The Organization, Promoter Analysis, and Expression of the Human PPARgamma Gene*

(Received for publication, January 27, 1997, and in revised form, May 2, 1997)

Lluis Fajas Dagger , Didier Auboeuf §, Eric Raspé Dagger , Kristina Schoonjans Dagger , Anne-Marie Lefebvre Dagger , Regis Saladin , Jamilla Najib Dagger , Martine Laville §, Jean-Charles Fruchart Dagger , Samir Deeb par , Antonio Vidal-Puig **, Jeffrey Flier **, Michael R. Briggs , Bart Staels Dagger Dagger Dagger , Hubert Vidal § and Johan Auwerx Dagger §§

From the Dagger  INSERM U325, Département d'Athérosclérose, Institut Pasteur, F-59019 Lille, France, the § INSERM U449, Faculté de Médecine René Laennec, Université Claude Bernard, F-69373 Lyon, France, the  Ligand Pharmaceuticals Inc., San Diego, California 92121, the par  Department of Medicine and Genetics, University of Washington, Seattle, Washington 98195, and the ** Department of Medicine, Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts 02215

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

PPARgamma is a member of the PPAR subfamily of nuclear receptors. In this work, the structure of the human PPARgamma cDNA and gene was determined, and its promoters and tissue-specific expression were functionally characterized. Similar to the mouse, two PPAR isoforms, PPARgamma 1 and PPARgamma 2, were detected in man. The relative expression of human PPARgamma was studied by a newly developed and sensitive reverse transcriptase-competitive polymerase chain reaction method, which allowed us to distinguish between PPARgamma 1 and gamma 2 mRNA. In all tissues analyzed, PPARgamma 2 was much less abundant than PPARgamma 1. Adipose tissue and large intestine have the highest levels of PPARgamma mRNA; kidney, liver, and small intestine have intermediate levels; whereas PPARgamma is barely detectable in muscle. This high level expression of PPARgamma in colon warrants further study in view of the well established role of fatty acid and arachidonic acid derivatives in colonic disease. Similarly as mouse PPARgamma s, the human PPARgamma s are activated by thiazolidinediones and prostaglandin J and bind with high affinity to a PPRE. The human PPARgamma gene has nine exons and extends over more than 100 kilobases of genomic DNA. Alternate transcription start sites and alternate splicing generate the PPARgamma 1 and PPARgamma 2 mRNAs, which differ at their 5'-ends. PPARgamma 1 is encoded by eight exons, and PPARgamma 2 is encoded by seven exons. The 5'-untranslated sequence of PPARgamma 1 is comprised of exons A1 and A2, whereas that of PPARgamma 2 plus the additional PPARgamma 2-specific N-terminal amino acids are encoded by exon B, located between exons A2 and A1. The remaining six exons, termed 1 to 6, are common to the PPARgamma 1 and gamma 2. Knowledge of the gene structure will allow screening for PPARgamma mutations in humans with metabolic disorders, whereas knowledge of its expression pattern and factors regulating its expression could be of major importance in understanding its biology.


INTRODUCTION

White adipose tissue is composed of adipocytes, which play a central role in lipid homeostasis and the maintenance of energy balance in vertebrates. These cells store energy in the form of triglycerides during periods of nutritional affluence and release it in the form of free fatty acids at times of nutritional deprivation. Excess of white adipose tissue leads to obesity (1-3), whereas its absence is associated with lipodystrophic syndromes (4). In contrast to the development of brown adipose tissue, which mainly takes place before birth, the development of white adipose tissue is the result of a continuous differentiation/development process throughout life (2, 5). During development, cells that are pluripotent become increasingly restricted to specific differentiation pathways. Adipocyte differentiation results from coordinate changes in the expression of several proteins, which are mostly involved in lipid storage and metabolism, that give rise to the characteristic adipocyte phenotype. The changes in expression of these specialized proteins are mainly the result of alterations in the transcription rates of their genes.

Several transcription factors including the nuclear receptor PPARgamma (6, 7), the family of CCAATT enhancer binding proteins (C/EBP)1 (8-13) and the basic helix-loop-helix leucine zipper transcription factor ADD1/SREBP1 (14, 15) orchestrate the adipocyte differentiation process (for reviews, see Refs. 1, 3, 16-18). In contrast to the wide tissue distribution of the various C/EBPs, PPARgamma has been shown to have an adipose-restricted pattern of expression in mouse. The currently favored hypothesis is that C/EBPbeta and delta  induce the expression of PPARgamma (11), which then triggers the adipogenic program. Terminal differentiation then requires the concerted action of both PPARgamma , C/EBPalpha , and ADD-1/SREBP1 (7, 15). Several arguments support the important role of PPARgamma in adipocyte differentiation. First, overexpression of PPARgamma by itself can induce adipocyte conversion of fibroblasts (6). In addition, PPARgamma together with C/EBPalpha can induce transdifferentiation of myoblasts into adipocytes (19). Second, the description of functional PPREs in the regulatory sequences of several of the genes that are induced during adipocyte differentiation, such as the genes coding for adipocyte fatty acid binding protein, aP2 (6), phosphoenolpyruvate carboxykinase (PEPCK) (20), acyl-CoA synthetase (ACS) (21, 22), and lipoprotein lipase (LPL) (23), is consistent with the crucial role attributed to PPARgamma in adipocyte differentiation. Finally, PPAR activators, such as fibrates (24, 25) and fatty acids (7, 26-28), or synthetic PPARgamma ligands, such as the thiazolidinediones (7, 28, 29), induce adipocyte differentiation. In this context, it is interesting to note that prostanoids, which are potent inducers of adipose differentiation programs (30-32), may be one of the natural ligands of PPARgamma . In addition to PPARgamma , PPARalpha , but not PPARdelta , has been shown to have some, albeit weaker, adipogenic activity (33).

