This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 100/5/e59    most recent 01.RES.0000260805.99076.22v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Brunelli, L. Articles by Baldini, A. Search for Related Content PubMed PubMed Citation Articles by Brunelli, L. Articles by Baldini, A. Related Collections Other Research Gene expression Gene regulation

(Circulation Research. 2007;100:e59.)
© 2007 American Heart Association, Inc.

UltraRapid Communication

Peroxisome Proliferator-Activated Receptor- Upregulates 14-3-3 in Human Endothelial Cells via CCAAT/Enhancer Binding Protein-ß

Luca Brunelli, Katarzyna A. Cieslik, Joseph L. Alcorn, Matteo Vatta, Antonio Baldini

From the Department of Pediatrics (Neonatal-Perinatal Medicine) (L.B., K.A.C., J.L.A.), The University of Texas at Houston Medical School; Department of Pediatrics (Cardiology) (M.V.), Baylor College of Medicine, Texas Children’s Hospital; and Center for Molecular Development and Disease (A.B.), Institute of Biosciences & Technology, Texas A&M University, Houston.

Correspondence to Luca Brunelli, MD, PhD, Division of Neonatal-Perinatal Medicine, The University of Texas at Houston Medical School, 6431 Fannin St, MSB 3.200, Houston, TX 77030-1503. E-mail Luca.Brunelli{at}uth.tmc.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Peroxisome proliferator-activated receptor (PPAR) agonists are promising new agents for treatment of the metabolic syndrome. Although they possess antiatherosclerotic properties in vivo and promote endothelial cell survival, their mechanism of action is incompletely understood. 14-3-3 is a critical component of the endothelial cell antiapoptotic machinery, which is essential to maintain homeostasis of the vascular wall. To test the hypothesis that PPAR targets 14-3-3 in endothelial cells, we studied the response of the gene that encodes 14-3-3 in humans, YWHAE, to PPAR ligands (L-165,041 and GW501516). We found that PPAR activates YWHAE promoter in a concentration and time-dependent manner. Consistent with these findings, L-165,041 increased 14-3-3 mRNA and protein level, whereas PPAR small interfering RNA suppressed both basal and L-165,041–dependent YWHAE transcription and 14-3-3 protein expression. Surprisingly, PPAR response elements in YWHAE promoter were not required for upregulation by PPAR, whereas a CCAAT/enhancer binding protein (C/EBP) site located at –160/–151 bp regulated both basal and PPAR-dependent promoter activity. Intriguingly, activation or knock down of endogenous PPAR regulated C/EBPß protein expression. Chromatin immunoprecipitation assays demonstrated that L-165,041 determines the localization of C/EBPß to the region spanning this C/EBP response element, whereas sequential chromatin immunoprecipitation analysis revealed that C/EBPß and PPAR form a transcriptional activating complex on this C/EBP site. Our work uncovers a novel role for C/EBPß as a mediator of PPAR-dependent 14-3-3 gene regulation in human endothelial cells and provides insight into the mechanism by which PPAR agonists may be beneficial in atherosclerosis.

Key Words: PPAR • C/EBP • 14-3-3 • endothelial cells • transcriptional regulation

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Peroxisome proliferator-activated receptors (PPARs) are ligand-inducible transcription factors that control fatty acid metabolism and are members of the nuclear receptor family. The 3 isotypes identified in vertebrates, PPAR (NR1C1), PPAR (NR1C2), and PPAR (NR1C3), regulate target gene expression by binding to specific PPAR response elements (PPREs) as a heterodimer with a retinoid X receptor.¹ This classical pathway has been recently challenged by the demonstration that PPRE-containing genes can be activated by the retinoid X receptor ligand 9-cis-retinoic acid and coactivators like steroid receptor coactivator-1, even in the absence of PPARs.² The ubiquitous and abundant PPAR is antiapoptotic in keratinocytes, kidney cells, and endothelial cells.^3–5 Exciting new evidence also suggests that PPAR agonists may possess antiatherosclerotic properties in vivo by decreasing the amount of nonliganded PPAR receptor and releasing the transcriptional repressor BCL-6 in foam cells.⁶ Therefore, PPAR agonists could represent a promising new treatment for the metabolic syndrome, also in view of their advantageous effects in obesity prevention and modulation of lipoprotein metabolism.^7–9 However, much remains to be learned about their mechanism of action on targets like the endothelial cell monolayer, which is uniquely exposed to the circulating inflammatory factors that predispose to atherosclerosis. As increasing evidence suggests that apoptosis of the endothelium might contribute to the development of atherosclerosis and acute coronary syndromes,¹⁰ further knowledge is needed about the targets of PPAR agonists in this tissue. The antiapoptotic PPAR might affect different proteins in endothelial cells, with 14-3-3 proteins being key candidates because of their ability to promote cell survival.¹¹

Because 14-3-3 proteins and 14-3-3 are antiapoptotic and antiinflammatory molecules in endothelial cells, they may play an important role in atherothrombosis.^12–14 Furthermore, recent evidence suggests that 14-3-3 proteins modulate crucial aspects of heart function both in vitro and in vivo.^15–18 In all eukaryotic species, 14-3-3 proteins comprise an abundant, ubiquitous and highly conserved 30-kDa protein family, which, in mammals, consists of at least 7 isoforms, each encoded by a distinct gene (ß, , , , , , and /).^19,20 Notably, 14-3-3 proteins were the first molecules identified as discrete binding partners of phosphoserine/threonine (pSer/Thr) residues in proteins, recognizing the sequence RSXpSXP or RXXXpSXP.^21,22 This specific targeting explains their critical role in signal transduction because 14-3-3 proteins modulate the recruitment of pSer/Thr-containing proteins into larger signaling complexes. Proteomic studies have determined that they bind a large number of proteins,^23–25 which explains their participation in processes as diverse as cell cycle regulation, cellular trafficking, and apoptosis.^11,26,27 The marked differences in 14-3-3– or 14-3-3–targeted deletion on mouse perinatal survival clearly demonstrate that 14-3-3 proteins have isoform-specific functions in different organs,^28,29 which suggest that differential gene expression may play a critical role in their regulation. However, relatively little is known regarding transcriptional control of these proteins, particularly in humans.^30,31 Taken together, these data emphasize the necessity to investigate which factors regulate 14-3-3 transcription and the mechanisms involved.

