Transcriptional regulation of the human, porcine and bovine OCTN2 gene by PPARα via a conserved PPRE located in intron 1
© Luo et al.; licensee BioMed Central Ltd. 2014
Received: 16 April 2014
Accepted: 6 August 2014
Published: 8 September 2014
The novel organic cation transporter 2 (OCTN2) is the physiologically most important carnitine transporter in tissues and is responsible for carnitine absorption in the intestine, carnitine reabsorption in the kidney and distribution of carnitine between tissues. Genetic studies clearly demonstrated that the mouse OCTN2 gene is directly regulated by peroxisome proliferator-activated receptor α (PPARα). Despite its well conserved role as an important regulator of lipid catabolism in general, the specific genes under control of PPARα within each lipid metabolic pathway were shown to differ between species and it is currently unknown whether the OCTN2 gene is also a PPARα target gene in pig, cattle, and human. In the present study we examined the hypothesis that the porcine, bovine, and human OCTN2 gene are also PPARα target genes.
Using positional cloning and reporter gene assays we identified a functional PPRE, each in the intron 1 of the porcine, bovine, and human OCTN2 gene. Gel shift assay confirmed binding of PPARα to this PPRE in the porcine, bovine, and the human OCTN2 gene.
The results of the present study show that the porcine, bovine, and human OCTN2 gene, like the mouse OCTN2 gene, is directly regulated by PPARα. This suggests that regulation of genes involved in carnitine uptake by PPARα is highly conserved across species.
The peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated transcription factor and serves as an important regulator of lipid metabolism and energy homeostasis [1-4]. Ligands of PPARα are fatty acids which are released from white adipose tissue during energy deprivation and taken up into tissues during this state or exogenous ligands such as fibrates (e.g., WY-14,643, clofibrate) . Transcriptional regulation of genes by PPARα is mediated by binding of activated PPAR/retinoid X receptor (RXR) heterodimers to specific DNA sequences, called peroxisome proliferator response elements (PPRE), thereby stimulating the expression of those genes. The PPRE consists of a direct repeat of two copies of a AGGTCA-like sequence separated by a single base, commonly called Direct Repeat 1 (DR1), and has been identified in the promoter, intron and 5′-untranslated region of PPAR target genes [6-10].
Interestingly, several earlier studies repeatedly reported that energy deprivation and treatment with fibrates causes a strong, up to five-fold elevation in the concentration of carnitine in the liver of rats [11-13]. Carnitine plays an important role in lipid and energy metabolism by acting as a shuttling molecule for the translocation of long-chain fatty acids from the cytosol into the mitochondrial matrix, where β-oxidation occurs. The mechanism underlying this phenomenon remained obscure until it was demonstrated many years later that activation of hepatic PPARα, which is induced by both energy deprivation and fibrate treatment, causes an up-regulation of the novel organic cation transporter 2 (OCTN2) in liver cells . OCTN2 is the physiologically most important carnitine transporter facilitating carnitine absorption in the intestine, carnitine reabsorption in the kidney, and carnitine distribution between tissues . In subsequent experiments, convincing evidence for the PPARα-dependency of this effect could be provided by demonstrating that the energy deprivation- or fibrate-induced increase in hepatic carnitine concentration and up-regulation of OCTN2 occurs only in wild-type mice but not in PPARα knockout mice ,. Moreover, it could be shown recently that the mouse gene encoding OCTN2 contains a functional PPRE in intron 1 and is therefore directly activated by PPARα .
Besides the abovementioned studies in rats and mice, studies in pigs ,, chicken  and cattle , demonstrated that PPARα activation increases hepatic or cellular carnitine content and up-regulates OCTN2 in the liver or hepatocytes, suggesting that the effect of PPARα as a regulator of carnitine uptake is well conserved across species . Despite the highly conserved role of PPARα as an important regulator of lipid metabolism in general, the specific genes under control of PPARα within each lipid metabolic pathway were shown to differ between species , and it is currently unknown whether the OCTN2 gene is also a PPARα target gene in pig, cattle and human. Comparative sequence analysis revealed that the functional PPRE identified in the mouse OCTN2 intron 1  is completely identical (100%) to a putative PPRE present in the porcine, bovine and human OCTN2 intron 1  indicating a high degree of conservation of this sequence and a similar regulation of the OCTN2 gene across these species. However, the functionality of a PPRE cannot be predicted from sequence comparison of closely related species with 100% certainty but rather has to be thoroughly established by cell culture, reporter gene and gel shift experiments. Based on this we hypothesized that the porcine, bovine, and human OCTN2 gene, like the mouse OCTN2 gene, are directly regulated by PPARα. In this study we therefore performed in silico-analyses and reporter gene and gel shift assays to identify a functional PPRE in the porcine, bovine, and human OCTN2 gene.