To better understand the physiological role of PPARgamma in human physiology, it is crucial that we gain insight into the regulation of PPARgamma gene expression in man. Therefore, we cloned the human PPARgamma cDNAs, determined the structure of the human PPARgamma gene, and studied the expression of the PPARgamma mRNAs and the regulation of their promoter. Both PPARgamma 1 and 2 are produced in human tissues but PPARgamma 2 appears to be the minor isoform in man. In addition to adipose tissue, which contains high levels of PPARgamma , we demonstrate high level expression of human PPARgamma in the colon. The structure of the gene encoding the mouse and human PPARgamma s is highly conserved. Furthermore our results demonstrate that 3 and 1 kb of DNA upstream of the transcription start sites of PPARgamma 1 and gamma 2, respectively, are sufficient to control basal and tissue-specific PPARgamma gene expression.


EXPERIMENTAL PROCEDURES

Materials and Oligonucleotides

The oligonucleotides used for various experiments in this manuscript are listed in Table I.

Table I. Oligonucleotides used in this study listed from 5' to 3'


Name Sequence

LF-2 TCTCCGGTGTCCTCGAGGCCGACCCAA
LF-14 AGTGAAGGAATCGCTTTCTGGGTCAAT
LF-18 AGCTGATCCCAAAGTTGGTGGGCCAGA
LF-20 CATTCCATTCACAAGAACAGATCCAGTGGT
LF-21 GGCTCTTCATGAGGCTTATTGTAGAGCTGA
LF-22 GCAATTGAATGTCGTGTCTGTGGAGATAA
LF-23 GTGGATCCGACAGTTAAGATCACATCTGT
LF-24 GTAGAAATAAATGTCAGTACTGTCGGTTTC
LF-25 TCGATATCACTGGAGATCTCCGCCAACAG
LF-26 ACATAAAGTCCTTCCCGCTGACCAAAGCAA
LF-27 CTCTGCTCCTGCAGGGGGGTGATGTGTTT
LF-28 GAAGTTCAATGCACTGGAATTAGATGACA
LF-29 GAGCTCCAGGGGTTGTAGCAGGTTGTCTT
LF-33 GACGGGCTGAGGAGAAGTCACACTCTGA
LF-35 AGCATGGAATAGGGGTTTGCTGTAATTC
LF-36 TAGTACAAGTCCTTGTAGATCTCC
LF-44 GTCGGCCTCGAGGACACCGGAGAG
LF-58 CACTCATGTGACAAGACCTGCTCC
LF-59 GCCGACACTAAACCACCAATATAC
LF-60 CGTTAAAGGCTGACTCTCGTTTGA
AII J PPRE GATCCTTCAACCTTTACCCTGGTAGA
ACO PPRE GATCCCGAACGTGACCTTTGTCCTGGTCCC
LPL PPRE GATCCGTCTGCCCTTTCCCCCTCTTCA
 gamma AS GCATTATGAGCATCCCCAC
 gamma S TCTCTCCGTAATGGAAGACC
 gamma 2S GCGATTCCTTCACTGATAC
CDS TTCTAGAATTCAGCGGCCGC(T)30(G/A/C)(G/A/C/T)

Isolation of the Human PPARgamma cDNA and Gene, Restriction Mapping, Determination of Intron/Exon Boundaries, and DNA Sequencing

A human adipose tissue lambda gt11 library was screened with a random primed 32P-labeled 200 bp fragment, covering the DNA-binding domain of the mouse PPARgamma cDNA. After hybridization, filters were washed in 2 × SSC, 0.1% SDS for 10 min at 20 °C and twice for 30 min in 1 × SSC, 0.1% SDS at 50 °C and subsequently exposed to x-ray film (X-OMAT-AR, Kodak). Of several positive clones, one clone 407 was characterized in detail. The insert of this clone, starting ±90 bp upstream of the ATG start codon and extending downstream into the 3'-untranslated region (UTR) sequence, was subcloned in the EcoRI site of pBluescript SK- to generate clone 407.2. Sequence analysis of 407.2 confirmed it as being the human homologue of the mouse PPARgamma 2 cDNA. While this work was in progress, other groups also reported the isolation of human PPARgamma 2 cDNA clones (34, 35).

To isolate genomic P1-derived artificial chromosome (PAC) clones containing the entire human PPARgamma gene, the primer pair LF-3 and LF-14 was used to amplify an 86-bp probe with human genomic DNA as template. This fragment was then used to screen a PAC human genomic library from human foreskin fibroblasts. Three positive clones, P-8854, P-8855, and P-8856, were isolated. Restriction digestion and Southern blotting were performed according to classical protocols as described by Sambrook et al. (36). Sequencing reactions were performed, according to the manufacturer instructions, using the T7 sequencing kit (Pharmacia Biotech Inc.).

Determination of the Transcription Initiation Site: Primer Extension and 5'-Rapid Amplification of cDNA Ends (5'-RACE)

Primer Extension

The oligonucleotide LF-35 was 32P-labeled with T4-polynucleotide kinase (Amersham Life Science, Inc) to a specific activity of 107 dpm/50 ng and purified by gel electrophoresis. For primer extension, 105 dpm of oligonucleotide was added in a final volume of 100 µl to 50 µg of adipose tissue total RNA isolated from different patients. Primer extension analysis was performed following standard protocols utilizing a mixture of 1.25 units of avian mycloblastosis virus reverse transcriptase (Life Technologies, Inc.) and 100 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.). A sequencing reaction and molecular mass standards were used to map the 5'-end of the extension products.

