Characterization of the Human PPAR
Promoter: Identification of a Functional Nuclear Receptor Response Element
Inés Pineda Torra,
Yalda Jamshidi,
David M. Flavell,
Jean-Charles Fruchart and
Bart Staels
U.545 Institut National de la Santé et de la Recherche Médicale (I.P.T., J.-C.F., B.S.), Département dAthérosclérose, Institut Pasteur de Lille, 59019 Lille, and the Faculté de Pharmacie, Université de Lille II, 59006 Lille, France; and Centre for Cardiovascular Genetics (Y.J., D.M.F.), Department of Medicine, Royal Free and University College London Medical School, The Rayne Institute, London WC1E 6JJ, United Kingdom
Address all correspondence and requests for reprints to: Bart Staels, U.545 INSERM, Institut Pasteur de Lille, 1 Rue Calmette BP245, 59019 Lille, France. E-mail: Bart.Staels{at}pasteur-lille.fr.
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ABSTRACT
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PPAR
is a nuclear receptor that controls lipid and glucose metabolism and exerts antiinflammatory activities. The factors regulating human PPAR
(hPPAR
) gene expression remain largely unexplored. To study the mechanisms controlling hPPAR
expression, the hPPAR
gene promoter was identified and characterized. First, an alternatively spliced exon within the 5'-untranslated region of the hPPAR
gene was identified by RT-PCR. Next, the transcription start site was mapped and the hPPAR
gene promoter was cloned and functionally analyzed. Because PPAR
levels are elevated in tissues expressing the hepatocyte nuclear factor-4 (HNF4), such as liver, the regulation of hPPAR
by HNF4 was examined. Transient transfections in HepG2 and Cos cells showed that HNF4 enhances hPPAR
promoter activity. 5'-Deletion and mutation analysis of the hPPAR
promoter identified a regulatory element (RE) consisting of a degenerate hexamer repeat with a single nucleotide spacer (direct repeat 1), termed
HNF4-RE. Gel shift assays demonstrated that HNF4 binds to this
HNF4-RE. Furthermore, HNF4 increased the activity of a heterologous promoter driven by two copies of the
HNF4-RE. The nuclear receptor COUP-TFII also bound this site and down-regulated basal as well as HNF4-induced hPPAR
promoter activity. Finally, PPAR
was shown to bind the
HNF4-RE, leading to an induction of PPAR
expression in hepatocytes. In summary, the organization of the 5'-flanking and untranslated region of the hPPAR
gene was characterized and the hPPAR
promoter region has been identified. Furthermore, these data demonstrate that the hPPAR
gene is regulated by nuclear receptors, such as HNF-4, COUP-TFII, and PPAR
.
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INTRODUCTION
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PPAR
[NR1C1] IS a ligand-activated transcription factor belonging to the nuclear receptor superfamily that regulates gene transcription by heterodimerizing with RXR. PPAR
/RXR heterodimers bind to PPAR response elements (PPREs) consisting of a direct repeat of the core AGGTCA sequence spaced by 1 or 2 bp [direct repeat 1 or 2 (DR1 or DR2)] preceded by a 5'-A/T-rich flanking sequence (1). In addition, PPAR
represses gene transcription by interfering with the nuclear factor
B (NF
B) and activating protein 1 (AP1) signaling pathways in a DNA binding-independent fashion (2).
PPAR
-mediated transcriptional regulation of target genes is tightly controlled by ligand activation. Fatty acid (FA) derivatives and the hypolipidemic fibrates are natural and synthetic PPAR
ligands, respectively (3). PPAR
mediates the action of fibrates on plasma lipid levels by regulating the transcription of genes controlling lipoprotein metabolism (4). Moreover, PPAR
is a pivotal modulator of intracellular lipid metabolism by regulating the gene expression of proteins involved in FA uptake, FA esterification, FA entry into the mitochondria, peroxisomal and mitochondrial FA catabolism, and ketone body synthesis (1). In addition to intra- and extracellular lipid metabolism, glucose and energy homeostasis as well as body weight were also reported to be influenced by PPAR
activators (5, 6). Furthermore, PPAR
is an important regulator of vascular inflammation. PPAR
inhibits the expression of various inducible factors implicated in the promotion of a local inflammatory response within the developing atherosclerotic plaque (2). In addition, PPAR
regulates the expression of lipoprotein receptors and cholesterol transporters involved in the reverse cholesterol transport pathway in macrophages (7, 8). PPAR
activators reduce plasma concentrations of inflammatory cytokines in vivo (9, 10) and reduce the progression of coronary atherosclerosis in humans (11, 12, 13, 14).
In addition to ligand activation, transcriptional control of PPAR
target genes depends on its expression levels. High levels of PPAR
expression are observed in tissues with elevated FA catabolic rates, such as liver, heart, kidney, muscle, and the large intestine (15, 16, 17). In rodents, PPAR
is regulated by stress (18) and by different hormones, such as GH, glucocorticoids, insulin, and leptin (19, 20, 21, 22). PPAR
expression oscillates diurnally (18), is diminished with age (23), and is induced during brown adipocyte differentiation (24). Furthermore, starvation increases PPAR
expression levels in liver leading to changes in mRNA levels of PPAR
target genes (25). Low PPAR
mRNA levels result in decreased PPAR
activity in pancreatic islets of obese Zucker diabetic (fa/fa) rats (22), in rat cardiomyocytes during cardiac hypertrophy (26), in livers of male Syrian hamsters upon lipopolysaccharide administration (27), and in intestinal epithelial cells during hypoxia (28). Moreover, PPAR
expression levels correlate with human apo AI gene induction in response to fibrate treatment as demonstrated in human apo AI transgenic mice crossed with PPAR
-deficient mice (29).
