Alternative splicing of human peroxisome proliferator-activated receptor delta (PPARdelta):effects on translation efficiency and trans-activation ability
© Lundell et al; licensee BioMed Central Ltd. 2007
Received: 12 December 2006
Accepted: 16 August 2007
Published: 16 August 2007
Peroxisome proliferator-activated receptor delta (PPARδ) is a member of the nuclear receptor superfamily. Numerous studies have aimed at unravelling the physiological role of PPARδ as a transcriptional regulator whereas the regulation of PPARδ gene expression has been less studied.
The principal transcription start site in the human PPARδ gene identified here is positioned upstream of exon 1, although four alternative 5'-ends related to downstream exons were identified. The demonstration of multiple 5'-UTR splice variants of PPARδ mRNA, with an impact on translation efficiency, suggests a translational regulation of human PPARδ expression. Five untranslated exons identified in this study contribute to the variability among the 5'-UTRs of human PPARδ mRNAs. Moreover, in vitro studies of a 3'-splice transcript encoding a truncated variant of PPARδ (designated PPARδ2) show that this isoform constitutes a potential dominant negative form of the receptor.
We propose that alternative splicing of human PPARδ constitutes an intrinsic role for the regulation of PPARδ expression and thus activity, and highlight the significance of alternative splicing of this nuclear receptor in physiology and disease.
Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors that activate target genes by binding to peroxisome proliferator response elements (PPREs) as heterodimers with the retinoid × receptor (RXR) (NR2B). Three PPAR isoforms encoded by different genes have been identified: α (PPARα or NR1C1), δ/β (PPARδ or NR1C2) and γ (PPARγ or NR1C3). The functional specificity among members of the PPAR subfamily is achieved by isoform-specific tissue expression as well as ligand binding specificity [1–5]. Endogenous ligands that activate PPARs are polyunsaturated fatty acids or their derivatives [6–8]. Ligand activation of PPARs induces conformational changes that promote binding of co-activators that are essential for the trans-activating function whereas binding of nuclear co-repressors is associated with transcriptional repression. PPARδ is the only isoform that maintains repressor activity when bound to DNA. It has been reported that unligated PPARδ can act as an intrinsic transcription repressor and inhibit the trans-activation activity of other PPARs [9–12]. Given that the PPARδ gene is ubiquitously expressed this suggests an additional role for PPARδ as a receptor whose relative level of expression can be used to repress the expression of PPRE target genes.
It was not until recently that the roles of PPARδ were disclosed through utilization of specific ligands together with relevant cells and animal models. PPARδ controls various physiological functions including lipid and glucose homeostasis, inflammation, cell proliferation and differentiation . PPARδ-deficiency in mice negatively affects growth, adipose stores, epidermal cell proliferation, and myelinisation of corpus callosum [14, 15]. Treatment of insulin-resistant obese rhesus monkeys with a PPARδ specific ligand has been shown to promote cholesterol efflux, increase HDL levels and decrease LDL, triglyceride and insulin levels . Further, PPARδ is highly expressed in skeletal muscle, and treatment with PPARδ specific ligands increases the proportion of oxidative slow-twitch myofibers and induces expression of genes involved in fatty acid utilization and oxidation as well as glucose uptake and metabolism [17–24].
Little is so far known considering how the expression of PPARδ itself is regulated. Here we report on new 5'-untranslated exons and multiple 5'-UTRs of PPARδ mRNA transcripts, with an impact on the translation efficiency. In addition, we report on the expression of a 3'-splice variant of human PPARδ (PPARδ2), encoding a potential dominant negative regulator of gene expression. Since alternative splicing contributes to the regulation of gene expression and constitutes an important source of evolutionary diversity, we hypothesize that spliced mRNA isoforms of human PPARδ play a role in controlling its function.
Multiple untranslated exons and alternative transcription start sites
Sequences of splice junctions of newly identified spliced-in exons in the 5'-UTR of human PPARδ
downstream of exon 2 (bp)
Sequences of identified alternative 5'-end exons in 5'-UTRs of human PPARδ transcripts
downstream of exon 2 (bp)
A bioinformatic approach to identify splice variants of human PPARδ using the NCBI human Genome Browser and the human EST-search tool, indeed, showed the presence of exons 2a, 2b, 2c and 2d among 5'-spliced transcripts of PPARδ, all with exons 1 and/or 2 upstream.
Expression of PPARδ 5'-UTR isoforms in human cell lines and tissues
TaqMan primers and probes used in this study
Analysis of promoter activities
Reporter gene constructs encompassing approximately 1 kb and 250 bp, respectively, of the 5'-regions of each of the four putative transcription start sites (summarized in Table 2 and illustrated in Figure 1B, 5'-UTR: H-K) showed no or weak basal promoter activity in Huh7 and Hela cells compared with the identified promoter region upstream of exon 1 (Figure 3B). In silico analysis revealed a potential PPRE located -86 to -74 bp upstream of the identified alternative 5'-end within exon 2a. However, the two reporter gene constructs (1 kb and 213 bp) containing this PPRE were neither affected by overexpression of PPARδ nor PPARα combined with isoform-specific ligands GW501516  and fibrates, respectively (results not shown).
Translation efficiency of PPARδ 5'-splice variants
Characterization of the truncated human PPARδ isoform (PPARδ2)
A 3'-spliced isoform of human PPARδ, formed by exon 9 skipping, has been reported from human placenta cDNA clones [EMBL: BC007578 and BC002715, RefSeq GenBank: NM_177435]. Intron 8 is retained in the primary transcript and an in-frame stop codon (UGA) in the extended exon 8 encodes a protein lacking the C-terminal 82 amino acids of PPARδ, which constitutes the end of the ligand-binding domain. The presence of a PPARδ2 transcript (identical to EMBL:BC007578 and BC002715) with a polyadenylation signal 583 nt downstream of the stop codon and a poly(A)tail attached was confirmed by 3'-RACE using cDNA from placenta and adipose tissue (data not shown). Adaptor linked transcripts with a 3'-end within intron 8 were also obtained from pancreas cDNA, however, poly(A)tail linked 3'-end transcripts could not be confirmed.