5'-RACE

The Marathon cDNA amplification kit (CLONTECH) was used to obtain a library of adaptor-ligated double-stranded cDNA from human adipose tissue. 1 µg of poly(A)+ RNA was used as a template for the first strand synthesis, with the 52-mer CDS primer and 100 units of the MMLV reverse transcriptase in a total volume of 10 µl. Synthesis was carried out at 42 °C for 1 h. Next, the second strand was synthetized at 16 °C for 90 min in a total volume of 80 µl containing the enzyme mixture (RNase H, Escherichia coli DNA polymerase I, and E. coli DNA ligase), the second strand buffer, the dNTP mix, and the first strand reaction. cDNA ends were then made blunt by adding to the reaction 10 units of T4 DNA polymerase and incubating at 16 °C for 45 min. The double-stranded cDNA was phenol/chloroform extracted, ethanol precipitated, and resuspended in 10 µl of water. Half of this volume was used to ligate the adaptor to the cDNA ends (adaptor sequence CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT) in a total volume of 10 µl using 1 unit of T4 DNA ligase. The ligation reaction was incubated 16 h at 16 °C. The resulting cDNA library was diluted to a final concentration of 0.1 mg/ml.

The 5'-end of PPARgamma 1 was PCR-amplified using 5 µl of the library as a template with the oligonucleotides AP-1 (binding to the adaptor) and LF-45 (binding antisense to the 5'-end of the PPARgamma 1). After an initial denaturing step at 95 °C for 3 min, 25 cycles were done at the following conditions: 10 s at 95 °C, 20 s at 60 °C, and 30 s at 72 °C. The resulting PCR product was reamplified for 30 additional cycles at the same conditions using the nested oligonucleotides AP2 (nested to AP1) and LF-2 (nested to LF-45). The PCR product was analyzed on a 2% agarose gel, treated with Pfu polymerase (Stratagene) and cloned into the EcoRV site of pBluescript SK+. A total of 20 white colonies were grown and sequenced from both ends using the oligonucleotides T3 and T7 (Dye Terminator Cycle sequencing kit, Applied Biosystems).

For the determination of the 5'-end of PPARgamma 2, the same procedure was followed except that the oligonucleotide LF-14 (specific for the PPARgamma 2 5'-UTR) was used in the first round PCR, and the oligonucleotide LF-35 (nested to LF-14) was used in the second round PCR with the same cycling conditions.

Tissue Biopsies and Cell Culture

Omental adipose tissue, small and large intestine, kidney, muscle, and liver biopsies were obtained from non-obese adult subjects undergoing elective surgery or endoscopy. All subjects had fasted overnight before surgery (between 8.00 p.m. and 10 a.m.) and received intravenous saline infusion. They had given informed consent, and the project was approved by the ethics committee of the University of Lille. All tissue was immediately frozen in liquid nitrogen until RNA preparation.

Standard cell culture conditions were used to maintain 3T3-L1 (obtained from ATCC), CV-1 (a kind gift from Dr. R. Evans, Salk Institute, La Jolla, CA), and Hep G2 cells (ATCC). BRL-49,653, supplied by Ligand Pharmaceuticals, San Diego, CA (in DMSO) and fatty acids (in ethanol) were added to the medium at the concentrations and times indicated. Control cells received vehicle only. Fatty acids were complexed to serum albumin contained in delipidated and charcoal-treated fetal calf serum by preincubation for 45 min at 37 °C.

mRNA Analysis by RT-Competitive PCR Assay

RNA preparation of total cellular RNA was performed as described previously (37). The absolute mRNA concentration of the differentially spliced PPARgamma variants was measured by reverse transcription reaction followed by competitive polymerase chain reaction (RT-competitive PCR) in the presence of known amounts of competitor DNA yielding amplicons of different size allowing the separation and the quantification of the PCR products. The competitor was constructed by deletion of a 74-bp fragment (nucleotides +433 to +507 by HindIII digestion) of PPARgamma 1 cloned into pBluescript KS+, yielding pBSCompPPARgamma . Working solution of the competitor was prepared by in vitro transcription followed by serial dilution in 10 mM Tris-HCl (pH 8.3), 1 mM EDTA buffer. For RT-competitive PCR, the antisense primer hybridized to the 3'-end of exon 3 (gamma AS:5'-GCATTATGAGCATCCCCAC-3', nt +600 to +620) and the sense primer to exon 1 (gamma S:5'-TCTCTCCGTAATGGAAGACC-3', nt +146 to +165) or to the B exon (gamma 2S:5'-GCGATTCCTTCACTGATAC-3', nt +41 to +59). Therefore, the same competitor served to measure either total PPARgamma mRNAs (gamma 1 + gamma 2; with primers gamma AS and gamma S) or, specifically, PPARgamma 2 mRNA (with primers gamma AS and gamma 2S). The gamma AS/gamma S primer pair gave PCR products of 474 and 400 bp for the PPARgamma mRNAs and competitor, respectively. The primer pair gamma AS/gamma 2S gave 580 bp for PPARgamma 2 mRNA and 506 bp for the competitor. For analysis of the PCR products, the sense primers gamma S and gamma 2S were 5'-end labeled with the fluorescent dye Cy-5 (Eurogentec, Belgium).

First-strand cDNA synthesis was performed from total RNA (0.1 µg) in the presence of the antisense primer gamma AS (15 pmol) and of thermostable reverse transcriptase (2.5 units; Tth DNA polymerase, Promega) as described (38). After the reaction, half of the RT volume was added to the PCR mix (90 µl) containing the primer pair gamma AS/gamma S for the assay of PPARgamma total mRNA, whereas the other half was added to a PCR mix (10 mM Tris-HCl, pH 8.3, 100 mM KCl, 0.75 M EGTA, 5% glycerol, 0.2 mM dNTP, 5 units of Taq polymerase) containing the primer pair gamma AS/gamma 2S for the assay of PPARgamma 2 mRNA. Four aliquots (20 µl) of the mixture were then transferred to microtubes containing a different, but known, amount of competitor. After 120 s at 95 °C, the samples were subjected to 40 PCR cycles (40 s at 95 °C, 50 s at 55 °C, and 50 s at 72 °C). The fluorescent-labeled PCR products were analyzed by 4% denaturing polyacrylamide gel electrophoresis using an automated laser fluorescence DNA sequencer (ALFexpress, Pharmacia, Uppsala, Sweden), and integration of the area under the curve using the Fragment manager software (Pharmacia) was performed as described (38).

