(Received for publication, January 31, 1995; and in revised form, April 20, 1995 )
From the
The long-chain acyl-coenzyme A synthetase (ACS) gene gives rise
to three transcripts containing different first exons preceded by
specific regulatory regions A, B, and C. Exon-specific oligonucleotide
hybridization indicated that only A-ACS mRNA is expressed in rat liver.
Fibrate administration induced liver C-ACS strongly and A-ACS mRNA to a
lesser extent. B-ACS mRNA remained undetectable. In primary rat
hepatocytes and Fa-32 hepatoma cells C-ACS mRNA increased after
treatment with fenofibric acid, -bromopalmitate,
tetradecylthioacetic acid, or
-linolenic acid. Nuclear run-on
experiments indicated that fenofibric acid and
-bromopalmitate act
at the transcriptional level. Transient transfections showed a 3.4-,
2.3-, and 2.2-fold induction of C-ACS promoter activity after
fenofibric acid,
-bromopalmitate, and tetradecylthioacetic acid,
respectively. Unilateral deletion and site-directed mutagenesis
identified a peroxisome proliferator activator receptor
(PPAR)-responsive element (PPRE) mediating the responsiveness to
fibrates and fatty acids. This ACS PPRE contains three imperfect half
sites spaced by 1 and 3 oligonucleotides and binds PPAR
retinoid X
receptor heterodimers in gel retardation assays. In conclusion, the
regulation of C-ACS mRNA expression by fibrates and fatty acids is
mediated by PPAR
retinoid X receptor heterodimers interacting
through a PPRE in the C-ACS promoter. PPAR therefore occupies a key
position in the transcriptional control of a pivotal enzyme controlling
the channeling of fatty acids into various metabolic pathways.
One of the general and essential features of cells is their
ability to metabolize fatty acids. This process can occur via different
pathways, depending on the nature of the cell and its metabolic
requirements(1) . Acyl-coenzymeA synthetases are crucial
enzymes that facilitate the uptake (2) and permit the
metabolism of intracellular lipids(3) . In fact, these enzymes
catalyze the first step of fatty acid metabolism by converting inactive
fatty acids into active acyl CoA derivatives(3, 4) .
Once activated, these intermediates can be utilized in two major
metabolic pathways: the catabolic pathway of fatty acid -oxidation
or the anabolic pathway, converting fatty acids into cellular lipids.
Several acyl-coenzymeA synthetases have been identified biochemically
by their different substrate specificity(1) . Recently, the rat
cDNA sequence (4) and the genomic structure (5) of
acyl-coenzyme A synthetase (EC 6.2.1.3) (ACS) (
)with
specificity for long-chain fatty acids have been described. The ACS
gene, a member of the luciferase gene superfamily(4) , also
shows some sequence similarity to the fatty acid transport
protein(2) . The ACS gene has a complex genomic structure; it
contains 3 different first exons 1A (53 bp), 1B (216 bp) and 1C (27
bp), giving rise to 3 different mRNA species through alternative
splicing (see Fig. 1A). The translation initiation
codon is, however, in the second exon, and therefore only one protein
is formed from these 3 different mRNAs. Transcription of these exons is
controlled by separate regulatory regions, termed A, B and C,
respectively, suggesting a complex transcriptional control (see Fig. 1A).
Figure 1: Structure of 5` upstream regulatory sequence of the ACS gene and the influence of fibrate treatment on the expression of the different ACS mRNA subspecies. A, scheme of the first and second exons of the rat ACS gene. Exons and promoters are not drawn in scale. B, influence of fenofibrate (FF) treatment on the expression of the different ACS mRNA subspecies. RNA was isolated from 3 adult control rats (lanes 1-3 and 7-9) and from 3 rats treated for 14 days with fenofibrate (0.5%, w/w, mixed in rat chow; lanes 4-6 and 10-12). Northern blots were prepared and hybridized with exon-specific oligonucleotides. RNA isolation, Northern blotting, and hybridization were performed as described under ``Experimental Procedures.''
