Research Article PPAR- Regulates Trophoblast Differentiation ...downloads.hindawi.com/journals/ppar/2014/637251.pdfPPAR- Regulates Trophoblast Differentiation in the BeWo Cell Model

Research ArticlePPAR-120574 Regulates Trophoblast Differentiationin the BeWo Cell Model

Khrystyna Levytska12 Sascha Drewlo3 Dora Baczyk1 and John Kingdom1245

1 Research Centre for Womenrsquos and Infantsrsquo Health Lunenfeld-Tanenbaum Research Institute Mount Sinai Hospital600 University Avenue Room 3-904 Toronto ON Canada M5G 1Z5

2Department of Laboratory Medicine and Pathobiology University of Toronto ON Canada3Department of Obstetrics and Gynecology C S Mott Center for Human Growth and DevelopmentWayne State University School of Medicine Detroit MI USA

4Maternal-Fetal Medicine Division Department of Obstetrics and Gynecology Mount Sinai Hospital600 University Avenue Room 3-904 Toronto ON Canada M5G 1Z5

5Department of Obstetrics and Gynecology University of Toronto Toronto ON Canada

Correspondence should be addressed to John Kingdom jkingdommtsinaionca

Received 26 September 2013 Revised 27 December 2013 Accepted 2 January 2014 Published 23 February 2014

Academic Editor Richard P Phipps

Copyright copy 2014 Khrystyna Levytska et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Commonpregnancy complications such as severe preeclampsia and intrauterine growth restriction disrupt pregnancy progressionand impairmaternal and fetal wellbeing Placentas from such pregnancies exhibit lesions principally within the syncytiotrophoblast(SCT) a layer in direct contact with maternal blood In humans and mice glial cell missing-1 (GCM-1) promotes differentiationof underlying cytotrophoblast cells into the outer SCT layer GCM-1 may be regulated by the transcription factor peroxisomeproliferator-activated receptor-gamma (PPAR-120574) in mice PPAR-120574 promotes labyrinthine trophoblast differentiation via Gcm-1and as we previously demonstrated PPAR-120574 activation ameliorates disease features in rat model of preeclampsia Here we aimedto characterize the baseline activity of PPAR-120574 in the human choriocarcinomaBeWocell line thatmimics SCT formation in vitro andmodulate PPAR-120574 activity to study its effects on cell proliferation versus differentiation We report a novel negative autoregulatorymechanism between PPAR-120574 activity and expression and show that blocking PPAR-120574 activity induces cell proliferation at theexpense of differentiation while these remain unaltered following treatment with the agonist rosiglitazone Gaining a deeperunderstanding of the role and activity of PPAR-120574 in placental physiology will offer new avenues for the development of secondaryprevention andor treatment options for placentally-mediated pregnancy complications

1 Introduction

During normal pregnancy the healthy developing placentaensures effective nourishment of the fetus by facilitating anexchange of gases nutrients and waste products betweenfetal andmaternal circulations [1]The critical cell type in thiscontext is the villous cytotrophoblast (VCT)which constantlyforms new syncytiotrophoblast (SCT) throughout pregnancyVCT cells are a heterogeneous population comprising ofprogenitors that divide repeatedly (symmetrically) and otherswhich divide asymmetrically to produce postmitotic cellsthat develop the potential to fuse into the overlying SCT

The process of cytotrophoblast proliferation differentiationand syncytial fusion is required to generate sufficient SCT tocover the developing placental villi [1]

The phenomenon of asymmetric cell division directed bythe transcription factor glial cell missing-1 (GCM-1) was firstidentified in Drosophila [2] In mice knock-out experimentsdemonstrated that Gcm-1 is critical for labyrinth forma-tion [3] whereas 119866119888119898-1+minus mice are viable with abnormalplacental development [4] Studies of human placental villireport analogous GCM-1 localization and function in villoustrophoblast to that seen in rodents [5 6] Interestingly levelsof GCM-1 are downregulated in the placentas of women

Hindawi Publishing CorporationPPAR ResearchVolume 2014 Article ID 637251 13 pageshttpdxdoiorg1011552014637251

2 PPAR Research

suffering from severe preeclampsia a pregnancy complica-tion characterized by impaired villous structure and placentaldevelopment [7 8]

The transcription factor peroxisome proliferator-acti-vated receptor gamma (PPAR-120574) may regulate the processof SCT formation since it is known to regulate Gcm-1expression [9 10] and placental development [11] in miceThe ability to control the transcriptional activity of PPAR-120574with highly specific drugs including the agonist rosiglitazone[12] and the antagonist T0070907 [13] has been utilized inan attempt to study its role in several features of placentaldevelopment We have recently shown that rosiglitazone-induced PPAR-120574 activation is able to ameliorate diseasecharacteristics in the rat model of preeclampsia via its down-stream target heme oxygenase-1 (HO-1) an enzyme whichproduces carbon monoxide (CO a potent vasodilator) andbilirubin an antioxidant [14 15] Furthermore other groupshave looked at the effect of PPAR-120574 activity induction oncell migration as well as analyzed expression profiles in VCTand extravillous trophoblast (EVT) cells [10 16] leading tothe conclusion that PPAR-120574 plays a role in human placentaldevelopment

In the present study we attempted to study the activityof this transcription factor in the human choriocarcinoma-derived cell line BeWo an established model of SCT forma-tion in vitro and primary trophoblast cells to establish theeffect of PPAR-120574 activity modulation on key features oftrophoblast physiology We hypothesized that stimulatingPPAR-120574 activity with a highly specific agonist rosiglitazonewill induce cell differentiation [resulting in increased expres-sion of the differentiation marker GCM-1 and augmentedrelease of cell fusionmarker human chorionic gonadotropin-120573 (120573-hCG)] while the opposite (ie proliferation) will befavored by the antagonist T0070907 To our surprise whenexamining the response of GCM-1 expression and 120573-hCGrelease we show that the baseline PPAR-120574 activity in theBeWo cell line is relatively high limiting our ability to stimu-late it further with the agonist rosiglitazone but presentingan opportunity to block it with the antagonist T0070907Interestingly our ability to stimulate theGCM-1 response wasaugmented when the endogenous levels of PPAR-120574 aredownregulated using siRNA adding support to the conceptthat PPAR-120574 regulates the differentiation axis in the BeWo cellline Although these findings outline the limitation of this cellline they nonetheless support the wide used of this modelin the study of molecular mechanisms present in the humanplacenta since the responses of target genes in isolatedhuman cytotrophoblast cells were found to be analogous tothose in BeWo cells

2 Materials and Methods

21 BeWo Cells The human choriocarcinoma-derived BeWocell line was purchased from ATCC (Burlington ONCanada) and fingerprinted at the Centre for Applied Genom-ics (SickKids Toronto ON Canada) markers were foundto be identical to those in the ATCC database BeWo cellsbetween passages 10 and 20were used For all treatments cells

were maintained in F12K medium (Wisent Inc St BrunoQC Canada) supplemented with 10 fetal bovine serum(FBS Canadian grade heat-inactivated Invitrogen Burling-ton ON Canada) 100 unitsmL penicillin 100 120583gmL strep-tomycin and 2 nM L-glutamine (Life Technologies Burling-ton ON Canada) in atmospheric O

25 CO

2at 37∘C

For treatments BeWo cells were seeded at 50000 cellsper 1mL of media and allowed to attach for 24 hours Thefollowing day cells were pretreated with the PPAR-120574 antag-onist T0070907 (Cayman Chemical Ann Arbor MI USA)for 30 minutes and were then treated with either the PPAR-120574 agonist rosiglitazone (Enzo Life Sciences BurlingtonON Canada) the antagonist T0070907 (Cayman Chemical)andor the weak agonist forskolin (Sigma-Aldrich OakvilleON Canada) Cell viability under all treatments was assessedat 48 hours of culture using CytoTox-ONE HomogeneousMembrane Integrity Assay (Promega Madison WI USA)No drug treatments resulted in significant cell toxicity at 48hours (data not shown)

Following treatment cells were washed in ice-cold D-PBS (Wisent Inc) and collected according to different down-stream applications For RNA analysis cells were collectedintoRLTPlus buffer (Qiagen TorontoONCanada)with 10120573-mercaptoethanol (Fisher Scientific Ottawa ON Canada)For protein analysis cells were scraped and collected in RIPABuffer (Thermo Scientific Ottawa ON Canada) with phos-phatase (Phosphatase Inhibitor Cocktail 2 Sigma-Aldrich)and protease inhibitors (Complete Mini EDTA-free ProteaseInhibitor Cocktail Tablets Roche Applied Science LavalQC Canada) Conditioned medium was collected and cen-trifuged for 5 minutes at 425 g at room temperature (RT) toremove cellular debris All collected samples were stored atminus80∘C for further analysis

22 Transfection of BeWo Cells The BeWo cells were trans-fected with commercially-available double-stranded siRNAoligonucleotides against the human PPAR-120574 sequence (San-taCruz Biotechnology Dallas TX USA) Nonsilencing con-trol (sequence 51015840-TTCTCCGAACGTGTCACGT-31015840) wasused as a negative control BeWo cells were plated into 12-well plates and transfected the following day according to themanufacturerrsquos specifications Briefly 50-confluent BeWocells were transfected with 30120583M of PPAR-120574-targeted siRNAor nonsilencing control with 25 120583L of PepMute siRNA trans-fection reagent (SignaGen Laboratories Ijamsville MD) perwell The following day media were changed and cells weretreated for another 48 hours Experiments with fluorescent-labeled siRNA established 80ndash90 transfection efficiency(data not shown) Toxicity of siRNA treatmentwasmonitoredwith Human Interferon Alpha ELISA kit (PBL BiomedicalLaboratories Brussels Belgium)

23 Human Primary Cytotrophoblast Cell Isolation Firstand second trimester (12ndash19 weeks) human placental tissuewas used for the isolation of primary cytotrophoblast (CT)cells Placentas were obtained from the Morgentaler Clinic(Toronto Canada) following elective legal terminations ofpregnancy Mount Sinai Hospital Research Ethics Board

PPAR Research 3

Table 1 Primer sequences

Gene Primer sequence (51015840 rarr 31015840) Number of bases Primer pair efficiency

GCM-1 Forward ATG GCA CCT CTA GCC CCT ACA 21 1025Reverse GCT CTT CTT GCC TCA GCT TCT AA 23

PPAR-120574 Forward CTC AGT GGA GAC CGC CCA GG 20 1092Reverse GCT CCA GGG CTT GTA GCA GG 20

HMOX-1 Forward CGG CTT CAA GCT GGT GAT GGC 21 1106Reverse CCT GCT CCA GGG CAG CCT TG 20

GAPDH Forward AGA TCA TCA GCA ATG CCT CC 20 1082Reverse CAT GAG TCC TCC CAC GAT AC 20

YWHAZ Forward ACT TTT GGT ACA TTG TGG CTT CAA 24 953Reverse CCG CCA GGA CAA ACC AGT AT 20

TBP Forward TGC ACA GGA GCC AAG AGT GAA 21 1104Reverse CAC ATC ACA GCT CCC CAC CA 20

