To identify human PPARs-responsive genes, we established each cell line that expresses any one of the PPAR subtypes (α, β/δ, γ1 or γ2) using the tet-regulated system. Previously, we established the tightly tet-regulatable HepG2 cell clone (HY-Toff) which was transfected with the pTet-off vector, and that this clone had a large induction/repression rate 22. The tet-off regulatable HY-Toff cells were transfected with the pBabepuro (for puromycin resistance) and pBI-EGFP vector harbouring the cDNA for human PPARα, PPARδ, PPARγ1 or PPARγ2. We picked out puromycin-resistant clones. Among these clones, we selected the strictly responsive cell lines (HepG2-tet-off-hPPAR cells) to the concentration of Dox. These cell lines were cultured in the presence of Dox at 0, 0.01, 0.1 or 1000 ng/ml for 5 days, and we found that PPARs expression in these cell lines was induced in a dose-dependent manner (Figure 1A–D). We then examined the time course experiment of each PPAR expression after the removal of Dox. The results showed that both the mRNA (Figure 2A–D) and protein (Figure 2E–H) levels of each PPAR were increased after the removal of Dox from the culture medium. Under these conditions, cell proliferation in these cell lines was not affected until 7 days after the removal of Dox. Based on these results, we decided that the expression of each PPAR on the 5th day was suitable for further analyses, such as the determination of the target genes of each PPAR subtype.

To characterize the regulation of gene expression by each PPAR subtype, we performed oligonucleotide microarray analyses. HepG2-tet-off-hPPAR cells were cultured in the presence or absence of Dox for 5 days, then treated with the PPAR ligands (100 μM fenofibric acid for PPARα, 100 nM GW501516 for PPARδ or 10 μM ciglitizone for PPARγ) or vehicle for 24 h. Total RNA samples were prepared from these cells, and oligonucleotide microarray analyses were performed using the Affymetrix HG-U133A arrays that contain >22,000 probe sets.

To identify human PPARs-responsive genes, we analysed the genes induced by ligand-activated PPARs. The criteria for selecting genes are described in the Methods section. Based on these criteria, 29, 21, 60 and 107 genes were up-regulated by ligand-activated PPARα, PPARδ, PPARγ1 and PPARγ2, respectively. As shown in Table 1 and the additional file 1, each PPAR affects the expression levels of several genes involved in lipid metabolism, glucose homeostasis, etc. PPARα tends to induce the expression of a number of genes involved in the β-oxidation of fatty acids by mitochondria, peroxisomal fatty acid oxidation, antioxidant and ketogenesis. Most of the genes that were induced by PPARδ were similar to those exhibited by PPARα. We observed that induced expression of PPARδ increased the expression in the presence of the ligand, while unliganded PPARδ repressed the expression of its target genes instead. On the other hand, PPARγ tends to induce the expression of several genes involved in gluconeogenesis, lipid storage, transport and metabolism. Moreover, PPARγ is likely to induce several genes involved in angiogenesis, cytoskeleton organization, protein modification, regulation of transcription, signal transduction, etc.

The expression of a subset of genes in response to treatment with PPAR ligands was further characterized using real-time quantitative PCR analyses. Total RNA samples were prepared from HepG2-tet-off-hPPAR cells, which were cultured in the presence or absence of Dox for 5 days, and subsequently treated with the PPAR ligands (100 μM fenofibric acid, 100 nM GW501516 or 10 μM ciglitizone for PPARα, PPARδ and PPARγ, respectively) or vehicle for 0, 8, 24, 48 or 72 h. Several PPAR-responsive genes (ADRP, 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2 (mitochondrial) (HMGCS2), hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolase/enoyl-Coenzyme A hydratase (trifunctional protein), α subunit (HADHA), phosphoenolpyruvate carboxykinase 1 (PEPCK), acyl-Coenzyme A dehydrogenase, C-4 to C-12 straight chain (MCAD), angiopoietin-like protein 4 (ANGPTL4), fatty acid binding protein 1, liver (L-FABP)) were up-regulated by the expression of PPARα and PPARγ (Figure 3). On the other hand, only ligand-activated PPARδ induced the expression of these genes, but unliganded PPARδ repressed them, in agreement with microarray analyses (Figure 3B and Table 1).

Although until now C-terminal linking and modulating protein/PDZ domain containing 1 (CLAMP/PDZK1) mRNA has not been reported as a target for PPARs, we observed that PPARs induced CLAMP/PDZK1 mRNA by microarray analyses (Table 1). We, therefore, verified the expression of CLAMP/PDZK1 mRNA using real-time quantitative PCR analysis. A similar result was observed on the expression pattern of CLAMP/PDZK1 mRNA (Figure 3).