Recently, considerable interest has been
shown for a group of chemicals called peroxisome proliferators. These
compounds, consisting of fibrate hypolipidemic drugs, certain
herbicides, and phtalate ester plasticizers, induce peroxisome
proliferation (6, 7, 8, 9) and
hepatomegaly, which may ultimately lead to hepatocarcinogenesis after
prolonged administration to rodents(10, 11) . The
peroxisome proliferation caused by these agents partially results from
a transcriptional induction of the enzymes of the -oxidation
pathway(12, 13, 14, 15, 16, 17) .
Lately, several studies have emphasized the importance of a group of
transcription factors called PPARs in mediating this transcriptional
activation (18, 19, 20, 21, 22, 23, 24, 25, 26) .
At present 4 distinct PPARs have been described:
,
,
,
and
(19, 26) . PPARs are members of the
superfamily of nuclear hormone receptors, which after ligand activation
regulate the expression of genes containing specific response elements
called PPREs in their regulatory sequences(27, 28) .
Functional PPREs have been characterized in several of the genes
encoding enzymes involved in the peroxisomal
-oxidation pathway,
such as acyl-CoA oxidase and the trifunctional
enzyme(27, 28, 29, 30, 31, 32) ,
in the cytochrome P450 4A6 gene(33, 34) , in the
3-hydroxy-3-methylglutaryl-CoA synthase gene(35) , in the
medium chain acyl-CoA dehydrogenase gene(36) , in the
apolipoprotein A-I gene(37) , and in the aP2 gene(24) .
In a previous report, we demonstrated that the initiating enzyme of
the -oxidation pathway, ACS, is induced by peroxisome
proliferators both in vivo in rat liver as well as in
vitro in hepatoma cell lines(38) . Given the pivotal role
of ACS in intracellular lipid metabolism, we initiated more detailed
studies to elucidate the molecular mechanisms underlying the regulation
of ACS gene expression in the rat hepatoma cell line Fa-32, as well as
in primary cultures of rat hepatocytes. In this report, we demonstrate
that fibrates, as well as fatty acids, induce the levels of the exon 1C
containing ACS mRNA in a time- and dose-responsive fashion in both
primary cultures of rat hepatocytes and the Fa-32 rat hepatoma cell
line. Furthermore, we show that this effect of fibrates is mediated
through binding of PPAR to a PPRE localized in the C promoter.
The expression vectors pSG5-mPPAR (a
kind gift of Dr. S. Green, Zeneca, UK) (18) and
pSG5-haPPAR
(25) , used in Fa-32 cell transfections, have
been described elsewhere. The haPPAR
, xPPAR
, mRXR
, and
mRXR
were used to synthesize the respective proteins (19, 25, 42) .
Rat hepatocytes were isolated
by collagenase perfusion of livers from male Sprague-Dawley rats (body
weight, 150-200 g) as described(46) . Cell viability was
higher than 85% as determined by trypan blue exclusion. The hepatocytes
were cultured in monolayer (1.5 10
cells/cm
) in Leibovitz-15 medium (Life Technologies,
Inc. BRL, Paisley, UK) supplemented with 10% (v/v) fetal calf serum,
0.2% (w/v) fatty acid free bovine serum albumin, 26 mM NaHCO
, 2 mM glutamine, 3 g/liter glucose, and
antibiotics at 37 °C in a humidified atmosphere of 5%
CO
/95% air. Fenofibric acid and fatty acids acid were added
immediately after seeding the cells. No morphological differences in
cell adhesion or cell toxicity (determined by the 3-(4,5-dimethyl
thiazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetric
test(47) ) were observed between control and treated
hepatocytes.
Nuclei were prepared from primary rat hepatocytes treated for 9 h
with fenofibric acid (500 µM) or -bromopalmitate (50
µM), or vehicle and transcription run-on assays were
performed as described by Nevins(50) . Equivalent counts of
nuclear RNA labeled with [
-
P]UTP (3000
Ci/mmol) were hybridized for 36 h at 42 °C to 5 µg of ACS
cDNA(4) , acyl-CoA oxidase cDNA(12) , albumin
cDNA(51) , and vector DNA immobilized on Hybond-C Extra filters
(Amersham). After hybridization, filters were washed at room
temperature for 10 min in 0.5
SSC and 0.1% SDS and twice for 30
min at 65 °C and subsequently exposed to x-ray film (X-OMAT-AR,
Kodak). Quantitative analysis was performed by scanning densitometry
(Bio-Rad GS670 densitometer).