HPRT Forward TGA CAC TGG CAA AAC AAT GCA 21 957Reverse GGT CCT TTT CAC CAG CAA GCT 21

approval (MSH REB no 04-0018-U) was obtained andprior to tissue collection all patients gave written informedconsent gestational age and viability were established preop-eratively Primary CT cells were isolated according to the fol-lowing protocol (modified from the original Kliman method[17]) Briefly placental tissue was separated frommembranesand subjected to a 40-min digestion in Trypsin Diges-tion Cocktail [25mM Hepes (Sigma-Aldrich) 50 unitsmLDNase I (Sigma-Aldrich) 425mM MgSO

4 0125 Trypsin

25 120583gmL fungizone 100 120583gmL gentamacin (all from Invit-rogen) and diluted in 1 1 mix of Hankrsquos buffered salt solution(HBSS) with and withoutMg2+ and Ca2+] to remove the SCTlayer Four 20-min digestions were then performed to col-lect subsequent layers containing primary cytotrophoblastfibroblast and endothelial cells Collected cells were washedand spun in Ficoll-Paque PLUS reagent (1077 gmL GEHealthcare Life Sciences Sweden) for 10minutes at 2000 rpmwithout a break to remove red blood cells and cell debrisFollowing separation from red blood cells the resultingring of cells was collected washed and resuspended inDMEMF12medium (Life Technologies) supplemented with10 FBS (Invitrogen) 100 unitsmL penicillin 100 120583gmLstreptomycin 2 nM L-glutamine (Life Technologies) and25 120583gmL fungizone (Invitrogen) All isolated cells wereseeded in 6-well plates at 50 confluency and maintainedin cell medium (described above) in 8 pO

2at 37∘C The

following day cells were washed once with D-PBS (WisentInc) to remove cellular debris and cultured for an additional24 hours under different drug treatments or vehicle controlsCells were collected for RNA analysis according to theprotocol described above

24 RNA Extraction Reverse Transcription and qRT-PCRBeWo and CT cell RNA was extracted using the RNeasyPlus Mini Kit (Qiagen) according to manufacturerrsquos instruc-tions Five hundred nanograms (ng) of RNA was reversetranscribed using iScript Reverse Transcription Supermix

(Bio-Rad Mississauga ON Canada) according to the fol-lowing protocol 5 minutes at 25∘C 30 minutes at 42∘Cand followed by 5 minutes at 85∘C Gene expression wasmeasured using quantitative real-time PCR (qRT-PCR) andrun on the CFX384 Real-Time PCR Detection System (Bio-Rad) with LuminoCt SYBR Green qPCR ReadyMix (Sigma-Aldrich) qRT-PCR reactions were performed according tothe following protocol initial activation at 95∘C for 5minutesfollowed by 38 thermal cycles of denaturation at 95∘C for5 seconds and annealingextension at 60∘C for 20 secondsGene expression was normalized to the geometric mean ofthree housekeeping genes (HPRT TBP and YWHAZ forBeWo cells HPRT GAPDH and YWHAZ for primary CTcells) Gene of interest expression in each treatment wasexpressed as fold change relative to its respective vehicle (setas 1) Primer sequences for all genes are listed in Table 1

25 Protein Isolation For protein analyses BeWo cells werecollected in 250 120583L of RIPA lysis buffer with phosphatase andprotease inhibitors Samples were homogenized and placedon a nutator for 1 hour at 4∘C after which they were spunat top speed for 10 minutes at 4∘C and the supernatantwas collected Protein concentration was measured usingPierce BCA Protein Kit (Thermo Scientific) according tomanufacturerrsquos instructions

26 Western Blotting Twenty 120583g of total protein (diluted inRIPA Buffer with inhibitors 4X Loading Dye [Invitrogen]and 10 120573-mercaptoethanol) was electrophoresed in 1x TG-SDS Buffer (Wisent Inc) at 50ndash100V on 4ndash20 Mini-PROTEAN TGX Gels (Bio-Rad) Following electrophore-sis proteins were transferred onto PVDF membrane (Bio-Rad) using the Trans-Blot Turbo Transfer System (Bio-Rad) Immediately following the transfer membranes wereblocked in 5 milkTBS-T (Blotting-Grade Blocker Bio-Rad TWEEN 20 Sigma-Aldrich) for 1 hour at RT All

4 PPAR Research

primary antibodies (See Table 1 in Supplementary Mate-rial available online at httpdxdoiorg1011552014637251)were applied overnight at 4∘C in 5 milkTBS-TThe follow-ing day membranes were washed with 0001 TBS-T (3 times20min) Membranes were incubated for 1 hour at RT withrespective secondary antibodies (GEHealthcare UK LimitedUK) diluted 1 3000 in 5 milkTBS-T Following washingmembranes were developed using Western Lightning Plus-ECL (Thermo Scientific) on Premium Autoradiography Film(Denville Scientific South Plainfield NJ USA) Band inten-sities within linear range were quantified using QuantityOne software (Bio-Rad) Protein levels were normalized toa housekeeping protein (120573-actin 120572-tubulin or lamin B)Protein expression for each condition was further comparedto its respective vehicle control (set as 1)

27 Cellular Fractionation Analysis Cellular fractionationwas performed using the Nuclear Extract Kit (Active MotifBurlington ON Canada) according to manufacturerrsquos pro-tocol Protein concentration was measured using the PierceBCA Protein Kit (Thermo Scientific) Protein amount wasnormalized and prepared forWestern blotting for PPAR-120574 120572-tubulin (cytoplasmic protein) and lamin B (nuclear protein)Both fractions were controlled for purity of separation bymeasuring the expression of the housekeeping protein fromthe other cellular compartment

28 Enzyme-Linked Immunosorbent Assay (ELISA) Free 120573-hCG ELISA Kit (Phoenix Pharmaceuticals Inc BurlingameCA USA) was used to measure 120573-hCG release accordingto manufacturerrsquos instructions 120573-hCG protein concentrationwas quantified using the standard curve and protein levelswere normalized to total released protein 120573-hCG releaseunder different treatments was further normalized to respec-tive vehicle controls (set as 1)

29 Fluorescent Immunohistochemistry (IHC) Immunofluo-rescence was used to visualize PPAR-120574 protein localizationand BeWo cell fusionThe following protocol was used for theexperiments Coverslips were submerged into 100 ethanoland left to dry under UV light for 30 minutes BeWo cellswere plated on coverslips at a density of 150000 cells perwell in 6-well plates Cells were incubated with treatmentsoutlined above Following treatment cells were washed inice-cold D-PBS (Wisent Inc) fixed in 1 1 methanol acetonesolution for 3 minutes on ice and washed with D-PBS Cellswere permeabilized in 02 TritonX-100 (Fisher Scientific)for 5 minutes on ice washed and blocked for 1 hour at RT inProtein Block Serum-Free Ready-To-Use (DAKO Carpinte-ria CA USA) Primary antibodies (Supplementary Table 1)were diluted in blocking solution and incubated overnight at4∘CThe following day the PPAR-120574 signal was amplified withanti-rabbit biotinylated antibody (diluted 1 300 in blockingsolution) for 1 hour at RT Lastly DAPI (1 1000 Sigma-Aldrich) anti-mouse Alexa546 antibody (1 200 Invitrogen)and SA-Alexa488 (1 1000 Invitrogen) all diluted in blockingsolution were incubated for 1 hour at RT Coverslips weremounted on slides using an Immu-Mountmountingmedium

(Thermo Scientific) Slides were left to dry in the dark andstored at 4∘C until further analysis

Fluorescent microscopy was performed using the Spin-ningDiscConfocalMicroscope (DMI6000B LeicaMicrosys-tems Concord ON Canada) Z stacks were taken usingVolocity software Version 530 (PerkinElmer WoodbridgeON Canada) and deconvolved using Huygens Essentialsoftware Version 422 (Scientific Volume Imaging Hilver-sum The Netherlands) All images were taken on the sameday under the same acquisition settings normalized to thehighest PPAR-120574 expression under T0070907 treatment tominimize variability and allow parallel comparison in proteinexpression across time points and treatments For fusionvisualization images were taken at 200x magnification forPPAR-120574 localization analysis Z stacks were taken at 630xmagnification

210 Luciferase Assay One kb region upstream of the humanGCM-1 promoter was analyzed for putative PPAR-120574 bindingsequences using the Gene2Promoter software (Version 63Genomix Germany Supplementary Figure 1(a)) two puta-tive binding sequences were identified Oligonucleotides ofthese sites were synthesised and linkers added for cloninginto the pGL410[luc2] cloning vector (binding sequences 1and 2 Promega) Sites were mutated to generate sequence-based controls (mutated sequences 1 and 2) Binding andmutated sequences are outlined in Supplementary Figure1(b) Oligonucleotides and vectors were digested inde-pendently purified and ligated One-Shot TOP10 Chemi-cally Competent E coli cells (Invitrogen) were transformedand plated on ampicillin-positive plates Antibiotic-resistantclones were isolated using Plasmid Plus Midi Kit (Qiagen)sequenced to confirm insertion and brought to similarconcentrations For transfection experiments BeWo cellswere seeded in tissue culture-suitable 96-well plates (Greinerbio-one Monroe NC USA) at 12500 cellswell to obtain30 confluency The following day cells were transfectedusing ExGene 500 transfection reagent (Fermentas Pitts-burgh PA USA) efficiency was confirmed using a GFP-expressing control plasmid (data not shown) Transfectionoptimization experiments established that 200ndash300 ng ofplasmid DNA were optimal for 70ndash90 transfection effi-ciency using 07120583L of transfection reagent Generated trans-fectionmixes (for binding andmutated sequences) containeda 10 1 composition of experimental vector to a coreportervector (renilla) Following their application culture plateswere centrifuged for 5 minutes at 300 g according to themanufacturerrsquos protocol Eight hours following transfectioncells were treated for 24 hours following which media wereremoved and cells were lysed with Passive Lysis Bufferaccording toDual-Luciferase ReporterAssay Systemprotocol(Promega) Luciferase activity was measured using an auto-mated photometer Renilla luciferase activity was an inter-nal calibrator relative luciferase activity with each plasmid(binding sequences 1 and 2) and drug was normalized toits corresponding vehicle control Furthermore to reducerandom allosteric sequence-dependent background signal

PPAR Research 5

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(b)

Figure 1 PPAR-120574 expression correlates inversely with its activity (a) PPAR-120574mRNA expression levels overtime in response to rosiglitazoneT0070907 their combination and forskolin treatment (b) Cellular PPAR-120574 protein expression at 48 hours of treatment (representative blotin the bottom panel) All treatments are compared to their respective vehicle controls (set as 1 red dashed line) Values are represented asmean plusmn SEM 119875 lt 005 versus vehicle control lowast119875 lt 005 lowastlowast119875 lt 0005 lowastlowastlowast119875 lt 0001 (119899 = 4) Rorosi rosiglitazone TT007 T0070907 Forforskolin

a signal obtainedwith each binding sequence was normalizedto the corresponding mutated sequence