Sequence identity scores of human PPARδ exons in comparable regions of orthologous genes
Chimpanzee Score (bp)
Macaque Score (bp)
Dog Score (bp)
Exon 2 (84)
Exon 2a (641)
Exon 2b (119)
Exon 2c (883)
Exon 2d (312)
Exon 2e (115)
Exon 3 (96)
The overall sequence conservation between human and chimpanzee PPARδ genes is pronounced. Moreover, the NCBI HomoloGene database displays two isoforms of chimpanzee PPARδ mRNA, denoted isoforms 3 [GenBank: XM_001172224] and 4 [GenBank: XM_001172238], which harbour spliced forms of exon 2a.
The major process whereby new exons evolve is considered to be exonization of intronic sequences [29, 30]. The primate specific Alu-elements can be exonized through a small number of mutations. Using the RepeatMasker to analyse the human PPARδ gene, exon 2b was found to fulfil the criteria of being an Alu-derived exon [31, 32]. Similar antisense Alu-elements are present in the corresponding region of the chimpanzee and macaque genes, both with mutations to enable exonization of orthologous exon 2b. A second exon-associated Alu-element is present in the 5'-end of exon 2c, close to the alternative 5'-end identified in pancreas. This Alu-element is conserved in the chimpanzee PPARδ gene. Exon 2d is located within a SVA element (SVA_D), named after its main components; short interspread nuclear element (SINE), variable number of tandem repeats (VNTR) and Alu . In general this subfamily of SVA elements is believed to predate the divergence of human and chimpanzee lineage but is not present in the chimpanzee PPARδ gene. Exon 2d is thus truly a human specific exon.
Homologues to the human PPARδ2 isoform are presented in the NCBI GenBank as isoform models for both chimpanzee and macaque [GenBank: XM_001172194 and XM_001116671]. The stop codon and the polyadenylation signal in intron 8 are fully conserved in the PPARδ genes of these primates. The potential 3'-UTRs of chimpanzee and macaque PPARδ2 show 99% and 94% overall sequence identity to the human counterpart, respectively. This pronounced sequence conservation indicates that PPARδ2 could be a primate specific isoform.
A diversity of 5'-UTRs derived from alternative splicing was identified among human PPARδ mRNAs in this study. The majority of 5'-UTRs have exon 1 in the 5'-end, and reporter gene analysis using Huh7 and HeLa cells suggests that the main promoter of human PPARδ is positioned upstream of exon 1. As previously reported , this promoter does not have a consensus TATA-box but is rich in potential Sp-1 binding elements, a typical feature for house-keeping genes. This region contains evolutionary conserved areas between the human and mouse PPARδ genes. Alternative promoters could, however, be present in the human PPARδ gene, as is the case for the mouse orthologous gene . Contrary to this assumption, reporter gene analysis using Huh7 and HeLa cells showed a low promoter activity related to only one of the four alternative promoter regions tested. Analysis of other cell types could, however, provide a different result. The identified alternative 5'-ends might represent tissue- or developmental-specific alternative transcription starts sites or be the products of as yet unknown post-transcriptional processing. A tissue-specific alternative transcription start site could apply to one alternative 5'-end, which was detected only in cDNA from pancreas.
Several levels of regulation of PPAR activity have been reported such as phosphorylation by kinases that affects the transactivation potential and ubiquitin-proteosome degradation, a mechanism to arrest transcriptional activation [26, 34–38]. The post-transcriptional regulation by 5'-splicing suggested here for human PPARδ and previously reported for the mouse orthologous gene  could represent one additional level of regulation. At least eight exons can be included in the 5'-UTR region of human PPARδ transcripts by alternative splicing; the previously identified exons 1, 2 and 3  and the five exons (2a-2e) identified in this study. The untranslated human PPARδ exons are not mutually exclusive but rather appear to be spliced-in forming a diversity of 5'-UTRs containing any combination of exons. In addition to the splice variants discussed and depicted in Figure 1B, ten more combinations involving the herein identified untranslated exons and the previously reported untranslated exons 1, 2 and 3 have been observed, either by sequencing of PCR products in this study or in data from the human EST-data base. The newly identified exons are positioned within the 63 kb long sequence separating exon 2 and the first coding exon. Interestingly, all PPAR isoforms share a similar gene organization with long introns separating untranslated exons from coding exons. The presence of several new splice variants affecting the 5'-UTR region of PPARγ was recently reported . Likewise multiple isoforms of human PPARα with variable 5'-UTRs are presented in the NCBI HomoloGene database.
Different combinations of 5'-untranslated human PPARδ exons form 5'-UTRs of various length and numbers of upstream AUGs. The originally postulated scanning mechanism for initiation of translation predicts that translation should be initiated at the first AUG codon closest to the 5'-end of the mRNA . However, upstream AUGs that are followed by a run of codons that ends in a termination codon within the 5'-UTR, so called upstream open reading frames (uORFs), play a role in translational regulation [41, 42]. The strong correlation between the number of upstream AUGs and the observed translation efficiency indicates that the regulation of the yield of PPARδ per unit mRNA involves uORFs. An influence on translation by GC rich regions with a potential to form stable secondary structures in the more extended 5'-UTRs cannot be ruled out. The possibility of an initiation of translation by internal ribosomal entry sites (IRES) that circumvents upstream AUG codons and secondary structures is intriguing. The presence of IRES in the 5'-UTRs of many growth regulatory genes is believed to provide a means for the tuning of their translation in time and space and in response to relevant stimuli .