To validate this technique, human PPARgamma 2 mRNA was synthesized by in vitro transcription from the expression vector pSG5hPPARgamma (Riboprobe system, Promega) and quantified by competitive PCR over a wide range of concentrations (0.25-25 attomole (amol) added in the RT reaction). Standard curves obtained when assaying PPARgamma total mRNA or PPARgamma 2 mRNA are shown in Fig. 2C. The linearity (r = 0.99) and the slopes of the standard curves (0.98 and 1.11) indicated that the RT-competitive PCR is quantitative and that all the mRNA molecules are copied into cDNA during the RT step. The lower limit of the assay was about 0.05 amol of mRNA in the RT reaction, and the interassay variation of the RT-competitive PCR was 7% with six separated determinations of the same amount of PPARgamma mRNA.


Fig. 2.

RT-competitive PCR method to measure PPARgamma mRNA levels. A, scheme highlighting the features of the vector pSG5hPPARgamma 2 and the vector derived from it, pBSCompPPARgamma , which served to synthesize competitor DNA. The primer pairs used in the RT and PCR reactions as well as the different-sized amplicons obtained are indicated. B, typical analysis of the fluorescence-labeled PCR products on an automated fluorescence DNA sequencer using a denaturing 4% polyacrylamide gel electrophoresis. C, validation of the RT-competitive PCR assay and standard curves obtained when assaying PPARgamma total mRNA or PPARgamma 2 mRNA. The linearity (r = 0.99) and the slopes of the standard curves (0.98 and 1.11) indicated that the RT-competitive PCR is really quantitative and that all the mRNA molecules are copied into cDNA during the RT step.


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Western Blot Analysis of PPARgamma

Cells and tissues were homogenized in a lysis buffer of PBS containing 1% Triton X-100 (Sigma). Tissues were homogenized in extraction buffer containing PBS and 1% Nonidet P-40 (Sigma), 0.5% sodium deoxycholate (Sigma), 0.1% SDS (Sigma). Fresh mixture protease inhibitor (ICN) was added (100 mg/ml AEBSF, 5 mg/ml EDTA, 1 mg/ml leupeptin, 1 mg/ml pepstatin). Protein extracts were obtained by centrifugation of the lysate at 4 °C, and concentration was measured with the Bio-Rad DC Protein colorimetric assay system.

Protein (100 µg) was separated by SDS-PAGE, transferred to nitrocellulose membrane (Amersham Life Science, Inc.), and blocked overnight in blocking buffer (20 mM Tris, 100 mM NaCl, 1% Tween-20, 10% skim milk). Filters were first incubated for 4 h at room temperature with rabbit IgG anti-mPPARgamma (10 mg/ml), raised against an N-terminal PPARgamma peptide (amino acids 20-104), and next developed for 1 h at room temperature with a goat anti-rabbit IgG (whole molecule) peroxidase conjugate (Sigma) diluted at 1/500. The complex was visualized with 4-chloro-1-naphtol as reagent.

Analysis of Promoter Activity

To test the activity of the human PPARgamma promoters several reporter constructs were made. A 1-kb fragment of PAC clone 8856 was isolated by PCR using the oligonucleotides LF-35 (binding antisense in the PPARgamma 2 5'-UTR) and the oligonucleotide LF-58 (binding sense at position -1000 of the PPARgamma 2), was sequenced, and was inserted into EcoRV site of pBluescript (Stratagene, La Jolla, CA). After digestion of plasmid pBSgamma 2p1000 with SmaI and KpnI, the insert was cloned into the reporter vector pGL3 (Promega), creating the expression vector pGL3gamma 2p1000. To isolate the PPARgamma 1 promoter, an 8-kb EcoRI fragment, which hybridized with the oligonucleotide LF-2 (corresponding to the 5'-UTR of gamma 1), was cloned into pBluescript. Partial mapping and sequencing of this clone revealed the presence of a 3-kb fragment upstream of the transcription initiation site. To test for promoter activity, a SacI/XhoI digestion of this clone containing the 3-kb promoter was inserted in the same sites of pGL3, resulting in the final vector pGL3gamma 1p3000. The pSG5-haPPARgamma (39) and pMSV-C/EBPalpha (10) expression vectors were described elsewhere. Transfections were carried out in 60-mm plates using standard calcium phosphate precipitation techniques (for 3T3-L1, CV-1, and COS cells) (22). Luciferase and beta -galactosidase assays were carried out exactly as described previously (22).

Electrophoretic Mobility Shift Assays (EMSA) and Oligonucleotide Sequences

haPPARgamma (39), hPPARgamma 2, and mRXRalpha (40) proteins were synthesized in vitro in rabbit reticulocyte lysate (Promega). Molecular weights and quality of the in vitro translated proteins were verified by SDS-PAGE. PPAR (2 µl) and/or RXR (2 µl) were incubated for 15 min on ice in a total volume of 20 µl with 1-ng probe, 2.5 µg of poly(dI-dC) and 1 µg of herring sperm DNA in binding 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 (from 10- to 200-fold molar excess) of cold oligonucleotide (AII-J-PPRE, 5'-GATCCTTCAACCTTTACCCTGGTAGA-3' (41); acyl-CoA oxidase (ACO)-PPRE, 5'-GATCCCGAACGTGACCTTTGTCCTGGTCCC-3' (42); or LPL-PPRE, 5'-GATCCGTCTGCCCTTTCCCCCTCTTCA-3') (23) were included just before adding T4-PNK end-labeled AII-J-PPRE oligonucleotide. DNA-protein complexes were separated by electrophoresis on a 4% polyacrylamide gel in 0.25 × TBE buffer at 4 °C (43).