In each EMSA 2
µl of PPAR and/or RXR were preincubated in a total volume of 20
µl for 15 min on ice with 2 µg of poly(dIdC) in TM buffer
(10 mM Tris-HCl, pH 7.9, 40 mM KCl, 10% glycerol,
0,05% Nonidet P-40, and 1 mM dithiothreitol). For competition
experiments, increasing amounts of cold oligonucleotide acyl-CoA
oxidase PPRE or ACS PPRE (from 25- to 200-fold molar excess) were
included just before adding T4-polynucleotide kinase (1
Tris
borate EDTA = 0.045 M Tris borate, 0.001 M EDTA) end-labeled oligonucleotides. For supershift experiments, an
antibody to xPPAR
(55) was used as described previously.
After 15 min of incubation at room temperature, DNA-protein complexes
were separated by electrophoresis on a 4% polyacrylamide gel in 0.25
TBE buffer at 4 °C(56) .
To further investigate the mechanisms controlling rat liver ACS gene induction after fibrate treatment, the exon-specific induction of ACS gene expression was studied in the rat hepatoma cell line, Fa-32, by Northern blot hybridization analysis. Similar to rat liver and unlike human HepG-2 cells, Fa-32 cells exhibit a remarkable increase in ACS gene expression after fenofibric acid administration(38) . Interestingly, although both A- and C-ACS mRNAs are induced by fenofibrate in Fa-32 cells, the level of induction of C-ACS mRNA is much more pronounced (Fig. 2A). Like in adult rat liver, B-ACS mRNA was not expressed in uninduced Fa-32 cells, nor could it be induced in fibrate-treated cells. The induction of C-ACS by peroxisome proliferators as determined by hybridizations with exon-specific oligonucleotides was confirmed by primer extension analysis (data not shown). These observations prompted us to focus on the mechanism underlying the stimulation of exon 1C containing ACS mRNA subspecies.
Figure 2: Influence of fenofibric acid treatment on the expression of the different ACS mRNA subspecies in the rat hepatoma cell line Fa-32. A, B-ACS mRNA is undetectable in Fa-32 cells by Northern blot hybridization. Exon 1A and 1C containing mRNAs are induced to a different extent by 100 µM fenofibric acid (FF) in Fa-32 cells. Hybridization of the same blot with GAPDH is shown in the bottom part of the panel. CON, control. B, dose-response of C-ACS mRNA induction after treatment of Fa-32 cells during 48 h with different doses of fenofibric acid (FF). Hybridization of the same blot with GAPDH is shown in the bottom part of the panel. C, the effect of the duration of treatment with fenofibric acid (500 µM) on C-ACS mRNA levels in Fa-32 cells was analyzed. Hybridization of the same blot with GAPDH is shown in the bottom part of the panel. Cell culture, RNA isolation, Northern blotting, and hybridizations were performed as described under ``Experimental Procedures.''
To determine the kinetics of
C-ACS mRNA accumulation, time course experiments were performed with
the optimal dose of fenofibric acid (500 µM) (Fig. 2C). C-ACS mRNA was already maximally induced
within 4-8 h. Similar kinetics of c-ACS mRNA induction were
observed with -bromopalmitate (50 µM) (data not
shown). Surprisingly, considerable variability in uninduced C-ACS mRNA
levels was observed during these experiments. In order to understand
the cause of this variability, the effects of different parameters like
the concentration of serum, the presence or absence of serum lipids,
and the degree of confluency on C-ACS gene expression were analyzed.
Neither serum nor lipids affected basal levels of C-ACS expression.
However, C-ACS mRNA steady state levels were positively correlated with
the degree of confluency of the cells (data not shown).
Similar
inductions of ACS gene expression were observed in primary rat
hepatocytes treated with fenofibric acid. A maximal 8-fold induction
was observed at concentrations between 50 and 500 µM of
fenofibric acid after 12 h (data not shown). -Linolenic acid
induced C-ACS mRNA weakly (1.5-fold), whereas
-bromopalmitate
induced C-ACS mRNA nearly 4-fold (data not shown), indicating that
fatty acids have weaker effects in primary hepatocytes when compared
with fenofibric acid.