However, to date, studies addressing the regulation of PPAR
gene expression have been largely performed either in rodent cells or in animal models. Although the regulation of PPAR
expression in humans remains largely unknown, the observation that hepatic PPAR
mRNA levels vary significantly among individuals (30, 31) suggests that PPAR
is also regulated at the transcriptional level in humans. Indeed, hPPAR
mRNA levels are induced during monocyte differentiation into macrophages (32), and high glucose concentrations up-regulate PPAR
gene expression in human macrophages from healthy control subjects as well as from type 2 diabetic patients (33).
To understand the molecular mechanisms governing PPAR
regulation, we decided to identify and functionally characterize the 5'-flanking region of the hPPAR
gene. In the present manuscript the transcription start site was determined, and the upstream 5'-flanking region of the hPPAR
gene was identified and cloned and its transcriptional activity was examined.1 In the course of these studies the sequence of the 5'-untranslated region (UTR) of the hPPAR
gene was completed and an alternatively spliced exon within the 5'-UTR of the gene was identified. Finally, hPPAR
promoter activity was shown to be regulated by the nuclear receptors hepatocyte nuclear factor 4 (HNF4), chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII), and PPAR
itself.
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RESULTS
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Characterization of the 5'-UTR Structure of the hPPAR
Gene and Identification of an Alternatively Spliced 5'-UTR Variant
The hPPAR
gene contains at least eight exons in which exons 1 and 2 and the 5'-end of exon 3 and the 3'-end of exon 8 are not translated (34, 35). The aim of our study was to determine the 5'-UTR structure of the hPPAR
gene and to identify and characterize the hPPAR
promoter. To identify noncoding exons within this region, different primers were designed and PCRs were carried out on reverse transcribed human liver RNA from two different subjects. Using this approach, a novel exon was discovered. When PCR was performed using a primer designed at the 3'-end of exon 2 (primer 13, Fig. 1A
) and a primer hybridizing the 3'-end of exon 1 (primer 15C, Fig. 1A
), a fragment of 158 bp containing both exon 1 and exon 2 was amplified (Fig. 1B
, left), as expected. Additionally, a second fragment 150 bp longer was repeatedly coamplified even at stringent PCR conditions. Similar results were obtained with four other human liver and adipose tissue mRNA samples (data not shown), indicating that the unexpected additional PCR product corresponded to a rather common transcript. Cloning and sequencing of this PCR fragment revealed the presence of 156 bp of novel sequence downstream of the sequence previously known as exon 1 (Fig. 1C
). Furthermore, when PCR was performed using a primer within the novel sequence (primer IP3) and the exon 1-specific primer 15C, a fragment was amplified, further indicating the existence of this additional exon (Fig. 1B
, right). The novel sequence was located between exon 1 and 2 in a bacterial artificial chromosome (BAC) clone containing the hPPAR
gene and was flanked by ag/gt consensus acceptor and donor splice sites, respectively (Fig. 1C
). The newly identified exon was named exon 1b, by analogy to the nomenclature used for the alternatively spliced exon present in the 5'-UTR of the mouse PPAR
gene (36), whereas the previously known exon 1 was renamed exon 1a (Fig. 1A
). In addition, when 5'-RACE (rapid amplification of cDNA ends) reactions were carried out using primer IP3 located in exon 1b as gene-specific primer, a fragment covering the novel exon plus a sequence corresponding to exon 1a was amplified (data not shown), corroborating the existence of a second alternative transcript containing exon 1b in addition to exon 1a. This suggests that exon 1b is spliced out alternatively giving rise to two different transcripts with distinct 5'-UTRs (Fig. 1A
). Thus, both mouse and human PPAR
(hPPAR
) genes display a similar 5'-UTR genomic organization. However, the noncoding exons do not show a significant degree of sequence homology among species (38% for exon 1a and 29% for exon 1b).

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Figure 1. Intron-Exon Structure of the 5'-UTR of the hPPAR Gene and Identification of an Alternative Spliced 5'-UTR Variant
A, Schematic representation of the 5'-UTR of the hPPAR gene and the identified spliced variants. The first coding exon (filled box) and the 5'-noncoding exons (hatched boxes) are depicted. Numbers over the boxes represent the exon length in base pairs. Oligonucleotides used for RT-PCR analysis are indicated by arrows. B, RT-PCR analysis on human liver RNA isolated from two different subjects. Total RNA was extracted, reverse transcribed, and amplified using primers 15C and 13 (left) or 15C and IP13 (right). -PCR corresponds to a PCR carried in the absence of RT product. C, Genomic sequence corresponding to the alternatively spliced exon 1b. Oligonucleotides IP3 and IP7 used for 5'-RACE analysis are indicated by arrows. The acceptor and donor splice sites are depicted in bold. Nucleotides in uppercase and lowercase letters correspond to exon and intron sequences, respectively.