Although all subtypes of PPARs could induce the expression of these genes, PPARα was more effective than other subtypes in the real-time PCR analyses (Figure 3).

To determine whether one of these genes was regulated by PPARs directly, we performed transient transfection experiments with the human ADRP promoter. It has been reported that the mouse ADRP promoter contains a functional PPRE 11. Examination of the corresponding region of the human adrp gene indicated that the essential features of this element are likely to be conserved, and we found that the human ADRP promoter contained a potential PPRE at positions -2361 to -2345 (Figure 4A).

To analyse whether this site can be regulated by PPARs, either the wild type 4-kb fragment of the human ADRP promoter (hADRP-4K), deletion promoter (hADRP-d1) whose potential PPRE is deleted or mutation promoter (hADRP-mut) whose potential PPRE is mutated, was cloned in front of the pGL3-luciferase reporter gene to construct reporter plasmids (Figure 4B). We co-transfected either the PPAR expression plasmid (pcDNA3-hPPARα, pcDNA3-hPPARδ, pcDNA3-hPPARγ1 or pcDNA3-hPPARγ2) or the vector plasmid (pcDNA3) as a control together with each reporter plasmid into HepG2 cells, and subsequently incubated these cells with or without ligands (100 μM fenofibric acid, 100 nM GW501516 or 10 μM ciglitizone for PPARα, PPARδ and PPARγ, respectively) for 24 h. Ligand-activated PPARs induced the promoter activity of the wild type 2 to 5-fold higher than the control (no PPAR expression without ligand) (Figure 4C–F). Interestingly, the expression of PPARδ without ligand diminished the wild type ADRP reporter gene expression to about 60% of the control in HepG2 cells (Figure 4D). We did not detect this observation when either deleted or mutated PPRE reporter plasmid was used. These data indicate that this PPRE in the ADRP promoter is a cis-acting element by which PPARs modulate human ADRP promoter activity.

To determine whether human PPARs/RXRα heterodimers bind this PPRE in the ADRP promoter, EMSAs were performed using this response element as a radiolabelled probe (Figure 5A–D). EMSAs revealed that all PPAR subtypes could bind the PPRE of the ADRP promoter in the presence of RXRα (closed arrowhead on lanes 2 to 4). Furthermore, these complexes were supershifted by anti-PPAR antibodies (open arrowhead on lane 5). This complex formation was competed by increasing amounts (10- and 100-fold excess) of unlabeled self-competitor (ADRP) and rat acyl CoA oxidase (ACO) PPRE fragments, but not by the mutated PPRE (ADRPmut) probes (lanes 6 to 11). Furthermore, no protein-DNA complex was observed when using the mutated PPRE (ADRPmut) probe (lane 12). Taken together, these data demonstrate that all PPARs bind to the same PPRE site at the position -2361 to -2345, and modulate human ADRP promoter activity.

To confirm that the PPARs/RXRα heterodimers bind to the PPRE in the ADRP promoter in vivo, we performed chromatin immunoprecipitation (ChIP) assays using an anti-PPARα, anti-PPARδ, anti-PPARγ, or anti-RXRα antibody. HepG2-tet-off-hPPAR cells were cultured in the absence of Dox for 5 days, and were subsequently treated with the PPAR ligands (100 μM fenofibric acid, 100 nM GW501516 or 10 μM troglitazone for PPARα, PPARδ and PPARγ, respectively) for 8 h and subsequently used for ChIP assays. PPARs/RXRα heterodimers bound to the PPRE in the ADRP promoter in vivo (Figure 6).

Fenofibric acid and GW501516 were synthesized as described previously 7. Ciglitizone was purchased from Sigma. Troglitazone was purchased from Cayman Chemical.

Construction of pcDNA3-hPPARα, pcDNA3-hPPARδ, pcDNA3-hPPARγ1 and pcDNA3-hPPARγ2 expression plasmids were described previously 42. Human PPARs fragments were excised from pcDNA3-hPPAR vectors. pBI-EGFP (Clontech) was digested with PvuII, and ligated with the PPAR fragment (termed pBI-EGFP-hPPAR).

To generate human ADRP promoter-reporter plasmids, human ADRP promoter containing -2981 to +1066 bp (hADRP-4K) and the deletion promoter containing -2345 to +1066 bp (hADRP-d1) were obtained by means of PCR with the bacterial artificial chromosome (BAC) clone plasmid (BACPAC resource center at Children's Hospital Oakland Research Institute) using a forward primer 5'-

GGTACC

GGTACC

AGATCT

Nucleotide sequences of these plasmids were confirmed by ABI PRISM^® 310 Genetic Analyzer (Applied Biosystems).

