Peroxisome Proliferator-Activated Receptor γ Regulates the Expression of Lipid Phosphate Phosphohydrolase 1 in Human Vascular Endothelial Cells

Hindawi Publishing Corporation PPAR Research Volume 2014, Article ID 740121, 6 pages http://dx.doi.org/10.1155/2014/740121 Research Article Peroxisome Proliferator-Activated Receptor 𝛾 Regulates the Expression of Lipid Phosphate Phosphohydrolase 1 in Human Vascular Endothelial Cells Yazi Huang, Beilei Zhao, Yahan Liu, and Nanping Wang Institute of Cardiovascular Science, Peking University Health Science Center, Beijing 100191, China Correspondence should be addressed to Nanping Wang; Received 27 November 2013; Accepted 1 April 2014; Published 12 May 2014 Academic Editor: Youfei Guan Copyright © 2014 Yazi Huang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Lipid phosphate phosphohydrolase 1 (LPP1), a membrane ectophosphohydrolase regulating the availability of bioactive lipid phosphates, plays important roles in cellular signaling and physiological processes such as angiogenesis and endothelial migration. However, the regulated expression of LPP1 remains largely unknown. Here, we aimed to examine a role of peroxisome proliferatoractivated receptor 𝛾 (PPAR𝛾) in the transcriptional control of LPP1 gene expression. In human umbilical vein endothelial cells (HUVECs), quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) demonstrated that activation of PPAR𝛾 increased the mRNA level of LPP1. Chromatin immunoprecipitation assay showed that PPAR𝛾 binds to the putative PPARresponsive elements (PPREs) within the 5󸀠 -flanking region of the human LPP1 gene. Genomic fragment containing 1.7-kilobase of the promoter region was cloned by using PCR. The luciferase reporter assays demonstrated that overexpression of PPAR𝛾 and rosiglitazone, a specific ligand for PPAR𝛾, could significantly upregulate the reporter activity. However, site-directed mutagenesis of the PPRE motif abolished the induction. In conclusion, our results demonstrated that PPAR𝛾 transcriptionally activated the expression of LPP1 gene in ECs, suggesting a potential role of PPAR𝛾 in the metabolism of phospholipids. 1. Introduction Lipid phosphate phosphohydrolases (LPPs), also known as phosphatidate phosphohydrolase-2 (PAP-2), are the Mg2+ -independent and N-ethylmaleimide-insensitive Nglycosylated integral membrane ectophosphohydrolase [1, 2]. LPPs catalyze the dephosphorylation of a range of lipid phosphates, such as lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) [3, 4]. Extracellular LPA and S1P bind to the G-protein-coupled receptors (GPCRs) and exert a number of pathophysiological actions, such as angiogenesis, platelet activation, inflammation, smooth muscle cells (SMCs) proliferation and migration, and cardiovascular remodeling [4, 5]. LPPs hydrolyze these lipid phosphates to terminate their signaling actions or generate new signaling molecules [6]. Three isoforms of LPPs (LPP1, LPP2, and LPP3) have been found [7]. LPP1 negatively regulates lysophospholipid signalings by degrading the bioactive lysophospholipids released from platelets and modulates their effects on the cell proliferation, migration, inflammation, coagulation, and wound healing [5, 6]. The activity of LPP1 is mainly regulated through de novo expression rather than posttranslational modification such as phosphorylation. Expression of LPP1 was induced by androgens in human prostatic adenocarcinoma cells and decreased in ovarian