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Determination of the Transcription Start Site
To determine the hPPAR
transcription start site, 5'RACE-PCR analysis was performed. Genomic sequence upstream of the previously reported exon 1a sequence (37) was obtained by primer walking the BAC clone containing the hPPAR
gene. Despite the overall low degree of homology shown by the PPAR
5'-UTRs among different species, a sequence of around 60 bp was found to be 88% homologous to a region close to the transcription start site in the mouse PPAR
5'-UTR (Fig. 2A
). This prompted us to examine whether the transcription start site in the human gene could also be upstream of this region. Accordingly, a primer close to that sequence (primer IP18, Fig. 2A
) was used as gene-specific primer in a 5'-RACE-PCR. A 213-bp DNA fragment was amplified from human liver RNA (Fig. 2B
) and subsequently sequenced. The most 5'-nucleotide before the poly-dC tail introduced by the 5'-RACE procedure mapped to a T and was designated as nucleotide +1 (Fig. 2A
). Similar results were obtained when a different gene-specific primer (primer 5-GSP) was used in the 5'-RACE-PCR (data not shown). To confirm the results of the 5'-RACE experiments, ribonuclease (RNase) protection analysis was performed. RNase mapping with a 337-nucleotide labeled cRNA probe complementary to bp -22 to +213 (Fig. 2C
) gave a major protected band of 213 bp (Fig. 2D
), thus confirming the transcription start site that had been identified by 5'-RACE. These data indicate that exon 1a is considerably longer than the partially sequenced exon 1 that had been previously reported (34, 35, 37) and than the exon 1a identified in the mouse PPAR
gene (36).

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Figure 2. Mapping of the Transcription Start Site of the hPPAR Gene by 5'-RACE and RNase Protection Analysis
A, Sequence corresponding to exon 1a in the 5'-UTR region. The oligonucleotides YGSP1, IP24, IP18, and 5-GSP used for 5'-RACE, RNase protection analysis, and cloning are indicated by arrows. The donor splice site is depicted in bold lowercase. The previously reported 5'-UTR sequence (37 ) is indicated in italics. Transcription start site (+1) is shown by an arrow. The sequence in bold uppercase represents a region showing 88% homology with both rat and mouse 5'-UTRs (see text for of the 5'-RACE products on an agarose gel. Total RNA from human liver was reverse transcribed and the cDNA was poly-(dC) tailed with terminal deoxynucleotide transferase (TdT) as described in Materials and Methods. dC-tailed cDNA (TdT+) or untailed cDNA (TdT-) were amplified using an universal primer (AAP) and a gene-specific primer (IP18). C, Schematic representation of the probe used for RNase protection analysis. A genomic DNA fragment (-22 to +213) was amplified with oligonucleotides IP24 and IP18 (depicted in panel A) and cloned into pBluescript SK+. Subsequently an antisense RNA probe was transcribed. The size of the protected fragment, as predicted by the 5'-RACE experiment shown in panel B, is indicated. D, Mapping of the transcription start site by RNase protection analysis. Autoradiograph of RNase protection analysis carried out on 20 µg of human liver total RNA extracted from two different subjects. The protected probe fragments (indicated by an arrow) correspond to those predicted in panel B.
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Cloning and Analysis of the 5'-Flanking Region of the hPPAR
Gene
To identify potential binding sites for transcription factors that may be important in the regulation of hPPAR
gene expression, 1.6 kb of hPPAR
sequence upstream of the transcription start site were cloned. No consensus TATA or CCAAT boxes were found within the first 100 bp, although a CCAAT box was observed at -472 bp (Fig. 3
). Furthermore, this region possesses the characteristics of a TATA-less promoter, namely, GC-rich sequences and multiple putative Sp1 binding sites. Computer-assisted analysis (38) revealed several potential transcription factor binding sites including AP2, Egr-1, Egr-2, E box, NF
B, and MZF-1 among others (Fig. 3
).

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Figure 3. Nucleotide Sequence of the 5'-Flanking Region of the hPPAR Gene
Numbers are relative to the transcription start site (+1). The putative HNF4-RE is underlined. Putative transcription factor binding sites are indicated by open boxes.
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Functional Characterization of the hPPAR
Promoter
To determine the ability of hPPAR
5'-flanking region to drive basal transcriptional activity, sequences were fused to a luciferase reporter gene. Because hPPAR
is highly expressed in liver (17), the 1,664-bp promoter-containing plasmid, p
(H-H)-pGL3, was transiently transfected into human HepG2 hepatoma cells. Activity of this construct was about 8-fold the activity of the parental promoterless construct (pGL3 basic) in these cells (Fig. 4
). Next, the promoter activity of different 5'-deletion constructs was evaluated. Deletion from -1,664 to -1,206 did not significantly affect the promoter activity. Truncation to -648 significantly reduced the luciferase activity in HepG2 cells. By contrast, in agreement with hPPAR
tissue-selective expression, luciferase activities of the p
(H-H)-pGL3 construct and its 5'-deletion mutants were only 2-fold higher than pGL3 basic in Cos-1 cells, a cell line that does not express PPAR
. These experiments suggest that these cells are devoid of the factors driving basal hPPAR
expression compared with HepG2 cells. The shortest construct, p
(P-H)-pGL3, containing the GC-rich proximal promoter was only 2-fold more active relative to pGL3 basic vector in both cell lines compared with the 6-fold activity shown by the p
(E-H)-pGL3 construct, which suggests that sequences between -536 and -1,664 may contain binding sites for key transcription factors driving basal hPPAR
gene expression.

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Figure 4. Functional Analysis of hPPAR Promoter 5'-Deletion Mutants
The region spanning 1,664 bp of promoter and the first 83 bp of exon 1a was progressively deleted from its 5'-end, fused to the pGL3 basic vector, and transfected into the indicated cell types as described in Materials and Methods. Values (mean ± SD) represent firefly luciferase activity normalized relative to a Renilla luciferase transfection internal control. Luciferase activities are shown relative to the activity of the pGL3 basic vector, which was arbitrarily set to 1.