Figure 3:
The induction of C-ACS mRNA is independent
of de novo protein synthesis. RNA obtained from control (CON, lane 1) or Fa-32 cells exposed for 6 h to 10
µM cycloheximide (CHX, lane 2), 10
µM cycloheximide + 500 µM fenofibric
acid (CHX + FF, lane 3), 10 µM cycloheximide + 100 µM -bromopalmitate (CHX + BP, lane 4), 500 µM fenofibric acid (FF, lane 5), or 100
µM
-bromopalmitate (BP, lane 6)
were analyzed for the expression of C-ACS mRNA. Hybridization of the
same blot with GAPDH is shown in the bottom part of the panel.
Cell culture, RNA isolation, Northern blotting, and hybridizations were
performed as described under ``Experimental
Procedures.''
To analyze whether C-ACS mRNA induction occurred at the
transcriptional level, nuclear run-on assays were performed on primary
cultures of adult rat hepatocytes treated with fenofibric acid (500
µM) or -bromopalmitate (50 µM) for 9 h,
a time when C-ACS mRNA steady state levels are still rising. In
comparison with control cells, the rate of C-ACS gene transcription was
respectively 10- and 3.5-fold higher in nuclei isolated from fenofibric
acid- and
-bromopalmitate-treated cells (Fig. 4). The
transcription rate of the gene for acyl-CoA oxidase, a key enzyme in
the peroxisomal
-oxidation pathway, was analyzed as a positive
control. Consistent with the increase in peroxisomal enzyme activity
after the addition of peroxisome proliferators, acyl-CoA oxidase
transcription rates increased more than 5-fold. Transcription of the
albumin gene, whose mRNA levels do not change upon treatment with
fibrates, did not change markedly under the conditions tested (Fig. 4). These relative increases in C-ACS transcription rates
were comparable with the induction in C-ACS mRNA steady state levels
observed after 24 h of fenofibric acid and
-bromopalmitate (data
not shown). These results together with the data from the cycloheximide
experiment suggest therefore that fibrates and fatty acids affect ACS
expression mainly at the transcriptional level by modification or
activation of already existing transcription factor(s), possibly PPAR.
Figure 4:
The induction of ACS mRNA is at the
transcriptional level. Transcription rates were determined for the
acyl-coA oxidase (ACO), acyl-coA synthetase (ACS),
and albumin (ALB) genes in nuclei from control (CON, open bars) or from rat hepatocytes cultured either in the
presence of 500 µM fenofibric acid (FF, black
bars) or 50 µM-bromopalmitate (BP, striped
bars). A pUC-20 template was used as a control. Densitometric
scanning of the results is depicted in the left
panel.
Figure 5:
The C-ACS regulatory elements contain the
sequence elements necessary to respond to fenofibric acid and PPAR. A, only the C-ACS promoter CAT construct is induced by
fenofibric acid. Fa-32 cells were transiently transfected with
different reporter plasmids and treated either with 500 µM fenofibric acid (FF) in MeSO or with vehicle
alone for 48 h. The plasmid construct used were the pCatEA-788
(pCatEA), pCatEB-728 (pCatEB), pCatEC-981 (pCatEC), or the cloning
vector pCat as negative control (pCAT). CAT activity was
measured and expressed as described under ``Experimental
Procedures.'' B, representative CAT assay showing the
effects of fenofibrate and/or mPPAR
on the 1C-ACS promoter. Fa-32
cells were transiently transfected with either the empty pSG5 vector (lanes 1, 2, 5, 6, 9, 10, 13, and 14) or the pSG5-mPPAR
(lanes 3, 4, 7, 8, 11, 12, 15, and 16) vectors and the indicated
reporter plasmids and treated either with 500 µM
fenofibric acid in Me
SO (lanes 2, 4, 6, 8, 10, 12, 14, and 16) or with vehicle alone (lanes 1, 3, 5, 7, 9, 11, 13, and 15) for 48 h. The plasmid constructs used were pCatEC-981,
pCatECRV (as control construct), SV40-KS-CAT, or 0-KS-CAT. CAT activity
was measured and expressed as described under ``Experimental
Procedures.'' CON, control.
In order to define whether PPAR could enhance the induction of
pCatEC-981 with fibrates, cotransfection experiments were performed.