The available evidence on PPAR and 14-3-3 proteins as antiapoptotic molecules raise pivotal questions regarding their regulatory interplay and led us to speculate about their functional relationship in endothelial cells. Does the transcription factor PPAR target 14-3-3? Which specific mechanisms might be involved in YWHAE transactivation? This investigation was designed to study the molecular mechanisms by which PPAR agonists regulate 14-3-3 expression in human endothelial cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
DMEM, high glucose, antibiotic–antimycotic solution, and TA Cloning Kit were obtained from Invitrogen (Carlsbad, Calif). Rabbit polyclonal anti–14-3-3, CCAAT/enhancer binding protein ß (C/EBPß), C/EBP, C/EBP, specificity protein (Sp1), and histone H3 antibodies, and goat polyclonal anti-PPAR antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif). Mouse monoclonal anti-actin antibody was obtained from EMD Biosciences (San Diego, Calif). Enhanced chemiluminescence solution and BCA reagent for protein assay were acquired from Pierce (Rockford, IL). SDS-PAGE Ready Gel Tris-HCl gels, 4% to 15% and plasmid purification kits were purchased from Bio-Rad (Hercules, Calif). L-165,041, FBS, phenylmethylsulfonyl fluoride (PMSF), leupeptin, aprotinin, ß-glycerophosphate, sodium orthovanadate, sodium fluoride, and streptavidin immobilized on 4% beaded agarose were obtained from Sigma (St Louis, Mo). Rosiglitazone, 15-deoxy--12,14-prostaglandin J₂ (15d-PGJ₂), WY14643, GW9662, and GW501516 were purchased from Cayman Chemical (Ann Arbor, Mich). Restriction enzymes (BglII and HindIII), dual-luciferase reporter assay system, phRL-CMV Renilla luciferase reporter vector, and pGL3-Basic promoter-less luciferase vector were purchased from Promega (Madison, Wis). Unmodified and biotinylated oligonucleotides were acquired from Integrated DNA Technologies (Coralville, Iowa). Genomic DNA purification kits and PCR purification kits were obtained from QIAGEN (Valencia, Calif). BD SMART RACE cDNA Amplification kit was acquired from Clontech (Mountain View, Calif). QuikChange II Site-Directed Mutagenesis kit was purchased from Stratagene (La Jolla, Calif). Protease inhibitor cocktail (Complete), and FuGENE 6 were acquired from Roche Applied Science (Indianapolis, Ind). Radioimmunoprecipitation assay lysis buffer and Protein A Agarose/Salmon Sperm DNA were obtained from Upstate (Lake Placid, NY). Adenoviral PPAR vector (Ad-PPAR) and a control vector (Ad-GFP) was kindly provided by Drs Kinzler and Vogelstein at The Johns Hopkins University (Baltimore, Md).³²

Cell Culture
Spontaneously transformed human umbilical vein endothelial cells (ECV-304) were prepared as previously described.³³ Briefly, cells were seeded at 50% to 60% confluence in a 100-mm tissue culture dish and cultured in DMEM containing 10% FBS, then washed and cultured in fresh serum-free medium for 24 hours before treatment. After washing, they were incubated in fresh serum-free medium with or without L-165,041. Human umbilical vein endothelial cells (HUVECs) were obtained from American Type Culture Collection (Manassas, Va) and cultured in F-12K Medium (American Type Culture Collection) supplemented with 0.1 mg/mL heparin, 0.03 mg/mL endothelial cell growth supplement (Sigma), and 10% FBS.

Plasmid Constructs and Site-Directed Mutagenesis
A 5'-flanking fragment of the YWHAE gene at nucleotide position –1624 to +36 (numbers are relative to the translation start site ATG) was obtained by polymerase chain reaction (PCR) using as template human genomic DNA isolated from human fibroblast Hs68 cells and synthetic oligomers as primers (5'-TCCCAAGCGCCAGAAGCTGA-3' and 5'-CTTCGCCTGGTACACCAGAT-3'). PCR products were purified from agarose gel, digested with BglII/HindIII and subcloned into the luciferase reporter vector pGL3-Basic. Progressive deletion mutants of the YWHAE –1624/+36 promoter fragment were created by PCR. The integrity of all constructs was confirmed by DNA sequencing. The 5'-deletion primers were as follow: –1311, 5'-CAATGGTGTGCTCTCGGTT-3'; –645, 5'-CAGCTACTCGAGAGGCTGA-3'; –295, 5'-TTCGGCAGTCGCAGTTCCC-3'; –164: 5'-GAGCGGTTGCCATAGAG-3'; –137, 5'-GTCCGCGTGCGCAGGCGG-3'; –102, 5'-GCCGCCATTTTTGCTGC-3'. A mutated human YWHAE promoter was prepared using the QuikChange II Site-Directed Mutagenesis kit (Stratagene) according to the protocol of the manufacturer. The wild-type plasmid DNA was PCR amplified with site-directed mutated primers (5'-GCGCCAGTTGCCAGGGAGCGGTGTAACTAGAGCTGAGCAGTTGTCCGCG-3' and 5'-CGCGGACAACTGCTCAGCTCTAGTTACACCGCTCCCTGGCAACTGGCGC-3' in which GTAAC was substituted for TGCCA at position –157/–153³⁴). The mutated DNA fragments were subcloned into pGL3-Basic and confirmed by DNA sequencing.

Mapping YWHAE Transcription Start Site
For analysis of transcription start site, total RNA from ECV-304 was isolated by TRIzol reagent (Invitrogen). 5'-Rapid amplification of cDNA ends (RACE) was performed using BD SMART RACE cDNA Amplification kit (Clontech) according to the protocol of the manufacturer. For 5'-RACE PCR, the following primers were used: 5'-TTTCGTCGTATCGCTCAGCCTGCTCGG-3' and 5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3'. 5'-RACE products (263 bp; Figure I in the online data supplement) were purified by Nucleo Trap Gel Extraction kit (Clontech) and cloned into a pCR2.1 vector using TA Cloning kit (Invitrogen). DH5-competent cells were transformed with ligation products and then performed blue/white screening. Fifteen white transformants were picked and plasmid DNA was isolated by Plasmid Miniprep kit (Bio-Rad). DNA samples were verified by restriction analysis (EcoRI), and 11 transformants were sent for sequencing. All 5'-RACE products sequences matched the sequence of the human chromosome region 17p13.3. For homology sequence search, we used the BLAST program (http://blast.genome.jp).

Transient Transfection and Luciferase Assay
ECV-304 at 60% confluence were transfected with 1 µg of pGL3 plasmid DNA expressing firefly luciferase under control of YWHAE promoter with 3 µL of FuGENE 6 per 35-mm tissue culture dish for 8 hours, as previously described.³⁵ To normalize transfection efficiency 10 ng of phRL-CMV Renilla luciferase reporter vector was cotransfected with YWHAE promoter plasmid DNA. Then, 18 hours after transfection, cells were washed and incubated in serum-free medium for 24 hours following stimulation for 24 hours in serum-free conditions. Afterward, cells were lysed by passive lysis buffer (Promega) and harvested, and luciferase activities were determined using dual luciferase reporter assay system according to protocol provided by manufacturer (Promega). Each experiment was repeated at least 3 times.

Whole Cell Lysate Preparation
Cells were washed with cold PBS containing 1x Complete (protease inhibitor cocktail), harvested, and spun down at 550g for 5 minutes. Cells were lysed using radioimmunoprecipitation assay lysis buffer containing 1x Complete. Cell pellet was then spun down at 6500g for 10 minutes. Supernatant was collected, and protein concentration was determined.

Western Blot Analysis
Western blotting was performed by a procedure described previously.³⁵ Protein (50 µg) was loaded to each lane (150 µg for PPAR), separated by 4% to 15% gradient SDS-PAGE, and transferred to a nitrocellulose membrane. Proteins were detected by enhanced chemiluminescence (Pierce). Antibodies dilutions: anti-PPAR, anti–14-3-3, anti-C/EBPß, anti-C/EBP, anti-Sp1, anti-histone H3 1:200; anti-actin 1:2000.