Our results and available human EST data suggest a ubiquitous occurrence of the newly identified exons in human PPARδ mRNAs. Quantification using real-time PCR, however, showed that the overall preferred 5'-UTR is short, containing only exons 1 and 2, thus representing the majority of the human PPARδ transcripts. Besides this, only mRNA isoforms containing exon 2a are present in measurable amounts. Considering that the presence of full-length exon 2a contributes substantially to the reduced translation efficiency, it is tempting to speculate that occurrence of exon 2a might be of importance for the functional roles of human PPARδ.
The generation rate of new exons has been calculated to approximately 2.71 × 10-3 per gene per million years . Most new exons are considered to originate from exonization of intronic sequences and appear in minor splice forms, which is in agreement with the finding in this study. An important question that remains to be answered is whether the low-abundant splice variants containing the newly identified exons represent biologically significant and functional isoforms. Comparative genomics was used as a guide to identify conserved sequence element, considering that functional regions generally are under strong selection pressure to stay unchanged. The previously identified untranslated exons 1 and 2 are highly conserved between species whereas the newly identified human PPARδ exons are either human- or primate-specific exons. Exon 2d was identified as a human-specific exon. All other novel exons are highly conserved in chimpanzee whereas only two exons (exons 2a and 2b) show the same high conservation in macaque. Likewise, all features related to the four alternative 5'-ends identified for the human PPARδ are fully conserved in chimpanzee whereas only one, the 5'-end related to exon 2a, is preserved in the macaque. At present, however, there is no available EST data to predict alternative splicing or promoter usage of PPARδ in chimpanzee or macaque.
The sequence of exon 2a appears to be the most conserved of the five new human PPARδ exons. All features related to this exon, including internal splice junctions and a putative 5'-end, are fully conserved in both the chimpanzee and macaque genes. Alternative splicing involving a homologous exon 2a in chimpanzee is suggested by available PPARδ isoforms for this species. The overall conservation of the exon 2a sequence is surprisingly high in the dog gene. However, it is highly unlikely that an orthologous exon is spliced in this species.
Taken together, it is likely that transcripts with a short 5'-UTR provide a constant basal level of PPARδ synthesis. Transcripts with extended 5'-UTRs, such as the mRNA isoforms containing full-length exon 2a, with multiple AUG codons and potential secondary structures, could provide a spatio-temporal regulation of protein expression during for example growth, development, differentiation and stress. It has been reported that dysregulation of key proteins regulating growth and differentiation via modulation of 5'-UTR activity plays a role in the development or progression of various forms of cancer . Whether mechanisms related to alternative splicing of PPARδ could contribute to the conflicting findings regarding the role of this nuclear factor in cancer development remains to be investigated . Likewise, the role of alternative promoters in the regulation of human PPARδ gene expression is a matter to consider in future studies.
The possibility of a dominant negative variant of the human PPARδ adds to the complexity of PPARδ as a transcriptional regulator. The alternative polyadenylation signal and premature stop-codon in human PPARδ intron 8 are not conserved in mouse, indicating that this isoform is human-specific, similar to the dominant negative variants of PPARα and PPARγ [45, 46]. Suggested orthologs of the human PPARδ2 in chimpanzee and macaque, on the other hand, indicate that this could be a primate-specific isoform. Investigation of 5'-UTRs among human PPARδ2 transcripts shows that this isoform, similar to full-length PPARδ, is preferentially transcribed from a promoter upstream of exon 1. Since the variability among 5'-UTRs of PPARδ2 is less pronounced, this isoform appears to be less subjected to alternative splicing and thus translational regulation.
Transcripts of PPARδ2 were identified in placenta and adipose tissue. The occurrence and functional implications of PPARδ2 transcripts translated into protein in different cells and tissues, however, remains to be elucidated. Here we have shown for the first time in vitro that the isoform PPARδ2 represses the trans- activating ability of full-length PPARδ. The mode of action of dominant negative nuclear receptors in general is either competition for binding of DNA, auxiliary factors and cofactors or an effect on subcellular localization. A dominant negative mutant of the full-length mouse orthologous of PPARδ (E114P) has been tested in a similar setting with a PPRE reporter gene construct and GW501516, and shown to dose-dependently inhibit PPARδ induced luciferase activity. Contrary to PPARδ2 this full-length mutant is suggested to bind ligand and PPRE but remains in a repressive form and acts by competing with wild type PPARδ for binding to DNA [47, 48]. The result of the mobility shift assay presented here indicates that PPARδ2 exerts its inhibitory properties by another mechanism than competition for binding to PPREs.
The main finding of this study is the demonstration of multiple 5'-UTR splice variants of PPARδ mRNA that can influence the translation capacity. Moreover, the alternative splicing of human PPARδ is also reflected by the generation of a dominant negative isoform. Considering that PPARδ is widely expressed, a master regulator of gene transcription and acts as a metabolic sensor, proper understanding of the factors affecting the expression level and activity of this nuclear receptor is of crucial importance. Dysregulation of alternative splicing might promote human disease . In the case of human PPARδ, this would entail dysregulation of protein expression with effects on both the trans- activating and repressor potentials of PPARδ.
Oligonucleotides and TaqMan probes
Oligonucleotide primers used for RACE and PCR in this study
intron 8 *
Rapid amplification of cDNA ends (RACE)
Marathon-Ready cDNAs (Clontech) from human placenta, pancreas and adipose tissue were utilized for RACE studies according to the manufacturer's instructions. Two sequential rounds of 5'-RACE were carried out with the reverse gene-specific primer (GSP) Ex7Rev, followed by Ex6Rev or Ex3Rev in combination with adapter primers (AP1 and AP-2; Clontech). Purified fragments were sequenced using Big Dye Terminator Kit and an automatic sequencer (Genetic Analyzer 3100, Applied Biosystems). Nested PCR was subsequently performed using primers Ex5Rev, Ex4:1Rev, Ex2a:1Rev, Ex2a:2Rev, Ex2cRev and Ex2eRev combined with the adapter primer AP2. Sequencing was performed either directly on the PCR products or on samples of gel-purified fragments when multiple bands were obtained.
