©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Induction of the Acyl-Coenzyme A Synthetase Gene by Fibrates and Fatty Acids Is Mediated by a Peroxisome Proliferator Response Element in the C Promoter (*)

(Received for publication, January 31, 1995; and in revised form, April 20, 1995 )

Kristina Schoonjans (1)(§) Mitsuhiro Watanabe (2) Hiroyuki Suzuki (2) Abderrahim Mahfoudi (1) Grigorios Krey (3)(¶) Walter Wahli (3) Paul Grimaldi (4) Bart Staels (1)(**) Tokuo Yamamoto (2) Johan Auwerx (1)(§§)

From the  (1)Laboratoire de Biologie des Régulations chez les Eucaryotes, Institut Pasteur, 1, Rue Calmette, 59019 Lille, France, the (2)Tohoku University Gene Research Center, Tohoku University, 1-1 Tsutsumidori-amamiya, Aoba, Sendai 981, Japan, the (3)Institut de Biologie Animale, Université de Lausanne, CH-1015 Lausanne, Switzerland, and the (4)Laboratoire de Biologie du Développement du Tissu Adipeux, Centre de Biochimie, Unité Mixte de Recherche 134 du CNRS, Parc Valrose, 06108 Nice, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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, alpha-bromopalmitate, tetradecylthioacetic acid, or alpha-linolenic acid. Nuclear run-on experiments indicated that fenofibric acid and alpha-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, alpha-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 PPARbulletretinoid X receptor heterodimers in gel retardation assays. In conclusion, the regulation of C-ACS mRNA expression by fibrates and fatty acids is mediated by PPARbulletretinoid 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.


INTRODUCTION

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 beta-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) (^1)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 beta-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: alpha, beta, , 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 beta-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 beta-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.


EXPERIMENTAL PROCEDURES

Materials

Fenofibric acid, pirixinic acid (Wy-14643), and tetradecylthioacetic acid (TTA) were kind gifts of Dr. Alan Edgar (Laboratoires Fournier, Daix, France), Dr. Sharon Burns (Wyeth-Ayerst, Princeton, NJ), and Dr. Jon Bremer (Oslo University, Oslo, Norway). Palmitate and alpha-linolenic acid were purchased from Sigma, and alpha-bromopalmitate was obtained from Aldrich.

Animals and Treatments

Male Wistar rats (90 days old) were treated for different periods of time with fenofibrate (Laboratoires Fournier) mixed at 0.5% (by mass) in standard rat chow. The food intake of the rats was recorded every 2 days throughout the treatment period. None of the treatments caused major changes in the amount of food consumed by the animals. At the end of the experiments, animals were sacrificed (at 9 a.m.) by exsanguination under ether anesthesia. The liver was removed immediately, rinsed with 0.9% NaCl, and frozen in liquid nitrogen until RNA was extracted.

Recombinant Plasmids

The construction of the reporter plasmids pCatEA, pCatEB, and pCatEC containing the 5`-flanking region preceding the different first exons 1A, 1B, and 1C of the ACS gene has been described elsewhere(5) . Deletion mutants of the C promoter of ACS, pCatEC-717, pCatEC-481, pCatEC-416, and pCatEC-282, were generated from pCatEC-981 by exonuclease III digestion (we use a numbering scheme in which the A of the AUG initiator codon of the ACS protein in exon 2 is designated as +1)(5) . The fragment from -981 to -51, preceding the 1C exon, has been cloned in the reverse orientation in the pCatECRV control CAT vector(5) . The SV-KS-CAT and 0-KS-CAT plasmids were described elsewhere(39) . Polymerase chain reaction fragments flanked by MluI and BglII restriction sites were amplified from pCatEC-981 and inserted in the corresponding sites of the pGL-2 basic vector (Promega), thereby creating the luciferase reporter genes C-ACS(-981)-LUC and C-ACS(-282)-LUC. Site-directed mutagenesis of PPRE was accomplished according to Kunkel (40) using the oligonucleotide 5`-TTTCAGGGCCCCAGCTGCATGCCGAG-3` as mutagenic primer (-159 to -184 on the sense strand of the C-ACS promoter) on pGL-2 single-stranded DNA templates. As an internal control for transfection efficiency in mammalian cells, a cytomegalovirus beta-galactosidase vector described by MacGregor and Caskey (41) was used.