Results and discussion
We have recently postulated that the physiological meaning of PPARα-mediated up-regulation of the OCTN2 gene is to supply cells with sufficient carnitine required for transport of excessive amounts of fatty acids into the mitochondrion, and therefore plays an important role in the adaptive response of cells to PPARα activation . In cattle and pigs, PPARα activation physiologically occurs in the liver during early lactation and peak lactation, respectively, because the negative energy balance during these states leads to the release of fatty acids from adipose tissues which are taken up into the liver and bind to and activate PPARα [26-28]. Thus, the observation from the present study that the porcine and bovine OCTN2 gene is regulated by PPARα provides a plausible explanation for the recent finding that OCTN2 in the liver is up-regulated during early lactation in high-producing dairy cows  and during peak lactation in sows . Our observation that the human OCTN2 gene is regulated by PPARα may be not only relevant with regard to carnitine homeostasis but also to tissue distribution and intestinal intestinal absorption of various other compounds, because OCTN2 is polyspecific and able to bind other monovalent cations and various drugs such as verapamil, spironolactone or mildronate [29-31].
The present results show that the intron 1 of porcine, bovine, and human OCTN2 gene contains a functional PPRE which is critical for PPARα-dependent regulation of the OCTN2 gene. This indicates that the porcine, bovine, and human OCTN2 gene, like the mouse OCTN2 gene , are directly regulated by PPARα. Given the fundamental role of OCTN2 for intestinal carnitine absorption and renal carnitine reabsorption and therefore carnitine homeostasis, our findings suggest that PPARα is a key regulator of carnitine homeostasis in these species. The similar regulation of the OCTN2 gene by PPARα between mouse, pig, cattle, and human suggests that regulation of genes involved in carnitine uptake by PPARα is highly conserved across species.
RPMI1640 GlutaMax-1 medium, fetal calf serum and gentamycin were obtained from Invitrogen. WY-14,643 was purchased from Sigma (Beijing, China).
The human hepatoma cell line HepG2 was obtained from Boster Biological Technology, Ltd. (Wuhan, China). The porcine kidney cell line PK-15 was purchased from Cell Lines Service GmbH (Eppelheim, Germany). HepG2 cells were grown in RPMI1640 GlutaMax-1 medium and PK-15 cells were cultivated in Hy-Clone Minimum Essential Medium/Earle’s Balanced Salt Solution (MEM/EBSS) medium. The media were supplemented with 10% fetal bovine serum and 0.05′mg/mL gentamicin. All cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2. Media were changed every 2 days. After reaching a confluence of 70-80%, the cells were either sub-cultivated or used for experiments.
RNA Isolation and qPCR analysis
Gene-specific primers used for qPCR
Oligonucleotide sequences (forward, reverse)
PCR product size (bp)
Western blot analysis
For western blot experiments, cells were seeded in 6-well culture plates at a cell density of 2 ± 105 (PK-15 cells) and 5 ± 105 (HepG2 cells) per well. After reaching confluence, cells were treated as described for qPCR experiments. Following treatment, cells were harvested and lysed with RIPA lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate) containing protease inhibitors (Sigma-Aldrich, Steinheim, Germany). Protein concentrations in cell lysates were determined by the BCA protein assay (Interchim, Montlucon, France). SDS-PAGE was performed with a 4% stacking gel and a 10% resolving gel. Afterwards proteins were transferred to a nitrocellulose membrane. The membranes were blocked overnight at 4°C in blocking solution (5% skim milk in Tris buffered saline with Tween-20), and then incubated with mouse monoclonal β-actin (1:500, Abcom, Cambridge, UK) or mouse polyclonal OCTN2 (1:500, Abcom, Cambridge, UK) primary antibodies for 2 h at room temperature and overnight at 4°C, respectively. The membranes were washed with TBS-T (50 mMol/L Tris, 150 mMol/L NaCl, pH 7.5, 0.2% Tween-20) and incubated with a horseradish peroxidase conjugated secondary monoclonal anti-mouse-IgG antibody (1:5000, Jackson Immuno Research, Suffolk, UK) for 1 h at room temperature. The blots were developed by using the Amersham™ ECL Plus Western Blotting Detection System (GE Healthcare, Munich, Germany) and detected by a chemiluminescence imager (Syngene, Cambridge, UK). The signal intensities of specific bands were detected with a Bio-Imaging system (Syngene, Cambridge, UK) and quantified using Syngene GeneTools software. For calculation of protein expression levels, the band intensity of OCTN2 was normalized by that of β-actin and normalized protein expression level of WY-14,643 treated cells was presented relative to that of DMSO-treated control cells, which was set to 1.0.