Another round of 5'-RACE was conducted on Marathon-Ready cDNAs using either a full-length PPARδ (PPARδ1) primer (Ex9:1Rev) or a PPARδ2 specific primer (In8:1Rev) in combination with the adapter primer AP1. Further rounds of PCR on these 5'-RACE products, thus enriched in either PPARδ1 or PPARδ2, were performed using exon/exon specific primer pairs specified in the Result section (Figure 5).
Two sequential rounds of 3'-RACE were carried out using Marathon-Ready cDNAs and the gene-specific forward primers Ex8:1Fw and In8Fw in combination with adapter primers AP1 and AP-2, respectively. Sequencing was performed on PCR products as described above.
RNA extraction, reverse transcription, PCR and real-time PCR analysis
Total RNA was extracted using RNeasy system (Qiagen) from a panel of human cell lines; the human hepatocellular carcinoma cell lines HepG2 and Huh7, the cervical epithelioid carcinoma cell line HeLa, the human acute monocytic leukaemia cell line THP-1 and kidney epithelial carcinoma cell line A498. One microgram of total RNA was reverse transcribed with a poly-dT primer using Superscript II (Invitrogen) according to the manufacturer's instructions. First strand cDNAs were subjected to PCR using exon specific primer pair combinations (Table 5). The PCR cycles were as follows: 94°C for 3 min followed by 36 cycles of 94°C for 20 sec, 55°C for 30 sec, 72°C for 30 sec, and finally 72°C for 10 min. Sequencing was performed on PCR products as described above. Real-time PCR (TaqMan analysis) was performed on cDNA from human cell lines and tissues according to the manufacturer's instructions (Applied Biosystem, USA) and the reaction conditions involved denaturation for 10 min at 95°C, and 45 cycles of amplification with 15 sec at 95°C and 1 min at 60°C. Primers and probes are listed in Table 3. Two different cDNAs obtained using total RNA from each cell line (described above) or tissue (Clontech) were analysed twice and determined in triplicates using an ABI prism 7000 (Applied Biosystem). Standard curves were run for all assays to ensure consistent amplification efficacy. All variants were normalised to the Ex8:9 assay using the comparative CT-method (User Bulletin #2, December 11, 1997 (updated 10/2001); ABI PRISM 7700 Sequence Detection System) and presented as relative expression levels.
Reporter gene constructs, transient transfections and luciferase assays
A luciferase reporter gene construct covering the proximal promoter region of the human PPARδ gene upstream of exon 1, from -733 to + 44 cloned into pGL3 Basic (Promega) (hereafter referred to as the -733 bp construct) has previously been described . Additional constructs covering longer (2.6 kb and 1.5 kb) and shorter sequences (171 bp, 61 bp and 48 bp) of this promoter were created from restriction enzyme digests of a genomic PPARδ clone  and the -733 bp construct. Likewise, genomic upstream sequences (approximately 1 kb and 250 bp) for each of the four alternative transcription start sites identified in this study were PCR amplified and cloned into pGL3 Basic.
The Huh7 and HeLa cell lines were maintained in Dulbecco's modified Eagle's medium (D-MEM 1g/ml glucose, Invitrogen) supplemented with 10% fetal bovine serum, penicillin (100 units/ml) and streptomycin (100 ug/ml) at 37°C in 5% CO2 in air. Approximately 1 × 105 cells/well were plated on a 24-well plate and transiently transfected after 24 h with 100 ng of the reporter gene constructs or empty vector using Lipofectamin (Invitrogen) according to the manufacturer's protocol. Plasmid pSV-β-galactosidase control vector (Promega) (260 ng) was co-transfected for normalization of the transfection efficiency. After 24 h, cells were washed, lysed and luciferase activity was measured as previously described . The β-galactosidase enzyme assay system (Promega) was used for measuring the β-galactosidase activity in cell lysates according to the manufacturer's instructions. The activities were determined in quadruplicates and the data presented are based on the mean (± SD) luciferase/β-galactosidase ratios of three independent transfections, setting the value of empty vector to 1.
A fragment covering the full-length PPARδ mRNA sequence from exon 1 to the 3'-UTR was amplified using Marathon placenta cDNA and primers Ex1:1Fw (introducing a KpnI restriction site in the 5'-end) and Ex9:2Rev. The fragment was cloned into the pTNT vector (Promega) using the restriction sites KpnI and SmaI in the vector and KpnI and MscI in PPARδ, respectively. Variable 5'-UTRs upstream of exon 4 were introduced using the primer- based KpnI site in the 5'-end of exon 1 and an NcoI site in exon 4. The 5'-UTR sequences were amplified using Marathon cDNAs and combinations of exon specific primers Ex1:1Fw, Ex2a:1Fw, Ex2a:2Fw, Ex2eFw, Ex2eRev, Ex3Rev and Ex4:2Rev. Longer 5'-UTRs were amplified in two pieces, subcloned in PGEM-T vector (Promega) and ligated together prior to being introduced in the 5'-end of PPARδ in pTNT vector. Coupled in vitro transcription and translation was performed on these plamids using a TNT Quick Coupled Transcription/Translation System (Promega) and [35S]methionine (Amersham) according to the manufacturer's instructions. The [35S]methionine labelled products were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), subjected to autoradiography and analysed on a PhosphorImager scanner (Fuji film Bas-2500, Australia). The in vitro translated products were also subjected to Western blot analysis under reducing conditions on 10% SDS-PAGE as described by Laemmli  using the polyclonal PPARδ specific antibody IMG-3297 (Nordic Biosite, Sweden) and a horseradish peroxidase-labelled secondary antibody. Detection was performed with the ECL Advanced Western blot detection system (Amersham). Both the autoradiographic and Western blot bands were quantified using the NIH ImageJ software . The amounts of plasmids used (1 μg) were within a range of saturating conditions where the outcome of the transcription/translation reactions was independent on the differences in target DNA concentration introduced by the different lengths of the 5'-UTRs.