The expression vectors pSG5-mPPARalpha (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, xPPARalpha, mRXRalpha, and mRXRbeta were used to synthesize the respective proteins (19, 25, 42) .

Cell Culture

Fa-32, a rat hepatoma cell line that is a subclone of Faza967 (43) was maintained in Dulbecco's modified Eagle's minimal essential medium supplemented with 10% fetal calf serum at 37 °C in a humidified atmosphere of 5% CO(2)/95% air(44, 45) . Fenofibric acid, Wy-14643, and fatty acids (alpha-linolenic acid, palmitate, TTA, and alpha-bromopalmitate) were dissolved in Me(2)SO and ethanol, respectively, and added to the medium at the appropriate concentration and time indicated. Only vehicle was added to control cells. In the case of fatty acids, preincubation with the medium containing fetal calf serum (and hence serum albumin) was carried out for 45 min at 37 °C.

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 10^5 cells/cm^2) 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(3), 2 mM glutamine, 3 g/liter glucose, and antibiotics at 37 °C in a humidified atmosphere of 5% CO(2)/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.

RNA Analysis and Transcriptional Rate Assay

RNA from cells and tissues was prepared as described by Chomczynski and Sacchi(48) . Northern blot hybridizations and quantification of total cellular RNA were performed as described previously(44) . Specific ACS mRNA species were measured using specific polynucleotide kinase-labeled antisense oligonucleotides (A-ACS, 40-mer 5`-CCCGGGCGCTCCGCAGGCGGCTGTCACTGCAATCCACGAA-3`; B-ACS, 40-mer 5`-GTTTGGATCTGTCCTCTA-TGGGGTGCCTGGTTGGATTGGT-3`; C-ACS, 24-mer 5`-CATCTGTGCCACCGACAGCTGACT-3`). A human GAPDH cDNA clone (49) labeled by random priming (Boehringer Mannheim) was used as control. For oligonucleotide hybridizations, blots were washed two times for 15 min at 37 °C and one time for 30 min at 55 °C.

Nuclei were prepared from primary rat hepatocytes treated for 9 h with fenofibric acid (500 µM) or alpha-bromopalmitate (50 µM), or vehicle and transcription run-on assays were performed as described by Nevins(50) . Equivalent counts of nuclear RNA labeled with [alpha-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).

Transient Transfections and Expression Assays

Transfections in Fa-32 cells were performed at 50-60% confluency by the calcium phosphate coprecipitation procedure with a mixture of plasmids that contained, in addition to the CAT or luciferase reporter plasmids and expression vector(s), 1 µg of thymidine kinase-driven beta-galactosidase expression vector as a control for transfection efficiency. All samples were complemented with pSG5 plasmid to an equal total amount of plasmid DNA. After 8 h, Fa-32 cells were washed with phosphate-buffered saline and incubated for another 38 h with fenofibric acid or fatty acids in fresh medium containing 10% fetal calf serum. After transfection, the cells were lysed by four freeze-thaw cycles in 100 µl of 0.25 M Tris-HCl, pH 8.0, 1 mM dithiothreitol. Luciferase activity was monitored in 50-µl extracts using a LKB 1250 luminometer (52) . For the CAT reporter vectors, CAT activity was determined as described by Gorman et al.(53) . Autoradiographs were quantified by densitometry. Transfection efficiencies were normalized with the beta-galactosidase activity assay (Promega Protocols and Applications Guide, 2nd Edition). All transfection experiments were performed at least 3 times.