Therefore, pCatEC-981 reporter plasmid was cotransfected in the
presence of the mouse PPAR expression vector pSG5-mPPAR and Fa-32
cells were treated with fenofibric acid (500 µM) or
vehicle only. In the absence of pSG5-mPPAR
, pCatEC-981 exhibited a
low level of CAT activity, which was induced 3.4-fold after the
addition of fenofibric acid. However, when pSG5-mPPAR
was
cotransfected with pCatEC-981, CAT activity was already increased
7.5-fold in the absence of fenofibric acid, and an 18-fold induction
relative to uninduced CAT activity was observed when fenofibric acid
was added (Fig. 5B). Similar results were obtained when
a haPPAR
expression vector was used instead of the mPPAR
,
suggesting that the effect was not limited to a specific PPAR subtype
(data not shown). These data suggest that PPAR contributes in directing
peroxisome proliferator-dependent transcriptional activation of the
C-ACS gene.
We further delineated the PPAR-responsive region by
transfecting a series of progressive 5` deletion mutants of the C-ACS
promoter in the presence or absence of either pSG5-mPPAR and/or
fenofibric acid (Fig. 6). Similarly, all these constructs
exhibited low activity in the absence of pSG5-mPPAR
and fenofibric
acid. The CAT activity of all these constructs was, however, induced by
fenofibric acid or pSG5-mPPAR
, and a similar synergistic induction
was observed in the presence of both pSG5-mPPAR
and fenofibric
acid. These data indicate that the potential PPRE is most likely
confined to a region comprising the first 282 5`-flanking sequences of
C-ACS. By computer homology searches, a potential PPRE
(TGACTGATGCCCTGAAAGACCT) could be localized around positions -175
and -154 in the C-ACS promoter. This sequence contains 3 copies
of a motif related to the consensus steroid hormone receptor binding
half-site TGACCT arranged in direct repeats with 1- and 3-nucleotide
spacing. It was hence investigated whether this element represents the
functional response element mediating the observed effects of PPAR on
ACS gene transcription. To this end, the wild-type PPRE in the
C-ACS(-282)-PPRE
-LUC construct was mutated (Fig. 7A). When activity of this
C-ACS(-282)-PPRE
-LUC construct was compared with the
activity of the C-ACS(-282)-PPRE
-LUC construct in
Fa-32 cells, a loss of inducibility of expression by fenofibric acid,
by PPAR, or by fenofibric acid and PPAR was observed for the mutated
ACS promoter (Fig. 7B). These data unequivocally
demonstrate the importance of the PPRE located between positions
-175 and -154 from the transcription initiation site of the
C promoter in mediating the inducibility of the ACS promoter by PPARs.
Figure 6:
The potential PPRE resides in the
sequences confined between -282 and -51 relative to the
translation initiation site of the C-ACS mRNA. A, schematic
structure of the different C-ACS promoter deletion constructs. All
constructs contained the entire C promoter region lying immediately
upstream of exon 1C. The numbering scheme takes the A in the first AUG
codon (translation initiation site) in exon 2 as +1. CON,
control; FF, fenofibrate; PPAR, mPPAR (PPAR); FF + PPAR, fenofibrate and mPPAR
. B, regulation of deletions in 5`-flanking sequences of the
C-ACS gene by fenofibric acid and mPPAR
. Fa-32 cells were
transfected with the C-ACS promoter deletion constructs (described in
the legend to Fig. 6A) in the presence of cotransfected
pSG5 vector plasmid and vehicle (CON), of cotransfected pSG5
vector plasmid in the presence of 500 µM fenofibric acid (FF), of cotransfected pSG5-mPPAR
expression vector and
vehicle (PPAR), or of cotransfected pSG5-mPPAR
in the
presence of 500 µM fenofibric acid (FF + PPAR). CAT activity was measured and expressed as described under
``Experimental Procedures.''
Figure 7:
Localization of the C-ACS PPRE by in
situ mutagenesis. A, scheme depicting the
C-ACS(-282)-PPRE-LUC and
C-ACS(-282)-PPRE
-LUC vectors. The bases mutated in
C-ACS(-282)-PPRE
-LUC are indicated in bold. The numbering scheme takes the A in the first AUG codon
(translation initiation site) in exon 2 as +1. B, the
C-ACS(-282)-PPRE
-LUC (black bars) and
C-ACS(-282)-PPRE
-LUC (shaded bars) vectors were
transfected in Fa-32 cells with the pSG5 plasmid (pSG5), with
pSG5 in the presence of 250 µM fenofibric acid (pSG5+FF), with pSG5-mPPAR
(PPAR), or with
pSG5-mPPAR
in the presence of 250 µM fenofibric acid (PPAR+FF). Experiments were performed as described under
``Experimental Procedures.'' The luciferase activity was
normalized to the value obtained after cotransfection of pSG5 vector,
which was given the arbitrary value of 100. The results represent the
mean ± S.D. of three independent
experiments.