Isolation of porcine and bovine genomic DNA
50-100 mg ear tissues of pig and cattle were obtained from Mashen pig and Jinnan yellow cattle of Shanxi. These experiments were approved by the Shanxi Administration Office of Laboratory Animal. The tissues were washed with 1× PBS followed by incubation in 500 μL lysis buffer containing 100 mM Tris hCl, 5′mM EDTA, 0.2% SDS, 200 mM NaCl and 100 μg/mL proteinase K at 55′C overnight. At the next day, the lysate was centrifuged for 5′min and the supernatant was transferred into a new tube. Subsequently, 500 μL of isopropanol was added and centrifuged again for 20 min. Finally, DNA pellets were washed with 70% ethanol and suspended in TE buffer.
Isolation of human genomic DNA
For isolation of human genomic DNA, HepG2 cells were seeded in 6-well culture plates at a cell density of 1 ± 106 per well. After reaching 70-80% confluence the medium was removed and the cells were washed with 1× PBS. Following, cells were collected by scraping and suspended in 500 μL lysis buffer containing 5% SDS, 250 mM Tris and 5′N NaOH. Subsequently, an equal volume of a 1:1 (v:v) mixture of phenol/chloroform was added and mixed. After a 15′min centrifugation step, the upper aqueous phase was transferred into a new tube containing 100% ethanol and centrifuged again for 10 min. Finally, DNA pellets were washed with 70% ethanol and resuspended in TE buffer.
Construction of the reporter genes and site-directed mutagenesis
Oligonucleotides used for cloning, mutagenesis and EMSA
Denomination of oligonucleotides
PCR product size (bp)
Oligos used for cloning
Oligos used for mutagenesis and EMSA-mut
Oligos used for EMSA
Transient transfection and dual-luciferase reporter assay
Transient transfection was carried out as described previously . Briefly, HepG2 cells were grown in RPMI1640 GlutaMax-1 medium supplemented with 10% fetal calf serum and 0.05′mg/mL gentamycin. After reaching 70-80% confluency, cells were seeded in 96-well culture plates at a cell density of 4.5 ± 104 per well. Then cells were transiently transfected with 50 ng of the generated reporter gene constructs and co-transfected with 50 ng of both, mouse PPARα expression plasmid (pCMX-mPPARα) and mouse RXRα expression plasmid (pCMX-mRXRα) or 100 ng empty vector (pCMX) using FuGENE 6 transfection reagent (Promega) according to the manufacturer’s protocol. Cells were co-transfected with 5′ng pGL4.74 plasmid (Promega) encoding the renilla luciferase reporter gene as internal control reporter vector to normalize for differences in transfection efficiency. A plasmid containing three copies of the functional PPRE from the acyl-CoA oxidase (ACO) promoter in front of a luciferase reporter gene (3xACO-PPRE plasmid) and the empty vector pGL4.23 were transfected as positive and negative controls, respectively. 12 h after transfection, the cells were treated with either 50 μM WY-14,643 to achieve activation of PPARα or vehicle only (DMSO = control) for 24 h. Luciferase activities were determined with the Dual-Luciferase Reporter Assay System from Promega according to the manufacturer’s instructions using a Mithras LB940 luminometer (Berthold Technologies, Bad Wildbad, Germany). For control of background luminescence Firefly- and Renilla-luciferase activities were also determined in the lysates of nontransfected control cells and substracted from total luminescence of transfected cells. Data were normalized for transfection efficiency by dividing Firefly-luciferase activity of the generated reporter constructs by that of Renilla-luciferase activity of the cotransfected pGL4.74 Renilla luciferase plasmid. Results represent normalized luciferase activities and are shown relative to cells transfected with the empty vector pGL4.23 and treated with DMSO only which was set to 1.
Electrophoretic mobility shift assay (EMSA)
EMSA was performed using DIG Gel Shift Kit, 2nd (Roche) according to the manufacturer’s protocol. An oligonucleotide containing the rat ACO-PPRE and a random oligonucleotide were used as specific and non-specific controls, respectively. Synthesized wild-type and mutated oligonucleotides used for EMSA are listed in Table 1. The mouse PPARα and RXRα proteins were generated from the expression vectors by in vitro transcription/translation using TNT® Quick Coupled Transcription/Translation Kit (Promega) according to the manufacturer’s protocol. The DNA-protein complexes were detected by chemiluminescence using Anti-Digoxigenin-AP Conjugate and CSPD (Roche) and quantified using a Bio-Imaging system (Syngene, Cambridge, UK) and Syngene GeneTools software.
Quantitative data are expressed as mean ± standard deviation (SD) based on at least three independent experiments performed in triplicate. Differences were analysed by one-way ANOVA using SAS 9.1.3 (SAS Institute, Inc., 2001). A P-value < 0.05 was considered to be significant.
This study was funded by 100 plans from Shanxi province, national 863 plans projects (2011AA100307-05), and natural science foundation of Shanxi (2012011035′2, 20130311024-1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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