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Identification of a Functional HNF4 Regulatory Element (HNF4-RE) in the hPPAR
Promoter
Because hPPAR
is highly expressed in liver and the hPPAR
5'-flanking region shows significant promoter activity in HepG2 cells, the regulation of hPPAR
promoter activity by HNF4, a nuclear receptor that is abundantly expressed in liver, was examined. To this purpose, transient transfection studies were performed with the p
(H-H)-pGL3 construct in HepG2 cells. In the presence of increasing concentrations of HNF4, hPPAR
promoter activity was induced in a dose-dependent fashion (Fig. 5A
). To localize the region that confers HNF4 responsiveness within the hPPAR
promoter, cotransfection assays were repeated with a series of 5'-deletion hPPAR
promoter constructs (Fig. 5B
). These experiments revealed that the HNF4 responsive region resides within the fragment flanked by HindIII and XhoI sites. Analysis of the sequence in this region of the hPPAR
promoter revealed two potential hexamer binding sites separated by a single nucleotide between nucleotide -1,492 to -1,483 (Fig. 3
), conforming to the DR1 response elements for HNF4 (HNF4-RE). To unequivocally characterize the imperfect DR1 as an HNF4 response element, HepG2 and Cos cells were transfected with a hPPAR
promoter construct bearing two nucleotide mutations in the 5'-hexamer of the DR1 element, p
(H-H)
HNF4-REmut-pGL3. In contrast to the wild-type promoter construct p
(H-H)-pGL3, the mutated promoter was not influenced by HNF4 cotransfection in both cell lines (Fig. 5C
), thus demonstrating that the hPPAR
HNF4-RE (
HNF4-RE) indeed confers HNF4 response to the hPPAR
promoter.
Binding Analysis of HNF4 to the DR1 Element
To demonstrate direct binding of HNF4 to the
HNF4-RE, EMSAs were performed with a radiolabeled DR1 oligonucleotide probe and in vitro translated HNF4 proteins. As shown in Fig. 6
, HNF4 bound the
HNF4-RE (Fig. 6A
, lane 4) and the well characterized C3P site present in the apo CIII promoter (39) with similar efficacy (Fig. 6A
, lanes 2 and 4). DNA binding was almost completely abolished when two-point mutations were introduced into the
HNF4-RE-labeled oligonucleotide (GCAGCAAAGTTCA instead of GGGGCAAAGTTCA where the underlined letters indicate the mutated nucleotides) (Fig. 6A
, lane 5). Furthermore, the specificity of the HNF4-DR1 interaction was demonstrated by competition studies. Inhibition of formation of the complex was shown by the addition of increasing concentrations of either the unlabeled
HNF4-RE (Fig. 6B
, lanes 35) or C3P (Fig. 6B
, lanes 911) oligonucleotides, whereas competition with an equivalent amount of unlabeled mutated
HNF4-RE did not significantly displace the labeled wild-type element (Fig. 6B
, lanes 68).
The
HNF4-RE Confers HNF4 Responsiveness to a Heterologous Promoter
To explore the activation of the
HNF4-RE by HNF4 in the context of a heterologous promoter, cotransfection studies were performed with the
HNF4-REx2S-TKpGL3 construct, which contains two copies of the DR1 in the sense orientation in front of the thymidine kinase (TK) promoter (Fig. 7A
). Upon cotransfection of HNF4 into both HepG2 and RK13 cells, the
HNF4-REx2S-TKpGL3 transcriptional activity was significantly enhanced (Fig. 7
, B and C). By contrast, the reporter activity of a similar heterologous promoter construct containing two copies of the mutated
HNF4-RE was not influenced by HNF4 cotransfection (Fig. 7C
). Taken together, these data show that the DR1 site is a bona fide HNF4-RE that mediates HNF4 induction of hPPAR
promoter activity.
COUP-TFII Binds the
HNF4-RE and Represses Basal and HNF4-Induced hPPAR
Promoter Activity
DR1 elements can be recognized not only by HNF4, but also by other members of the nuclear receptor superfamily, including the transcription factor COUP-TFII, also known as apo A-1 regulatory protein 1 or ARP1 (40). In addition, COUP-TFII has been shown to actively repress or modulate the effects of HNF4 on the transcription of several genes in liver (39, 41, 42, 43, 44). Therefore, the ability of COUP-TFII to bind the
HNF4-RE was evaluated by gel shift analysis. COUP-TFII proteins bound to the radiolabeled
HNF4-RE probe, and competition with 50-fold molar excess of unlabeled
HNF4-RE oligonucleotide completely inhibited the formation of the COUP-TFII -DNA complex (Fig. 8A
, lanes 2 and 3).
The influence of COUP-TFII on the hPPAR
promoter activity was then investigated by cotransfection experiments in HepG2 and Cos cells. Transient transfection of COUP-TFII alone decreased the activity of the hPPAR
promoter construct containing the
HNF4-RE site in both HepG2 and Cos cells (Fig. 8B
). As previously observed, upon cotransfection of HNF4, hPPAR
promoter activity was induced in both cell lines. However, overexpression of COUP-TFII in the presence of HNF4 abrogated the HNF4-induced transcriptional activity. On the contrary, when identical experiments were performed with a promoter construct bearing a mutation in the
HNF4-RE, promoter activity was not affected by cotransfection of either COUP-TFII or HNF4 alone or added together (Fig. 8B
). These data demonstrate that the repression of the PPAR
promoter by COUP-TFII and the antagonism of the HNF4 induction are dependent on the
HNF4-RE. Altogether these results indicate that COUP-TFII negatively regulates PPAR
gene transcription.
hPPAR
Binds the
HNF4-RE and Regulates Its Own Expression
Next, direct binding of PPAR
/RXR
heterodimers to the hPPAR
HNF4-RE was examined. To this purpose, gel shift analysis was performed with the radiolabeled
HNF4-RE probe and in vitro translated PPAR
and RXR
proteins. PPAR
/RXR
heterodimers bound the
HNF4-RE oligonucleotide (Fig. 9A
, lane 2). DNA binding could be competed with increasing amounts of unlabeled
HNF4-RE oligonucleotide (Fig. 9A
, lanes 35), whereas incubation with a mutated
HNF4-RE oligonucleotide did not compete for binding (Fig. 9A
, lanes 68).