Figure 8:
PPARRXR heterodimers bind to the
C-ACS PPRE. A, PPAR
RXR heterodimers bind to the C-ACS
PPRE. EMSAs were performed on end-labeled C-ACS PPRE oligonucleotide in
the presence of unprogrammed reticulocyte lysate (Retic, lane 1), in vitro translated mRXR
(lane
2), haPPAR
(lane 3), or haPPAR
and mRXR
(lanes 4 and 5) or as described under
``Experimental Procedures.'' In lane 4 100-fold
molar excess cold C-ACS oligonucleotide was added as competitor. The
C-ACS PPRE is shown at the bottom of this panel. Details
regarding the experimental protocols can be found under
``Experimental Procedures.'' B, an anti-PPAR
antibody inhibits binding of PPAR/RXR. EMSAs were performed on
end-labeled C-ACS PPRE oligonucleotide in the presence of vaccinia
virus-produced xPPAR
and in vitro translated mRXR
as
described under ``Experimental Procedures.'' Lane 1,
xPPAR
and mRXR
; lane 2, xPPAR
and mRXR
in
the presence of an anti-xPPAR
antibody; lane 3,
xPPAR
and mRXR
in the presence of preimmune serum. C, competition experiments for binding vaccinia virus-produced
xPPAR
and in vitro translated mRXR
. Competition
experiments were performed using either end-labeled C-ACS PPRE
oligonucleotide (top panel) or acyl-CoA oxidase PPRE
oligonucleotide (bottom panel). EMSAs were performed either in
the presence of a nonspecific oligonucleotide (NS, lanes 1 and 8) or in the presence of 5-, 10-, 15-, 20-, 30-, 50-,
or 100-fold molar excess of cold C-ACS PPRE (lanes 5-7 and 14-18) or acyl-CoA oxidase oligonucleotide (lanes 2-4 and 9-13). The part of the gel
showing the free probe is not shown.
To demonstrate that
the binding of PPAR and RXR to the ACS PPRE is similar to that of the
acyl-CoA oxidase PPRE, cross-competition experiments were performed
next. In the first experiment, we tested whether cold acyl-CoA oxidase
and C-ACS PPRE oligonucleotide could compete with the binding of
xPPARmRXR
heterodimers to the labeled C-ACS PPRE
oligonucleotide (Fig. 8C, top panel). Both the
acyl-CoA oxidase and C-ACS PPRE sequences competed with the binding of
xPPAR
mRXR
heterodimers to the C-ACS PPRE probe. The
competition was, however, more efficient with the cold acyl-CoA oxidase
PPRE oligonucleotide. In a second experiment, cross-competition was
performed, using acyl-CoA oxidase PPRE as a probe. Whereas the addition
of a 100-fold molar excess of cold acyl-CoA oxidase oligonucleotide
could almost completely prevent xPPAR
mRXR
heterodimers
from binding to the acyl-CoA oxidase PPRE, an approximately 2-fold
lower level of competition was observed with the cold C-ACS PPRE,
thereby confirming that the 1C-ACS PPRE is a lower affinity PPRE,
compared with the acyl-CoA oxidase PPRE.
Fibric acid derivatives, ambiguously known for their
therapeutic purpose as hypolipidemic agents in man and for their
cancerigenic peroxisomal proliferative action in rodents, induce
several enzymes important in intracellular fatty acid metabolism, such
as enzymes of the peroxisomal -oxidation pathway (acyl-CoA
oxidase, bifunctional enzyme, and thiolase). The induction of these
enzymes by fibrates has been shown to be mediated by a family of
ligand-activated transcription factors termed PPARs. The growing list
of enzymes and proteins implicated in lipid metabolism, whose
responsiveness to peroxisome proliferators is under control of PPAR,
points not only to a crucial and general role of PPAR in lipid
metabolism, but suggests also that the expression of many more genes
might be modulated by the PPAR signal transduction pathway. Indeed, in
a previous paper, we reported that the expression of ACS is responsive
to fibrates(38) . In the present paper, our interest was
focused on the mechanisms controlling the induction of ACS gene
expression upon fibrate and fatty acid treatment in liver, hepatocytes,
and hepatoma cell lines.