211 Cell Proliferation Assay Relative BeWo cell numberswere assessed using the CellTiter-Fluor Cell Viability Assay(Promega) In 96-well plates (Greiner bio-one) cells wereseeded at 10000 cellswell and treated the following day withdrugs as described above Cell number was measured at 48hours of culture according to the manufacturerrsquos protocolThe plate was read using the spectrometer and recordedOD values were blanked using the media-only control toaccount for background fluorescence Fluorescent measure-ments obtained with this assay represented an indication ofthe relative BeWo cell number such that cell proliferationcould be assessed between different drug treatments relativeto their respective controls (set as 1)

212 Statistical Analysis Experiments were performed intechnical duplicates of at least three biological replicates Dataare represented as mean plusmn standard error of the mean (SEM)Studentrsquos 119905-test was used to make comparison drug treat-ments to corresponding vehicle controls One-way ANOVAfollowed by the Newman-Keuls Multiple Comparison Testwas used to compare between treatment groups All statistical

calculations were performed using GraphPad Prism 52software 119875 values lt 005 were considered significant

3 Results

31 PPAR-120574 Expression Inversely Correlates with Its ActivityIn the first set of experiments we attempted to study theresponse of PPAR-120574 expression to the modulation of itsactivity We assessed the mRNA and protein expressionfollowing rosiglitazone (the agonist) and T0070907 (theantagonist) treatment at different time-points (Figure 1)When the mRNA expression profile was assessed at 3 6 and24 hours we found that induction of PPAR-120574 activity withrosiglitazone resulted in a decrease of receptor expressionwhile treatment with T0070907 had the opposite effectwith the most pronounced changes observed 24 hoursafter treatment (Figure 1(a)) Following activation of PPAR-120574 with rosiglitazone PPAR-120574 mRNA expression decreasedsignificantly to 47 plusmn 34 by 24 hours compared to vehiclecontrol (119875 lt 005 119899 = 4) Conversely blocking PPAR-120574activity with T0070907 resulted in a significant upregulationof its expression by 24 hours (20 plusmn 01-fold 119875 lt 005)Forskolin alone did not have a significant effect on PPAR-120574expression at 24 hours Coadministration of T0070907 withrosiglitazone significantly ameliorated rosiglitazone-induced

6 PPAR Research

PPAR-120574 downregulation at 6 (119875 lt 001) and 24 (119875 lt 0001)hours of treatment (versus rosiglitazone alone)

Next we examined cellular PPAR-120574 protein expression at48 hours of treatment using Western blotting (Figure 1(b))Total PPAR-120574 protein levels were decreased significantly to34 plusmn 98 and to 22 plusmn 63 following treatment with thelower and higher dose of rosiglitazone respectively (119875 lt 005119899 = 4) On the contrary PPAR-120574 expression rose significantlyby 183 plusmn 018-fold following treatment with T0070907(119875 lt 005) PPAR-120574 levels also decreased significantlyfollowing treatment with forskolin a weak agonist of PPAR-120574 (119875 lt 005) Although the combination of rosiglitazoneand T0070907 resulted in a downward trend of PPAR-120574expression levels when compared to vehicle control thisfailed to reach significance (119875 = 00623) Based on ourmRNAand protein analyses we observed that PPAR-120574 expressionand activity are inversely related such that inducing receptoractivity results in a downregulation of its expression and viceversa

32 PPAR-120574 Protein within the Nuclear CompartmentResponds to PPAR-120574 Activity-Modulating Drugs PPAR-120574cellular localization and expression was assessed usingfluorescent IHC and cellular fractionation analysis Proteinlocalization was visualized at 3 6 and 24 hours of treatmentwith rosiglitazone (10120583M) T0070907 (1 120583M) or vehicle(Figure 2(a)) PPAR-120574 was found to mainly localize inthe nucleus with some expression seen in the cytoplasmConcurrent with the PPAR-120574 protein analysis using Westernblotting there was an increased expression of PPAR-120574 afterT0070907 treatment and a decrease in staining levels afterrosiglitazone treatment

Cellular fractionation analysis was also used to studychanges in PPAR-120574 protein localization after 1 6 and 24 hoursof treatment (Figures 2(b) and 2(c)) Although no changes inprotein expression were seen after 1 hour following 6 hoursof rosiglitazone treatment nuclear PPAR-120574 levels decreasedsignificantly by 39 plusmn 108 (119875 lt 005 119899 = 7ndash9) whileT0070907 treatment led to a significant rise in PPAR-120574 levelsby 18 plusmn 03-fold (119875 lt 005) Combining both drugs didnot alter PPAR-120574 protein expression compared to vehicle(Figure 2(b)) On the contrary no expression changes wereseen in the cytoplasmic fraction at 6 hours of treatment(Figure 2(c))

PPAR-120574 expression changes were pronounced in bothcellular compartments after 24 hours of treatment Withinthe nuclear fraction the rosiglitazone-induced decrease inprotein expression remained significant (down by 53 plusmn 86119875 lt 005) while the effect of T0070907 was less pronouncedFurthermore at 24 hours of treatment rosiglitazone led tolower levels of PPAR-120574 within the cytoplasm (05 plusmn 01-fold119875 lt 005) as did the combination of rosiglitazone andT0070907 (05 plusmn 002-fold versus vehicle 119875 lt 005) Col-lectively our results illustrate that following agonist andorantagonist treatments protein expression changes within thecell nucleus were more pronounced compared to changes inthe cell cytoplasm

33 Blocking PPAR-120574Activity Decreases GCM-1 TranscriptionThe BeWo cell model was used to study the effect ofpharmacological PPAR-120574 activity modulation on the pro-liferationdifferentiation balance an important physiologicprocess within the villous trophoblast layerWe assessed theseevents by studying markers of syncytial differentiation (suchasGCM-1 expression and free 120573-hCG release) andmeasuringBeWo cell proliferation

It has been previously described that lack of Ppar-120574 resultsin lower Gcm-1 expression in mouse trophoblast stem cells[9] therefore we studied the response of GCM-1 in BeWocells to rosiglitazone and T0070907 treatments at 3 6 and 24hours of treatment (Figure 3(a)) Activation of PPAR-120574 withthe agonist led to a transient induction of GCM-1 expressionsignificant only at 3 hours of treatment (15 plusmn 01-fold 119875 lt005 119899 = 4) Independent evidence of induction of PPAR-120574 activity was inferred by observing a robust increase inanother PPAR-120574 target heme oxygenase-1 (HO-1) at boththe mRNA and protein levels (Supplementary Figure 2)Conversely blocking PPAR-120574 activitywithT0070907 resultedin a prolonged and significant reduction in GCM-1 mRNAexpression (60 plusmn 54 decrease) which persisted at 24 hours(119875 lt 005) When both rosiglitazone and T0070907 werecombined a significant 34plusmn82 reduction inGCM-1mRNAexpression was observed at 24 hours versus vehicle (119875 lt005) Forskolin alone promoted a much greater upregulationofGCM-1 expression in BeWo cells evident as early as 3 hoursafter treatment (29 plusmn 02-fold increase 119875 lt 005) this effectwas also significant at 6 (23 plusmn 01-fold rise 119875 lt 005) and 24hours (29 plusmn 04-fold change 119875 lt 005) Importantly changesin GCM-1 PPAR-120574 andHO-1mRNA levels as described thusfar were found to be analogous to those in isolated humanprimary cytotrophoblast cells supporting the notion that theresponses seen in the BeWo cell line are representative ofthe true human placental phenotype and are neither cultureeffects nor confined to a cell line (Supplementary Figure 3)

34 Baseline PPAR-120574 Activity within the GCM-1 Promoter isHigh in BeWo Cells Despite being able to block the activityof PPAR-120574 and decrease gene expression of its downstreamtargets in BeWo cells it was considerably more challengingto induce its activity above baseline thereby upregulatingthe expression of GCM-1 Because of this inability to induceGCM-1 expression over a prolonged period of time wedecided to study the transcriptional activity of PPAR-120574 inBeWo cells using the luciferase reporter assay to test thehypothesis that BeWo cells operate in a state of a high baselinePPAR-120574 activity (Figures 3(b) and 3(c)) We studied PPAR-120574 binding to two PPREs (binding sequences 1 and 2) inthe upstream 1kb region of the human GCM-1 gene (seeSupplementary Figure 1 for gene map)

Following a 24-hour treatment of BeWo cells with rosigli-tazone (10 120583M) luciferase activity for binding sequence 1 wasincreased by 32 plusmn 127 while failing to reach statisticalsignificance (119875 = 01274 119899 = 4 Figure 3(b)) Thehigher dose of rosiglitazone (100 120583M) significantly increasedluciferase activity by 46 plusmn 118 (119875 lt 005) and althoughthere was no response to T0070907 treatment alone incombination with the higher dose of rosiglitazone T0070907

PPAR Research 7

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Figure 2 Nuclear not cytoplasmic PPAR-120574 expression changes upon drug treatment (a) PPAR-120574 expression was visualized at 3 (top) 6(middle) and 24 (bottom) hours following treatment with vehicle rosiglitazone or T0070907 PPAR-120574 shown in green e-cadherin (cellsurface marker) in red DAPI (nuclear marker) in blue 630X magnification Nuclear (b) and cytoplasmic (c) PPAR-120574 protein expression wasassessed at 1 6 and 24 hours of treatment (representative images below) PPAR-120574 protein levels were assessed usingWestern blotting nuclearexpression was normalized to lamin B cytoplasmic expression normalized to 120572-tubulin Each treatment was further normalized to vehicle(set as 1 red dashed line) Values are represented as mean plusmn SEM 119875 lt 005 versus vehicle control lowast119875 lt 005 (119899 = 7ndash9) Rosi rosiglitazoneT007 T0070907

8 PPAR Research

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Rela

tive p

rom

oter

activ

ity(n

orm

aliz

ed to

veh

icle

)

Ro10

120583M

Ro100120583

M

Vehi

cle

T01120583

M

T05120583

M

Ro100120583

M+

T05120583

M

lowastlowast

(b)

00

05

10

15

20

Binding sequence 2

Rela

tive p

rom

oter

activ

ity(n

orm

aliz

ed to

veh

icle

)

Ro10

120583M

Ro100120583

M

Vehi

cle

T01120583

M

T05120583

M

Ro100120583

M+

T05120583

M

lowastlowastlowast

(c)

Figure 3 Activity of PPAR-120574 within the 1kb region of human GCM-1 promoter is relatively high (a) GCM-1 expression levels overtimein response to rosiglitazone T0070907 their combination and forskolin treatment PPAR-120574 activity within the GCM-1 promoter [PPAR-120574 response element 1 (b) and 2 (c)] measured by the luciferase reporter assay at 24 hours of treatment Treatments are compared to theirrespective vehicle (set as 1 red dashed line) Values are represented asmeanplusmn SEM 119875 lt 005 versus vehicle control lowastlowast119875 lt 0005 lowastlowastlowast119875 lt 0001(119899 = 3-4) Rorosi rosiglitazone TT007 T0070907

significantly blocked the effect of rosiglitazone (119875 lt 001versus rosiglitazone alone)