Expression vectors for PPARδ, transient transfections and Luciferase assays
The expression vector for full-length PPARδ (PPARδ1) under the control of the human cytomegalovirus (CMV) promoter was a kind gift of Dr CN Palmer and is described elsewhere . The expression vector for PPARδ2 was created using an EcoRV site in the coding region, common to both PPARδ1 and 2, and replacing the downstream sequence of the expression vector with the corresponding 3'-sequence of PPARδ2. The 3'-sequence of PPARδ2 was obtained by PCR using a genomic clone of PPARδ as template , and the forward primer Ex8:2Fw and reverse primer In8:2Rev. The reporter construct pFABPLUC containing four copies of the PPRE from the human liver FABP gene in front of the HSV-TK promoter is described elsewhere . Growth and transfection of HeLa cells, Luciferase assay, and β-galactosidase assay were performed as described above. Nuclear extract prepared as previously described  from HeLa cells transfected with the expression vectors for PPARδ1 and PPARδ2, respectively, was subjected to Western blot analysis as described above, using the PPARδ antibody sc-7197 (Santa Cruz Biotechnology). Nuclear extract prepared from untransfected HeLa cells was used as a negative control.
Electrophoretic mobility shift assay, EMSA
The binding properties of PPARδ1 and PPARδ2, respectively, to the functional PPRE of the rat acyl-CoA oxidase gene with and without the heterodimer partner RXR were investigated by EMSA. Human PPARδ1 and PPARδ2, cloned into the TNT vector, and TNT-hRXRα (a kind gift from Dr Glinghammar, AZ, Sweden) were in vitro transcribed/translated using the rabbit reticulocyte lysate system (Promega) as previously described but with unlabelled methionine. Incubation for EMSA was conducted as previously described  using 6 μl of in vitro produced PPARδ1 or PPARδ2 with 2 μl of RXRα or mock lysate produced using an empty vector, in a total volume of 20 μl (2 μg poly(dI-dC), 0.75 mM EDTA pH 8.0, 18 mM HEPES pH 7.9, 0.5 mM dithiothreitol, 4% Ficoll), and [γ-32P]-end labelled double stranded ACO-oligo (5'-ggaccAGGACAaAGGTCAcgttcgg-3'). For competition, a 100-fold molar excess of unlabelled double stranded ACO-oligo was added. A PPARδ antibody sc-7197 was used for supershift. This antibody, directed against the N-terminal region (epitope 2–75) of human PPARδ, was shown to detect both variants of in vitro translated PPARδ using Western blot (data not shown). DNA-protein complexes were applied to a 6% polyacrylamide gel and run for 4 hours at 200V in 0.25 × TBE at 4°C. The gel was dried, subjected to autoradiography and analysed on a PhosphoImager scanner.
Bioinformatics and comparative genomics
The NCBI human Genome Browser, the human EST-search tool and homologue RefSeq of PPARδ were provided by NCBI . The Evolutionary Conserved Regions Browser tool (ECR Browser)  was used to identify sequence conservation between species, ClustalW  for sequence alignments and sequence identity scores and the RepeatMasker  to unravel repetitive elements in PPARδ genes. Localization and extraction of relevant sequences for comparative analysis between species was performed using the ECR Browser tool and defined regions were subsequently extracted from the PPARδ gene RefSeq for each species and aligned to the relevant human exon sequences using ClustalW.
peroxisome proliferator-activated receptor
peroxisome proliferator response element
rapid amplification of cDNA ends
gene specific primer
polymerase chain reaction
sodium dodecyl sulfate
open reading frame
upstream open reading frame
expressed sequence tags
We thank Dr Tobias Cassel for his help in producing reporter gene constructs used in this study. This study was supported by the Swedish Medical Research Council (12659) (EE and KL), the Swedish Heart-Lung Foundation (EE and AH), the Swedish Diabetes Foundation (EE and KL), Professor Nanna Svartz Foundation (EE), Magn. Bergvalls Foundation (KL), the Foundation for Old Servants (EE), the Stockholm County Council (AH), and the Karolinska Institutet (EE).
