Jeffrey D. Coleman, 1 Jerry T. Thompson, 1 Russell W. Smith III, 1 Bogdan Prokopczyk, 2,3 and John P. Vanden Heuvel 1,3,4. 1.

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1 PPAR Reserh Volume 213, Artile ID , 11 pges Reserh Artile Role of Peroxisome Prolifertor-Ativted Reeptor β/δ nd B-Cell Lymphom-6 in Regultion of Genes Involved in Metstsis nd Migrtion in Pnreti Cner Cells Jeffrey D. Colemn, 1 Jerry T. Thompson, 1 Russell W. Smith III, 1 Bogdn Prokopzyk, 2,3 nd John P. Vnden Heuvel 1,3,4 1 Deprtment of Veterinry nd Biomedil Sienes nd Center for Moleulr Toxiology nd Crinogenesis, Penn Stte University, 325 Life Sienes Building, University Prk, PA 1682, USA 2 Deprtment of Phrmology, Penn Stte University, Hershey, PA 1733, USA 3 Penn Stte Cner Institute, Hershey, PA 1733, USA 4 Indigo Biosienes In., Stte College, PA 1681, USA Correspondene should e ddressed to John P. Vnden Heuvel; jpv2@psu.edu Reeived 9 Jnury 213; Revised 18 Mrh 213; Aepted 7 April 213 Ademi Editor: Annmri Cimini Copyright 213 Jeffrey D. Colemn et l. This is n open ess rtile distriuted under the Cretive Commons Attriution Liense, whih permits unrestrited use, distriution, nd reprodution in ny medium, provided the originl work is properly ited. PPARβ/δ is lignd-tivted trnsription ftor tht regultes vrious ellulr funtions vi indution of trget genes diretly or in onert with its ssoited trnsriptionl repressor, BCL-6. Mtrix remodeling proteinses re frequently over-expressed in pnreti ner nd re involved with metstsis. The present study tested the hypothesis tht PPARβ/δ is expressed in humn pnreti ner ells nd tht its tivtion ould regulte MMP-9, deresing ner ells ility to trnsverse the sement memrne. In humn pnreti ner tissue there ws signifintly higher expression of MMP-9 nd PPARβ/δ,nd lower levels of BCL-6 mrna. PPARβ/δ tivtion redued the TNFα-indued expression of vrious genes implited in metstsis nd redued the invsion through sement memrne in ell ulture models. Through the use of short hirpin RNA inhiitors of PPARβ/δ, BCL-6, nd MMP-9, it ws evident tht PPARβ/δ ws responsile for the lignd-dependent effets wheres BCL-6 dissoition upon GW51516 tretment ws ultimtely responsile for deresing MMP-9 expression nd hene invsion tivity. These results suggest tht PPARβ/δ plys role in regulting pnreti ner ell invsion through regultion of genes vi lignd-dependent relese of BCL-6 nd tht tivtion of the reeptor my provide n lterntive therpeuti method for ontrolling migrtion nd metstsis. 1. Introdution Pnreti ner is the fourth leding use of ner-relted deths of men nd women in the United Sttes. The Amerin Cner Soiety estimtes for 29 predited pproximtely 42,47 new ses of pnreti ner nd tht 35,24 of those ses would result in deth. Lk of identifile symptoms or iomrkers omined with 4% five-yer survivl rte mkes pnreti ner one of the dedliest mlignnies [1]. Although pnreti ner is diffiult to detet in its erly stges, severl known risk ftors exist, with smoking eing the most well-doumented etiologi gent [2]. Severl other risk ftors inlude ge, diets high in ft [3], exessive lohol onsumption [4], dietes mellitus [5], nd hroni pnretitis [6]. Common hemotherpeuti tretments hve hd little suess in improving survivl rtes or restrining the highly metstti mlignnies [7] withthemedinsurvivl rte of less thn six months nd surgil resetion s the only effetive tretment [8]. Prevention strtegies nd lterntive tretments for pnreti ner re sorely needed. Peroxisome prolifertor-tivted reeptor-β/δ (PPARβ/ δ) is memer of the nuler reeptor superfmily of lignd-tivted trnsription ftors. The PPARs onsist of three isoforms: PPARα (NR1C1), PPARβ/δ (NR1C2; NUC1;