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Figure 9. PPAR Positively Regulates Its Own Expression
A, PPAR binds the HNF4-RE. EMSA performed on end-labeled HNF4-RE oligonucleotide in the presence of in vitro translated PPAR and RXR. Competition experiments were performed in the presence of 10-, 25-, and 50-fold molar excess of the indicated unlabeled oligonucleotides. B, PPAR modulates hPPAR promoter activity via the HNF4-RE. Cos cells were transfected with either the p (H-H)-pGL3 hPPAR or the p (H-H) HNF4-REmut-pGL3 hPPAR promoter construct, with or without a PPAR expression plasmid in the presence or absence of 100 µM Wy 14,643 as indicated. Relative luciferase activities (mean ± SD) for each construct are shown relative to the promoter activity in the absence of PPAR and activator, which was arbitrarily set to 1. C, The HNF4-REx2S-TKpGL3 plasmid was transfected in Cos cells with or without PPAR in the presence or absence of 50 µM Wy 14,643 as indicated. Relative luciferase activities (mean ± SD) are shown as in panel B. D, Fenofibric acid enhances hPPAR mRNA levels in primary cultures of human hepatocytes. Human hepatocytes were isolated and treated for 24 h with (FF) or without (CON) 250 µM fenofibric acid. One microgram of total RNA was reversed transcribed and subjected to Real Time PCR analysis as described in Materials and Methods. Specific hPPAR mRNA normalized to 28S content is expressed relative to untreated cells (mean ± SD, n = 3).
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To determine the effect of PPAR
on hPPAR
promoter activity, transient transfection assays were performed using the hPPAR
promoter construct containing the
HNF4-RE. Cotransfection of PPAR
induced hPPAR
reporter activity approximately 2-fold, an effect that was enhanced in the presence of Wy 14,643. By contrast, no effect by either PPAR
or its ligand could be observed when a construct containing the mutated
HNF4-RE was employed, indicating that an intact
HNF4-RE site is required for the PPAR
-mediated induction. To investigate whether the
HNF4-RE could function as a PPRE in front of a heterologous promoter, transient transfection assays were performed using the
HNF4-REx2S-TKpGL3 construct (Fig. 9C
). Incubation of cells with Wy 14,643 in the presence of cotransfected PPAR
resulted in a 5-fold activation of the
HNF4-REx2S-TKpGL3 construct, indicating that the
HNF4-RE site is a functional PPRE.
Finally, to analyze whether hPPAR
mRNA expression is regulated by PPAR
agonists, primary hepatocytes isolated from human liver were cultured for 24 h in medium containing fenofibric acid. hPPAR
mRNA levels were markedly up-regulated upon treatment with fenofibric acid (Fig. 9D
). Taken together, these data show that PPAR
positively autoregulates its expression.
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DISCUSSION
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PPAR
is a transcription factor that regulates lipid, glucose, and energy homeostasis and controls body weight and vascular inflammation. Thus, PPAR
may play a modulatory role in the onset and evolution of different metabolic and vascular disorders such as dyslipidemia, obesity, insulin resistance, and atherosclerosis (1). Dysregulation of PPAR
expression may contribute to the development of these disorders as well as the response to its fibrate agonists. Indeed, PPAR
expression levels have been shown to influence its transcriptional activity as well as the response to fibrate treatment in rodents (22, 25, 26, 27). However, the study of the molecular mechanisms governing PPAR
regulation in humans has not been addressed to date.
To study PPAR
regulation in humans, we characterized the 5'-flanking region of the hPPAR
gene and identified the hPPAR
promoter. The genomic organization of hPPAR
was recently reported (34, 35). The hPPAR
gene contains at least eight exons in which exons 1 and 2, the 5'-end of exon 3, and the 3'-end of exon 8 are not translated (34, 35). As an initial step in our study, the 5'-UTR of the hPPAR
gene was further characterized, resulting in the completion of the 5'-end sequence of exon 1, renamed exon 1a, and in the identification of an alternatively spliced 5'-variant. An alternatively spliced exon in the mouse PPAR
5'-UTR has been previously reported (36). However, alignment of both mouse and human 5'-UTR sequences revealed no significant degree of homology between them despite the coding exons being very similar between species.
The transcription start site of the hPPAR
gene was mapped. The proximal promoter of the hPPAR
gene contains no typical TATA or CCAAT boxes and has a high GC content, including several consensus Sp1 sites, features that are also present in the mouse PPAR
promoter (36). Excluding the proximal GC-rich region, both promoters do not show a high degree in sequence homology (
40%). However, only a small fragment of the mouse PPAR
promoter has been sequenced to date (36). This sequence disparity in both the 5'-UTR and the promoter regions may indicate that PPAR
gene expression among species could be regulated in a different manner although sufficient data to establish a thorough comparison between species are lacking at this stage.