Quantitative Real-Time RT-PCR Analysis
Quantitative real-time RT-PCR (Q-PCR) was performed by use of an ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) and a TaqMan 2-step Q-PCR method, as previously described.³⁶ Briefly, total cellular RNA was isolated from cell culture by using RiboPure kit from Ambion (Austin, Tex) according to the protocol of the manufacturer. For the reverse-transcription reaction, 1 µg of total RNA (High Capacity cDNA Archive kit, Applied Biosystems) was used, and 10 ng of transcribed DNA was spent for each Q-PCR. As a target probe, TaqMan MGB human YWHAE labeled with 6-carboxy-fluorescein dye (FAM) (Applied Biosystems) was used. As an endogenous control, we used TaqMan MGB human ß-actin probe labeled with FAM (Applied Biosystems). We applied comparative C_T method to calculate the amount of target mRNA normalized to an endogenous reference (ß-actin) run in the same Q-PCR. Each sample was tested in triplicates to assure reproducibility. The following PCR conditions were used: 95°C for 10 minutes, 95°C for 15 seconds, and 60°C for 1 minute for 40 cycles in an ABI Prism 7000 (Applied Biosystems). Evaluation of threshold cycle, amplification plot, and spectra was performed using an ABI PRISM 7000 Sequence Detection System Version 1.0 (Applied Biosystems).

Small Interfering RNA
Small interfering RNA of PPAR (siPPAR) and nontargeting control siRNA (siRNA control no. 1) were purchased from Dharmacon (Chicago, Ill). Experiments were performed using DharmaFECT1 (Dharmacon) as a transfection agent and siRNA at 100 nmol/L concentration. For promoter analysis, cells at 80% confluence were first cotransfected with YWHAE promoter and phRL-CMV Renilla reporter vectors (see below, under Transient Transfection and Luciferase Assay) using FuGENE6 in serum-free medium. Medium was removed and replaced with complete medium 8 hours after lipid–DNA complexes were introduced to cells. Sixteen hours later, cells were transfected again using DharmaFECT1 (3.5 µL/35-mm tissue culture dish) with siRNA reagent. After 24 hours, medium was changed and cells were treated with or without 50 µmol/L L-165,041. Promoter activity was assessed after 24 hours of incubation with L-165,041. For mRNA or Western blot analysis, cells were transfected with target gene siRNA or control nontargeting siRNA at 100 mmol/L concentrations using 17.5 µL of DharmaFECT1 per 100-mm tissue culture dish. Twenty-four hours after transfection, cells were washed and incubated in serum-free medium for the following 24 hours. Next, cells were treated with or without L-165,041 for 24 hours and then processed.

Nuclear Extract Preparation
Nuclear extracts were prepared by a procedure previously described.³⁵ Cells were harvested in 1 mL PBSI buffer (PBS buffer containing multiple protease inhibitors: 1 mmol/L sodium orthovanadate; 10 mmol/L sodium fluoride; 25 mmol/L ß-glycerophosphate; 0.1 mmol/L PMSF; 1 µg/mL aprotinin; 1 µg/mL leupeptin; and 0.5 mmol/L dithiothreitol) and spun down at 550g for 5 minutes. Pellet was resuspended in buffer A (10 mmol/L Hepes [pH 7.9], 1.5 mmol/L MgCl₂, 10 mmol/L KCl, 300 mmol/L sucrose, 0.5% Nonidet P-40, 0.1 mmol/L PMSF, 1 µg/mL aprotinin, 1 µg/mL leupeptin, and 0.5 mmol/L dithiothreitol), kept on ice for 10 minutes, and then centrifuged at 2600g for 20 seconds. Pellet was dissolved in PBSI and sonicated for 5 seconds. The sample was spun down at 10 400g for 5 minutes, and the supernatant was aliquoted and kept at –80°C.

Streptavidin-Agarose Pull-Down Assay
Binding of nuclear extract proteins to biotinylated probes were assayed by streptavidin pull down as previously described.³⁵ Briefly, 400 µg of nuclear extract proteins in 500 µL of PBSI buffer were incubated with a mixture of 4 µg of double-stranded biotinylated 20-bp oligonucleotides containing either a human YWHAE promoter (–164/–145) within C/EBP enhancer element (shown underlined), 5'-GAGCGGTTGCCATAGAGCTG-3', or a human YWHAE promoter (–164/–145) with mutated C/EBP sequence, (shown underlined), 5'-GAGCGGTGTAACTAGAGCTG-3' (TGCCA substituted by GTAAC³⁴) and 40 µL of 4% beaded-agarose conjugated with streptavidin for 2 hours on a rocking platform at room temperature. The beads were collected by centrifugation at 550g for 1 minute, washed 3 times with PBSI, and resuspended in 40 µL of Laemmli sample buffer. Nuclear proteins bound to the beads were dissociated by incubating the mixture at 95°C for 5 minutes and analyzed by Western blotting. The same procedure was used to study binding of nuclear proteins to a double-stranded biotinylated 21-bp oligonucleotide probe containing the –606/–578 bp YWHAE promoter sequence containing a Sp1-binding element (shown underlined), 5'-CCGGGAAGGCGGACGTTGTGGTAACCCGA-3'.

Chromatin Immunoprecipitation Assays
For chromatin immunoprecipitation (ChIP) assay, ECV-304 (80% confluent) were cross-linked with 1% (final concentration) formaldehyde for 10 minutes at room temperature. To quench nonreacted formaldehyde, cells were incubated in 125 mmol/L glycine for 5 minutes in room temperature. Then cells were washed in cold PBS and harvested in PBS containing protease inhibitors (1 µg/mL aprotinin, 1 µg/mL leupeptin, and 1 mmol/L PMSF), spun down at 700g for 5 minutes. Cell pellet was resuspended in SDS buffer (1% SDS, 10 mmol/L EDTA, 50 mmol/L Tris [pH 8.1]) containing protease inhibitors and lysed on ice for 10 minutes. Cell lysate was then sonicated and spun down at 10 000g for 10 minutes. Supernatant was divided in 5 tubes for input DNA, normal rabbit IgG, and anti-protein of interest, then diluted 10 times with dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mmol/L EDTA, 16.7 mmol/L Tris-HCl [pH 8.1], 167 mmol/L NaCl, protease inhibitors). Lysate was precleared with 50 µL of Protein A Agarose/Salmon Sperm DNA for 30 minutes at 4°C. The precleared chromatin was incubated overnight at 4°C with 2.5 µg of each antibody, and the immunocomplexes were collected on Protein A Agarose/Salmon Sperm DNA beads. Immunoprecipitated chromatin was washed with 1 mL of each of low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mmol/L EDTA, 20 mmol/L Tris-HCl [pH 8.1], 150 mmol/L NaCl), high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mmol/L EDTA, 20 mmol/L Tris-HCl [pH 8.1], 0.5 mol/L NaCl), lithium chloride wash buffer (0.25 mol/L LiCl, 1% NP-40, 1% deoxycholic acid, 1 mmol/L EDTA, 10 mmol/L Tris-HCl [pH 8.1]), and TE buffer (10 mmol/L Tris-HCl [pH 8.1], 1 mmol/L EDTA); and eluted with elution buffer (1% SDS, 0.1 mol/L NaHCO₃). Cross-linking was reversed by heating samples to 65°C for 4 hours. DNA was recovered by phenol/chloroform extraction and ethanol precipitation. For PCR detection, 4% of purified DNA was used as a template with the following primers: 5'-TCTTTTGAAACACAGTCTCACA-3', 5'-AATTAGCTTGGCATGGTGG-3' for promoter fragment between –1175 and –1019; and 5'-CGTTACAGCCTCCGTCGTTC-3', 5'-ATAGTGTCTCCGACTCTCTCAGCC-3' for promoter fragment located between –432 and –45 bp. DNA samples were amplified by PCR using the following conditions: 95°C for 5 minutes followed by 35 cycles of heating to 95°C for 1 minute, annealing at 59°C for 1 minute (for fragment –432/–45) or 54°C for 1 minute (for fragment –1175/–1019), and extension at 72°C for 2 minutes.