RESULTS

Cloning of the human PPARgamma cDNA

A cDNA probe containing a 200-bp (KpnI-BglII) fragment encoding the DNA binding domain of the mouse PPARgamma (44) was used to screen a human adipose tissue cDNA library. Several independent human PPARgamma cDNA clones, representing both the PPARgamma 1 and PPARgamma 2 subtypes, were isolated and sequenced (Fig. 1A). The human PPARgamma protein shows a 99% similarity and a 95% identity on the amino acid level with mouse PPARgamma (Fig. 1A). Interestingly, the initiation codon for human PPARgamma 1 is different from the mouse PPARgamma 1 (Fig. 1B). Therefore, human PPARgamma 1 is 2 amino acid residues longer than its mouse homologue.


Fig. 1. Sequence of the human PPARgamma cDNA and comparison with the mouse PPARgamma sequence. A, sequence comparison of mouse and human PPARgamma . Identical amino acids are indicated by a vertical line and conservative changes are indicated by a dot. B, splicing of exon A2 and exon B with exon 1. hPPARgamma 1 contains two extra amino acids relative to mPPARgamma 1. The presence of a promoter in front of exon A and B is indicated by an arrow. Nucleotides in capitals are located in exons, whereas the nucleotides in the intron are in lowercase.
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Expression of PPARgamma mRNA and Protein

To analyze the expression pattern of the two PPARgamma isoforms, we developed a sensitive RT-competitive PCR assay in which relative amounts of PPARgamma 1 and gamma 2 mRNA could be measured from minute quantities of RNA (0.1 µg total RNA). This method relies on the co-amplification in the same tube of known amounts of competitor DNA (Fig. 2A) with PPARgamma cDNA, obtained after reverse transcription from total tissue RNA. The competitor and the target use the same fluorescently labeled PCR primers but yield amplicons with a different size (Fig. 2, A and B), allowing their separation and quantification on an automated sequencing gel at the end of the reaction (Fig. 2C). All tissue preparations were carefully dissected, and the RNA was shown to be free of contamination with adipose tissue as evidenced by the absence of human leptin mRNA by RT-competitive PCR assay (38) (data not shown). PPARgamma 1 mRNA was the predominant PPARgamma isoform in all human tissues analyzed (Fig. 3). PPARgamma 2 was detected in both liver and adipose tissue where it accounted for 15% of all PPARgamma mRNA. Interestingly, in addition to the high level of expression of PPARgamma mRNA expected in adipose tissue, we found a very high level of PPARgamma 1 in large intestine. In contrast to adipose tissue, large intestine contained no PPARgamma 2 mRNA. Kidney, liver, and small intestine contained intermediate levels of PPARgamma mRNA, whereas PPARgamma mRNA was barely detectable in skeletal muscle (Fig. 3).


Fig. 3. Expression of PPARgamma 1 and gamma 2 mRNA. PPARgamma mRNA levels were measured by RT-competitive PCR as described in samples of the indicated tissues obtained from three different normal individuals. The results represent the mean ± S.D. of the three analyses.
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Next, the expression of the human PPARgamma protein was analyzed in human adipose tissue. A PPARgamma specific antibody, raised against a peptide corresponding to amino acids 20-104 of mPPARgamma , was used. This antibody is highly specific for PPARgamma and does not cross-react with PPARalpha and delta  in Western blot experiments (Fig. 4, A and B). Using this antibody in a Western blot of protein extracts from human adipose tissue, we detected a band (potentially representing a doublet) with an approximate molecular mass of 60 kDa, consistent with the predicted mass of PPARgamma 1 and 2 and with the protein product generated by in vitro transcription/translation in the presence of [35S]methionine (Fig. 4, B and C).


Fig. 4. In vitro translation and immunoblot of human PPARgamma in adipose tissue. A, SDS-PAGE of in vitro translated 35S-labeled proteins. B, validation of a PPARgamma antibody. mPPARalpha , mPPARgamma , haPPARgamma , and hPPARgamma were synthesised in rabbit reticulocyte lysate and electrophoresed on a 10% SDS-PAGE gel. The proteins were transferred to nitrocellulose and developed with a rabbit anti-PPARgamma antibody. As indicated, the antibody specifically recognized PPARgamma but did not recognize PPARalpha . C, Western blot of three different human adipose tissue samples. Two different protein extracts of the same adipose tissue sample were run side-by-side. WAT, white adipose tissue.
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PPARgamma 2 Binds and Transactivates through a PPRE

To analyze whether PPARgamma could bind to a PPRE, classically composed of direct repeats spaced by one intervening nucleotide (DR-1), EMSA was performed using in vitro transcribed/translated PPARgamma 2 protein. An oligonucleotide containing a high affinity PPRE, previously identified in the apoA-II promoter J site, was used in EMSA (29). This oligonucleotide was capable of binding both human and hamster PPARgamma /mRXRalpha heterodimers in EMSA (Fig. 5, lanes 5 and 6). Homodimers of either hPPARgamma or mRXRalpha , however, were incapable of binding to this oligonucleotide. When increasing concentrations of unlabeled apoA-II J site were added as competitor, binding of the hPPARgamma /mRXRalpha heterodimer to the labeled PPRE was almost completely inhibited (Fig. 5, lanes 7-9). In addition, oligonucleotides corresponding to the PPRE elements of the ACO or LPL genes competed, albeit less efficiently (Fig. 5, compare lanes 7 with 10 and 13).