Nuclear Extracts and Gel Retardation Assays

haPPAR(25) , mRXRalpha (42) and mRXRbeta (42) proteins were synthesized in vitro using rabbit reticulocyte lysate (Promega). Vaccinia virus-expressed xPPARalpha was a kind gift of A. Hihi (Université de Lausanne). The quality of the in vitro translated or vaccinia virus-made proteins were verified by SDS-polyacrylamide gel electrophoresis. To study the C-ACS PPRE, a synthetic double-stranded 41-bp oligonucleotide spanning nucleotides -183 to -147 of the rat C-ACS gene 5` upstream regulatory sequence was used (5`-GATCCGGCATGTGACTGATGCCCTGAAAGACCTTGGCAGGA-3`). A 30-bp double-stranded oligonucleotide containing the PPRE of the rat acyl-CoA oxidase gene and spanning sequences -634 to -607 (5`-`GATCCCGAACGTGACCTTTGTCCTGGTCCC-3`) was used as a control PPRE. An oligonucleotide binding CP-1 (5`-TCGACTACACCTATAAACCAATCACCT-3`) corresponding to the adenovirus major late promoter served as a nonspecific control in EMSA reactions(54) .

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(dIbulletdC) 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 xPPARalpha (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) .


RESULTS

Fenofibrate Induces Exon 1A and Exon 1C Containing ACS mRNA in Rat Liver and Fa-32 Cells

Recent work from Yamamoto and colleagues (5) demonstrated the existence of three distinct ACS mRNA species differing by an alternatively used first exon, each preceded by its own regulatory region (Fig. 1A). In order to determine to which of the three ACS mRNA species the previously observed induction of rat liver ACS mRNA after fibrate administration (38) could be attributed, a Northern blot obtained from rats treated for 14 days with 0.5% fenofibrate was hybridized with oligonucleotides specific for each of the first three exons. Under basal conditions only the A-ACS mRNA was expressed in rat liver, whereas the B- and C-ACS mRNAs were undetectable (Fig. 1B). Treatment with fenofibrate provoked a moderate (2.8-fold) induction of the A-ACS mRNA, whereas C-ACS mRNA was strongly induced (Fig. 1B). In accordance with the data obtained by Suzuki et al.(5) , B-ACS mRNA was undetectable in control or in fibrate-treated rat liver by Northern blot hybridization.

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.''



Fenofibric Acid and Fatty Acids Induce Exon 1C Containing ACS mRNA in a Dose- and Time-dependent Fashion in Fa-32 Cells and Primary Hepatocytes

When Fa-32 cells were treated for 48 h with different doses of fenofibric acid, a dose-dependent increase of C-ACS mRNA was observed with a maximal 29-fold induction at 500 µM fenofibric acid (Fig. 2B). A similar induction pattern of C-ACS gene expression was observed after the addition of fatty acids, such as alpha-linolenic acid (100 µM, 9-fold induction). Furthermore, alpha-bromopalmitate (up to 100 µM) and TTA (80 µM), two nonmetabolized fatty acids, induced C-ACS gene expression to a higher extent than alpha-linolenic acid (21-fold for alpha-bromopalmitate, 17-fold for TTA) (data not shown).

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 alpha-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). alpha-Linolenic acid induced C-ACS mRNA weakly (1.5-fold), whereas alpha-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.

Induction of C-ACS mRNA by Fenofibric Acid and Bromopalmitate Is Independent on de Novo Protein Synthesis and Is Mediated at the Transcriptional Level

Next, it was investigated whether C-ACS mRNA gene induction was dependent on de novo protein synthesis. Treatment of Fa-32 cells with cycloheximide alone during 6 h did not change C-ACS mRNA levels in comparison with control cells (Fig. 3, lanes 1 and 2). When cells were treated with cycloheximide combined with either fenofibric acid or alpha-bromopalmitate, C-ACS mRNA levels increased to similar levels compared with fenofibric acid or alpha-bromopalmitate alone (Fig. 3, compare lanes 3-6). This suggests that the observed C-ACS mRNA induction does not require ongoing protein synthesis.