Computer-assisted analysis of the hPPAR
promoter sequence identified various putative transcription factor binding sites, such as sites for members of the early growth response (Egr) gene family, which are involved in the induction of monocytic differentiation (45). Egr proteins could thus be involved in the up-regulation of hPPAR
expression upon monocyte differentiation into macrophages (32) although further studies are needed to confirm this hypothesis. Furthermore, potential sites for NF
B and AP1, factors involved in the transduction of several cytokine and proinflammatory signals, as well as for the myeloid zinc finger gene, MZF-1, were found in the hPPAR
promoter. PPAR
inhibition of NF
B- and AP1-induced expression of inflammatory response genes (46) lies at the basis of its antiinflammatory activities. It is tempting to speculate that hPPAR
expression, in its turn, is controlled by inflammatory cytokines. Because rodent PPAR
mRNA levels are increased by glucocorticoids (20, 21), putative GR response elements were searched for. No obvious GR response element could be detected within the hPPAR
regulatory region analyzed; however, regulation studies in human tissues or cell lines would be necessary to determine whether hPPAR
expression is also modulated by glucocorticoids in humans.
HNF4 is a nuclear receptor that plays a key role in liver-specific gene expression (47) and is required for mammalian hepatocyte differentiation and for normal metabolic regulation in liver (48). HNF4 is expressed at high levels in liver, kidney, and intestine (47). It binds as homodimers to DR1-like response elements, thereby modulating the expression of several liver-enriched genes encoding apolipoproteins, coagulation factors, serum proteins, proteins of the cytochrome P450 family, and genes involved in FA and glucose metabolism (49). Regulation of hPPAR
by HNF4 is consistent with the tissue-expression pattern shown by PPAR
. In the present study induction of hPPAR
promoter activity by HNF4 is reported. A HNF4-RE was identified at nt -1,492 to -1,483 within the hPPAR
promoter. This element closely resembles several HNF4-REs found in other genes and the GGGGCA A AGGTCA consensus HNF4 binding site (50). Moreover, the regulation of PPAR
gene expression by HNF4 in vivo in mice was recently demonstrated using HNF4-deficient mice (51). These mice display significantly lower PPAR
mRNA levels compared with the wild-type mice, thus demonstrating that PPAR
expression is regulated by HNF4 also in mice. Nevertheless, the promoter experiments performed with the hPPAR
promoter indicate that the HNF4 site identified is not responsible for the activity of the hPPAR
promoter in liver because the promoter construct lacking this site still possesses a significant activity in HepG2 cells (Fig. 4
). This may suggest that this site binds as well other factors, such as COUP-TF, present in these cells that negatively regulate the hPPAR
promoter either as a result of a direct repression of the promoter or due to competition with factors, such as HNF4, for binding to the site. In addition, negative factors may bind to sites located between -1,664 and -1,206 bp in the hPPAR
promoter. Future studies on the regulation of this promoter will be required to elucidate the factors governing hPPAR
gene expression in liver.
Members of the COUP-TF family of orphan nuclear receptors have been reported to bind DR1 sites that are also recognized by HNF4 (52). COUP-TF actively represses or antagonizes HNF4-mediated transcriptional activation of several genes (39, 41, 42, 43, 44). COUP-TF II has been shown to play a role in cardiac energy metabolism and heart development (53, 54). In this study, COUP-TFII was also shown to bind the HNF4-RE within the hPPAR
regulatory region and to repress both basal and HNF4-induced hPPAR
promoter activity. The COUP-TFII antagonism may finely tune HNF4-dependent gene expression. The expression level of a COUP-TFII/HNF4 target gene would therefore depend on the relative concentrations of both transcription factors in a given cell type or during a certain condition. For instance, the age-dependent diminution of apo AI gene expression has been shown to occur at the transcriptional level and to be associated with a decrease in the binding capacity and expression of HNF4 and a parallel increase in COUP-TFII binding and concentration levels (55). Interestingly, PPAR
gene expression is also diminished in aged rats (23). Further studies are required to demonstrate whether the molecular mechanisms described for the age-dependent decline of apo AI can also explain the decreased expression of PPAR
with age.
Finally, PPAR
is also capable of binding to the
HNF4-RE. Induction of PPAR
gene expression by PPAR
agonists has been reported in different rodent cell lines (22, 24, 56). Our data indicate that PPAR
agonists, such as fenofibrate, induce hPPAR
mRNA levels in primary cultures of human hepatocytes as well as hPPAR
promoter activity, suggesting that these effects are exerted at the transcriptional level. Furthermore, induction of hPPAR
promoter activity is dependent on the identified
HNF4-RE. Autoregulation is a common feature among nuclear receptors. For instance, GRs are regulated by glucocorticoids, and glucocorticoids exert these actions mainly at the level of transcription (57). AR is another nuclear receptor that is autoregulated (58), and three different ER
promoters have been shown to be regulated by estrogens (59).
The coding exons of the hPPAR
gene have been screened for polymorphisms. A leucine-to-valine missense mutation at codon 162 in exon 5 of the hPPAR
gene (L162V) was recently identified (34, 35). This polymorphism shows an enhanced transactivation activity (34) and has been associated with altered plasma lipid and lipoprotein concentrations in type 2 diabetic subjects (34), supporting a role for PPAR
in dyslipidemia and diabetes in humans also. Variants have been identified in other exons and introns of the hPPAR
gene (Ref. 60 and Flavell, D., I. Pineda Torra, and B. Staels, unpublished observations). However, these polymorphisms either show low frequency in the populations studied or they do not directly affect PPAR
activity due to their location in intronic sequences of the gene. Thus, it will be interesting to search for polymorphisms in the promoter sequences of the gene that may affect PPAR
expression and function.