Luciferase activity under the control of binding sequence2 is shown in Figure 3(c) Here rosiglitazone treatment didnot have an effect on PPAR-120574 activity but T0070907 treat-ment led to a significant 38 plusmn 36 reduction in enzymeactivity (119875 lt 005 119899 = 4) Interestingly a combination ofrosiglitazone and T0070907 significantly increased receptoractivity when compared to T0070907 alone (119875 lt 0001)indicating that rosiglitazone was able to exert its effects ontranscription

35 Lower Endogenous PPAR-120574 Levels Lead to a StrongerRosiglitazone-Induced GCM-1 Response To assess whether

the changes in levels of target genes were PPAR-120574-specific andnot artifacts of cell culture we performed a series ofPPAR-120574 silencing experiments (Figure 4) Using siRNAoligonucleotides targeted against the human PPAR-120574 genewe successfully downregulated the expression of PPAR-120574 by63plusmn53 (119875 = 00003 119899 = 4 Figure 4(a)) Interestingly whendrug treatments were compared to vehicle controls withinrespective siRNA-treated and nonsilencing control-treatedgroups the gene expression profiles were analogous in bothconditions indicating that the system remained functionaloverall regardless of whether the levels of PPAR-120574 weresignificantly downregulated or not (data not shown)

To elucidate the contribution of PPAR-120574 itself thetarget gene mRNA expression in response to each drug

PPAR Research 9

NS control siRNA0

1

2

3

4

5

expr

essio

nAb

solu

tePP

AR-120574

lowast

(a)

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

00

05

10

(nor

mal

ized

to N

S co

ntro

l)PP

AR-120574

expr

essio

n

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Vehi

cle

(b)

00

05

10

(nor

mal

ized

to N

S co

ntro

l)

lowastlowast

lowast

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

HO

-1ex

pres

sion

(c)

00

05

10

15

20

(nor

mal

ized

to N

S co

ntro

l)lowastlowast

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

GCM

-1ex

pres

sion

(d)

Figure 4 Effect of PPAR-120574 downregulation on gene expression following treatment with PPAR-120574 activity-modulating drugs (a) PPAR-120574expression was significantly downregulated after 48 hours of treatment with a specific siRNA sequence PPAR-120574 (b) HO-1 (c) and GCM-1(d) expression in the siRNA-treated cells was compared to the same drug treatment in the nonsilencing control-treated cells to elucidatethe contribution of PPAR-120574 to changes in target gene expression All drug treatments (vehicle rosiglitazone and T0070907) under siRNAtreatment were normalized to the same treatment under nonsilencing control (red dashed line set as 1) Values are represented as mean plusmnSEM lowast119875 lt 005 lowastlowast119875 lt 0005 lowastlowastlowast119875 lt 0001 lowastlowastlowastlowast119875 lt 00001 (119899 = 4) NS nonsilencing rosi rosiglitazone T007 T0070907

in the siRNA-treated sample was compared to the samedrug treatment in the nonsilencing (ns) control-treatedsample (ns control was set as 1) Such an approach allowed usto calculate whether and by howmuch themRNA responseswere ameliorated or augmented by the downregulation ofendogenous PPAR-120574 levels Together our findings showthat the responses seen following agonist and antagonisttreatment are indeed PPAR-120574-dependent

First we studied the expression of PPAR-120574 under suchconditions (Figure 4(b)) As predicted the expression ofPPAR-120574 was significantly decreased under all treatments(vehicle rosiglitazone and T0070907) in the siRNA-treatedgroup when compared to the nonsilencing controls (119875 lt0001 119899 = 4) Next we examined the effect of PPAR-120574downregulation on expression levels of a potent and easily-inducible downstream target HO-1 mRNA analysis sup-ported the notion that responses ofHO-1 to drugs are PPAR-120574-dependent such that PPAR-120574 downregulation resulted inlower levels ofHO-1 transcription under vehicle control con-dition and both doses of rosiglitazone (Figure 4(c)) Under

baseline (ie vehicle) conditions PPAR-120574 downregulationled to a significant 27 plusmn 68 decrease in HO-1 levelswhile treatment with lower and higher doses of rosiglitazoneameliorated HO-1 response by 38 plusmn 77 and 42 plusmn 61respectively when compared to analogous treatments in thenon-silenced group (119875 lt 005 119899 = 4) Response to T0070907did not differ between siRNA-treated and non-treated cells

Lastly we analyzed the response of GCM-1 transcriptionto PPAR-120574 downregulation and treatment with PPAR-120574activity-modulating drugs (Figure 4(d)) Our findings illus-trate that GCM-1 response to rosiglitazone is augmented bysiRNA treatment compared to the same drug treatment in thenon-silenced group at 48 hours By decreasing endogenousPPAR-120574 levels we observed a trend towards a strongerGCM-1 response with the lower dose of rosiglitazone as well as asignificant reduction of GCM-1 expression under T0070907treatment (119875 lt 0005 119899 = 4) Such findings support ourobservations of high baseline PPAR-120574 transcriptional activitywithin the GCM-1 promoter region assessed by the luciferasereporter assay (described in Figures 3(b) and 3(c))

10 PPAR Research

36 Blocking PPAR-120574 Activity Inhibits Differentiation andInduces Proliferation of BeWo Cells To complete the studyof the role of PPAR-120574 on the maintenance of prolifera-tiondifferentiation balance we assessed differentiation ofBeWo cells using free 120573-hCG as a marker (Figure 5(a))Furthermore we assessed cell fusion morphologically usinge-cadherin as a cell surface marker (Supplementary Figure4) In our experiments forskolin a known inducer of free120573-hCG secretion [18] was used as a positive control for celldifferentiation A 209 plusmn 28-fold induction in 120573-hCG releasefollowing forskolin treatment was seen at 48 hours (119875 lt 005119899 = 7) Lower dose of rosiglitazone (10 120583M) did not affectfree 120573-hCG release while a higher concentration of the drugshowed only an upward trend in 120573-hCG release (34 plusmn 204increase 119875 = 019 119899 = 4) On the contrary blocking PPAR-120574 activity with T0070907 significantly downregulated 120573-hCGsecretion by 41 plusmn 73 (119875 lt 005 119899 = 7) Coadministrationof both the inhibitor and the activator resulted in no changeof 120573-hCG protein release compared to vehicle

The effect of PPAR-120574 activity modulation on the BeWocell number was assessed after 48 hours of treatment(Figure 5(b)) Forskolin treatment which is known to inducedifferentiation and decrease cell proliferation [5] signifi-cantly decreased BeWo cell numbers at 48 hours by 16plusmn14(119875 lt 005 119899 = 4) Treatment of BeWo cells with T0070907significantly increased BeWo cell numbers by 39 plusmn 128when compared to vehicle control (119875 lt 005) Rosiglitazonedid not have an effect on the BeWo cell number as predicted

4 Discussion

The present study has established the regulatory role ofPPAR-120574 on the differentiation of the villous trophoblast layerrepresented by the BeWo cell line model of SCT formationin vitro together with supporting evidence from primaryisolated human cytotrophoblast cells We report a novelfinding of a negative autoregulatory mechanism of PPAR-120574expression in response to its activitymodulationwith the ago-nist rosiglitazone and the antagonist T0070907 Furthermorewe show the role of PPAR-120574 in the differentiation of BeWocells into confluent syncytialized structures that mimic theSCT layer in vivo Importantly these findings are analogousto those found in isolated human primary CT cells suggestingthat the BeWo cell line represents molecular mechanismswithin the human placenta In our BeWo model blockingPPAR-120574 activity with an antagonist T0070907 promoted cellproliferation at the expense of fusion reflected by a decreasein both mRNA expression of the transcription factor GCM-1and free 120573-hCG release into the overlying media Converselyinduction of PPAR-120574 activity in BeWo cells with the agonistrosiglitazone did not produce the opposite effects suggestingthat PPAR-120574 activity is maximal in BeWo cells to drive theprocess of syncytialization although induction of this activityis possible in a different pathway as demonstrated by arise in another target gene (HO-1) expression Furthermorethis effect was found to be PPAR-120574-specific such that theseresponses were partially ameliorated by the downregulationof endogenous PPAR-120574 levels

We tested the hypothesis that PPAR-120574 activity is modu-lated via a negative feedback loop We observed that PPAR-120574 participates in a negative autoregulation feedback mecha-nism whereby the induction of PPAR-120574 activity is accom-panied by a decrease in its expression while the oppositeoccurs following treatment with the antagonistThis suggeststhat certain molecular mechanisms in the BeWo cell line arein place to ensure fine-tuning of PPAR-120574 activity inductionof activity is compensated for by a decrease in transcriptionfactor expression while a decrease in its activity is compli-mented with a rise in receptor levels Although it has beenreported that PPAR-120574 cofactors (such as PPAR-120574Coactivator-1120573) [19 20] and coreceptors (such as Liver X Receptor-120572) [21]participate in positive autoregulatory loops simultaneousfindings by us andKnabl et al [22] are the first reports to shownegative PPAR-120574 autoregulation in the human cell line BeWoThis has important implications considering that PPAR-120574 isa transcription factor with an array of functions and playsa role in lipid metabolism cell growth differentiation andso forth carefully regulating its activity is crucial for cellularhemostasis and timely cell cycle progression

We further analyzed PPAR-120574 expression changes in bothcellular compartments (nucleus and the cytoplasm) followingdrug treatments We observed that nuclear fluctuations inreceptor levels were more robust when cells were treated withthe agonist and the antagonist while changes in the cyto-plasmic compartment were more subtle and delayed Thissuggests that changes in the nuclear compartment consistentwith its primary action as a nuclear receptor and transcriptionfactor are mostly responsible for fluctuations seen in wholecell lysates These changes were confirmed using fluorescentimmunohistochemistry assessments and thus were not anartifact of the cellular fractionation technique caused byleaking or active transport of PPAR-120574 out of the nucleus uponcell lysis This finding leads us to speculate that changes inthe nucleus may be due to altered protein stability (possiblyaffected by ubiquitination or unfolded protein response) orshunting between the two cell compartments following drugtreatment Collectively our findings suggest that there aredifferential PPAR-120574 protein regulation mechanisms in thenucleus and the cytoplasm

Furthermore we studied the effect of PPAR-120574 activitymodulation on the balance between continued BeWo cellproliferation as opposed to the commitment to terminaldifferentiation via syncytialization bymonitoring the expres-sion of differentiation-promoting transcription factor GCM-1 together with the release of free 120573-hCG into the overlyingmedia To our surprise we found that attempts to stimulatePPAR-120574 with rosiglitazone did not induce GCM-1 expressionbeyond 3 hours of treatment and did not result in a rise in120573-hCG release This was true despite both GCM-1 inductionand a rise of free 120573-hCG secretion by forskolin treatmentand possibility of stimulation of PPAR-120574 transcriptionalactivity as seen by a robust rise in levels of another targetHO-1 following rosiglitazone treatment By contrast theantagonist T0070907 had a pronounced effect causing asignificant reduction in both GCM-1 expression and 120573-hCGrelease this suppression of differentiation was accompaniedby a predicted rise in cell proliferation indicating that