2 2 PPAR Reserh FAAR ftty id-tivted reeptor), nd PPARγ (NR1C3). The PPARs effet gene trnsription in response to vrious stimuli, suh s ftty ids nd their metolites, xenoiotis nd isoform-speifi drugs, through heterodimeriztion with retinoidx reeptors (RXRs) nd susequent reognition nd inding to peroxisome prolifertor-responsive elements (PPREs) within the promoter regions of trget genes [9, 1]. PPARβ/δ,unlike PPARα or PPARγ whih hve distint tissue expressionptternsndsynthetilignds,isuiquitously expressed, often t higher levels thn the other isoforms. This reeptor regultes ftty id oxidtion nd lipid homeostsis [11], ell prolifertion nd differentition [12], ell survivl [13], ndtheinflmmtoryresponse[14]. The ltter response my e vi its ssoition with the trnsriptionl repressor BCL-6, whih is relesed upon tivtion of PPARβ/δ [15]. In the pnres, PPARβ/δ is expressed in islet ells to greter extent thn either PPARα or PPARγ ndinetells where it regultes the inflmmtory response [16]. Expression profiling nlyses in the mouse demonstrted high PPARβ/δ expression in the ytoplsm of delt ells of the islet of Lngerhns, suggesting potentil role for the reeptor in the regultion of gluose metolism [17]. Pnreti dutl denorinoms re y fr the most ommon of pnreti mlignnies [18], nd the role(s) of PPARβ/δ in pnreti dutl ells is poorly understood. The mtrix metlloproteinses re fmily of zindependent proteolyti enzymes tht degrde extrellulr mtrix (ECM) proteins nd re well-known regultors of pnreti ner ell metstsis nd invsion [19, 2]. Mtrix metlloproteinse-9 (MMP-9,lso known s geltinse B)in prtiulr is highly expressed in oth linil nd experimentl models of pnreti ner [21]. Furthermore, pnreti ner ells disply extremely high sl MMP-9 expression, whih is further induile y phorol 12-myristte 13-ette (PMA) [22]. Reently, severl studies hve linked PPARβ/δ to MMP-9; in PPARβ/δ null mrophges, sl MMP-9 expression is redued [15], nd in vsulr smooth musle ells (VSMCs) PPARβ/δ tivtion suppressed the expression of oth MMP-2 nd MMP-9, with further inhiition on VSMC migrtion nd prolifertion [23]. The role(s) of PPARs, prtiulrly PPARβ/δ, in tumorigenesis nd ner ell invsion remins ontroversil. For exmple, inhiition of PPARγ suppressed pnreti ner ell motility in Cpn-1 nd Pn-1 ells [24], while its tivtion in AsPC-1 ells y the speifi lignd rosiglitzone inresed levels of the tumor suppressor PTEN nd deresed levels of phosphorylted Akt [25] nd indued spsemedited poptosis in Mip-2 ells [26]. PPARβ/δ is n APC-regulted trget of nonstreroidl nti-inflmmtory drugs (NSAIDs), suggesting tht NSAIDs inhiit tumorigenesis vi PPARβ/δ inhiition [27], nd geneti disruption of PPARβ/δ ontriutes to the growth-inhiitory effets of APC [28]. Opposing evidene exists suggesting tht PPARβ/δ tivtion inreses [29 31] nd dereses ell prolifertion[32, 33] in vrious ell types. Previous evidene, however, estlishes ler link etween PPARβ/δ,BCL-6,nd MMP-9,nd we sought to eluidte the role(s) of