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 alpha-bromopalmitate (CHX + BP, lane 4), 500 µM fenofibric acid (FF, lane 5), or 100 µM alpha-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 alpha-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 alpha-bromopalmitate-treated cells (Fig. 4). The transcription rate of the gene for acyl-CoA oxidase, a key enzyme in the peroxisomal beta-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 alpha-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 µMalpha-bromopalmitate (BP, striped bars). A pUC-20 template was used as a control. Densitometric scanning of the results is depicted in the left panel.



Delineation of a PPRE in the Regulatory Sequences of the ACS Gene

To delineate the cis-acting regulatory sequences within the 5` upstream region of the ACS gene implicated in the induction of C-ACS mRNA upon treatment with fenofibric acid and fatty acids, transient transfection assays were carried out in Fa-32 cells using the CAT reporter plasmids, pCatEA-788, pCatEB-728, and pCatEC-981, which contain the A, B, and C promoter regions, respectively (Fig. 5A). Under uninduced conditions the reporter plasmid pCatEA-788 was strongly expressed in Fa-32 cells, whereas pCatEC-981 was expressed only weakly and pCatEB-728 was not expressed at all. Furthermore, when pCatEA-788 was transfected in Fa-32 cells, no significant induction of CAT activity was observed after addition of fenofibric acid, indicating that the A promoter is constitutively expressed. The pCatEB-728 was not expressed either in the presence or absence of fenofibric acid. By contrast, only the pCatEC-981 reporter plasmid was induced upon addition of fenofibric acid (Fig. 5B, compare lanes 13 and 14). In the presence of palmitate and alpha-linolenic acid, only a limited induction of pCatEC-981-driven CAT activity was observed (1.6- and 1.8-fold, respectively). However, natural fatty acids, such as palmitate, are very quickly metabolized by liver cells (t, 8 h as measured by pulse-chase experiments using ^3H-labeled palmitate in the presence of an appropriate concentration of unlabeled fatty acid; data not shown). Therefore, additional transfection experiments with chemically modified fatty acids, such as alpha-bromopalmitate and TTA, were performed. These fatty acids induced the pCatEC-981-driven CAT activity significantly (2.5-fold for alpha-bromopalmitate and 2.2-fold for TTA).


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 Me(2)SO 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 mPPARalpha 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-mPPARalpha (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(2)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-mPPARalpha and Fa-32 cells were treated with fenofibric acid (500 µM) or vehicle only. In the absence of pSG5-mPPARalpha, pCatEC-981 exhibited a low level of CAT activity, which was induced 3.4-fold after the addition of fenofibric acid. However, when pSG5-mPPARalpha 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 mPPARalpha, 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-mPPARalpha and/or fenofibric acid (Fig. 6). Similarly, all these constructs exhibited low activity in the absence of pSG5-mPPARalpha and fenofibric acid. The CAT activity of all these constructs was, however, induced by fenofibric acid or pSG5-mPPARalpha, and a similar synergistic induction was observed in the presence of both pSG5-mPPARalpha 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, mPPARalpha (PPAR); FF + PPAR, fenofibrate and mPPARalpha. B, regulation of deletions in 5`-flanking sequences of the C-ACS gene by fenofibric acid and mPPARalpha. 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-mPPARalpha expression vector and vehicle (PPAR), or of cotransfected pSG5-mPPARalpha 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-mPPARalpha (PPAR), or with pSG5-mPPARalpha 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.