PPAR Research 11

00

05

10

15

20

15

20

25

(r

elat

ive t

o ve

hicle

cont

rol)

Free

120573-h

CG se

cret

ion

Ro10

120583M

Vehi

cle

Ro100120583

M

T1120583

M

Ro10

120583M

+T1

120583M

For2

0120583

M

lowast

(a)

00

05

10

15

20

Relat

ive c

ell p

rolif

erat

ion

(nor

mal

ized

to v

ehic

le co

ntro

l)

Ro10

120583M

Vehi

cle

Ro100120583

M

T1120583

M

Ro10

120583M

+T1

120583M

For2

0120583

M

(b)

Figure 5 Effect of PPAR-120574manipulation on free 120573-hCG release from BeWo cells and cell number (a) Free 120573-hCG release was measured at48 hours after-treatment and its levels were normalized to total protein content in the media (b) BeWo cell numbers were measured usingCellTiter-Fluo Cell Viability Assay at 48 hours of treatment Each drug treatment was compared to its respective vehicle control and expressedas fold-change (vehicle set as 1 red dashed line) Values are represented asmeanplusmn SEM 119875 lt 005 versus vehicle control lowast119875 lt 005 (119899 = 4ndash7)Ro rosiglitazone T T0070907 For forskolin

the antagonist was able to exert a strong repressive effecton the PPAR-120574GCM-1syncytialization axis We found aplausible explanation for this observation using the luciferasereporter system whereby we observed that rosiglitazonetreatment could not further stimulate PPAR-120574 binding to theGCM-1 promoter whereas the antagonist T0070907 signif-icantly decreased the interaction between PPAR-120574 and itsresponse element Downregulation of PPAR-120574 expressionusing siRNA oligonucleotides further supported this obser-vation as GCM-1 rise due to rosiglitazone treatment was aug-mented under lower endogenous PPAR-120574 levels Althoughthese results did not reach statistical significance they maybe explained by the incomplete receptor knockdown as wellas the presence of the autoregulatory PPAR-120574 feedbackmech-anism Collectively these findings suggest that the baselineactivity of PPAR-120574 in the BeWo cell line is relatively highlimiting the potential for its further induction with an agonisttreatment

Our studies support a role for PPAR-120574 in mediating keyfunctions of the trophoblast layer that is in direct contactwithmaternal blood In this location PPAR-120574maypotentiallynavigate the balance between the need to retain a proliferatingpopulation ofCT lineage-restricted progenitorswith the needto constantly form an overlying SCT layer The majority ofpregnancy complications requiring preterm delivery mainlysevere preeclampsia and intrauterine growth restrictionexhibit structural abnormalities of placental villi [8 23]including increased apoptosis of the villous trophoblastcompartment [24] depletion of proliferating CT cells andpatchy areas of apoptosis and necrosis in the SCT layer [25]Our capacity to pharmacologically induce PPAR-120574 activityposes a potential avenue for improving placental trophoblast

physiology via upregulation of GCM-1 expression in residualtrophoblast progenitors thereby restoring the process ofsyncytiotrophoblast formation and thus normal placentalfunction in pregnancies characterized by abnormal placentaldevelopment

5 Conclusions

PPAR-120574 is one of key metabolic regulators in the humanbody and has recently been suggested to play a role in phys-iologic placental development and thus normal pregnancyprogression Its role has been implicated in several pregnancycomplications including preeclampsia [14 26] gestationaldiabetes [22] and hypoxia-induced fetal growth restrictionat high altitudes [27] Elucidating the role of this receptorin human trophoblast cell lineage differentiation and healthyplacentation is instrumental to the utilization of these path-ways for advancement of therapeutics Our ability to phar-macologically manipulate this metabolic regulator is aninvaluable tool for the development of possible prophylacticandor treatment options for women at risk of developing andsuffering from common pregnancy complications

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors thank Drs Sarah Keating Theodore Brown SLee Adamson (University of Toronto) and Dr Asif Ahmed

12 PPAR Research

(University of Edinburgh) for their helpful discussions andsupervision of Ms Levytska in her MSc studies The authoralso thank Dr Caroline Dunk and Dr Lubna Nadeem fortheir help with fluorescent IHC and primary cell isolationAdditionally the authors thank the donors and the ResearchCentre for Women and Infantrsquos Health Biobank Program ofthe CIHRGroup inDevelopment and Fetal Health (CIHRnoMGC-13299) the Lunenfeld-Tanenbaum Research Instituteand the MSHUHN Department of Obstetrics and Gyne-cology for the human specimens used in this study DrKingdomrsquos research is supported by CIHR (no 64302) andthe Rose Torno Chair at Mount Sinai Hospital Toronto ONCanada

References

[1] K Benirschke K K P and R Baergen Pathology of the HumanPlacenta Springer New York NY USA 5th edition 2005

[2] T Hosoya K Takizawa K Nitta and Y Hotta ldquoglial cells miss-ing a binary switch between neuronal and glial determinationin Drosophilardquo Cell vol 82 no 6 pp 1025ndash1036 1995

[3] L Anson-Cartwright K Dawson D Holmyard S J Fisher RA Lazzarini and J C Cross ldquoThe glial cells missing-1 proteinis essential for branching morphogenesis in the chorioallantoicplacentardquo Nature Genetics vol 25 no 3 pp 311ndash314 2000

[4] S A Bainbridge A Minhas K J Whiteley et al ldquoEffects ofreduced Gcm1 expression on trophoblast morphology fetopla-cental vascularity and pregnancy outcomes in micerdquoHyperten-sion vol 59 no 3 pp 732ndash739 2012

[5] D Baczyk S Drewlo L Proctor C Dunk S Lye and JKingdom ldquoGlial cell missing-1 transcription factor is requiredfor the differentiation of the human trophoblastrdquoCell Death andDifferentiation vol 16 no 5 pp 719ndash727 2009

[6] D Baczyk A Satkunaratnam B Nait-Oumesmar B HuppertzJ C Cross and J C P Kingdom ldquoComplex patterns of GCM1mRNA and protein in villous and extravillous trophoblast cellsof the human placentardquo Placenta vol 25 no 6 pp 553ndash5592004

[7] C-P Chen C-Y Chen Y-C Yang T-H Su and H ChenldquoDecreased placental GCM1 (glial cells missing) gene expres-sion in pre-eclampsiardquoPlacenta vol 25 no 5 pp 413ndash421 2004

[8] M G Walker B Fitzgerald S Keating J G Ray R Windrimand J C P Kingdom ldquoSex-specific basis of severe placentaldysfunction leading to extreme preterm deliveryrdquo Placenta vol33 no 7 pp 568ndash571 2012

[9] M M Parast H Yu A Ciric M W Salata V Davis and D SMilstone ldquoPPARgamma regulates trophoblast proliferation andpromotes labyrinthine trilineage differentiationrdquo PloSONE vol4 no 11 article e8055 2009

[10] M Ruebner M Langbein P L Strissel et al ldquoRegulation ofthe human endogenous retroviral Syncytin-1 and cell-cell fusionby the nuclear hormone receptors PPARgammaRXRalpha inplacentogenesisrdquo Journal of Cellular Biochemistry vol 113 no 7pp 2383ndash2396 2012

[11] Y Barak M C Nelson E S Ong et al ldquoPPAR120574 is required forplacental cardiac and adipose tissue developmentrdquo MolecularCell vol 4 no 4 pp 585ndash595 1999

[12] JM Lehmann L BMoore T A Smith-OliverWOWilkisonT M Willson and S A Kliewer ldquoAn antidiabetic thiazolidine-dione is a high affinity ligand for peroxisome proliferator-activated receptor 120574 (PPAR120574)rdquo Journal of Biological Chemistryvol 270 no 22 pp 12953ndash12956 1995

[13] G Lee F Elwood JMcNally et al ldquoT0070907 a selective ligandfor peroxisome proliferator-activated receptor 120574 functions asan antagonist of biochemical and cellular activitiesrdquo Journal ofBiological Chemistry vol 277 no 22 pp 19649ndash19657 2002

[14] F P McCarthy S Drewlo J Kingdom E J Johns S KWalsh and L C Kenny ldquoPeroxisome proliferator-activatedreceptor-120574 as a potential therapeutic target in the treatment ofpreeclampsiardquo Hypertension vol 58 no 2 pp 280ndash286 2011

[15] P R Ortiz De Montellano ldquoThe mechanism of heme oxyge-naserdquo Current Opinion in Chemical Biology vol 4 no 2 pp221ndash227 2000

[16] M Bilban P Haslinger J Prast et al ldquoIdentification of noveltrophoblast invasion-related genes heme oxygenase-1 con-trols motility via peroxisome proliferator-activated receptor 120574rdquoEndocrinology vol 150 no 2 pp 1000ndash1013 2009

[17] H J Kliman J E Nestler and E Sermasi ldquoPurification charac-terization and in vitro differentiation of cytotrophoblasts fromhuman term placentaerdquo Endocrinology vol 118 no 4 pp 1567ndash1582 1986

[18] I Knerr S W Schubert C Wich et al ldquoStimulation of GCMaand syncytin via cAMP mediated PKA signaling in humantrophoblastic cells under normoxic and hypoxic conditionsrdquoFEBS Letters vol 579 no 18 pp 3991ndash3998 2005

[19] T Deng D H Sieglaff A Zhang et al ldquoA peroxisomeproliferator-activated receptor 120574 (PPAR120574)PPAR120574 coactivator1120573 autoregulatory loop in adipocyte mitochondrial functionrdquoJournal of Biological Chemistry vol 286 no 35 pp 30723ndash30731 2011

[20] E Hondares O Mora P Yubero et al ldquoThiazolidinediones andrexinoids induce peroxisome proliferator-activated receptor-coactivator (PGC)-1120572 gene transcription an autoregulatoryloop controls PGC-1120572 expression in adipocytes via peroxisomeproliferator-activated receptor-120574 coactivationrdquo Endocrinologyvol 147 no 6 pp 2829ndash2838 2006

[21] B A Laffitte S B Joseph R Walczak et al ldquoAutoregulation ofthe human liver X receptor 120572 promoterrdquoMolecular and CellularBiology vol 21 no 22 pp 7558ndash7568 2001

[22] J Knabl R Huttenbrenner S Hutter et al ldquoPeroxisomeproliferator-activated receptor-gamma (PPARgamma) is downregulated in trophoblast cells of gestational diabetes mellitus(GDM) and in trophoblast tumour cells BeWo in vitro afterstimulation with PPARgamma agonistsrdquo Journal of PerinatalMedicine pp 1ndash9 2013

[23] C Franco M Walker J Robertson et al ldquoPlacental infarctionand thrombophiliardquo Obstetrics and Gynecology vol 117 no 4pp 929ndash934 2011