Although three different ACS mRNAs are
transcribed from the ACS gene, it is of particular interest that the
induction of ACS gene expression after the addition of both fenofibric
acid and fatty acids was most reflected by a strong induction of mRNA
containing exon 1C both in rat liver, isolated rat hepatocytes, and in
rat hepatoma cells. The A-ACS mRNA, which is expressed to a much higher
level under basal conditions in the liver, is induced to a lesser
extent by peroxisome proliferators, whereas the B-ACS mRNA could not be
detected by hybridization techniques in cells of hepatic origin.
Although exon-specific nuclear run-on assays were technically not
feasible due to the short nature of exon 1C, the induction of C-ACS
mRNA steady state levels is most likely caused by an increased
transcription rate of the C-ACS mRNA as evidenced by the transient
transfection assays using the C promoter. In addition, because the
induction in total ACS mRNA after peroxisome proliferators could
largely be accounted for by the induction of the C-ACS species, the
result from the run-on assay using the entire ACS cDNA suggests that
this induction is caused by an enhanced transcription. Dose-response
and time course experiments showed a maximum increase of C-ACS mRNA
expression 4-8 h after the addition of fenofibric acid (500
µM). Interestingly, a similar induction pattern of C-ACS
mRNA was observed when fatty acids were added to the cell culture
medium. Whereas palmitate and -linolenic acid were rather weak
inducers of C-ACS gene expression,
-bromopalmitate and
tetradecylthioacetic acid, which are nonmetabolized fatty acids, could
induce ACS mRNA almost to a similar extent as fenofibric acid. This
effect of modified fatty acids was consistent with the previously
reported inductive effects on total long-chain ACS mRNA levels in
response to alkyl thioacetic acids (3-thia fatty acids) in the 7800 C1
Morris hepatoma cell line(57) .
The induction of C-ACS mRNA in the presence of fenofibric acid and fatty acids was apparently an early and direct event, because inhibition of de novo protein synthesis by cycloheximide treatment did not suppress the observed increase of ACS mRNA by both agents. Therefore, we suggest that the effect of fenofibric acid and fatty acids on ACS gene expression in the liver must involve the activation of factors already present under basal conditions.
The strong increase of C-ACS mRNA steady state
levels suggested that the regulatory sequences preceding the 1C exon
are functionally implicated in this induction. Indeed, transfection
studies analyzing the different 5` regulatory sequences flanking the
1A, 1B, and 1C exons for enhancer activity in the presence of
fenofibric acid supported this hypothesis. Only the expression of the C
promoter could be significantly induced, whereas the A promoter was not
regulated by peroxisome proliferators and the B promoter was not
expressed at all. The discordance between the weak induction of A-ACS
mRNA and the absence of an inductive effect of peroxisome proliferators
on the pCatEA-788 vector might be explained by the absence of the
required regulatory elements in the first 788 bases preceding the
transcription initiation site of the A-ACS mRNA. In contrast to the
situation for the 1A exon, the regulatory sequences 5` of the 1C exon
are clearly capable of mediating an inductive response to fibrates.
Moreover, by cotransfection of a PPAR expression vector, we
demonstrated that PPAR mediates the fenofibric acid-dependent
transcriptional activation of 1C-ACS. It is noteworthy that 1C-ACS is
also transcriptionally activated through PPAR in the absence of
fenofibric acid. This could be due to inherent activity of the
transcriptional activating functions of PPAR (58) or
alternatively and perhaps more likely by the presence of natural
ligand(s) constitutively activating the nuclear receptor. It is
furthermore not excluded that both mechanisms can act together. By
using unilateral deletions of the C-ACS 5`-flanking region, we defined
the responsive region to the first 282 bp upstream of the translation
initiation site. A region localized in these first 282 bp and spanning
-175 to -154 termed C-ACS PPRE was found to contain three
copies of a motif related to the consensus steroid hormone receptor
half-site TGACCT arranged with a spacing of 1 and 3 nucleotides. Point
mutations introduced in this region defined unequivocally that these
motifs are involved in mediating the effect of PPAR and fibrates on
C-ACS gene expression, whereas EMSA experiments indicated that the
C-ACS PPRE could in fact bind PPARRXR heterodimers. The
similarity between the acyl-CoA oxidase PPRE and the C-ACS PPRE could
furthermore be confirmed by cross-competition experiments using
radioactive C-ACS PPRE and cold acyl-CoA oxidase PPRE and vice versa.