HepG2 cells were cultured in DMEM (Nacalai tesque) containing 10% heat-inactivated charcoal/dextran treated foetal bovine serum (FBS) (HyClone), 100 IU/ml penicillin and 100 μg/ml streptomycin. The tightly tet-regulatable HepG2 cell clone (HY-Toff) which was transfected with the pTet-off vector, was isolated as previously described 22. HY-Toff cells were co-transfected with pBI-EGFP-hPPAR and pBabepuro using TransIT^®-LT1 Transfection Reagents (Mirus). The cells were cultured in a medium containing 600 μg/ml G418 (Nacalai tesque), 1 μg/ml puromycin (Sigma) and 1 μg/ml Dox (Clontech). G418- and puromycin-resistant clones were isolated. These clones were further screened for well-inducible clones by checking the expression of EGFP using fluorescence microscopy and the expression of PPAR proteins using immunoblot analysis in the absence of Dox.

Total RNA was isolated using an RNA preparation kit (Isogen; Nippon Gene Corp.). First strand cDNA was synthesized from 5 μg of total RNA of each cell sample using the SuperScript™ First-Strand Synthesis System for RT-PCR (Invitrogen) with oligo(dT)_12–18 primer. The cDNAs were then used as templates for individual PCR reactions using specific primer sets (Table 2), which were designed by the Primer3 program written by the Whitehead Institute 43. PCR reactions were carried out using QuantiTect™ SYBR^® Green PCR Kit (Qiagen). The quantitative PCR analysis was performed using the DNA Engine Opticon™ System (Bio-Rad Laboratories). Amplification specificity was verified by visualizing PCR products on an ethidium bromide-stained 3% agarose gel. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalizing each expression data.

HepG2-tet-off-hPPAR cells were cultured in the presence (Dox) or absence of Dox for 5 days. In the case of the absence of Dox, the cells were treated with PPAR ligands (100 μM fenofibric acid for PPARα (Feno), 100 nM GW501516 for PPARδ (GW) or 10 μM ciglitizone for PPARγ (Cig)) or vehicle (DMSO) for 24 h. Total RNA samples were prepared from these cells using an RNA preparation kit (Isogen). Hybridization samples were prepared according to the Affymetrix protocol as previously described 7. Briefly, 10 μg total RNA was used to generate first-strand cDNA. After second-strand cRNA synthesis, biotinylated and amplified RNAs were purified using RNeasy (Qiagen) and quantitated by a spectrophotometer. Biotinylated cRNA samples were then hybridized to Affymetrix Human Genome U133A arrays. These arrays contain probe sets for >22,000 transcripts and EST clones. After hybridization, microarrays were washed, scanned, and analysed with the GeneChip^® software Microarray Suite (MAS) Ver.5.0 (Affymetrix). The criteria for selecting genes that were induced by ligand-activated PPARs were as follows: (1) in the presence of ligand, the average difference of gene was ≥ 100 and the gene represented as "presence"; (2) the ratio of the expression level in the presence of ligand to the expression level in the presence of Dox was greater than two; and (3) the ratio of the expression level in the presence of ligand to the expression level in the absence of ligand was greater than 1.5. Raw data are available at NCBI GEO, web page accession number GSE2699.

HepG2 cells were transfected using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's instructions. HepG2 cells (3 × 10⁴ cells/well) were seeded in 96-well plates 14–18 h before transfection. The cells were transfected with 50 ng of human ADRP reporter plasmid, 50 ng of phRL-TK (Promega) and either 5 ng of pcDNA3, pcDNA3-hPPARα, pcDNA3-hPPARδ, pcDNA3-hPPARγ1 or the pcDNA3-hPPARγ2 expression vector. Twenty-four hours following transfection, the cells were incubated with a medium containing dimethylsulfoxide (DMSO) (vehicle), 100 μM fenofibric acid, 100 nM GW501516 or 10 μM ciglitizone. Following a period of 24 h, both firefly and Renilla luciferase activities were quantified using a Dual-Luciferase^® Reporter Assay System (Promega) according to manufacturer's instructions.

