted HNF-4-enhanced
transcription rather than inhibited basal transcription of the apoC-III
gene. It is noteworthy that the C3P sequence of the apoC-III gene is
homologous to the peroxisomal acyl-CoA oxidase promoter sequence which
binds PPAR in the course of inducing transcriptional activation of
peroxisomal genes by HD/PP
(23, 24, 25) .
Nevertheless, as noted above, basal transcription is not affected by
binding of PPAR to the C3P element in the context of the
-854/+22 apoC-III promoter, whereas transcriptional
transactivation is induced by binding of PPAR to the C3P element in the
context of peroxisomal acyl-CoA oxidase
promoter
(23, 24, 25) . Thus, the selective
effect of PPAR binding presumably reflects the promoter context of the
particular concerned gene.
Figure 4:
Transcriptional activity of the apoC-III
promoter in HNF-4, PPAR, and PPAR/RXR transfected cells and as a
function of Medica 16. A, HepG2 cells were transfected as
described under ``Experimental Procedures'' with
(-854/+22)h-apoC-III-CAT construct (5 µg) and
cotransfected with expression vectors for HNF-4 (as indicated) and PPAR
(1 µg). Representative experiment out of 2 independent experiments.
B, HeLa cells were transfected as described under
``Experimental Procedures'' with
(-854/+22)h-apoC-III-CAT gene (5 µg) and cotransfected
with 0.2 µg each of expression vectors for HNF-4, PPAR and RXR.
Representative experiment out of three independent experiments. CAT
activity determined in the presence of HNF-4 expression vector is
presented as -fold induction compared to CAT activity determined in its
absence. , HNF-4;
, HNF-4 + RXR;
,
HNF-4 + PPAR;
, HNF-4 + PPAR + Medica 16;
, HNF-4 + PPAR + RXR;
, HNF-4 + PPAR
+ RXR + Medica 16.
The putative binding of PPAR, PPAR-RXR,
or HNF-4-PPAR to the rat (-91/-77) or human
(-87/-66) apoC-III C3P element was studied by gel shift
using the concerned transcription factors translated in vitro in rabbit reticulocytes or expressed in transfected Cos cells
(Fig. 5). HNF-4 and the PPAR-RXR heterodimer, but not PPAR or RXR
alone, were strongly bound by the rat or human apoC-III C3P element.
The binding affinity of the human apoC-III C3P element to HNF-4 or
PPAR-RXR was determined by mobility shift analysis using labeled
h-apoC-III C3P element and increasing concentrations of nonradioactive
C3P in the presence of Cos extracts derived from cells transfected with
expression vectors for either HNF-4 or PPAR-RXR. The binding affinity
of the h-apoC-III C3P element to HNF-4 and PPAR-RXR was found to be
0.48 ± 0.1 nM and 0.67 ± 0.06 nM,
respectively. Binding of Cos extracts to the apoC-III C3P element was
not affected by the presence of HD/PP, either during transfection of
Cos cells with expression vectors for the respective transcription
factors or during gel shift (data not shown). The respective band
formed in the presence of both HNF-4 and PPAR was not shifted by an
anti-PPAR antibody under conditions where binding of PPAR was
completely displaced, thus indicating that no heterodimer was formed in
the presence of HNF-4 and PPAR. These binding experiments thus further
indicate that transcriptional suppression of the apoC-III gene by HD/PP
may be mediated by displacement of HNF-4 from its apoC-III response
element by PPAR-RXR binding to this promoter element.
Figure 5:
Mobility shift analysis of human and rat
C3P elements by PPAR, RXR, and HNF-4. The respective transcription
factors were transcribed and translated in vitro in rabbit
reticulocytes (A) or in transfected Cos cells (B) as
described under ``Experimental Procedures.'' Mobility shift
analysis was as described under ``Experimental Procedures''
using either the human (5`-agctGCAGGTGACCTTTGCCCAGCGCC-3`) or rat
(5`-agctGCAGGTGACCTTTGACCAGCTc-3`) apoC-III P-labeled C3P
elements, respectively. Where denoted (+Ab), anti-m-PPAR
antibodies (1 µl of immune serum) were added to the incubation
mixture.
-oxidation
genes
(19, 20) , P-4504A1
(26) , liver thyroid
hormone-dependent genes
(50, 51) ), all mediated by
HD/PP-dependent binding of PPAR to C3P elements in the respective
promoters, may form the basis for the pleiotropic effect of xenobiotic
HD/PP as well as of putative natural amphipathic carboxylates (e.g. long chain fatty acids
(52, 53) ) acting as broad
spectrum pharmacological or physiological modulators of gene
expression.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.