PPARbulletRXR Heterodimers Bind to the C-ACS PPRE

To investigate whether the PPRE defined by transfection experiments is capable of binding PPAR in vitro, an oligonucleotide spanning sequences -175 to -154 relative to the transcription initiation site of C-ACS was used in EMSAs. This oligonucleotide was capable of binding haPPAR and mRXRalpha heterodimers in EMSAs (Fig. 8A). haPPAR homodimers or mRXRalpha homodimers were incapable of binding to this oligonucleotide. When a 100-fold molar excess cold C-ACS PPRE oligonucleotide was added as competitor in the EMSA experiment, binding of the haPPARbulletmRXRalpha heterodimer to the labeled C-ACS PPRE was completely inhibited (Fig. 8A, lane 4), whereas addition of similar concentrations of an unrelated cold oligonucleotide (Fig. 8C, lanes 1 and 8) or the mutated C-ACS PPRE (data not shown), used in the transfection experiments, did not result in competition. Similar binding data were obtained when xPPARalpha was used instead of haPPAR, and mRXRalpha was replaced by mRXRbeta (Fig. 8B, lane 1), demonstrating that the C-ACS PPRE element was capable of binding different types of PPARbulletRXR heterodimers. The addition of an antibody against xPPARalpha (55) in the EMSA reaction mixture containing xPPARalpha and mRXRbeta reduced the intensity of the specific complex and induced the formation of a supershifted complex (Fig. 8B, lane 2). This pattern was not observed in the control sample or in the presence of preimmune serum (Fig. 8B, lanes 1 and 3). Together these data suggest that PPAR and RXR indeed takes part in the formation of the protein DNA complex in EMSA reactions.


Figure 8: PPARbulletRXR heterodimers bind to the C-ACS PPRE. A, PPARbulletRXR 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 mRXRalpha (lane 2), haPPAR (lane 3), or haPPAR and mRXRalpha (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 xPPARalpha and in vitro translated mRXRbeta as described under ``Experimental Procedures.'' Lane 1, xPPARalpha and mRXRbeta; lane 2, xPPARalpha and mRXRbeta in the presence of an anti-xPPARalpha antibody; lane 3, xPPARalpha and mRXRbeta in the presence of preimmune serum. C, competition experiments for binding vaccinia virus-produced xPPARalpha and in vitro translated mRXRbeta. 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 xPPARalphabulletmRXRbeta 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 xPPARalphabulletmRXRbeta 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 xPPARalphabulletmRXRbeta 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.


DISCUSSION

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 beta-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 alpha-linolenic acid were rather weak inducers of C-ACS gene expression, alpha-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 PPARbulletRXR 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 PPARbulletRXR 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 alpha-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 alpha-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.


FOOTNOTES

*
This research was sponsored by grants from INSERM, National Fonds voor Wetenschapelijk Guderzoek ``levenslijn'' 7.0022.91, Fondation pour la Recherche Médicale, Association pour la Recherche sur le Cancer (ARC), ARCOL, the Swiss National Science Foundation, and the Bioavenir program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by fellowships of the Association pour la Recherche sur le Cancer (ARC) and Institut Francais de Nutrition (IFN).

Supported by a BIOMED-1 fellowship.

**
Research associate of CNRS.

§§
Research director of CNRS. To whom correspondence should be addressed. Tel.: 33-20-87-77-88; Fax: 33-20-87-73-60.

(^1)
The abbreviations used are: ACS, long-chain acyl-coenzyme A synthetase; CAT, chloramphenicol acetyl transferase; CoA, coenzyme A; EMSA, electrophoretic mobility shift assay; FF, fenofibric acid; PPAR, peroxisome proliferator activated receptor; PPRE, peroxisome proliferator-response element; RXR, retinoid X receptor; TTA, tetradecylthioacetic acid; bp, base pair(s).


ACKNOWLEDGEMENTS

The excellent technical assistance of R. Saladin and D. Cayet are kindly acknowledged. We are grateful to A. Hihi for the vaccinia virus-expressed xPPARalpha, to Dr. P. Chambon for the mRXRalpha and beta plasmids, to Dr. C. Dreyer for the xPPARalpha antibody, to Dr T. Osumi for the acyl-CoA oxidase cDNA, to Dr. S. Green for pSG5-mPPARalpha vector, and to Dr. C. Szpirer for the kind gift of the Fa-32 cells. We kindly acknowledge the gift of fenofibrate by Fournier Pharmaceuticals (Dr. A. Edgar), the gift of Wy-14643 by Wyeth-Ayerst (Dr. S. Burns), and the gift of tetradecylthioacetic acid by Dr. Jon Bremer.