Re-ChIP of Chromatin Bound Proteins
Sequential ChIP assays used a similar protocol with the following exceptions. Primary immunocomplex was eluted with elution buffer supplemented with 10 mmol/L dithiothreitol. The eluate was then diluted 20 times with buffer (1 mmol/L EDTA, 50 mmol/L NaCl, 1% Triton X-100, 20 mmol/L Tris-HCl [pH 8.1]) and immunoprecipitated with the secondary antibody.

Statistical Analysis
ANOVA software (Smith’s Statistical Package; http://www.economics.pomona.edu/StatSite/framepg.html) was used to determine statistical differences of luciferase activity between groups. A probability value of <0.05 was considered to be statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
To study the elements that regulate transcription of YWHAE, the gene that encodes 14-3-3, we cloned the 5'-flanking region of the human YWHAE gene (GenBank accession number NM_006761) up to 1624 bp upstream of the translation initiation site ATG (Figure 1). The cloned YWHAE promoter was placed upstream of the luciferase reporter pGL3 Basic. We also generated serial 5'-deletion constructs of the YWHAE promoter (–1311/+36, –645/+36, –295/+36, –164/+36, and –102/+36) and subcloned them into pGL3 Basic. The full-length promoter nucleotide sequence does not contain a canonical TATA box and is CG rich. It harbors putative binding sites for transactivators such as PPAR, C/EBP, Sp1, and nuclear factor kappa B (NF-B) (Figure 1). These features are characteristic of a housekeeping gene that may be regulated by multiple trans-acting elements. The bona fide transcription start site of YWHAE was determined by 5'-RACE analysis in ECV-304 cells, as shown in supplemental Figure I. Cloning and nucleotide sequence analysis of eleven 5'-RACE products revealed 2 transcription start sites (–110 bp and –106 bp) as a major and minor transcription start site, respectively (Figure 1).

View larger version (25K): [in this window] [in a new window]   Figure 1. Promoter region of human YWHAE gene. The nucleotide sequence of the human YWHAE promoter region was cloned by PCR. Numbers are relative to the translation initiation site ATG, which is marked in bold characters. Location of putative response elements are underlined. The major (–110) and minor (–106) transcription start sites, as determined by 5'-RACE analysis, are marked by an asterisk.

To ascertain the promoter function of the 5'-flanking region of YWHAE gene, and test whether PPAR regulates its activity, we transfected ECV-304 cells with either a vector containing the luciferase reporter pGL3 Basic driven by the full-length YWHAE promoter construct (–1624/+36) or the promoter-less reporter pGL3 Basic. Cells were treated with increasing concentrations of the PPAR agonist L-165,041 (1, 10, and 50 µmol/L) for 24 hours. In time-course experiments, cells were exposed to 50 µmol/L L-165,041 for 4, 8, and 24 hours. L-165,041 enhanced YWHAE promoter activity in a concentration and time-dependent manner, with a 2.5- to 3-fold maximal increase after a 24-hour exposure (Figure 2A and 2B), whereas the promoter-less reporter pGL3 Basic displayed no significant upregulation (Figure 2A). We also tested the specific PPAR ligand GW501516 and obtained similar concentration and time-dependent results, with a maximal 2-fold increase in promoter activity after exposure to 10 µmol/L GW501516 for 24 hours (supplemental Figure IIA and IIB). Next, we evaluated the effects of PPAR overexpression. ECV-304 cells were transfected with adenoviral PPAR vector (Ad-PPAR), whereas adenoviral green fluorescent protein vector (Ad-GFP) was used as a control. After confirming that Ad-PPAR increases PPAR protein expression in ECV-304 (Figure 2C), we determined that Ad-PPAR enhances YWHAE promoter activity 2-fold by cotransfecting ECV-304 with the –1624/+36 construct and Ad-PPAR (Figure 2D). Concurrent exposure of Ad-PPAR–treated cells to 50 µmol/L L-165,041 for 24 hours did not augment significantly the activity of the promoter vector (Figure 2D). To further assess the specificity of this response, we tested the effects of PPAR and PPAR agonists on YWHAE promoter. ECV-304 cells were transfected with the –1624/+36 construct and then exposed for 24 hours to increasing concentrations of either the PPAR agonists 15d-PGJ₂ and rosiglitazone or the PPAR agonist WY14643. Whereas 2.5 µmol/L 15d-PGJ₂ was sufficient to suppress YWHAE promoter activity (Figure 3A), rosiglitazone had moderate suppressive effects only at a concentration of 50 µmol/L (Figure 3B). Interestingly, the PPRA antagonist GW9662 did not reverse the suppressive effects of either PPAR agonist (Figure 3C), implying that the action of 15d-PGJ₂ and 50µmol/L rosiglitazone are PPAR independent. Importantly, the specific PPAR agonist rosiglitazone does not affect YWHAE promoter up to a dose of 10 µmol/L (Figure 3B). The response of the promoter to increasing concentrations of the PPAR agonist WY14643 was also evaluated. We found that WY14643 had no detectable effect up to 50 µmol/L for 24 hours (Figure 3D). Taken together, these data provide evidence that the promoter region of YWHAE is regulated by PPAR in endothelial cells, whereas selective activation of either PPAR or PPAR has no apparent effect under our experimental conditions.

View larger version (16K): [in this window] [in a new window]   Figure 2. PPAR agonists and PPAR overexpression increase YWHAE promoter activity in endothelial cells. ECV-304 cells were transfected with –1624/+36 YWHAE promoter construct. In dose-response experiments, cells were treated with increasing concentrations of L-165,041 for 24 hours. In time-course experiments, cells were exposed to 50 µmol/L L-165,041 for the indicated period of time. A, Upregulation of promoter by L-165,041 in a concentration-dependent manner, illustrated as a black bar. The response of the promoter-less construct pGL3 Basic is shown as a white bar. Each bar is the mean±SEM of 4 to 6 experiments. B, Time-dependent promoter regulation by L-165,041. Each bar is the mean±SEM of 4 to 6 experiments. C, PPAR adenoviral vector (Ad-PPAR) increases expression of PPAR protein. ECV-304 cells were transfected with Ad-PPAR (50 multiplicities of infection [moi]) or green fluorescent protein adenoviral vector (Ad-GFP; 50 moi) as a control. Twenty-four hours posttransfection, cells were harvested, lysed, and immunoblotted with anti-PPAR or anti-actin antibody. A representative blot of 3 experiments is shown. D, Ad-PPAR augments YWHAE promoter activity. ECV-304 cells were cotransfected with luciferase reporter containing YWHAE promoter and Ad-PPAR (50 moi) or Ad-GFP (50 moi) as a control. Cells were then treated with or without 50 µmol/L L-165,041 for 24 hours, and luciferase activity was determined. Each bar is the mean±SEM of 3 to 4 experiments. *P<0.05.