[24] M S Longtine B Chen A O Odibo Y Zhong and D M Nel-son ldquoVillous trophoblast apoptosis is elevated and restricted tocytotrophoblasts in pregnancies complicated by preeclampsiaIUGR or preeclampsia with IUGRrdquo Placenta vol 33 no 5 pp352ndash359 2012

[25] B Fitzgerald K Levytska J Kingdom M Walker D Baczykand S Keating ldquoVillous trophoblast abnormalities in extremelypreterm deliveries with elevated second trimester maternalserum hCG or inhibin-Ardquo Placenta vol 32 no 4 pp 339ndash3452011

PPAR Research 13

[26] F P McCarthy S Drewlo F A English et al ldquoEvidence impli-cating peroxisome proliferator-activated receptor-gamma Inthe pathogenesis of preeclampsiardquo Hypertension vol 58 no 5pp 882ndash887 2011

[27] C G Julian I V Yang V A Browne et al ldquoInhibition of per-oxisome proliferator-activated receptor gamma a potential linkbetween chronic maternal hypoxia and impaired fetal growthrdquoThe FASEB Journal 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 PPAR Research

suffering from severe preeclampsia a pregnancy complica-tion characterized by impaired villous structure and placentaldevelopment [7 8]

The transcription factor peroxisome proliferator-acti-vated receptor gamma (PPAR-120574) may regulate the processof SCT formation since it is known to regulate Gcm-1expression [9 10] and placental development [11] in miceThe ability to control the transcriptional activity of PPAR-120574with highly specific drugs including the agonist rosiglitazone[12] and the antagonist T0070907 [13] has been utilized inan attempt to study its role in several features of placentaldevelopment We have recently shown that rosiglitazone-induced PPAR-120574 activation is able to ameliorate diseasecharacteristics in the rat model of preeclampsia via its down-stream target heme oxygenase-1 (HO-1) an enzyme whichproduces carbon monoxide (CO a potent vasodilator) andbilirubin an antioxidant [14 15] Furthermore other groupshave looked at the effect of PPAR-120574 activity induction oncell migration as well as analyzed expression profiles in VCTand extravillous trophoblast (EVT) cells [10 16] leading tothe conclusion that PPAR-120574 plays a role in human placentaldevelopment

In the present study we attempted to study the activityof this transcription factor in the human choriocarcinoma-derived cell line BeWo an established model of SCT forma-tion in vitro and primary trophoblast cells to establish theeffect of PPAR-120574 activity modulation on key features oftrophoblast physiology We hypothesized that stimulatingPPAR-120574 activity with a highly specific agonist rosiglitazonewill induce cell differentiation [resulting in increased expres-sion of the differentiation marker GCM-1 and augmentedrelease of cell fusionmarker human chorionic gonadotropin-120573 (120573-hCG)] while the opposite (ie proliferation) will befavored by the antagonist T0070907 To our surprise whenexamining the response of GCM-1 expression and 120573-hCGrelease we show that the baseline PPAR-120574 activity in theBeWo cell line is relatively high limiting our ability to stimu-late it further with the agonist rosiglitazone but presentingan opportunity to block it with the antagonist T0070907Interestingly our ability to stimulate theGCM-1 response wasaugmented when the endogenous levels of PPAR-120574 aredownregulated using siRNA adding support to the conceptthat PPAR-120574 regulates the differentiation axis in the BeWo cellline Although these findings outline the limitation of this cellline they nonetheless support the wide used of this modelin the study of molecular mechanisms present in the humanplacenta since the responses of target genes in isolatedhuman cytotrophoblast cells were found to be analogous tothose in BeWo cells

2 Materials and Methods

21 BeWo Cells The human choriocarcinoma-derived BeWocell line was purchased from ATCC (Burlington ONCanada) and fingerprinted at the Centre for Applied Genom-ics (SickKids Toronto ON Canada) markers were foundto be identical to those in the ATCC database BeWo cellsbetween passages 10 and 20were used For all treatments cells

were maintained in F12K medium (Wisent Inc St BrunoQC Canada) supplemented with 10 fetal bovine serum(FBS Canadian grade heat-inactivated Invitrogen Burling-ton ON Canada) 100 unitsmL penicillin 100 120583gmL strep-tomycin and 2 nM L-glutamine (Life Technologies Burling-ton ON Canada) in atmospheric O

25 CO

2at 37∘C

For treatments BeWo cells were seeded at 50000 cellsper 1mL of media and allowed to attach for 24 hours Thefollowing day cells were pretreated with the PPAR-120574 antag-onist T0070907 (Cayman Chemical Ann Arbor MI USA)for 30 minutes and were then treated with either the PPAR-120574 agonist rosiglitazone (Enzo Life Sciences BurlingtonON Canada) the antagonist T0070907 (Cayman Chemical)andor the weak agonist forskolin (Sigma-Aldrich OakvilleON Canada) Cell viability under all treatments was assessedat 48 hours of culture using CytoTox-ONE HomogeneousMembrane Integrity Assay (Promega Madison WI USA)No drug treatments resulted in significant cell toxicity at 48hours (data not shown)

Following treatment cells were washed in ice-cold D-PBS (Wisent Inc) and collected according to different down-stream applications For RNA analysis cells were collectedintoRLTPlus buffer (Qiagen TorontoONCanada)with 10120573-mercaptoethanol (Fisher Scientific Ottawa ON Canada)For protein analysis cells were scraped and collected in RIPABuffer (Thermo Scientific Ottawa ON Canada) with phos-phatase (Phosphatase Inhibitor Cocktail 2 Sigma-Aldrich)and protease inhibitors (Complete Mini EDTA-free ProteaseInhibitor Cocktail Tablets Roche Applied Science LavalQC Canada) Conditioned medium was collected and cen-trifuged for 5 minutes at 425 g at room temperature (RT) toremove cellular debris All collected samples were stored atminus80∘C for further analysis

22 Transfection of BeWo Cells The BeWo cells were trans-fected with commercially-available double-stranded siRNAoligonucleotides against the human PPAR-120574 sequence (San-taCruz Biotechnology Dallas TX USA) Nonsilencing con-trol (sequence 51015840-TTCTCCGAACGTGTCACGT-31015840) wasused as a negative control BeWo cells were plated into 12-well plates and transfected the following day according to themanufacturerrsquos specifications Briefly 50-confluent BeWocells were transfected with 30120583M of PPAR-120574-targeted siRNAor nonsilencing control with 25 120583L of PepMute siRNA trans-fection reagent (SignaGen Laboratories Ijamsville MD) perwell The following day media were changed and cells weretreated for another 48 hours Experiments with fluorescent-labeled siRNA established 80ndash90 transfection efficiency(data not shown) Toxicity of siRNA treatmentwasmonitoredwith Human Interferon Alpha ELISA kit (PBL BiomedicalLaboratories Brussels Belgium)

23 Human Primary Cytotrophoblast Cell Isolation Firstand second trimester (12ndash19 weeks) human placental tissuewas used for the isolation of primary cytotrophoblast (CT)cells Placentas were obtained from the Morgentaler Clinic(Toronto Canada) following elective legal terminations ofpregnancy Mount Sinai Hospital Research Ethics Board

PPAR Research 3











4 0125 Trypsin


2at 37∘C The






4 PPAR Research








PPAR Research 5

lowastlowast


10120583

M)

T(1120583

M)

+T

3

6

24

Rela

tiveP

PAR-120574

mRN

A

(a)

120574

Ro10

120583M

Ro120583

M

T1120583

M

Ro10

120583M

20120583

M

Ro10

120583M

Ro120583

M

T1120583

M

Ro10

120583M

+T1

120583M

20120583

M

lowastlowastlowast

120574

120573

55

42

+T1

120583M

(b)






3 Results


6 PPAR Research










PPAR Research 7

120574

(a)

1 6 240

1

2

3lowast

120574

10120583

M)

T(1120583

M)

+T

10120583

M)

T(1120583

M)

+T

10120583

M)

T(1120583

M)

+T

1 hr 6 24

55

67

10120583M)T (1120583M)

+ T

Relat

ive n

ucle

ar P

PAR-120574

expr

essio

n

(b)

1 6 24

10120583M)T (1120583M)

+ T

lowast

10120583

M)

T(1120583

M)

+T

10120583

M)

T(1120583

M)

+T

10120583

M)

T(1120583

M)

+T

1 hr 6 24

55

55


Relat

ive c

ytop

lasm

ic P

PAR-120574

expr

essio

n

(c)


8 PPAR Research

0

1

2

3

4

lowastlowast

lowastlowast

3 hrs

6 hrs

24 hrs


expr

essio

n le

vels

Rela

tiveG

CM-1

mRN

A

(a)

00

05

10

15

20

Binding sequence 1

Rela

tive p

rom

oter

activ

ity(n

orm

aliz

ed to

veh

icle

)

Ro10

120583M

Ro100120583

M

Vehi

cle

T01120583

M

T05120583

M

Ro100120583

M+

T05120583

M

lowastlowast

(b)

00

05

10

15

20

Binding sequence 2

Rela

tive p

rom

oter

activ

ity(n

orm

aliz

ed to

veh

icle

)

Ro10

120583M

Ro100120583

M

Vehi

cle

T01120583

M

T05120583

M

Ro100120583

M+

T05120583

M

lowastlowastlowast

(c)







PPAR Research 9

NS control siRNA0

1

2

3

4

5

expr

essio

nAb

solu

tePP

AR-120574

lowast

(a)

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

00

05

10

(nor

mal

ized

to N

S co

ntro

l)PP

AR-120574

expr

essio

n

Rosi10

120583M

Rosi100120583

M

T007

1120583

M


lowastlowastlowast

lowastlowastlowast

Vehi

cle

(b)

00

05

10

(nor

mal

ized

to N

S co

ntro

l)

lowastlowast

lowast

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

HO

-1ex

pres

sion

(c)

00

05

10

15

20

(nor

mal

ized

to N

S co

ntro

l)lowastlowast

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

GCM

-1ex

pres

sion

(d)






10 PPAR Research



4 Discussion





PPAR Research 11

00

05

10

15

20

15

20

25

(r

elat

ive t

o ve

hicle

cont

rol)

Free

120573-h

CG se

cret

ion

Ro10

120583M

Vehi

cle

Ro100120583

M

T1120583

M

Ro10

120583M

+T1

120583M

For2

0120583

M

lowast

(a)

00

05

10

15

20

Relat

ive c

ell p

rolif

erat

ion

(nor

mal

ized

to v

ehic

le co

ntro

l)

Ro10

120583M

Vehi

cle

Ro100120583

M

T1120583

M

Ro10

120583M

+T1

120583M

For2

0120583

M

(b)





5 Conclusions




Acknowledgments


12 PPAR Research


References


























PPAR Research 13








of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















PPAR Research 3











4 0125 Trypsin


2at 37∘C The






4 PPAR Research








PPAR Research 5

lowastlowast


10120583

M)

T(1120583

M)

+T

3

6

24

Rela

tiveP

PAR-120574

mRN

A

(a)