REFERENCES

  1. Groot, P. H. E., Scholte, H. R., and Hulsman, W. C. (1976) Adv. Lipid Res. 14,75-126 [Medline] [Order article via Infotrieve]
  2. Schaffer, J. E., and Lodish, H. F. (1994) Cell 79,427-436 [Medline] [Order article via Infotrieve]
  3. Yamamoto, T., Fujino, T., Abe, T., and Suzuki, H. (1990) Prog. Obesity Res. 219-224
  4. Suzuki, H., Kawarabayasi, Y., Kondo, J., Abe, T., Nishikawa, K., Kimura, S., Hashimoto, T., and Yamamoto, T. (1990) J. Biol. Chem. 265,8681-8685 [Abstract/Free Full Text]
  5. Suzuki, H., Watanabe, M., Fujino, T., and Yamamoto, T. (1995) J. Biol. Chem. 270,9676-9682 [Abstract/Free Full Text]
  6. Lock, E. A., Mitchell, A. M., and Elcombe, C. R. (1989) Annu. Rev. Pharmacol. Toxicol. 29,145-163 [CrossRef][Medline] [Order article via Infotrieve]
  7. Reddy, J. K., Warren, J. R., Reddy, M. K., and Lalwani, M. D. (1982) Ann. N. Y. Acad. Sci. 386,81-110 [Medline] [Order article via Infotrieve]
  8. van den Bosch, H., Schutgens, R. B. H., Wanders, R. J. A., and Tager, J. M. (1992) Annu. Rev. Biochem. 61,157-197 [CrossRef][Medline] [Order article via Infotrieve]
  9. Tolbert, N. E. (1981) Annu. Rev. Biochem. 50,133-157 [CrossRef][Medline] [Order article via Infotrieve]
  10. Reddy, J., Azarnoff, D., and Hignite, C. (1980) Nature 283,397-398 [Medline] [Order article via Infotrieve]
  11. Svoboda, D. J., Azarnoff, D. L., and Hignite, C. E. (1966) J. Cell Biol. 30,442-450 [Free Full Text]
  12. Osumi, T., Ozasa, H., and Hashimoto, T. (1984) J. Biol. Chem. 259,2031-2034 [Abstract/Free Full Text]
  13. Chatterjee, B., Demyan, W. F., Lalwani, N. D., Reddy, J. K., and Roy, A. K. (1983) Biochem. J. 214,879-883 [Medline] [Order article via Infotrieve]
  14. Chatterjee, B., Murty, C. V. R., Olson, M. J., and Roy, A. K. (1987) Eur. J. Biochem. 166,273-278 [Abstract]
  15. McQuaid, S., Russel, S. E. H., Withe, S. A., Pearson, C. M., Elcombe, C. R., and Humphries, P. (1987) Cancer Lett. 37,115-124 [Medline] [Order article via Infotrieve]
  16. Reddy, J. K., Goel, S. K., Nemali, M. R., Carrino, J. J., Laffler, T. G., Reddy, M. K., Sperbeck, S. J., Osumi, T., Hashimoto, T., Lalwani, N. D., and Rao, M. S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83,1747-1751 [Abstract]
  17. Hijikata, M., Ishii, N., Kagamiyama, H., Osumi, T., and Hashimoto, T. (1987) J. Biol. Chem. 262,8151-8158 [Abstract/Free Full Text]
  18. Isseman, I., and Green, S. (1990) Nature 347,645-650 [CrossRef][Medline] [Order article via Infotrieve]
  19. Dreyer, C., Krey, G., Keller, H., Givel, F., Helftenbein, G., and Wahli, W. (1992) Cell 68,879-887 [Medline] [Order article via Infotrieve]
  20. Gottlicher, M., Widmark, E., Li, Q., and Gustafsson, J. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,4653-4657 [Abstract]
  21. Schmidt, A., Endo, N., Rutledge, S. J., Vogel, R., Shinar, D., and Rodan, G. A. (1992) Mol. Endocrinol. 6,1634-1641 [Abstract]