View larger version (18K): [in this window] [in a new window]   Figure 3. Effect of PPAR and PPAR agonists on YWHAE promoter. Cells were transfected with –1624/+36 construct and treated with indicated concentration of either PPAR (15d-PGJ2 and rosiglitazone) or PPAR ligands (WY14643) for 24 hours; then promoter activity was measured. A, 15d-PGJ2 downregulates YWHAE promoter activity. B, Rosiglitazone does not affect YWHAE promoter up to 10 µmol/L but depresses it slightly at 50 µmol/L. C, PPAR antagonist GW9662 does not reverse suppression of YWHAE promoter caused by 15d-PGJ2 or 50 µmol/L rosiglitazone. Cells were pretreated with 10 µmol/L GW9662 for 1 hour before exposure to either 15d-PGJ2 or rosiglitazone at the indicated concentrations. D, PPAR agonist WY14643 does not affect YWHAE promoter activity. Each bar is the mean±SEM of 3 experiments. *P<0.05.

Before focusing our efforts on the transcriptional mechanisms that mediate the effects of PPAR on YWHAE promoter, we elected to determine the biological significance of these findings by studying whether PPAR activation regulates YWHAE transcription and 14-3-3 protein expression in human endothelial cells. As L-165,041 appeared to recapitulate the induction of YWHAE by PPAR, this agonist was used in the following experiments. ECV-304 cells and HUVECs were treated with 50 µmol/L L-165,041 for 24 hours, and then YWHAE mRNA and 14-3-3 protein level were assessed by quantitative Q-PCR and western blotting, respectively. In both endothelial cell lines, YWHAE transcripts were significantly augmented by L-165,041 (Figure 4A and 4C). Furthermore, 14-3-3 protein was constitutively expressed, and its level was upregulated 2- to 2.5-fold by L-165,041 (Figure 4B and 4D). Cotreatment of ECV-304 with 50 µmol/L L-165,041 and 5 µmol/L 15d-PGJ₂ suppressed 14-3-3 upregulation, implying that the PPAR-independent pathways used by 15d-PGJ₂ can overcome PPAR-mediated activation of 14-3-3 (Figure 4B). To further evaluate the role of endogenous PPAR on 14-3-3 protein expression, we undertook a siRNA approach to reduce the level of PPAR and then study the effect of L-165,041 on YWHAE promoter and mRNA, and 14-3-3 protein expression. We identified a siRNA oligonucleotide that specifically abrogated PPAR expression in ECV-304, both at baseline and after exposure to 50 µmol/L L-165,041 for 24 hours (Figure 5A). The reduced levels were specific because actin was not affected. We found that siPPAR suppresses YWHAE mRNA transcripts and 14-3-3 protein expression in the resting state (Figure 5C and 5D). Furthermore, siPPAR depressed YWHAE promoter and mRNA, and 14-3-3 protein, expression on stimulation with L-165,041 (Figure 5B, 5C and 5D). These data, combined with the previous findings on YWHAE promoter (Figure 2), not only demonstrate that PPAR activation targets 14-3-3 by regulating its transcription but also reveal that the intracellular level of PPAR protein is important in the modulation of YWHAE gene expression.

View larger version (14K): [in this window] [in a new window]   Figure 4. PPAR activation augments YWHAE transcription and 14-3-3 protein expression in endothelial cells. ECV-304 cells and HUVECs were treated with 50 µmol/L L-165,041 for 24 hours; then either total RNA was isolated and quantitative Q-PCR analysis performed or Western blot analysis of 14-3-3 protein was performed. A and B, L-165,041 upregulates YWHAE transcription (A) and 14-3-3 protein expression (B) in ECV-304 cells. Coexposure of cells to L-165,041 and 5 µmol/L 15d-PGJ2 depressed PPAR-dependent upregulation of 14-3-3 expression. C and D, L-165,041 upregulates YWHAE transcription (C) and 14-3-3 protein expression (D) in HUVECs. For Q-PCR, 14-3-3 and ß-actin TaqMan probes were used. 14-3-3 expression level was calculated using CT comparative method (see Materials and Methods for details). Each bar is the mean±SD of 3 experiments. *P<0.05. Western blots are representative of 3 independent experiments. Actin levels were measured as a control and the mean densitometry (expressed in arbitrary units) was calculated as a ratio to actin. C indicates control; L, L-165,041.

View larger version (15K): [in this window] [in a new window]   Figure 5. Knock down of endogenous PPAR suppresses YWHAE transcription and 14-3-3 protein expression. ECV-304 cells were transfected with siPPAR or nontargeting siRNA for 24 hours and then treated with or without 50 µmol/L L-165,041 for 24 hours. A, siPPAR suppresses basal and L-165,041–dependent PPAR protein expression. Immunoblots were performed using anti-PPAR antibody. B, siPPAR downregulates L-165,041–dependent YWHAE promoter activity. ECV-304 cells were transfected with YWHAE luciferase vector before transfection with siPPAR or nontargeting siRNA. Promoter activity was measured as luciferase assay. Each bar is the mean±SEM of 3 experiments. C, siPPAR depresses baseline and abrogates L-165,041–dependent YWHAE transcription. Total RNA was isolated and subjected to quantitative Q-PCR analysis, where 14-3-3 and ß-actin TaqMan probes were used. 14-3-3 expression level was calculated using CT comparative method (see Materials and Methods for details). Each bar is the mean±SD of 3 experiments. D, siPPAR suppresses baseline and abolishes L-165,041–dependent 14-3-3 protein expression. Western blots were performed using anti–14-3-3 antibody and are representative of 3 independent experiments. Actin levels were measured as a control and the mean densitometry (expressed in arbitrary units) was calculated as a ratio to actin. *P<0.05. C indicates control; L, L-165,041.

We then turned our attention back to YWHAE promoter to determine the cis-regulatory region that responds to PPAR agonists. We transfected ECV-304 cells with serial 5'-deletion promoter constructs and determined their response to 50 µmol/L L-165,041 for 24 hours. To our surprise, the –1624- to –1311-bp region harboring 3 potential PPREs was not required for promoter response to L-165,041. In fact, constructs –1311/+36 and –645/+36 exhibited a higher activity, both basal and L-165,041–induced, compared with the full-length construct (Figure 6A). Interestingly, a significant decrease in luciferase activity was detected in 2 regions: –645/–295 and –164/–102 bp (Figure 6A). Similar results were also obtained using the PPAR agonist GW501516 (data not shown). Evaluation of promoter activity as fold induction clearly showed that, although the activity of the –1624/+36 construct may increase up to 3-fold, response of the promoter remains robust up to –164/+36, but is abolished in the –102/+36 construct (supplemental Figure III). These findings suggest the involvement of other transactivators. As the region between –164 and –102 bp contains a sole putative C/EBP binding element at –160/–151 bp (Figure 1A), we elected to investigate its role by mutating this site and comparing its response to that of the wild-type promoter. We constructed a luciferase reporter vector containing a mutated promoter (–1311/+36 MT) by substituting TGCCA with GTAAC at –157/–153 bp.³⁴ We then transfected ECV-304 with either a wild-type (–1311/+36) or mutated (–1311/+36 MT) construct and treated cells with or without 50 µmol/L L-165,041 for 24 hours. Mutation of the C/EBP site decreased basal YWHAE promoter activity by 50% and L-165,041–induced promoter activity by 70% (Figure 6B). These results demonstrate that PPREs are not required for the response of YWHAE promoter to PPAR agonists. As a specific C/EBP site appears to be necessary both for basal and PPAR-dependent upregulation of promoter activity, these data also imply that C/EBP proteins might be involved in the regulation of 14-3-3 by PPAR.