cancers [8, 9]. However, transcriptional mechanism underlying the regulation expression of the LPP1 remains largely unclear. Peroxisome proliferator-activated receptors (PPARs) are a family of ligand-activated nuclear receptors and transcription factors [10]. Among three PPAR isoforms (𝛼, 𝛽/𝛿, and 𝛾), PPAR𝛾 is predominantly expressed in adipose tissue and also in vasculature including vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) [11, 12]. PPAR𝛾 forms a heterodimer with RXR and binds to the PPAR response elements (PPREs) in the promoter region of target genes [13]. When activated by various natural and synthetic ligands such as prostaglandin metabolite 15d-PGJ2 [14] and the 2 insulin sensitizer rosiglitazone [15], PPAR𝛾 transactivates the gene expression and regulates adipogenesis [16] and insulin response [17]. In addition, PPAR𝛾 possesses antiatherogenic and anti-inflammatory actions in ECs [18, 19]. Therefore, we attempted to examine a role of PPAR𝛾 in the regulation of LPP1 gene expression in ECs. PPAR Research Table 1: The sequences of the primers for ChIP assay. hLPP1 PPRE1 hLPP1 PPRE2 hLPP1 PPRE3 5󸀠 -AGGTGACGGTGGATGGAA-3󸀠 5 -CCTTTGTTGTAGAAGCCCTT-3󸀠 5󸀠 -AGGGCTTCTACAACAAAGG-3󸀠 5󸀠 -ATCATCCATCCTCGATACCT-3󸀠 5󸀠 -CGAGGATGGATGATTTAGCA-3󸀠 5󸀠 -GAGCCCTTTCTCACTTAGG-3󸀠 󸀠 2. Materials and Methods 2.1. Cell Culture and Reagents. Human umbilical vein endothelial cells (HUVECs) were cultured as previously described [20]. Bovine aortic endothelial cells (BAECs) were harvested from bovine aorta and maintained in DMEM with 10% FBS [21]. Rosiglitazone, GW501516, and GW9662 were obtained from Cayman Chemical. Polyclonal rabbit anti-PPAR𝛾 and rabbit IgG were from Santa Cruz Biotechnology. Luciferase assay reagent, MMLV reverse transcriptase, Taq polymerase, restriction enzymes (XhoI, NheI), and DNA ligase were from Promega Corporation. Lipofectamine 2000 and Trizol reagent were obtained from Invitrogen. The QuikChange sitedirected mutagenesis kit was from Stratagene Corporation. 2.2. Adenoviral Infection. Cells were infected with adenoviruses encoding the wild type human PPAR𝛼, 𝛽/𝛿, or 𝛾1 (AdWT-PPAR𝛼 or Ad-WT-PPAR𝛽/𝛿, Ad-WT-PPAR𝛾) together with Ad-tTA, which encodes a tetracycline-responsive transactivator. These viral constructs were previously described and used at 50 multiplicities of infection [22, 23]. Infected cells were maintained in the presence or absence of tetracycline (0.1 𝜇g/mL, a tet-off expression) for 48 hours as described [20]. 2.3. RNA Extraction and Real-Time Quantitative RT-PCR (qRT-PCR). Total RNA was extracted with Trizol reagent and reverse transcribed into cDNA with M-MLV reverse transcriptase with oligo-dT as a primer. Real-time PCR was performed with SYBR-green dye and Taq polymerase in the DNA Engine Opticon real-time system (Bio-Rad Laboratories Inc.). GAPDH was used as an internal control. The primer sequences are: LPP1 5󸀠 -TCAACTGCAGCGATGGTTAC (forward), 5󸀠 -GCCCACATAAATGGATACGG (reverse); GAPDH 5󸀠 -ACCACAGTCCATGCCATCAC (forward), 5󸀠 -TCCACCACCCTGTTGCTGTA (reverse). 2.4. Plasmids, Mutation, Transfection, and Reporter Assay. The genomic fragment containing −1921 to −221 bp upstream of the transcription start site of human LPP1 gene was PCR amplified from human genomic DNA with the primers (5󸀠 -CTTGATAGTACAACAGGGTCA and 5󸀠 -TCAGGTGGTCTCCGAACT) with flanking sites of NheI and XhoI. The amplified product was subcloned into the pGL3basic luciferase vector to generate the pGL3/LPP1-l (...truncated)