120574

Ro10

120583M

Ro120583

M

T1120583

M

Ro10

120583M

20120583

M

Ro10

120583M

Ro120583

M

T1120583

M

Ro10

120583M

+T1

120583M

20120583

M

lowastlowastlowast

120574

120573

55

42

+T1

120583M

(b)






3 Results


6 PPAR Research










PPAR Research 7

120574

(a)

1 6 240

1

2

3lowast

120574

10120583

M)

T(1120583

M)

+T

10120583

M)

T(1120583

M)

+T

10120583

M)

T(1120583

M)

+T

1 hr 6 24

55

67

10120583M)T (1120583M)

+ T

Relat

ive n

ucle

ar P

PAR-120574

expr

essio

n

(b)

1 6 24

10120583M)T (1120583M)

+ T

lowast

10120583

M)

T(1120583

M)

+T

10120583

M)

T(1120583

M)

+T

10120583

M)

T(1120583

M)

+T

1 hr 6 24

55

55


Relat

ive c

ytop

lasm

ic P

PAR-120574

expr

essio

n

(c)


8 PPAR Research

0

1

2

3

4

lowastlowast

lowastlowast

3 hrs

6 hrs

24 hrs


expr

essio

n le

vels

Rela

tiveG

CM-1

mRN

A

(a)

00

05

10

15

20

Binding sequence 1

Rela

tive p

rom

oter

activ

ity(n

orm

aliz

ed to

veh

icle

)

Ro10

120583M

Ro100120583

M

Vehi

cle

T01120583

M

T05120583

M

Ro100120583

M+

T05120583

M

lowastlowast

(b)

00

05

10

15

20

Binding sequence 2

Rela

tive p

rom

oter

activ

ity(n

orm

aliz

ed to

veh

icle

)

Ro10

120583M

Ro100120583

M

Vehi

cle

T01120583

M

T05120583

M

Ro100120583

M+

T05120583

M

lowastlowastlowast

(c)







PPAR Research 9

NS control siRNA0

1

2

3

4

5

expr

essio

nAb

solu

tePP

AR-120574

lowast

(a)

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

00

05

10

(nor

mal

ized

to N

S co

ntro

l)PP

AR-120574

expr

essio

n

Rosi10

120583M

Rosi100120583

M

T007

1120583

M


lowastlowastlowast

lowastlowastlowast

Vehi

cle

(b)

00

05

10

(nor

mal

ized

to N

S co

ntro

l)

lowastlowast

lowast

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

HO

-1ex

pres

sion

(c)

00

05

10

15

20

(nor

mal

ized

to N

S co

ntro

l)lowastlowast

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

GCM

-1ex

pres

sion

(d)






10 PPAR Research



4 Discussion





PPAR Research 11

00

05

10

15

20

15

20

25

(r

elat

ive t

o ve

hicle

cont

rol)

Free

120573-h

CG se

cret

ion

Ro10

120583M

Vehi

cle

Ro100120583

M

T1120583

M

Ro10

120583M

+T1

120583M

For2

0120583

M

lowast

(a)

00

05

10

15

20

Relat

ive c

ell p

rolif

erat

ion

(nor

mal

ized

to v

ehic

le co

ntro

l)

Ro10

120583M

Vehi

cle

Ro100120583

M

T1120583

M

Ro10

120583M

+T1

120583M

For2

0120583

M

(b)





5 Conclusions




Acknowledgments


12 PPAR Research


References


























PPAR Research 13








of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















4 PPAR Research








PPAR Research 5

lowastlowast


10120583

M)

T(1120583

M)

+T

3

6

24

Rela

tiveP

PAR-120574

mRN

A

(a)

120574

Ro10

120583M

Ro120583

M

T1120583

M

Ro10

120583M

20120583

M

Ro10

120583M

Ro120583

M

T1120583

M

Ro10

120583M

+T1

120583M

20120583

M

lowastlowastlowast

120574

120573

55

42

+T1

120583M

(b)






3 Results


6 PPAR Research










PPAR Research 7

120574

(a)

1 6 240

1

2

3lowast

120574

10120583

M)

T(1120583

M)

+T

10120583

M)

T(1120583

M)

+T

10120583

M)

T(1120583

M)

+T

1 hr 6 24

55

67

10120583M)T (1120583M)

+ T

Relat

ive n

ucle

ar P

PAR-120574

expr

essio

n

(b)

1 6 24

10120583M)T (1120583M)

+ T

lowast

10120583

M)

T(1120583

M)

+T

10120583

M)

T(1120583

M)

+T

10120583

M)

T(1120583

M)

+T

1 hr 6 24

55

55


Relat

ive c

ytop

lasm

ic P

PAR-120574

expr

essio

n

(c)


8 PPAR Research

0

1

2

3

4

lowastlowast

lowastlowast

3 hrs

6 hrs

24 hrs


expr

essio

n le

vels

Rela

tiveG

CM-1

mRN

A

(a)

00

05

10

15

20

Binding sequence 1

Rela

tive p

rom

oter

activ

ity(n

orm

aliz

ed to

veh

icle

)

Ro10

120583M

Ro100120583

M

Vehi

cle

T01120583

M

T05120583

M

Ro100120583

M+

T05120583

M

lowastlowast

(b)

00

05

10

15

20

Binding sequence 2

Rela

tive p

rom

oter

activ

ity(n

orm

aliz

ed to

veh

icle

)

Ro10

120583M

Ro100120583

M

Vehi

cle

T01120583

M

T05120583

M

Ro100120583

M+

T05120583

M

lowastlowastlowast

(c)







PPAR Research 9

NS control siRNA0

1

2

3

4

5

expr

essio

nAb

solu

tePP

AR-120574

lowast

(a)

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

00

05

10

(nor

mal

ized

to N

S co

ntro

l)PP

AR-120574

expr

essio

n

Rosi10

120583M

Rosi100120583

M

T007

1120583

M


lowastlowastlowast

lowastlowastlowast

Vehi

cle

(b)

00

05

10

(nor

mal

ized

to N

S co

ntro

l)

lowastlowast

lowast

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

HO

-1ex

pres

sion

(c)

00

05

10

15

20

(nor

mal

ized

to N

S co

ntro

l)lowastlowast

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

GCM

-1ex

pres

sion

(d)






10 PPAR Research



4 Discussion





PPAR Research 11

00

05

10

15

20

15

20

25

(r

elat

ive t

o ve

hicle

cont

rol)

Free

120573-h

CG se

cret

ion

Ro10

120583M

Vehi

cle

Ro100120583

M

T1120583

M

Ro10

120583M

+T1

120583M

For2

0120583

M

lowast

(a)

00

05

10

15

20

Relat

ive c

ell p

rolif

erat

ion

(nor

mal

ized

to v

ehic

le co

ntro

l)

Ro10

120583M

Vehi

cle

Ro100120583

M

T1120583

M

Ro10

120583M

+T1

120583M

For2

0120583

M

(b)





5 Conclusions




Acknowledgments


12 PPAR Research


References


























PPAR Research 13








of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















PPAR Research 5

lowastlowast


10120583

M)

T(1120583

M)

+T

3

6

24

Rela

tiveP

PAR-120574

mRN

A

(a)

120574

Ro10

120583M

Ro120583

M

T1120583

M

Ro10

120583M

20120583

M

Ro10

120583M

Ro120583

M

T1120583

M

Ro10

120583M

+T1

120583M

20120583

M

lowastlowastlowast

120574

120573

55

42

+T1

120583M

(b)






3 Results


6 PPAR Research










PPAR Research 7

120574

(a)

1 6 240

1

2

3lowast

120574

10120583

M)

T(1120583

M)

+T

10120583

M)

T(1120583

M)

+T

10120583

M)

T(1120583

M)

+T

1 hr 6 24

55

67

10120583M)T (1120583M)

+ T

Relat

ive n

ucle

ar P

PAR-120574

expr

essio

n

(b)

1 6 24

10120583M)T (1120583M)

+ T

lowast

10120583

M)

T(1120583

M)

+T

10120583

M)

T(1120583

M)

+T

10120583

M)

T(1120583

M)

+T

1 hr 6 24

55

55


Relat

ive c

ytop

lasm

ic P

PAR-120574

expr

essio

n

(c)


8 PPAR Research

0

1

2

3

4

lowastlowast

lowastlowast

3 hrs

6 hrs

24 hrs


expr

essio

n le

vels

Rela

tiveG

CM-1

mRN

A

(a)

00

05

10

15

20

Binding sequence 1

Rela

tive p

rom

oter

activ

ity(n

orm

aliz

ed to

veh

icle

)

Ro10

120583M

Ro100120583

M

Vehi

cle

T01120583

M

T05120583

M

Ro100120583

M+

T05120583

M

lowastlowast

(b)

00

05

10

15

20

Binding sequence 2

Rela

tive p

rom

oter

activ

ity(n

orm

aliz

ed to

veh

icle

)

Ro10

120583M

Ro100120583

M

Vehi

cle

T01120583

M

T05120583

M

Ro100120583

M+

T05120583

M

lowastlowastlowast

(c)







PPAR Research 9

NS control siRNA0

1

2

3

4

5

expr

essio

nAb

solu

tePP

AR-120574

lowast

(a)

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

00

05

10

(nor

mal

ized

to N

S co

ntro

l)PP

AR-120574

expr

essio

n

Rosi10

120583M

Rosi100120583

M

T007

1120583

M


lowastlowastlowast

lowastlowastlowast

Vehi

cle

(b)

00

05

10

(nor

mal

ized

to N

S co

ntro

l)

lowastlowast

lowast

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

HO

-1ex

pres

sion

(c)

00

05

10

15

20

(nor

mal

ized

to N

S co

ntro

l)lowastlowast

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

GCM

-1ex

pres

sion

(d)






10 PPAR Research



4 Discussion





PPAR Research 11

00

05

10

15

20

15

20

25

(r

elat

ive t

o ve

hicle

cont

rol)

Free

120573-h

CG se

cret

ion

Ro10

120583M

Vehi

cle

Ro100120583

M

T1120583

M

Ro10

120583M

+T1

120583M

For2

0120583

M

lowast

(a)

00

05

10

15

20

Relat

ive c

ell p

rolif

erat

ion

(nor

mal

ized

to v

ehic

le co

ntro

l)

Ro10

120583M

Vehi

cle

Ro100120583

M

T1120583

M

Ro10

120583M

+T1

120583M

For2

0120583

M

(b)





5 Conclusions




Acknowledgments


12 PPAR Research


References


























PPAR Research 13








of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















6 PPAR Research










PPAR Research 7

120574

(a)

1 6 240

1

2

3lowast

120574

10120583

M)

T(1120583

M)

+T

10120583

M)

T(1120583

M)

+T

10120583

M)

T(1120583

M)

+T

1 hr 6 24

55

67

10120583M)T (1120583M)

+ T

Relat

ive n

ucle

ar P

PAR-120574

expr

essio

n

(b)

1 6 24

10120583M)T (1120583M)

+ T

lowast

10120583

M)

T(1120583

M)

+T

10120583

M)

T(1120583

M)

+T

10120583

M)