View larger version (15K): [in this window] [in a new window]   Figure 6. Specific C/EBP response element is necessary for PPAR-dependent YWHAE promoter activation. A, PPREs are not required for L-165,041–dependent YWHAE promoter activation. ECV-304 cells were transfected with serial 5'-deletion constructs of the YWHAE promoter and treated with 50 µmol/L L-165,041 for 24 hours; then luciferase activity was measured. The activity of 5'-deletion YWHAE promoter fragments is expressed as percentage in relation to the promoter activity of –1311/+36 construct, which was designated as 100%. The activity of the promoter-less construct pGL3 Basic is also shown. On the schematic representation of the –1624/+36 bp fragment, black squares denote 3 PPREs and the white square denotes C/EBP response element at –160/–151 bp. B, C/EBP at –160/–151 bp is necessary for L-165,041–dependent YWHAE promoter activation. A mutated YWHAE promoter construct (–1311/+36 MT) was generated by site-directed mutagenesis. The wild-type (WT) (–1311/+36) and mutated (–1311/+36 MT) constructs were transfected into ECV-304 cells, and then cells were treated with or without 50 µmol/L L-165,041 for 24 hours, and promoter activity was assessed as luciferase expression. Each bar is the mean±SEM of 4 to 6 experiments. *P<0.05.

Next, we reasoned that the expression of C/EBPs might be modulated by L-165,041 and that these events would precede the upregulation of 14-3-3. To test our hypothesis, we treated ECV-304 cells with 50 µmol/L L-165,041 and assessed PPAR, C/EBPß, C/EBP, C/EBP, and 14-3-3 protein level at 0, 6, 12, 18, and 24 hours after treatment by western blotting. Low expression of PPAR was detected in ECV-304 cells at baseline, but the level rose by 6 hours and reached a plateau by 18 hours (Figure 7A). C/EBPß and C/EBP proteins increased by 12 hours, peaked at 18 hours, and decreased toward baseline by 24 hours (Figure 7A). C/EBP was almost undetectable at baseline, and no response to L-165,041 was detected (data not shown). Interestingly, we found that 14-3-3 protein expression appeared to peak at 24 hours (Figure 7B). Taken together, these findings are consistent with a model in which C/EBPs mediate the effects of PPAR on YWHAE promoter. To further assess which specific C/EBP might be involved, we collected nuclear proteins from ECV-304 treated with 50 µmol/L L-165,041 for 24 hours and resolved PPAR, C/EBP, and C/EBPß. Although C/EBP expression did not change, both PPAR and C/EBPß protein levels increased significantly in the nucleus at 24 hours (Figure 7C). To determine whether C/EBPß may be regulated by PPAR, we again undertook a siRNA approach to knock down the endogenous level of PPAR and then studied the effects of L-165,041 on C/EBPß protein expression. Consistent with our previous findings, we discovered that L-165,041–dependent upregulation of C/EBPß expression was abolished by siPPAR (Figure 7D). These data demonstrate that PPAR regulates expression of C/EBPß and suggest that C/EBPß may be involved in L-165,041–dependent transcriptional regulation of 14-3-3.

View larger version (19K): [in this window] [in a new window]   Figure 7. PPAR activation and knockdown control C/EBPß and 14-3-3 protein expression. A, L-165,041 regulates expression of PPAR, C/EBPß, C/EBP, and 14-3-3 proteins in a time-dependent manner. After ECV-304 cells were exposed to 50 µmol/L L-165,041 for the indicated period of time, cytosolic proteins were isolated and Western blot analysis performed for PPAR, C/EBPß, C/EBP, and 14-3-3. Actin levels were measured as a control. These blots are representative of 3 experiments. B, L-165,041 time-dependently regulates expression of 14-3-3 protein. Cells were treated as indicated in A, but Western blot analysis was performed using an anti–14-3-3 antibody. Actin levels were measured as a control, and the mean densitometry (expressed in arbitrary units) was calculated as a ratio to actin. These blots are representative of 3 experiments. C, L-165,041 increases expression of PPAR and C/EBPß proteins in the nucleus. ECV-304 cells were treated with 50 µmol/L L-165,041 for 24 hours. Nuclear proteins were then isolated and resolved with antibodies for PPAR, C/EBPß, and C/EBP. Western blot analysis of histone H3 expression was used as a control. These blots are representative of 3 to 4 experiments. D, Endogenous PPAR regulates L-165,041–dependent C/EBPß expression. ECV-304 cells were transfected with siPPAR or nontargeting siRNA for 24 hours, then treated with or without 50 µmol/L L-165,041 for 24 hours. Afterward, cells were harvested, and immunoblots for C/EBPß were performed. Actin levels were measured as a control, and the mean densitometry (expressed in arbitrary units) was calculated as a ratio to actin. These blots are representative of 3 independent experiments. C indicates control, L, L-165,041.

To test the participation of C/EBPß in these events, we performed in vitro DNA binding studies. Nuclear proteins, collected from ECV-304 treated with of without 50 µmol/L L-165,041 for 24 hours, were incubated with a 5'-biotin–labeled probe containing the specific –160/–151 C/EBP response element of YWHAE. Protein–DNA complexes were then pulled down by streptavidin–agarose, and the resolved transactivators were identified by Western blotting. Trace C/EBPß was detected in resting conditions and L-165,041 increased its level significantly (Figure 8A). Importantly, we did not detect PPAR binding to the same C/EBP probe (data not shown). Experiments were also conducted to evaluate C/EBPß binding after mutation of the C/EBP biotinylated probe, where TGCCA was substituted by GTAAC.³⁴ Both basal and L-165,041–dependent C/EBPß binding was suppressed when the C/EBP-mutated probe was used in the binding assay (Figure 8A). Because the –645/+36 fragment confers a much higher promoter activity compared with the –295/+36 fragment (Figure 6A), we also investigated whether Sp1 binds to a probe containing the Sp1 response element (–599/–595) of YWHAE promoter. Although a robust Sp1 signal was detected in resting conditions, L-165,041 did not increase Sp1 protein level (data not shown), implying that Sp1 is not involved in YWHAE promoter upregulation by PPAR. Altogether, these findings suggest that C/EBPß binding plays an important role in the transcriptional response of YWHAE to PPAR.