The C-ACS PPRE, however, showed a weaker affinity for the PPAR
RXR
heterodimers than the acyl-CoA oxidase PPRE.
Interestingly, for the
different PPAR activators tested, a remarkable parallel exists between
their capacity to induce C-ACS mRNA and the expression of the pPcat EC
reporter vectors. Palmitate and -linolenic acid, two naturally
occurring fatty acids, induced C-ACS mRNA levels to a lower extent than
fibrates and nonmetabolized fatty acids and correspondingly induced the
C promoter only weakly in transfection experiments. In contrast,
fibrates and the nonmetabolized fatty acids
-bromopalmitate and
TTA, which induce C-ACS mRNA levels more strongly, also showed a much
stronger induction of C promoter activity. The weaker capacity of
natural fatty acids to activate the C-ACS promoter in Fa-32 cells can
be explained by several mechanisms. First, naturally occurring fatty
acids are much more rapidly metabolized and hence may have a more
limited and transient capacity to activate genes than nonmetabolized
fatty acids or other peroxisome proliferators. Alternatively, their
rapid elimination and metabolism may prevent the induction of the true
PPAR ligands. Second, the effect of various fatty acids will also vary
from cell to cell. It is likely that cell types with an active lipid
metabolism, such as hepatocytes or adipocytes will behave different
from cells that are less specialized in handling lipids, such as COS or
HeLa cells, which have at present been most widely used in PPAR
research. Third, because it has been demonstrated that in yeast the
induction of peroxisomal proliferation in response to oleate is
mediated by another control element as PPRE (59) and because in
prokaryotes fatty acids also control gene expression through response
elements unrelated to a PPRE (60) , it is not unreasonable to
hypothesize that certain fatty acids might modulate ACS gene expression
independent from PPAR. Furthermore, as reviewed by Clarke and
Jump(61) , ample evidence is also accumulating that fatty acids
in higher eukaryotes might affect the redox or phosphorylation state (62) of specific trans-acting factors or affect the activity of
transcriptional auxiliary proteins that do not bind to DNA directly but
interact with DNA-binding proteins. Therefore, unlike their xenobiotic
counterparts, which act predominantly via PPAR, the effects of fatty
acids on gene expression may be determined by a delicate balance
between PPAR-dependent and PPAR-independent effects. Finally, it is
possible that the different effects of the various activators can be
found at the level of activation specificity of the receptor itself.
The homology between the ligand domains of the different PPARs is not
very high, suggesting the existence of PPAR subtype-specific
activators. Hence, it is conceivable that the different members of the
PPAR subfamily each respond with a different affinity to a distinct
ligand or group of ligands. Based on this assumption, the different
effects of fenofibric acid and nonmetabolized fatty acids on the one
hand and natural fatty acids on the other hand would implicate that
certain PPARs are more specifically activated by one activator relative
to others. Consequently, the responsiveness of a given cell to
different peroxisomal proliferators and fatty acids would depend on the
intracellular relative abundance of the different subtype receptors.
In conclusion, in liver the regulation of the expression of the different ACS transcripts is complex. Although it is clear that ACS gene expression is undoubtedly regulated by fibrates and by fatty acids in the intact animal, in cultured hepatocytes, and in hepatoma cell lines, the different ACS mRNA species are not regulated by the same mechanisms. For instance, the exon 1A containing ACS mRNA is expressed to a relatively high extent under basal conditions, but exon 1C containing mRNA is much more sensitive to the inductive effect of fibrates and fatty acids. Data in this paper support the hypothesis that fibrates and certain fatty acids regulate C-ACS gene expression by activating PPARs, which in their turn interact with specific response elements such as the C-ACS PPRE.