Human PPARα, human PPARδ, human PPARγ2 and human RXRα proteins were prepared using the IMPACT™-CN system (New England Biolabs). Human PPARγ1 protein was synthesized in vitro using the TNT^® Quick Coupled Transcription/Translation Systems (Promega). Double-strand oligonucleotides were labelled with [α-³²P]dCTP and a Klenow fragment and were used as probes. PPARα, PPARδ, PPARγ1, PPARγ2 and/or RXRα proteins were incubated with ³²P-labelled probe in a total volume of 12.5 μl binding buffer (10 mM Tris-HCl (pH 7.5), 5% glycerol, 1 mM DTT, 1 mM EDTA, 1 μg poly (dI-dC), 30 μg BSA) at room temperature for 30 min, followed by an incubation at 4°C for 30 min. Supershift assays were performed by adding antibodies 1 h before incubation with an oligonucleotide probe at room temperature. All monoclonal antibodies (PPARα [H0723], PPARδ [K9418], PPARγ [A3408A]) were obtained from Perseus Proteomics. In competition studies, the proteins were pre-incubated with 10- or 100-fold molar excess of unlabelled wild-type or mutant oligonucleotides. Protein-DNA complexes were resolved on a 5% nondenaturing polyacrylamide gel in 1 × TAE buffer. The loaded gel was fixed with 10% methanol and 10% acetic acid, and then the gel was dried and autoradiographed. Double-stranded oligonucleotides composed of the following sequences were used for the binding and competition assays: human ADRP PPRE wild type, 5'-GCATTTTGTAGGTGAAAGGGCGAGAGTC-3'; ADRP PPRE mutant, 5'-GCATTccGcAaGcttAAGGGCGAGAGTC-3', and rat acyl-CoA oxidase (ACO) PPRE wild type, 5'-GCGGACCAGGACAAAGGTCACGTTC-3' (Mutated bases are shown as lowercase letters).

HepG2-tet-off-hPPAR cells were cultured in the absence of Dox for 5 days. The cells were treated with PPAR ligands (100 μM fenofibric acid for PPARα, 100 nM GW501516 for PPARδ, or 10 μM troglitazone for PPARγ) for 8 h. Cells were fixed in vivo at room temperature for 10 min by the addition of formaldehyde at a final concentration of 1% directly onto the cell culture media. Fixation was completed following the addition of glycine with a 0.125 M final concentration and the incubation was continued for a further 5 min. The cells were washed twice using ice-cold phosphate-buffered saline and collected. The cell pellets were washed with cell lysis buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 3 mM MgCl₂, 0.5% NP-40, and a protease inhibitor cocktail (Sigma)) three times, and dissolved in SDS lysis buffer (10 mM Tris-HCl (pH 7.5), 270 mM NaCl, 3 mM MgCl₂, 1 mM CaCl₂, 4% NP-40, 1.3% SDS, and a protease inhibitor cocktail) and remained on ice for 10 min. The cell lysates were sonicated to shear chromosomal DNA with an average length of 1000 bp. After centrifugation to remove insoluble materials, the chromatin solution was diluted 10-fold in an IP dilution buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.01% SDS, and a protease inhibitor cocktail), and the diluted solution was pre-cleared with protein G Sepharose beads on a rotating wheel at 4°C for 1 h. Beads were removed by centrifugation and the supernatants were incubated with 2 μg of antibodies to PPARα (H-98, Santa Cruz), PPARδ (H-74, Santa Cruz), PPARγ (H-100, Santa Cruz), or RXRα (D-20, Santa Cruz) at 4°C overnight. For a negative control, pre-immune rabbit IgG (Santa Cruz) was incubated with the supernatant. The complexes were immunoprecipitated with protein G Sepharose beads. The beads were washed once with IP dilution buffer, twice with wash buffer 1 (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, and a protease inhibitor cocktail), once with wash buffer 2 (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, and a protease inhibitor cocktail), once with wash buffer 3 (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.25 M LiCl, 1% NP-40, 1% deoxycholate), and twice with TE buffer. Immune complexes were eluted from the beads in the elution buffer (25 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.5% SDS, 10 mM DTT) for 15 min. The proteins were removed from DNA by digesting with 1.5 mg/ml pronase at 42°C for 2 h. The crosslink was reversed by adding 5 M NaCl to a final concentration of 200 mM followed by incubation at 65°C for 6 h. The sample DNAs were then extracted with phenol-chloroform-isoamyl alcohol (25:24:1), precipitated with ethanol in the presence of glycogen, and resuspended in TE buffer. Similarly purified DNA fragments from the chromatin extracts (input) were used as a control for PCR reactions. Precipitated DNAs were analysed by PCR of 32 cycles using primers 5'-GCAAAAAGAAGCTTGCTCAG-3' and 5'-TGTTGCCATCTTCAGTGTTT-3' that flanked the PPRE of the ADRP promoter or primers 5'-ATGGTTGCCACTGGGGATCT-3' and 5'-TGCCAAAGCCTAGGGGAAGA-3' that are located about 6-kb upstream of the GAPDH promoter (negative control). PCR products were separated on a 2% agarose gel and stained with ethidium bromide.