Fig. 5. Electrophoretic mobility shift analysis demonstrating binding of hPPARgamma 2/RXR heterodimers to a PPRE. hPPARgamma 2/RXR heterodimers bind to the AII-Jwt site. EMSA was performed on end-labeled AII-Jwt site oligonucleotide in the presence of in vitro transcribed/translated hPPARgamma , haPPARgamma , and mRXRalpha or unprogrammed reticulocyte lysate (RL unprogr.) in the absence or presence of increasing concentrations of unlabeled wild-type AII-Jwt, ACO-PPRE, and LPL-PPRE oligonucleotides as described under "Experimental Procedures."
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We next verified that the human PPARgamma 2 cDNA was capable of activating gene transcription through a PPRE. Therefore, 3T3-L1 preadipocytes were cotransfected with the PPARgamma 2 expression vector pSG5hPPARgamma 2 and a PPRE-driven luciferase reporter gene. The luciferase gene was under the control of a multimerized ACO-PPRE site and the TK promoter (Fig. 6). hPPARgamma 2 was capable of activating this PPRE-based reporter 2-fold, an effect which was substantially enhanced when hPPARgamma 2 was cotransfected together with RXRalpha . Upon the addition of the PPARgamma ligand BRL-14653, luciferase expression was increased 6-fold when the transfection was done with hPPARgamma 2 alone or at least 10-fold when the cells were co-transfected with both hPPARgamma 2 and mRXRalpha . Similar results were obtained when prostaglandin J2 was used as a PPARgamma ligand (data not shown).


Fig. 6. Human PPARgamma efficiently transactivates a reporter gene driven by a PPRE. Undifferentiated 3T3-L1 cells were cotransfected with the Jwt3-TK-CAT plasmid in the presence of hPPARgamma , mRXRalpha , and both hPPARgamma and mRXRalpha together, or pSG5 vector plasmids. Cells were treated with 9c-RA (1 µM; RA), BRL-49653 (1 µM; BRL), or vehicle (DMSO; Control), and CAT activity was measured and expressed as described under "Experimental Procedures."
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Characterization of the Transcription Initiation Site of the Human PPARgamma Gene

To unambiguously identify the 5'-end of the cDNA, several approaches were undertaken. First, primer extension experiments were performed, utilizing different human adipose tissue RNA samples, and results were independently confirmed by using 5'-RACE. Several primer extension products were seen for the PPARgamma 1 mRNA using primer LF2 (Fig. 7, A and C). The relative positions of the transcription initiation sites as determined by the 5'-RACE were in agreement with the results for primer extension.


Fig. 7. Determination of transcription initiation site of the human PPARgamma 1 and 2 A and B, DNA sequences of the 5'-UTR and part of the A (panel A) and B (panel B) exons of the human PPARgamma gene. Transcription initiation sites as determined by primer extension (long arrows) and 5'-RACE (asterisks) are indicated. C, primer extension. Total human adipose tissue was used in primer extension. The major extension products are indicated by arrows. Size standards indicated on the right consist of a sequencing reaction. The sequence corresponding to the 5'-UTR is shown in panel A.
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One major extension product of 62 bp was observed consistently with the primer LF-35 for PPARgamma 2. A second extension product of 96 bp was found using the same primer (Fig. 7, B and C). The results of 5'-RACE were consistent with the primer extension (Fig. 7B). The transcription initiation sites identified correlated well with the transcription initiation sites observed for the mouse PPARgamma 2 mRNA (45). A striking feature of the human PPARgamma 2 5'-UTR is its high degree of sequence conservation with the mouse 5'-UTR (see Fig. 9). It awaits further study to determine the exact implications of this conservation.


Fig. 9. Sequence of the proximal promoter of the human and mouse PPARgamma 1 (A) and gamma 2 (B) genes. The relevant consensus binding sites of transcription factors (TATA sequence, the GC-rich sequences, the C/EBP, and AP-1 consensus sites) are indicated by boldface letters. Numbering is relative to the transcription initiation site. The asterisks indicate the transcription initiation site of PPARgamma .
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Structural Organization of the Human PPARgamma Gene

To clone the human PPARgamma gene and to determine its promoter sequence, we screened a PAC human genomic library derived from human foreskin fibroblasts. Three positive clones (P-8854, P-8855, and P-8856), each spanning >100 kb of genomic sequence, were isolated. All three clones were next shown to hybridize with the oligos LF-14 (corresponding to exon B) and LF-36 (exon 6), which indicates that they span most of the PPARgamma coding region. More importantly, clone P-8856 also hybridized to oligo LF-2 and, hence, contains the transcription initiation site for PPARgamma 1 and 2. This clone was further characterized by Southern blotting and partial sequence analysis, which allowed the construction of a physical map of the human PPARgamma locus (Fig. 8). The human PPARgamma gene spans more than 100 kb. The PPARgamma 1 and PPARgamma 2 mRNAs are encoded by 8 and 7 exons, respectively. The 5'-untranslated region of the PPARgamma 1 mRNA is encoded by two exons, which we, in analogy to the nomenclature used for the mouse gene, named exon A1 and A2. The coding region of PPARgamma 1 is contained in the next six exons (exons 1 to 6). Exons 1 to 6 also encode the majority of PPARgamma 2 mRNA. The additional 28 amino acids of PPARgamma 2 as well as the 5'-UTR are encoded by the B exon, which is located between exons A2 and A1.


Fig. 8. Comparison of the gene organization of the mouse and human PPARgamma genes. The genes are shown in 5' to 3' orientation and are drawn to scale. Exons are denoted by gray or black rectangles and introns by a solid line. Restriction site for BamHI, is indicated by a B. The location of the ATG start-codon is indicated. The asterisk indicates the different ATG used in mPPARgamma 1 (see Fig. 1).
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The length of the introns was determined by long-range PCR (CLONTECH Tth polymerase mix) using the oligonucleotide pairs LF-3/LF-18, LF-20/LF-21, LF-22/LF-23, LF-24/LF-25, LF-26/LF-27, and LF-28/LF-29 and the PAC clone P-8856 as a template. The intron-exon boundaries were sequenced using genomic DNA as template. The 5' donor and 3' acceptor splice sites were found to be conforming to the consensus splice donor and acceptor sequences (Table II). The DNA binding domain of the receptors is encoded by exons 2 and 3, each encoding a separate zinc finger. The entire ligand binding domain is encoded by exons 5 and 6, which are separated by 16.3 kb of intron sequence.