- van Bilsen M, van der Vusse GJ, Gilde AJ, Lindhout M, van der Lee KA: Peroxisome proliferator-activated receptors: lipid binding proteins controling gene expression. Mol Cell Biochem 2002,239(1–2):131-138. 10.1023/A:1020553428294View ArticlePubMedGoogle Scholar
- Hihi AK, Michalik L, Wahli W: PPARs: transcriptional effectors of fatty acids and their derivatives. Cell Mol Life Sci 2002,59(5):790-798. 10.1007/s00018-002-8467-xView ArticlePubMedGoogle Scholar
- Kota BP, Huang TH, Roufogalis BD: An overview on biological mechanisms of PPARs. Pharmacol Res 2005,51(2):85-94. 10.1016/j.phrs.2004.07.012View ArticlePubMedGoogle Scholar
- Desvergne B, Wahli W: Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocr Rev 1999,20(5):649-688. 10.1210/er.20.5.649PubMedGoogle Scholar
- Ferre P: The biology of peroxisome proliferator-activated receptors: relationship with lipid metabolism and insulin sensitivity. Diabetes 2004,53(Suppl 1):S43-50. 10.2337/diabetes.53.2007.S43View ArticlePubMedGoogle Scholar
- Xu HE, Lambert MH, Montana VG, Plunket KD, Moore LB, Collins JL, Oplinger JA, Kliewer SA, Gampe RT Jr, McKee DD, Moore JT, Willson TM: Structural determinants of ligand binding selectivity between the peroxisome proliferator-activated receptors. Proc Natl Acad Sci USA 2001,98(24):13919-13924. 10.1073/pnas.241410198PubMed CentralView ArticlePubMedGoogle Scholar
- Forman BM, Chen J, Evans RM: Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors alpha and delta. Proc Natl Acad Sci USA 1997,94(9):4312-4317. 10.1073/pnas.94.9.4312PubMed CentralView ArticlePubMedGoogle Scholar
- Xu HE, Lambert MH, Montana VG, Parks DJ, Blanchard SG, Brown PJ, Sternbach DD, Lehmann JM, Wisely GB, Willson TM, Kliewer SA, Milburn MV: Molecular recognition of fatty acids by peroxisome proliferator-activated receptors. Mol Cell 1999,3(3):397-403. 10.1016/S1097-2765(00)80467-0View ArticlePubMedGoogle Scholar
- Shi Y, Hon M, Evans RM: The peroxisome proliferator-activated receptor delta, an integrator of transcriptional repression and nuclear receptor signaling. Proc Natl Acad Sci USA 2002,99(5):2613-2618. 10.1073/pnas.052707099PubMed CentralView ArticlePubMedGoogle Scholar
- Jow L, Mukherjee R: The human peroxisome proliferator-activated receptor (PPAR) subtype NUC1 represses the activation of hPPAR alpha and thyroid hormone receptors. J Biol Chem 1995,270(8):3836-3840. 10.1074/jbc.270.8.3836View ArticlePubMedGoogle Scholar
- Krogsdam AM, Nielsen CA, Neve S, Holst D, Helledie T, Thomsen B, Bendixen C, Mandrup S, Kristiansen K: Nuclear receptor corepressor-dependent repression of peroxisome-proliferator-activated receptor delta-mediated transactivation. Biochem J 2002,363(Pt 1):157-165. 10.1042/0264-6021:3630157PubMed CentralView ArticlePubMedGoogle Scholar
- Muoio DM, MacLean PS, Lang DB, Li S, Houmard JA, Way JM, Winegar DA, Corton JC, Dohm GL, Kraus WE: Fatty acid homeostasis and induction of lipid regulatory genes in skeletal muscles of peroxisome proliferator-activated receptor (PPAR) alpha knock-out mice. Evidence for compensatory regulation by PPAR delta. J Biol Chem 2002,277(29):26089-26097. 10.1074/jbc.M203997200View ArticlePubMedGoogle Scholar
- Barish GD, Narkar VA, Evans RM: PPAR delta: a dagger in the heart of the metabolic syndrome. J Clin Invest 2006,116(3):590-597. 10.1172/JCI27955PubMed CentralView ArticlePubMedGoogle Scholar
- Peters JM, Lee SS, Li W, Ward JM, Gavrilova O, Everett C, Reitman ML, Hudson LD, Gonzalez FJ: Growth, adipose, brain, and skin alterations resulting from targeted disruption of the mouse peroxisome proliferator-activated receptor beta(delta). Mol Cell Biol 2000,20(14):5119-5128. 10.1128/MCB.20.14.5119-5128.2000PubMed CentralView ArticlePubMedGoogle Scholar
- Barak Y, Liao D, He W, Ong ES, Nelson MC, Olefsky JM, Boland R, Evans RM: Effects of peroxisome proliferator-activated receptor delta on placentation, adiposity, and colorectal cancer. Proc Natl Acad Sci USA 2002,99(1):303-308. 10.1073/pnas.012610299PubMed CentralView ArticlePubMedGoogle Scholar
- Oliver WR Jr, Shenk JL, Snaith MR, Russell CS, Plunket KD, Bodkin NL, Lewis MC, Winegar DA, Sznaidman ML, Lambert MH, Xu HE, Sternbach DD, Kliewer SA, Hansen BC, Willson TM: A selective peroxisome proliferator-activated receptor delta agonist promotes reverse cholesterol transport. Proc Natl Acad Sci USA 2001,98(9):5306-5311. 10.1073/pnas.091021198PubMed CentralView ArticlePubMedGoogle Scholar
- Dressel U, Allen TL, Pippal JB, Rohde PR, Lau P, Muscat GE: The peroxisome proliferator-activated receptor beta/delta agonist, GW50 regulates the expression of genes involved in lipid catabolism and energy uncoupling in skeletal muscle cells. Mol Endocrinol 1516,17(12):2477-2493. 10.1210/me.2003-0151View ArticleGoogle Scholar
- Wang YX, Zhang CL, Yu RT, Cho HK, Nelson MC, Bayuga-Ocampo CR, Ham J, Kang H, Evans RM: Regulation of muscle fiber type and running endurance by PPARdelta. PLoS Biol 2004,2(10):e294. 10.1371/journal.pbio.0020294PubMed CentralView ArticlePubMedGoogle Scholar