View larger version (15K): [in this window] [in a new window]   Figure 8. PPAR activation colocalizes CEBPß and PPAR on YWHAE promoter in endothelial cells in vivo. A, PPAR activation increases C/EBPß binding to a specific C/EBP response element on YWHAE promoter in vitro. Mutation of this C/EBP site suppresses L-165,041–dependent C/EBPß binding. ECV-304 cells were treated either with or without 50 µmol/L L-165,041 for 24 hours, nuclear proteins were collected, and C/EBPß binding to a wild-type (TGCCA) or mutant-type (GTAAC) probe containing the specific –160/–151 C/EBP response element of YWHAE was determined by streptavidin pull-down binding assay as described under Materials and Methods. These blots are representative of 3 to 4 experiments. B, L-165,041 determines binding of CEBPß to YWHAE promoter in vivo. After ECV-304 cells were treated with or without 50 µmol/L L-165,041 for 24 hours, histones were cross-linked to DNA in vivo. Chromatin was then sheared by sonication and immunoprecipitated with antibodies against C/EBPß, C/EBP, and PPAR. DNA fragments in the immunoprecipitated chromatin were amplified by a set of primers bordering the YWHAE promoter region between –432 and –45 bp, and PCR products of the expected size (387 bp) were generated. Note that only C/EBPß binding requires L-165,041 treatment, as C/EBP and PPAR bind both at baseline and after treatment. This promoter fragment does not contain PPREs. C, PPAR activation does not induce localization of C/EBPß, C/EBP, or PPAR to a YWHAE region, which does not contain either C/EBP sites or PPREs. ECV-304 cells were treated with 50 µmol/L L-165,041 for 24 hours, and immunoprecipitated DNA was amplified by means of a set of primers flanking the –1175/–1019 promoter region (expected PCR product size of 156 bp), which does not contain either C/EBP sites or PPREs. D, L-165,041 colocalizes CEBPß and PPAR on YWHAE promoter in vivo. ECV-304 cells were treated with or without 50 µmol/L L-165,041 for 24 hours. Protein/chromatin complexes were subjected to C/EBPß antibody and then, after elution from agarose beads, immunoprecipitated with PPAR antibody. The precipitated DNA fragments were amplified by the same set of primers used in B. Normal rabbit IgG was used as a negative control. Input DNA represents 10% of the total chromatin extract.

To verify in vivo the role of C/EBPß in the transcriptional induction of YWHAE by PPAR, ChIP assays were performed in ECV-304 cells treated with or without 50 µmol/L L-165,041 for 24 hours. DNA fragments encompassing the –160/–151 C/EBP response element or a control sequence of the human YWHAE promoter were amplified by PCR in the chromatin immunoprecipitated with anti-C/EBPß antibody. As shown in Figure 8B, no C/EBPß binding was detected in the –432/–45 YWHAE promoter region under resting conditions. However, C/EBPß was readily detected on treatment with 50 µmol/L L-165,041 for 24 hours. Interestingly, PCR amplification of chromatin immunoprecipitated with anti-PPAR and anti-C/EBP antibodies, consistently localized these transcription factors to this region both under quiescent conditions and after PPAR activation (Figure 8B). The –1175/–1019 YWHAE promoter region, which contains neither C/EBP binding sites nor PPRE, was used as control and confirmed no binding of these transcription factors after treatment with L-165,041 (Figure 8C). One question of considerable interest is whether 2 transcription factors colocalize on the same DNA element. To address this point, we performed 2 sequential immunoprecipitations, where chromatin that has been resolved by 1 antibody (ChIP) is eluted and immunoprecipitated with a second antibody (re-ChIP) before being amplified by PCR (see schematic of experiment in supplemental Figure IV). As shown in Figure 8D, amplification of the promoter region encompassing the C/EBP response element was detected following immunoprecipitation with anti-C/EBPß antibody and subsequent immunoprecipitation with anti-PPAR antibody. No amplification was observed after a second immunoprecipitation with IgG immunoglobulins. Altogether, these results clearly demonstrate that L-165,041 induces a transcriptional activating complex between C/EBPß and PPAR on a specific C/EBP binding site of YWHAE promoter.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Here, we investigated the mechanisms by which PPAR agonists control expression of 14-3-3, a key antiinflammatory protein in endothelial cells.¹² Our data not only provide evidence that PPAR modulates expression of YWHAE gene and 14-3-3 protein under resting conditions but also demonstrate that this nuclear receptor upregulates 14-3-3 expression by targeting transcription via a PPRE-independent pathway involving colocalization of C/EBPß and PPAR on YWHAE promoter. Several lines of evidence support these conclusions. First, PPAR agonists regulated YWHAE promoter activity in a concentration- and time-dependent manner. Concordantly, YWHAE promoter was upregulated by PPAR overexpression, whereas specific PPAR and PPAR ligands had no effect on YWHAE promoter under our experimental conditions. Second, PPAR activation increased 14-3-3 mRNA and protein expression in both primary and spontaneously transformed endothelial cell lines, whereas PPAR knockdown depressed basal and L-165,041–dependent YWHAE transcription and 14-3-3 protein level. Third, PPREs were not required for PPAR-dependent YWHAE promoter upregulation. In fact, a specific C/EBP response element was necessary for YWHAE promoter activation by PPAR. Fourth, L-165,041 not only determined maximal upregulation of C/EBPß and PPAR proteins before 14-3-3 but also increased their expression in the nucleus. Concordantly, siPPAR suppressed L-165,041–dependent upregulation of C/EBPß protein expression. Fifth, in vitro binding studies established that PPAR agonist increases C/EBPß binding to a specific C/EBP response element in YWHAE promoter. Finally, in vivo analysis demonstrated that PPAR agonist is required for C/EBPß binding to the YWHAE promoter region encompassing the C/EBP response element, whereas sequential ChIP clearly showed the formation of a transcriptional activating complex between C/EBPß and PPAR on this site. These data provide a novel mechanism by which PPAR regulates gene expression in human endothelial cells. PPAR agonists have an antiatherosclerotic effect in vivo,⁶ whereas 14-3-3 proteins modulate endothelial cell survival and inflammation.^12–14 Therefore, our findings provide an mechanism through which PPAR agonists may maintain homeostasis of the vascular environment and prevent or delay the development of atherosclerosis.³⁷

Although the nature of endogenous PPAR ligands is a partly unresolved issue in PPAR biology, L-165,041 and GW501516 are well-established and highly selective synthetic ligands.^9,38,39 Concentrations of L-165,041 and GW501516 were used to stimulate YWHAE promoter based on our dose-response experiments. In 2 recent studies, 10 µmol/L GW501516 selectively activated PPAR over PPAR and PPAR in colorectal cancer cells,⁴⁰ and up to 100 µmol/L L-165,041 were used to specifically bind and stimulate PPAR in endothelial cells.⁴¹ Alternatively, we selectively overexpressed PPAR by adenoviral vector construct and demonstrated that endogenous PPAR upregulates YWHAE promoter by 2-fold regardless of L-165,041 (Figure 2D). Interestingly, our findings demonstrate that the intracellular level of PPAR is important in regulating YWHAE/14-3-3 (Figure 5B through 5D) and that L-165,041 augments PPAR protein expression (Figure 7A). The molecular mechanisms of this induction are unclear, but this pathway could provide an amplification signal to increase intracellular availability of PPAR on activation by a ligand. Although siPPAR did not depress YWHAE promoter activity under quiescent conditions, the triple transfection (siRNA, Renilla and YWHAE luciferase reporters) required for this experiment may prevent efficient siRNA delivery (L.B., communication with Dharmacon). Importantly, siPPAR did depress YWHAE mRNA and 14-3-3 protein under basal condition. Therefore, PPAR protein level is necessary for YWHAE transcription in the resting state.