T(1120583

M)

+T

1 hr 6 24

55

55


Relat

ive c

ytop

lasm

ic P

PAR-120574

expr

essio

n

(c)


8 PPAR Research

0

1

2

3

4

lowastlowast

lowastlowast

3 hrs

6 hrs

24 hrs


expr

essio

n le

vels

Rela

tiveG

CM-1

mRN

A

(a)

00

05

10

15

20

Binding sequence 1

Rela

tive p

rom

oter

activ

ity(n

orm

aliz

ed to

veh

icle

)

Ro10

120583M

Ro100120583

M

Vehi

cle

T01120583

M

T05120583

M

Ro100120583

M+

T05120583

M

lowastlowast

(b)

00

05

10

15

20

Binding sequence 2

Rela

tive p

rom

oter

activ

ity(n

orm

aliz

ed to

veh

icle

)

Ro10

120583M

Ro100120583

M

Vehi

cle

T01120583

M

T05120583

M

Ro100120583

M+

T05120583

M

lowastlowastlowast

(c)







PPAR Research 9

NS control siRNA0

1

2

3

4

5

expr

essio

nAb

solu

tePP

AR-120574

lowast

(a)

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

00

05

10

(nor

mal

ized

to N

S co

ntro

l)PP

AR-120574

expr

essio

n

Rosi10

120583M

Rosi100120583

M

T007

1120583

M


lowastlowastlowast

lowastlowastlowast

Vehi

cle

(b)

00

05

10

(nor

mal

ized

to N

S co

ntro

l)

lowastlowast

lowast

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

HO

-1ex

pres

sion

(c)

00

05

10

15

20

(nor

mal

ized

to N

S co

ntro

l)lowastlowast

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

GCM

-1ex

pres

sion

(d)






10 PPAR Research



4 Discussion





PPAR Research 11

00

05

10

15

20

15

20

25

(r

elat

ive t

o ve

hicle

cont

rol)

Free

120573-h

CG se

cret

ion

Ro10

120583M

Vehi

cle

Ro100120583

M

T1120583

M

Ro10

120583M

+T1

120583M

For2

0120583

M

lowast

(a)

00

05

10

15

20

Relat

ive c

ell p

rolif

erat

ion

(nor

mal

ized

to v

ehic

le co

ntro

l)

Ro10

120583M

Vehi

cle

Ro100120583

M

T1120583

M

Ro10

120583M

+T1

120583M

For2

0120583

M

(b)





5 Conclusions




Acknowledgments


12 PPAR Research


References


























PPAR Research 13








of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















PPAR Research 7

120574

(a)

1 6 240

1

2

3lowast

120574

10120583

M)

T(1120583

M)

+T

10120583

M)

T(1120583

M)

+T

10120583

M)

T(1120583

M)

+T

1 hr 6 24

55

67

10120583M)T (1120583M)

+ T

Relat

ive n

ucle

ar P

PAR-120574

expr

essio

n

(b)

1 6 24

10120583M)T (1120583M)

+ T

lowast

10120583

M)

T(1120583

M)

+T

10120583

M)

T(1120583

M)

+T

10120583

M)

T(1120583

M)

+T

1 hr 6 24

55

55


Relat

ive c

ytop

lasm

ic P

PAR-120574

expr

essio

n

(c)


8 PPAR Research

0

1

2

3

4

lowastlowast

lowastlowast

3 hrs

6 hrs

24 hrs


expr

essio

n le

vels

Rela

tiveG

CM-1

mRN

A

(a)

00

05

10

15

20

Binding sequence 1

Rela

tive p

rom

oter

activ

ity(n

orm

aliz

ed to

veh

icle

)

Ro10

120583M

Ro100120583

M

Vehi

cle

T01120583

M

T05120583

M

Ro100120583

M+

T05120583

M

lowastlowast

(b)

00

05

10

15

20

Binding sequence 2

Rela

tive p

rom

oter

activ

ity(n

orm

aliz

ed to

veh

icle

)

Ro10

120583M

Ro100120583

M

Vehi

cle

T01120583

M

T05120583

M

Ro100120583

M+

T05120583

M

lowastlowastlowast

(c)







PPAR Research 9

NS control siRNA0

1

2

3

4

5

expr

essio

nAb

solu

tePP

AR-120574

lowast

(a)

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

00

05

10

(nor

mal

ized

to N

S co

ntro

l)PP

AR-120574

expr

essio

n

Rosi10

120583M

Rosi100120583

M

T007

1120583

M


lowastlowastlowast

lowastlowastlowast

Vehi

cle

(b)

00

05

10

(nor

mal

ized

to N

S co

ntro

l)

lowastlowast

lowast

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

HO

-1ex

pres

sion

(c)

00

05

10

15

20

(nor

mal

ized

to N

S co

ntro

l)lowastlowast

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

GCM

-1ex

pres

sion

(d)






10 PPAR Research



4 Discussion





PPAR Research 11

00

05

10

15

20

15

20

25

(r

elat

ive t

o ve

hicle

cont

rol)

Free

120573-h

CG se

cret

ion

Ro10

120583M

Vehi

cle

Ro100120583

M

T1120583

M

Ro10

120583M

+T1

120583M

For2

0120583

M

lowast

(a)

00

05

10

15

20

Relat

ive c

ell p

rolif

erat

ion

(nor

mal

ized

to v

ehic

le co

ntro

l)

Ro10

120583M

Vehi

cle

Ro100120583

M

T1120583

M

Ro10

120583M

+T1

120583M

For2

0120583

M

(b)





5 Conclusions




Acknowledgments


12 PPAR Research


References


























PPAR Research 13








of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















8 PPAR Research

0

1

2

3

4

lowastlowast

lowastlowast

3 hrs

6 hrs

24 hrs


expr

essio

n le

vels

Rela

tiveG

CM-1

mRN

A

(a)

00

05

10

15

20

Binding sequence 1

Rela

tive p

rom

oter

activ

ity(n

orm

aliz

ed to

veh

icle

)

Ro10

120583M

Ro100120583

M

Vehi

cle

T01120583

M

T05120583

M

Ro100120583

M+

T05120583

M

lowastlowast

(b)

00

05

10

15

20

Binding sequence 2

Rela

tive p

rom

oter

activ

ity(n

orm

aliz

ed to

veh

icle

)

Ro10

120583M

Ro100120583

M

Vehi

cle

T01120583

M

T05120583

M

Ro100120583

M+

T05120583

M

lowastlowastlowast

(c)







PPAR Research 9

NS control siRNA0

1

2

3

4

5

expr

essio

nAb

solu

tePP

AR-120574

lowast

(a)

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

00

05

10

(nor

mal

ized

to N

S co

ntro

l)PP

AR-120574

expr

essio

n

Rosi10

120583M

Rosi100120583

M

T007

1120583

M


lowastlowastlowast

lowastlowastlowast

Vehi

cle

(b)

00

05

10

(nor

mal

ized

to N

S co

ntro

l)

lowastlowast

lowast

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

HO

-1ex

pres

sion

(c)

00

05

10

15

20

(nor

mal

ized

to N

S co

ntro

l)lowastlowast

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

GCM

-1ex

pres

sion

(d)






10 PPAR Research



4 Discussion





PPAR Research 11

00

05

10

15

20

15

20

25

(r

elat

ive t

o ve

hicle

cont

rol)

Free

120573-h

CG se

cret

ion

Ro10

120583M

Vehi

cle

Ro100120583

M

T1120583

M

Ro10

120583M

+T1

120583M

For2

0120583

M

lowast

(a)

00

05

10

15

20

Relat

ive c

ell p

rolif

erat

ion

(nor

mal

ized

to v

ehic

le co

ntro

l)

Ro10

120583M

Vehi

cle

Ro100120583

M

T1120583

M

Ro10

120583M

+T1

120583M

For2

0120583

M

(b)





5 Conclusions




Acknowledgments


12 PPAR Research


References


























PPAR Research 13








of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















PPAR Research 9

NS control siRNA0

1

2

3

4

5

expr

essio

nAb

solu

tePP

AR-120574

lowast

(a)

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

00

05

10

(nor

mal

ized

to N

S co

ntro

l)PP

AR-120574

expr

essio

n

Rosi10

120583M

Rosi100120583

M

T007

1120583

M


lowastlowastlowast

lowastlowastlowast

Vehi

cle

(b)

00

05

10

(nor

mal

ized

to N

S co

ntro

l)

lowastlowast

lowast

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

HO

-1ex

pres

sion

(c)

00

05

10

15

20

(nor

mal

ized

to N

S co

ntro

l)lowastlowast

Rosi10

120583M

Rosi100120583

M

T007

1120583

M

Vehi

cle

GCM

-1ex

pres

sion

(d)






10 PPAR Research



4 Discussion





PPAR Research 11

00

05

10

15

20

15

20

25

(r

elat

ive t

o ve

hicle

cont

rol)

Free

120573-h

CG se

cret

ion

Ro10

120583M

Vehi

cle

Ro100120583

M

T1120583

M

Ro10

120583M

+T1

120583M

For2

0120583

M

lowast

(a)

00

05

10

15

20

Relat

ive c

ell p

rolif

erat

ion

(nor

mal

ized

to v

ehic

le co

ntro

l)

Ro10

120583M

Vehi

cle

Ro100120583

M

T1120583

M

Ro10

120583M

+T1

120583M

For2

0120583

M

(b)





5 Conclusions




Acknowledgments


12 PPAR Research


References


























PPAR Research 13








of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















10 PPAR Research



4 Discussion





PPAR Research 11

00

05

10

15

20

15

20

25

(r

elat

ive t

o ve

hicle

cont

rol)

Free

120573-h

CG se

cret

ion

Ro10

120583M

Vehi

cle

Ro100120583

M

T1120583

M

Ro10

120583M

+T1

120583M

For2

0120583

M

lowast

(a)

00

05

10

15

20

Relat

ive c

ell p

rolif

erat

ion

(nor

mal

ized

to v

ehic

le co

ntro

l)

Ro10

120583M

Vehi

cle

Ro100120583

M

T1120583

M

Ro10

120583M

+T1

120583M

For2

0120583

M

(b)





5 Conclusions




Acknowledgments


12 PPAR Research


References


























PPAR Research 13








of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















PPAR Research 11

00

05

10

15

20

15

20

25

(r

elat

ive t

o ve

hicle

cont

rol)

Free

120573-h

CG se

cret

ion

Ro10

120583M

Vehi

cle

Ro100120583

M

T1120583

M

Ro10

120583M

+T1

120583M

For2

0120583

M

lowast

(a)

00

05

10

15

20

Relat

ive c

ell p

rolif

erat

ion

(nor

mal

ized

to v

ehic

le co

ntro

l)

Ro10

120583M

Vehi

cle

Ro100120583

M

T1120583

M

Ro10

120583M

+T1

120583M

For2

0120583

M

(b)





5 Conclusions




Acknowledgments


12 PPAR Research


References


























PPAR Research 13








of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















12 PPAR Research


References


























PPAR Research 13








of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















PPAR Research 13








of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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