Taken together, the 5'-flanking region of the hPPAR
gene has been characterized, cloned, and functionally studied. HNF4 induces hPPAR
expression via a DR1 element present in the hPPAR
promoter, whereas this effect is antagonized by the orphan receptor COUP-TFII. In addition, PPAR
itself modulates its own expression. Knowledge of the hPPAR
promoter structure will allow further studies of the molecular mechanisms governing hPPAR
gene regulation.
 |
MATERIALS AND METHODS
|
---|
DNA Sequencing
Sequencing of a BAC clone containing the hPPAR
gene (kind gift of B.Wilkinson; Glaxo Welcome, Research Triangle Park, NC) was performed using the dRhodamine terminator cycle sequencing kit on an ABI Prism 377 DNA sequencer (Perkin-Elmer Corp., La Jolla, CA).
Rapid Amplification of cDNA Ends (5'-RACE) PCR and RT-PCR Analysis
Total human liver RNA was isolated using the guanidinium isothiocyanate method (61). 5'-RACE was performed on 1 µg total RNA using the 5' RACE-System for Rapid Amplification of cDNA Ends kit (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturers instructions. For the determination of the 5'-end of exon 1a, first-strand synthesis of hPPAR
cDNA was carried out using the IP7 oligonucleotide (5'-CAAGCAGTCCTCCTACCTCAGCCTTCCAAG-3') located in exon 1b (Fig. 1C
) as a gene-specific primer. After RNase H treatment and poly(dC)-tailing of the cDNA with a terminal deoxynucleotide transferase, the resulting cDNA (5 µl) was amplified in a PCR using the Abridged Anchor Primer (AAP) and the internal gene-specific primer YGSP1 (5'-AGTTCTGAGCTTCTCTTGCCACAAACAGTTGAGTCC-3') located in exon 1a (Fig. 2D
). PCR cycling parameters were as follows: initial denaturation at 95 C for 5 min followed by 35 cycles of 1 min at 95 C, 40 sec at 57 C, and 40 sec at 72 C with a final elongation at 72 C for 3 min. The PCR product was then diluted 100-fold, and the diluted PCR (5 µl) was used for a nested PCR under the same conditions described above with the Abridged Universal Amplification Primer (AUAP) (nested to AAP) and oligonucleotide IP18 (5'-AGAGTCGCTGCGGTCCCCAGAC-3') (nested to YGSP1). Oligonucleotides AUAP and AAP were provided in the 5'-RACE kit. PCR products were visualized on a 2% agarose gel and sequenced as described above. RT-PCR amplification of the hPPAR
5'-UTR was performed as described (31) using oligonucleotides 15C (5'-CAGGGCCCTGTCTGCTCTGTGCA-3'), 13 (5'-CTCTTATCTATGAAGCAGGAAGCACG-3'), and IP3.
RNase Protection Analysis
The RNase protection probe used to map the transcription initiation site of the hPPAR
gene was amplified by PCR from the BAC-hPPAR
clone using oligonucleotides IP24 (5'-CGCGGGGCCCGGGGTCTCGGGGTCTCCG-3') and IP18 and cloned into the EcoRV site of pBluescript SK+ (Stratagene, La Jolla, CA). After linearizing with XhoI, antisense RNA was transcribed with T3 polymerase using the Maxiscript Kit (Ambion, Inc., Austin, TX) as described by the manufacturer. This 337-nucleotide probe contains cRNA complementary to the hPPAR
5'-flanking region (bp -22 to +213) and to the polylinker of the pBluescript SK+ vector (102 bp). RNase protection analysis was carried out using the Hybspeed RNase protection Kit (Ambion, Inc.) as described previously (31).
Real-Time PCR mRNA Quantification
PPAR
and 28S mRNAs were quantified by reverse transcription followed by Real-Time PCR using a LightCycler (Roche Diagnostics, Meylan, France) with oligonucleotide primers specific for hPPAR
(5'-ATATCTCCCTTTTTGTGGCTGCTA-3' and 5'-TCCGACTCCGTCTTCTYGATGA-3') and 28S (62). Reactions were carried out in a 20-µl reaction containing a 500-nM concentration of each primer, 4 mM MgCl2, and the LightCycler-FastStart DNA Master SYBR Green I mix as recommended by the manufacturer with the following conditions: 95 C for 8 min, followed by 40 cycles of 10 sec at 95 C, 10 sec at 55 C and 14 sec (for hPPAR
) or 15 sec (for 28S) at 72 C. hPPAR
mRNA levels were normalized to 28S mRNA.
Cloning of the hPPAR
Promoter and Construction of Reporter Plasmids
Genomic sequences between -3,593 and +83 (numbered on the basis of the distance from the transcription initiation site) obtained by direct sequencing of the BAC-hPPAR
clone were amplified by PCR using oligonucleotides IP34 (5'-GCCAGGAGCAGCCACCAGGGAAATC-3') and 5-GSP (5'-AGTCCTCGGTGTGTGTCCTCGCTCCTC-3') from BAC clone DNA. The resulting product was cloned blunt into the EcoRV site of pBluescript SK+ (Stratagene). Reporter constructs were generated thereafter by digestion using restriction sites within the hPPAR
promoter. The pBS(IP345GSP) construct was digested with HindIII or both HindIII and XhoI, and the resulting promoter digestion products were gel purified and cloned into a HindIII or XhoI plus HindIII-digested pGL3 basic plasmid (Promega Corp., Madison, WI), creating the reporter vectors p
(H-H)-pGL3 and p
(X-H)-pGL3, respectively. Plasmids p
(E-H)-pGL3 and p
(P-H)-pGL3 were generated by digestion of p
(X-H)-pGL3 with EcoRI plus XhoI or PstI plus XhoI, Klenow fill in of cohesive ends, and subsequent ligation of the obtained blunt ends.