- Luquet S, Lopez-Soriano J, Holst D, Fredenrich A, Melki J, Rassoulzadegan M, Grimaldi PA: Peroxisome proliferator-activated receptor delta controls muscle development and oxidative capability. Faseb J 2003,17(15):2299-2301.PubMedGoogle Scholar
- Al-Khalili L, Chibalin AV, Kannisto K, Zhang BB, Permert J, Holman GD, Ehrenborg E, Ding VD, Zierath JR, Krook A: Insulin action in cultured human skeletal muscle cells during differentiation: assessment of cell surface GLUT4 and GLUT1 content. Cell Mol Life Sci 2003,60(5):991-998.PubMedGoogle Scholar
- Kramer DK, Al-Khalili L, Perrini S, Skogsberg J, Wretenberg P, Kannisto K, Wallberg-Henriksson H, Ehrenborg E, Zierath JR, Krook A: Direct activation of glucose transport in primary human myotubes after activation of peroxisome proliferator-activated receptor delta. Diabetes 2005,54(4):1157-1163. 10.2337/diabetes.54.4.1157View ArticlePubMedGoogle Scholar
- Chevillotte E, Rieusset J, Roques M, Desage M, Vidal H: The regulation of uncoupling protein-2 gene expression by omega-6 polyunsaturated fatty acids in human skeletal muscle cells involves multiple pathways, including the nuclear receptor peroxisome proliferator-activated receptor beta. J Biol Chem 2001,276(14):10853-10860. 10.1074/jbc.M008010200View ArticlePubMedGoogle Scholar
- Holst D, Luquet S, Nogueira V, Kristiansen K, Leverve X, Grimaldi PA: Nutritional regulation and role of peroxisome proliferator-activated receptor delta in fatty acid catabolism in skeletal muscle. Biochim Biophys Acta 2003,1633(1):43-50.View ArticlePubMedGoogle Scholar
- Tanaka T, Yamamoto J, Iwasaki S, Asaba H, Hamura H, Ikeda Y, Watanabe M, Magoori K, Ioka RX, Tachibana K, Watanabey Y, Uchiyama Y, Sumi K, Iguchi H, Ito S, Doi T: Activation of peroxisome proliferator-activated receptor delta induces fatty acid beta-oxidation in skeletal muscle and attenuates metabolic syndrome. Proc Natl Acad Sci USA 2003, 100: 15924-15929. 10.1073/pnas.0306981100PubMed CentralView ArticlePubMedGoogle Scholar
- Skogsberg J, Kannisto K, Roshani L, Gagne E, Hamsten A, Larsson C, Ehrenborg E: Characterization of the human peroxisome proliferator activated receptor delta gene and its expression. Int J Mol Med 2000,6(1):73-81.PubMedGoogle Scholar
- Pelton P: GW-501516 GlaxoSmithKline/Ligand. Curr Opin Investig Drugs 2006,7(4):360-370.PubMedGoogle Scholar
- Larsen LK, Amri EZ, Mandrup S, Pacot C, Kristiansen K: Genomic organization of the mouse peroxisome proliferator-activated receptor beta/delta gene: alternative promoter usage and splicing yield transcripts exhibiting differential translational efficiency. Biochem J 2002,366(Pt 3):767-775.PubMed CentralView ArticlePubMedGoogle Scholar
- Ovcharenko I, Nobrega MA, Loots GG, Stubbs L: ECR Browser: a tool for visualizing and accessing data from comparisons of multiple vertebrate genomes. Nucleic Acids Res 2004, 32: W280-286. 10.1093/nar/gkh355PubMed CentralView ArticlePubMedGoogle Scholar
- Batzer MA, Deininger PL: Alu repeats and human genomic diversity. Nat Rev Genet 2002,3(5):370-379. 10.1038/nrg798View ArticlePubMedGoogle Scholar
- Zhang XH, Chasin LA: Comparison of multiple vertebrate genomes reveals the birth and evolution of human exons. Proc Natl Acad Sci USA 2006,103(36):13427-13432. 10.1073/pnas.0603042103PubMed CentralView ArticlePubMedGoogle Scholar
- Sorek R, Lev-Maor G, Reznik M, Dagan T, Belinky F, Graur D, Ast G: Minimal conditions for exonization of intronic sequences: 5' splice site formation in alu exons. Mol Cell 2004,14(2):221-231. 10.1016/S1097-2765(04)00181-9View ArticlePubMedGoogle Scholar
- Lev-Maor G, Sorek R, Shomron N, Ast G: The birth of an alternatively spliced exon: 3' splice-site selection in Alu exons. Science 2003,300(5623):1288-1291. 10.1126/science.1082588View ArticlePubMedGoogle Scholar
- Wang H, Xing J, Grover D, Hedges DJ, Han K, Walker JA, Batzer MA: SVA elements: a hominid-specific retroposon family. J Mol Biol 2005,354(4):994-1007. 10.1016/j.jmb.2005.09.085View ArticlePubMedGoogle Scholar
- Lazennec G, Canaple L, Saugy D, Wahli W: Activation of peroxisome proliferator-activated receptors (PPARs) by their ligands and protein kinase A activators. Mol Endocrinol 2000,14(12):1962-1975. 10.1210/me.14.12.1962PubMed CentralView ArticlePubMedGoogle Scholar
- Diradourian C, Girard J, Pegorier JP: Phosphorylation of PPARs: from molecular characterization to physiological relevance. Biochimie 2005,87(1):33-38. 10.1016/j.biochi.2004.11.010View ArticlePubMedGoogle Scholar
- Hansen JB, Zhang H, Rasmussen TH, Petersen RK, Flindt EN, Kristiansen K: Peroxisome proliferator-activated receptor delta (PPARdelta)-mediated regulation of preadipocyte proliferation and gene expression is dependent on cAMP signaling. J Biol Chem 2001,276(5):3175-3182. 10.1074/jbc.M005567200View ArticlePubMedGoogle Scholar
- Blanquart C, Barbier O, Fruchart JC, Staels B, Glineur C: Peroxisome proliferator-activated receptors: regulation of transcriptional activities and roles in inflammation. J Steroid Biochem Mol Biol 2003,85(2–5):267-273. 10.1016/S0960-0760(03)00214-0View ArticlePubMedGoogle Scholar