Table II. Intron-exon boundaries of the PPARgamma exons

The nucleotides in the exon are indicated in uppercase letters, whereas the flanking nucleotides in the intron are in lowercase. The approximate size of the introns are indicated in kilobases, and the exact length in base pairs of the exons are indicated between brackets. Amino acids encoded by the nucleotides flanking the intron/exon border are indicated by their letter symbol. The stop codon is indicated by an asterisk.

Exon (bp) Donor Intron in kb Acceptor Exon

A1 (97) CGCAG gtcagagt.. >20 ..ttgttaag ATTTG A2
A2 (74) TAACG gtaagtaa.. >20 ..cctttcag AA ATG 1
               M
B (211) CAA G gtaaagtt.. 21 ..cctttcag AA ATG 1
 Q             E  M
1 (231) CAA A gtatgatg.. 1.6 ..atacacag GT GCA 2
 Q             S  A
2 (170) C AAG gtaattaa.. 9.5 ..ctttgcag GGT T 3
   K             G
3 (139) AAT G gtaagtaa.. 10.7 ..ctctatag CC ATC 4
 N             A  I
4 (203) A TCA gttagttc.. 10 ..atttgcag CCA T 5
   S             P
5 (451) GGA G gtaagatt.. 16.3 ..ttccccag AC CGC 6
 G             D  R
6 (248) TAC TAG cagaga..
 Y   *

Tissue-specific Determinants of the Human PPARgamma Promoter

We next subcloned the region 5' to the transcription initiation sites of PPARgamma 1 and gamma 2 and sequenced the proximal promoters (Figs. 7 and 9). No canonical TATA box was found in the PPARgamma 1 promoter region close to the transcription initiation site (Fig. 7). The sequence immediately upstream of the transcription initation site is extremely GC-rich, including several consensus Sp1 binding sites. Also a CCAAC box was found in the proximal promoter. Whether any of these factors are important for the regulation of PPARgamma 1 gene expression awaits further study. The PPARgamma 2 promoter contains a TATA-like element at position -68, relative to the transcription initiation site. Furthermore, sequence analysis identified a potential CAAT-like consensus C/EBP protein binding site at -56 (CCAATT) and a perfect AP-1 site at +10 (TGACTCA) (Figs. 7 and 9).

In experiments to evaluate the tissue specificity of these promoters, DNA fragment extending from about -3 kb to +110 bp and -1 kb to +122 bp relative to the transcription initiation sites of PPARgamma 1 and PPARgamma 2 were inserted into the pGL3-basic luciferase vector (Promega) to generate the constructs pGL3-gamma 1p3000 and pGL3-gamma 2p1000 (Fig. 10). These vectors were then transfected into mouse 3T3-L1 and Hep G2 cells. Transfection efficiency of the various cell lines was monitored by evaluation of the activity of control vectors. Relative to the promoterless parent vector, the human PPARgamma 1 promoter fragment stimulated luciferase expression up to 3.5-fold in 3T3-L1 cells, maintained under non-differentiating conditions. In Hep G2 cells, luciferase expression was 9-fold higher with the pGL3-gamma 1p3000 vector relative to the pGL3-basic vector (Fig. 10). Similar results were obtained with COS cells (data not shown). The expression of the pGL3-gamma 2p1000 construct containing the PPARgamma 2 promoter was not different from the pGL3-basic promoterless vector in Hep G2 cells. In undifferentiated 3T3-L1 cells, the PPARgamma 2 promoter induced luciferase expression 2-fold relative to the promoterless control.


Fig. 10. Tissue-specific activity of the PPARgamma promoter. A, normalized luciferase activity of the pGL3-gamma 1p3000 construct containing 3000 bp of regulatory sequence of the human PPARgamma 1 gene after transfection in 3T3-L1 and Hep G2 cells. Transfections were performed as described under "Experimental Procedures." Scheme of the reporter constructs pGL3-gamma 1p3000 used in transfection assays is shown above the graphic. B, normalized luciferase activity of the pGL3-gamma 2p1000 construct containing 1000 bp of regulatory sequence of the human PPARgamma 2 gene after transfection in 3T3-L1 and Hep G2 cells. Transfections were performed as described under "Experimental Procedures." Scheme of the reporter constructs pGL3-gamma 2p1000 used in transfection assays is shown above the graphic.
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DISCUSSION

Two important findings recently underlined the importance of the PPARgamma transcription factor. First, PPARgamma has been identified as one of the key factors controlling adipocyte differentiation and function in rodent systems (6, 7). Second, the recent identification of prostaglandin J2 derivatives and antidiabetic thiazolidinediones as natural and synthetic PPARgamma ligands, respectively (28, 29, 46-48). Thiazolidinediones are a new group of anti-diabetic drugs which improve insulin-resistance (for review, see Refs. 49 and 50). The identification of thiazolidinediones as PPARgamma ligands together with the central role that adipose tissue plays in the pathogenesis of important metabolic disorders, such as obesity and non-insulin-dependent diabetes mellitus (NIDDM), have generated a major interest to determine the role of this PPAR subtype in normal and abnormal adipocyte function in humans.

The PPARgamma gene spans about 100 kb and is composed of 9 exons, which give rise to PPARgamma 1 and PPARgamma 2 mRNAs by differential promoter usage and differential splicing. The gene structure as well as the sequence of the encoded protein are well conserved between human and mice (45) (99% similarity and 95% identity). Relative to the mouse, hamster, and Xenopus PPARgamma (6, 39, 51), the human protein contains two additional amino acids. This is in agreement with the previous reports on the human PPARgamma cDNA (34, 35, 52). The availability of the structure of the human PPARgamma gene and protein will now allow for genetic studies, evaluating its role in disorders such as insulin resistance, NIDDM, and diseases characterized by altered adipose tissue function such as obesity or lipodystrophic syndromes.