  22. Sher, T., Yi, H. F., McBride, O. W., and Gonzalez, F. J. (1993) Biochemistry 32,5598-5604 [Medline] [Order article via Infotrieve]
  23. Zhu, Y., Alvares, K., Huang, G., Rao, M. S., and Reddy, J. K. (1993) J. Biol. Chem. 268,26817-26820 [Abstract/Free Full Text]
  24. Tontonez, P., Hu, E., Graves, R. A., Budavari, A. I., and Spiegelman, B. M. (1994) Genes & Dev. 8,1224-1234
  25. Aperlo, C., Pognonec, P., Saladin, R., Auwerx, J., and Boulukos, K. (1995) Gene ( Amst. ), in press
  26. Kliewer, S. A., Forman, B. M., Blumberg, B., Ong, E. S., Borgmeyer, U., Mangelsdorf, D. J., Umesono, K., and Evans, R. M. (1994) Proc. Nat. Acad. Sci. U. S. A. 91,7355-7359 [Abstract]
  27. Osumi, T., Wen, J. K., and Hashimoto, T. (1991) Biochem. Biophys. Res. Commun. 175,866-871 [Medline] [Order article via Infotrieve]
  28. Tugwood, J. D., Isseman, I., Anderson, R. G., Bundell, K. R., McPheat, W. L., and Green, S. (1992) EMBO J. 11,433-439 [Abstract]
  29. Zhang, B., Marcus, S. L., Sajjadi, F. G., Alvares, K., Reddy, J. K., Subramani, S., Rachubinski, R. A., and Capone, J. P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,7541-7545 [Abstract]
  30. Marcus, S. L., Miyata, K. S., Zhang, B., Subramani, S., Rachubinski, R. A., and Capone, J. P. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,5723-5727 [Abstract]
  31. Alvarez, K., Fan, C., Daddras, S. S., Yelandi, A. V., Rachubinski, R. A., Capone, J. P., Subramani, S., Iannaccone, P. M., Rao, M. S., and Reddy, J. K. (1994) Cancer Res. 54,2303-2306 [Abstract]
  32. Bardot, O., Aldridge, T. C., Latruffe, N., and Green, S. (1993) Biochem. Biophys. Res. Commun. 192,37-45 [CrossRef][Medline] [Order article via Infotrieve]
  33. Muerhoff, A. S., Griffin, K. J., and Johnson, E. F. (1992) J. Biol. Chem. 267,19051-19053 [Abstract/Free Full Text]
  34. Palmer, C. N. A., Hsu, M.-H., Muerhoff, A. S., Griffin, K. J., and Johnson, E. F. (1994) J. Biol. Chem. 269,18083-18089 [Abstract/Free Full Text]
  35. Rodriguez, J. C., Gil-Gomez, G., Hegardt, F. G., and Haro, D. (1994) J. Biol. Chem. 269,18767-18772 [Abstract/Free Full Text]
  36. Gulick, T., Cresci, S., Caira, T., Moore, D. D., and Kelly, D. P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,11012-11016 [Abstract/Free Full Text]
  37. Vu-Dac, N., Schoonjans, K., Laine, B., Fruchart, J. C., Auwerx, J., and Staels, B. (1994) J. Biol. Chem. 269,31012-31018 [Abstract/Free Full Text]
  38. Schoonjans, K., Staels, B., Grimaldi, P., and Auwerx, J. (1993) Eur. J. Biochem. 216,615-622 [Abstract]
  39. Tsonis, P. A., Manes, T., Milan, J. L., and Goetinck, P. F. (1988) Nucleic Acids Res. 16,7745 [Medline] [Order article via Infotrieve]
  40. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,488-492 [Abstract]
  41. MacGregor, G. R., and Caskey, C. T. (1989) Nucleic Acids Res. 17,2365 [Medline] [Order article via Infotrieve]