- Gelman L, Michalik L, Desvergne B, Wahli W: Kinase signaling cascades that modulate peroxisome proliferator-activated receptors. Curr Opin Cell Biol 2005,17(2):216-222. 10.1016/j.ceb.2005.02.002View ArticlePubMedGoogle Scholar
- Chen Y, Jimenez AR, Medh JD: Identification and regulation of novel PPAR-gamma splice variants in human THP-1 macrophages. Biochim Biophys Acta 2006,1759(1–2):32-43.PubMed CentralView ArticlePubMedGoogle Scholar
- Kozak M: Initiation of translation in prokaryotes and eukaryotes. Gene 1999,234(2):187-208. 10.1016/S0378-1119(99)00210-3View ArticlePubMedGoogle Scholar
- Iacono M, Mignone F, Pesole G: uAUG and uORFs in human and rodent 5'untranslated mRNAs. Gene 2005, 349: 97-105. 10.1016/j.gene.2004.11.041View ArticlePubMedGoogle Scholar
- Wang XQ, Rothnagel JA: 5'-untranslated regions with multiple upstream AUG codons can support low-level translation via leaky scanning and reinitiation. Nucleic Acids Res 2004,32(4):1382-1391. 10.1093/nar/gkh305PubMed CentralView ArticlePubMedGoogle Scholar
- van der Velden AW, Thomas AA: The role of the 5' untranslated region of an mRNA in translation regulation during development. Int J Biochem Cell Biol 1999,31(1):87-106. 10.1016/S1357-2725(98)00134-4View ArticlePubMedGoogle Scholar
- Wang W, Zheng H, Yang S, Yu H, Li J, Jiang H, Su J, Yang L, Zhang J, McDermott J, Samudrala R, Wang J, Yang H, Yu J, Kristiansen K, Wong GK: Origin and evolution of new exons in rodents. Genome Res 2005,15(9):1258-1264. 10.1101/gr.3929705PubMed CentralView ArticlePubMedGoogle Scholar
- Gervois P, Torra IP, Chinetti G, Grotzinger T, Dubois G, Fruchart JC, Fruchart-Najib J, Leitersdorf E, Staels B: A truncated human peroxisome proliferator-activated receptor alpha splice variant with dominant negative activity. Mol Endocrinol 1999,13(9):1535-1549. 10.1210/me.13.9.1535PubMedGoogle Scholar
- Sabatino L, Casamassimi A, Peluso G, Barone MV, Capaccio D, Migliore C, Bonelli P, Pedicini A, Febbraro A, Ciccodicola A, Colantuoni V: A novel peroxisome proliferator-activated receptor gamma isoform with dominant negative activity generated by alternative splicing. J Biol Chem 2005,280(28):26517-26525. 10.1074/jbc.M502716200View ArticlePubMedGoogle Scholar
- Bastie C, Luquet S, Holst D, Jehl-Pietri C, Grimaldi PA: Alterations of peroxisome proliferator-activated receptor delta activity affect fatty acid-controlled adipose differentiation. J Biol Chem 2000,275(49):38768-38773. 10.1074/jbc.M006450200View ArticlePubMedGoogle Scholar
- Pesant M, Sueur S, Dutartre P, Tallandier M, Grimaldi PA, Rochette L, Connat JL: Peroxisome proliferator-activated receptor delta (PPARdelta) activation protects H9c2 cardiomyoblasts from oxidative stress-induced apoptosis. Cardiovasc Res 2006,69(2):440-449. 10.1016/j.cardiores.2005.10.019View ArticlePubMedGoogle Scholar
- Caceres JF, Kornblihtt AR: Alternative splicing: multiple control mechanisms and involvement in human disease. Trends Genet 2002,18(4):186-193. 10.1016/S0168-9525(01)02626-9View ArticlePubMedGoogle Scholar
- Skogsberg J, Kannisto K, Cassel TN, Hamsten A, Eriksson P, Ehrenborg E: Evidence that peroxisome proliferator-activated receptor delta influences cholesterol metabolism in men. Arterioscler Thromb Vasc Biol 2003,23(4):637-643. 10.1161/01.ATV.0000064383.88696.24View ArticlePubMedGoogle Scholar
- Glinghammar B, Skogsberg J, Hamsten A, Ehrenborg E: PPARdelta activation induces COX-2 gene expression and cell proliferation in human hepatocellular carcinoma cells. Biochem Biophys Res Commun 2003,308(2):361-368. 10.1016/S0006-291X(03)01384-6View ArticlePubMedGoogle Scholar
- Laemmli U: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227: 660-685. 10.1038/227680a0View ArticleGoogle Scholar
- Research Services Branch[http://rsb.info.nih.gov]
- Vosper H, Patel L, Graham TL, Khoudoli GA, Hill A, Macphee CH, Pinto I, Smith SA, Suckling KE, Wolf CR, Palmer CN: The peroxisome proliferator-activated receptor delta promotes lipid accumulation in human macrophages. J Biol Chem 2001,276(47):44258-44265. 10.1074/jbc.M108482200View ArticlePubMedGoogle Scholar
- Andrews NC, Faller DV: A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res 1991,19(9):2499. 10.1093/nar/19.9.2499PubMed CentralView ArticlePubMedGoogle Scholar
- Dawson SJ, Wiman B, Hamsten A, Green F, Humphries S, Henney AM: The two allele sequences of a common polymorphism in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene respond differently to interleukin-1 in HepG2 cells. J Biol Chem 1993,268(15):10739-10745.PubMedGoogle Scholar
- Juge-Aubry C, Pernin A, Favez T, Burger AG, Wahli W, Meier CA, Desvergne B: DNA binding properties of peroxisome proliferator-activated receptor subtypes on various natural peroxisome proliferator response elements. Importance of the 5'-flanking region. J Biol Chem 1997,272(40):25252-25259. 10.1074/jbc.272.40.25252View ArticlePubMedGoogle Scholar
- National Center for Biotechnology Information[http://www.ncbi.nlm.nih.gov]
- ECR Browser[http://ecrbrowser.dcode.org]
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