HNF4-REx2S-TKpGL3 and
HNF4-REmutx2S-TKpGL3 were created by ligating two copies of the corresponding double-stranded oligonucleotides (5'-GATCCTGGAGGGTGGGGCAAAGTTCACCATAGGTA-3' for
HNF4-REx2S-TKpGL3 and 5'-GATCCTGGAGGGTGCAGCAAAGTTCACCATAGGTA-3' for
HNF4-REmutx2S-TKpGL3) in the sense orientation into the BamHI site of TK-pGL3. All constructs were verified by restriction site digestion and sequence analysis. Transcription factor analysis of the promoter was performed using the MatInspector program (http://genomatix.gsf.de/cgi-bin/matinspector/matinspector.pl).
Site-Directed Mutagenesis
To create p
(H-H)
HNF4-REmut-pGL3, mutation of the
HNF4-RE was generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) and oligonucleotide (5'-GGTGTCTGGAGGCTGCAGCAAAGTTCACCATAG-3') and its complementary oligonucleotide according to the manufacturers recommendations.
Cell Culture and Transient Transfection Assays
Human liver specimens were collected for transplantation at the Moscow Center, and hepatocytes were obtained by a two-step collagenase perfusion as previously described (63). Donors were physically healthy subjects who died after traumatic brain injury. Permission to use the remaining untransplanted donor liver for scientific research purposes was obtained from the Ministry of Health of the Russian Federation. After 20 h of culture the medium was discarded and 250 µM fenofibric acid (Laboratories Fournier, Daix, France) or vehicle was added at the indicated concentration in serum-free medium for 24 h. HepG2 (human hepatoblastoma), RK13 (rabbit kidney), and Cos cells were cultured as previously described (31). HepG2 (105), RK13 (8 x 104), and Cos (4 x 104) cells were transfected using the cationic lipid RPR 120535B (kind gift of Aventis, Vitry, France) as described previously (64). One hundred nanograms of the indicated pGL3 basic firefly luciferase reporter constructs or 10 ng of the TK-pGL3 reporters were cotransfected with or without 3050 ng of pSG5-hHNF4
1 (termed HNF4 throughout this manuscript, kindly provided by B. Laine), pcDNA3-COUP-TFII [subcloned from pMT2-COUP-TFII (65) into pcDNA3] or pSG5-hPPAR
, and either 20 ng or 5 ng of the internal control Renilla luciferase reporter plasmids, pRL-Null (when basal promoter activities of the different deletion constructs were examined) (Promega Corp.) and pRL-C+ (for the remaining transfection experiments), respectively. pRL-C+ was obtained by replacing the luc + cassette in pGL3-C+ (Promega Corp.) with the Renilla gene. When either HNF4 or COUP-TFII plasmid was not cotransfected, pSG5 (Stratagene) or pcDNA3 (Invitrogen) empty vectors were added to the transfection mixture. Luciferase activity was assayed 36 h later using the Dual-Luciferase Reporter Assay System (Promega Corp.). Transfections were carried out in triplicate, and each experiment was repeated at least twice.
EMSAs
HNF4, COUP-TFII, PPAR
, and RXR
were synthesized in vitro using the TNT Quick Coupled Transcription/Translation System (Promega Corp.). Double-stranded oligonucleotides were end-labeled with [
-32P]ATP by using T4-polynucleotide kinase. Proteins (2 µl) were incubated for 15 min at room temperature in a total volume of 20 µl with 2.5 µg poly (dI-dC) and 1 µg herring sperm DNA in binding buffer (10 mM HEPES-NaOH, pH 7.8; 100 mM NaCl; 0.1 mM EDTA; 10% glycerol; 1 mg/ml BSA; and 0.5 mM dithiothreitol) before the radiolabeled probe (0.5 ng) was added. Binding reactions were further incubated for 15 min and resolved by 4% nondenaturing polyacrylamide gel electrophoresis in 0.25x Tris-borate-EDTA buffer at room temperature. For competition experiments, increasing amounts of unlabeled oligonucleotide
HNF4-RE,
HNF4-REmut, or C3P (39) were included in the binding reaction just before addition of the labeled oligonucleotide.
 |
ACKNOWLEDGMENTS
|
---|
We thank Christian Duhem, Odile Vidal, and Eric Raspe for their expert technical assistance. We are grateful to Lluis Fajas, Raphael Darteil, and Olivier Barbier for their helpful advice and discussions.
 |
FOOTNOTES
|
---|
This work was supported by European Community Grant ERBFMBICT983214 (to I.P.T.) and BIOMED 2 Grant PL963324 (to Y.J. and B.S.). Y.J. is also supported by British Heart Foundation Grant FS/98058.
Abbreviations: AP1 or 2, Activating protein 1 or 2; AAP, abridged anchor primer; BAC, bacterial chromosomal clone, COUP-TFII, chicken ovalbumin upstream promoter-transcription factor II; DR1, direct repeat-1, FA, fatty acid; HNF4, hepatocyte nuclear factor 4; HNF4-RE, HNF4 regulatory element; NF
B, nuclear factor
B; PPRE, PPAR response element; RACE, rapid amplification of cDNA ends; RNase, ribonuclease; TK, thymidine kinase; UTR, untranslated region.
1 The sequence data for the hPPAR
exon 1a, 1b, and promoter have been submitted to the GenBank database under GenBank accession numbers AF323915, AF323916, and AF323917, respectively. 
Received for publication August 31, 2001.
Accepted for publication January 17, 2001.
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