  42. Leid, M., Kastner, P., Lyons, R., Nakshatri, H., Saunders, M., Zacharewski, T., Chen, J. Y., Staub, A., Garnier, J. M., Mader, S., and Chambon, P. (1992) Cell 68,377-395 [Medline] [Order article via Infotrieve]
  43. Deschatrette, J., and Weiss, M. C. (1974) Biochimie (Paris) 56,1603-1612 [Medline] [Order article via Infotrieve]
  44. Auwerx, J., Deeb, S., Brunzell, J. D., Peng, R., and Chait, A. (1988) Biochemistry 27,2651-2655 [Medline] [Order article via Infotrieve]
  45. Auwerx, J., Deeb, S., Brunzell, J. D., Wolfbauer, G., and Chait, A. (1989) Biochemistry 28,4563-4567 [Medline] [Order article via Infotrieve]
  46. Staels, B., Vu-Dac, N., Kosykh, V., Saladin, R., Fruchart, J. C., Dallongeville, J., and Auwerx, J. (1995) J. Clin. Invest. 95,705-712 [Medline] [Order article via Infotrieve]
  47. Mosmann, T. (1983) J. Immunol. Methods 65,55-63 [CrossRef][Medline] [Order article via Infotrieve]
  48. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162,156-159 [CrossRef][Medline] [Order article via Infotrieve]
  49. Tokunaga, K., Nakaruma, Y., Sakata, K., Fujimoro, K., Ohkubo, M., Sawada, K., and Sakiyama, S. (1987) Cancer Res. 47,5616-5619 [Abstract]
  50. Nevins, J. R. (1987) Methods Enzymol. 152,234-241 [Medline] [Order article via Infotrieve]
  51. Sanger, T. D., Yang, M., and Bonner, J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78,243-246 [Abstract]
  52. de Wet, J. R., Wood, K., DeLuca, M., Helinski, D. R., and Subramani, S. (1987) Mol. Cell. Biol. 7,725-737 [Medline] [Order article via Infotrieve]
  53. Gorman, C. M., Moffat, L. F., and Howard, B. H. (1982) Mol. Cell. Biol. 2,1044-1051 [Medline] [Order article via Infotrieve]
  54. Chodish, L. A., Baldwin, A. S., Carthew, R. W., and Sharp, P. A. (1988) Cell 53,11-24 [Medline] [Order article via Infotrieve]
  55. Dreyer, C., Keller, H., Mahfoudi, A., Laudet, V., Krey, G., and Wahli, W. (1993) Biol. Cell 77,67-77 [Medline] [Order article via Infotrieve]
  56. Fried, M. G., and Crothers, D. M. (1983) Nucleic Acids Res. 11,141-158 [Abstract]
  57. Wu, P., Skrede, S., Hvattum, E., and Bremer, J. (1993) Biochim. Biophys. Acta 1170,118-124 [Medline] [Order article via Infotrieve]
  58. Tora, L., White, J., Brou, C., Tasset, D., Webster, N., Scheer, E., and Chambon, P. (1989) Cell 59,477-487 [Medline] [Order article via Infotrieve]
  59. Einerhand, A. W. C., Kos, W. T., Distel, B., and Tabak, H. F. (1993) Eur. J. Biochem. 214,323-331 [Abstract]
  60. Henry, M. F., and Cronan, J. E., Jr. (1992) Cell 70,671-679 [CrossRef][Medline] [Order article via Infotrieve]
  61. Clarke, S. D., and Jump, D. B. (1994) Annu. Rev. Nutr. 14,83-98 [CrossRef][Medline] [Order article via Infotrieve]
  62. Khan, W. A., Blobe, G. C., and Hannun, Y. A. (1992) J. Biol. Chem. 267,3605-3612 [Abstract/Free Full Text]

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