To determine tissue-specific patterns of expression of the human PPARgamma gene, we developed an RT-competitive PCR assay. Unlike results of previous reports, which used commercially available blots or single RNA samples (34, 35), we used multiple independent samples to base our conclusions on. As was observed in rodents (6, 7), we found PPARgamma to be strongly expressed in adipose tissue. In addition to adipose tissue, the large intestine had high levels of PPARgamma expression. Several other tissues, such as liver, kidney, and small intestine contained lower but nevertheless considerable levels of PPARgamma RNA. Skeletal muscle, in contrast, contained only trace amounts of PPARgamma mRNA.

In adipose tissue and liver, about 15% of all PPARgamma mRNA was of the PPARgamma 2 type, whereas in the remaining tissues no PPARgamma 2 mRNA was detected. These observations have several important implications. First, our data question the relative importance of PPARgamma 2. Indeed, our results in humans as well as the data by Xue et al. (53) in rodent adipocytes show consistently lower levels of PPARgamma 2 mRNA and protein relative to the PPARgamma 1 subtype. These observations are in line with the previous observations that the N-terminal domain of PPARgamma was dispensable, both regarding transcriptional activation and capacity to induce adipocyte differentiation in vitro (7). However, the N-terminal domain is highly conserved between different species, suggesting it might have an important function in vivo. Second, PPARgamma expression is much more widespread than previously realized, which implies that PPARgamma controls gene expression in several tissues in addition to adipose tissue. Especially striking is the high level of PPARgamma expression in the human large intestine. These reports are consistent with the reported high level expression of PPARgamma in colonic mucosa in mouse (54). It is interesting to note that fatty acids, potential PPAR activators, have been shown to play an important role in modulating the function of the large intestine. For instance diets enriched in saturated lipids have been shown to predispose to the development of colon cancer (55). Furthermore, it has been shown that diets enriched in omega -3 fatty acids, powerful PPAR activators, have a beneficial response on inflammatory diseases of the gastrointestinal tract such as colitis ulcerosa and Crohn's disease (56, 57). Since the high level expression of PPARgamma suggest that it might play an important role in normal and abnormal colonic function, further studies aimed at exploring this are definitely needed. Finally, the low levels of PPARgamma expression in skeletal muscle cells also deserve some reflection. Muscle is responsible for clearance of the majority of glucose in the body and abnormal muscle glucose uptake is one of the prime features of insulin resistance and NIDDM. The low levels of PPARgamma in muscle argue, therefore, that the beneficial effects of thiazolidinedione antidiabetic agents are not likely to be due to a direct effect of these agents on PPARgamma present in the muscle. In fact, even though the liver has considerably higher levels of PPARgamma relative to muscle, thiazolidinediones do not seem to affect PPAR responsive genes in liver tissue at the concentrations commonly used to lower glucose levels (23). This observation together with the observed tissue distribution of PPARgamma suggests that the glucose lowering effects of the thiazolidinedione PPARgamma ligands are primarily a result of their activity on adipose tissue, which then, via a secreted signal, might influence muscle glucose uptake.

To identify the molecular circuitry underlying tissue-specific expression of PPAR, we cloned and performed an initial characterization of the human PPARgamma promoters. As shown, 3000 bp of the PPARgamma 1 and 1000 bp of the PPARgamma 2 promoter account for substantial levels of basal promoter activity. Further functional studies are underway to determine elements necessary for tissue-specific and regulated expression of the PPARgamma gene. In this context, it will be interesting to determine the effects of transcription factors known to induce adipocyte differentiation on PPARgamma expression in this tissue and to define the hierachical role that PPARgamma plays in this process. PPARgamma is not the only transcription factor involved in adipocyte differentiation. In addition to PPARgamma , the basic helix-loop-helix leucine zipper factor ADD-1/SREBP1 and transcription factors of the C/EBP family also play a role in determining adipocyte differentiation. It is interesting to note that, as in the mouse PPARgamma 2 promoter (45), a potential consensus C/EBP response element could be identified in the human PPARgamma 2 promoter by homology searches. This observation fits well with the previous observation that forced expression of C/EBPbeta could induce PPARgamma expression and further studies on this subject are underway (11, 12).

In conclusion, we report the characterization of the human PPARgamma gene structure and furthermore define the structure of the PPARgamma 1 and gamma 2 promoter. In addition, our data show that human PPARgamma has a similar structure and similar transactivation function as the rodent PPARs. The expression patterns of PPARgamma 1 and gamma 2 show that in man, PPARgamma 1 is the predominant form. Our results furthermore demonstrate that, in addition to adipose tissue, human colon expresses high levels of PPARgamma . It is expected that the gene structure will facilitate our analysis of eventual PPARgamma mutations in humans, whereas knowledge of expression patterns and sequence elements, as well as factors regulating PPARgamma gene expression, could be of major importance in understanding PPAR biology.


FOOTNOTES

*   This work was supported in part by grants from ARC, INSERM, and Institut Pasteur.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger Dagger    Research associate from the CNRS.
§§   Research director from the CNRS and to whom correspondence should be addressed: INSERM U 325, Département d'Athérosclérose, Institut Pasteur, 1 Rue Calmette, F-59019 Lille, France. Fax 33-320-87 73 60; E-mail: Johan.Auwerx{at}pasteur-lille.fr.
1   The abbreviations used are: C/EBP, CCAATT enhancer binding protein; LPL, lipoprotein lipase; kb, kilobase(s); bp, base pair(s); UTR, untranslated region; RACE, rapid amplification of cDNA ends; EMSA, electrophoretic mobility shift assays; ACO, acyl-CoA oxidase; PAC, P1-derived artificial chromosome; PPRE, peroxisome proliferator response element; NIDDM, non-insulin-dependent diabetes mellitus.

ACKNOWLEDGEMENTS

The technical help of Delphine Cayet, Veerle Beelen, and Odile Vidal and the support and/or discussion with Drs. Rich Heyman and James Paterniti from Ligand Pharmaceuticals are kindly acknowledged. We acknowledge the gift of materials from Drs. Ronald Evans and Alex Nadzan. Access to an automatic sequencer by Drs. Philippe Froguel and Jorg Hager in the initial phases of this project are greatly appreciated.


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