The ligand 15d-PGJ₂ suppressed YWHAE promoter and 14-3-3 protein. However, the more specific PPAR agonist rosiglitazone had no effect up to a concentration of 10 µmol/L, whereas 50 µmol/L caused only moderate depression of the promoter (Figure 3B). Importantly, the suppressive effects of both 15d-PGJ₂ and rosiglitazone appear to be PPAR independent, as suggested by the inability of GW9662 to rescue promoter downregulation (Figure 3C). PPAR agonists are known to regulate gene expression both via PPAR-dependent and independent mechanisms,⁴² as elegantly shown for 15d-PGJ₂ directly hindering NF-B signaling by inhibition of IB kinase and NF-B DNA binding.^43,44 Although PPAR alone had no effect on YWHAE promoter, further assessment of both PPAR and PPAR with respect to 14-3-3 may necessitate overexpression of these nuclear receptors in endothelial cells. Along these lines, one could also ask whether these 2 nuclear receptors may act in concert in respect to 14-3-3 regulation. We believe that clarification of the interplay among alternative PPAR isoforms in the regulation of 14-3-3 will require additional studies. These investigations have important implications because of the widespread use of PPAR ligands for the treatment of diabetes mellitus and their ability to attenuate the development of atherosclerosis.^45,46

We describe a novel transactivating mechanism of PPAR involving a C/EBPß-PPAR transcriptional complex in vivo (Figure 8B and 8D). Canonical pathways of PPAR-dependent stimulation of target genes involve their activation by a ligand, recruitment of coactivators, heterodimerization with retinoid X receptor, and binding to PPREs. Data published during the final stages of preparation of this report provide evidence that PPAR activation upregulates YWHAE expression through upstream PPAR binding sites.¹⁴ However, these studies focused on an early time point of YWHAE promoter activation, and 5'-deletion studies were performed using a complex viral system overexpressing both PPAR and cyclooxygenase-1/prostacyclin synthase. Different mechanisms may regulate YWHAE promoter expression at different times, whereas viral vectors could have paradoxical promoter activation patterns. Analysis performed by FirstEF, a software developed by Davuluri et al to identify promoters in the human genome, suggests that the promoter region of YWHAE is between –626 and –58 bp (data available on request).⁴⁷ This region does not contain PPREs but spans the regulatory C/EBP binding site we describe in this report (Figure 1). Interestingly, the 90 bp encompassing this site, specifically between –181 and –91 bp, also represent the only region of complete homology between human and murine YWHAE promoter. The 5'-deletion studies reported here provide clear evidence that PPREs are not required for L-165,041–dependent YWHAE promoter regulation (Figure 6A and supplemental Figure III).

PPRE-independent transcriptional mechanisms are increasingly recognized as key regulatory pathways in PPAR biology. Data in murine cells demonstrate that GW501516 leads to increased binding of C/EBPß to the prostaglandin E₂ receptor, subtype EP4, promoter region, thereby upregulating its transcription.⁴⁸ PPAR has been shown to positively regulate IB promoter via a PPRE-independent pathway mediated by NF-B1 and Sp1.⁴⁹ Also, activation of PPAR inhibits C/EBPß-dependent 1-acid glycoprotein expression by physical interaction with C/EBPß.⁵⁰ However, the interplay between C/EBPß and PPAR may be more intricate than anticipated. As we did not detect binding of PPAR to the –160/–151 C/EBP site in our in vitro experiments using a naked fragment of DNA, additional in vivo regulatory elements and proteins may be required in the stabilization of the C/EBPß-PPAR transcriptional complex. Clearly, further studies are needed to delineate these molecular events.

Results from our in vivo binding studies demonstrate a critical role of different C/EBP isoforms. C/EBP may be important in YWHAE gene regulation under quiescent conditions, whereas C/EBPß mediates the effects of PPAR activation by localizing on YWHAE promoter (Figure 8B). In fact, L-165,041–dependent C/EBPß expression reaches its highest level concurrently with the initial increase of 14-3-3 protein, which peaks at 24 hours (Figure 7A and 7B). Interestingly, a time-dependent switch of C/EBP and ß binding to cyclooxygenase-2 promoter has been shown to differentially regulate cyclooxygenase-2 expression.⁵¹ C/EBPß and C/EBP are members of the ubiquitous C/EBP family of basic leucine zipper transcription factors, which mediate transcription of genes relevant to prevention of apoptosis, cellular differentiation, inflammation and development (reviewed elsewhere⁵²). They form homodimers as well as heterodimers with other C/EBP isoforms, and the dimers bind to a specific DNA sequence at the promoter region of target genes. Because C/EBPß contains an intramolecular negative regulatory region that restricts its binding to DNA unless a critical activation step takes place via phosphorylation,^53,54 it also remains to be established how C/EBPß may be activated by PPAR ligands. As our data show an increased concentration of C/EBPß in the nucleus of endothelial cells, PPAR-dependent YWHAE promoter activation may be explained in the context of C/EBPß phosphorylation promoted by a PPAR agonist. Kinase cascades are increasingly recognized as critical modulators of PPAR activation.⁵⁵ However, current knowledge regarding specific kinases that mediate the effects of PPAR are limited to the downregulation of protein kinase C/mitogen-activated protein kinase pathways via ubiquitin-dependent degradation of protein kinase C,⁵⁶ and the activation of the phosphatidylinositol 3-kinase pathway in keratinocytes and lung carcinoma cells.^4,48

In conclusion, we identified a novel regulatory mechanism in endothelial cells where PPAR targets the human YWHAE promoter via a C/EBPß–PPAR transcriptional complex. These data imply that PPAR agonists might modulate the development of atherosclerosis and acute coronary syndromes, not only by targeting foam cells⁶ and lipoprotein metabolism,^8,9 or protecting against obesity,⁷ but also by promoting endothelial cell survival via 14-3-3. Because the function of 14-3-3 proteins is a field of active and intense investigation, our findings may also be relevant to other tissues, such as the heart, in which 14-3-3 proteins and 14-3-3 have been shown to play important roles.^15–18

	Acknowledgments

 
We are grateful to Drs Schwartz, Barton, Di-Poi, and Wahli for useful advice. We are also indebted to Dr Taegtmeyer for use of the ABI PRISM 7000 Sequence Detection System and for helpful discussion.

Sources of Funding

This work was supported by internal funds from the Department of Pediatrics at The University of Texas at Houston Medical School.

Disclosures

None.

	Footnotes

 
Original received December 15, 2006; revision received February 5, 2007; accepted February 7, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Berger J, Moller DE. The mechanisms of action of PPARs. Annu Rev Med. 2002; 53: 409–435.[CrossRef][Medline] [Order article via Infotrieve]
2. Ijpenberg A, Tan NS, Gelman L, Kersten S, Seydoux J, Xu J, Metzger D, Canaple L, Chambon P, Wahli W, Desvergne B. In vivo activation of PPAR ta