PPARβ/δ tivtion on potentil trget genes involved in pnreti ner invsion nd metstsis. The PPARβ/δ-speifi tivtor GW51516 nd shrnas to derese expression of PPARβ/δ,BCL-6,nd MMP-9 were used in two humn pnreti ner ell lines, Mip-2 (COX-2 negtive) nd BxP-3 (COX-2 positive). The experiments show tht lignd-dependent tivtion of PPARβ/δ uses BCL6-dependent repression of MMP-9 nd other genes involved in ner metstsis nd dereses indies of ell migrtion, suggesting tht PPARβ/δ gonists my e enefiil tool in the prevention nd tretment of pnreti ner. 2. Mterils nd Methods 2.1. Cells nd Regents. Humn pnreti ner ells, Mip-2 (COX-2 negtive, CRL-142) nd BxP-3 (COX-2 positive, CRL-1687), were purhsed from the ATCC (Mnsss, VA) nd ultured in high-gluose DMEM ontining 1% FBS nd 1% peniillin/streptomyin in humidified tmosphere t 37 C ontining 5% CO 2.Humnemryoni kidney 293 ells were ultured in DMEM ontining 1% FBS nd 1% peniillin/streptomyin. All medi omponents nd fetl ovine serum (FBS) were purhsed from Gio BRL/Life Tehnologies (Crlsd, CA). Ciprofirte (Cipro), purhsed from Sigm Chemil Co. (St Louis, MO), ws used s the positive ontrol for PPARα. GW51516(GW), purhsed from Sigm Chemil Co., ws used s the positive ontrol for PPARβ/δ. Rosiglitzone (rosi), purhsed from Cymn Chemils (Ann Aror, MI), ws used s the positive ontrol for PPARγ. Reominnt humn TNFα nd humn MMP-9 ELISAs were purhsed from Invitrogen (Crlsd, CA) nd used ording to the mnufturer s instrutions. Humn pnreti ner, hroni pnretitis, nd pnres tissue smples were otined from Dr. Gerhrd Leder, (At. Allgemein-und Viszerlhirurgie, St. Josef Hospitl Klinikum der Ruhr, University of Bohum, Germny). MISSION shrna teril glyerol stoks trgeted ginst humn PPARβ/δ, Bl6, MMP-9, s well s the nontrgeting vetor, were purhsed diretly from Sigm- Aldrih. High Cpity DNA Arhive Kit nd ABI73 reltime PCR system were purhsed from Applied Biosystems (Foster City, CA). The ppackh1 pkging plsmids were kindly provided y Dr. Curtis J. Omieinski (Penn Stte University). CytoSelet 96-well ell invsion ssy (sement memrne, fluorometri formt) ws purhsed from Cell Biols, In. (Sn Diego, CA) nd used ording to the mnufturer s instrutions Isoltion of Totl RNA nd Rel-Time Quntittive RT- PCR. TotlRNAwsisoltedfromMip-2ndBxP-3 ells using Tri-Regent nd the mnufturer s reommended protool (Sigm). Humn pnreti tissue smples were riefly homogenized in 1 ml Tri-Regent, nd totl RNA ws isolted. One μg of totl RNA ws reverse-trnsried using the High Cpity DNA Arhive Kit (Applied Biosystems, Foster City, CA). PCR primers for quntittive rel-time RT-PCR were designed sed on pulished sequenes in GenBnk nd re shown in Tle 1 in Supplementry Mteril ville online t The housekeeping gene β-tin ws used to normlize ll the

3 PPAR Reserh 3 tested genes. The dt shown re representtive of three independent experiments with triplite smples Quntifition of MMP-9 Protein y ELISA. MMP-9 protein levels were quntified using the humn MMP-9 ELISA ording to the mnufturer s instrutions (Invitrogen). Briefly, ontrol Mip-2 ells or shrna knokdown ells were plted in 6-well tissue ulture pltes nd treted with 1ng/mLTNFα with or without 5 nm GW51516 for 24 h. At the end of the inution time, the medi ws removed nd diluted 1 : 4 in stndrd diluent uffer. Diluted medi smples nd MMP-9 stndrds were dded to 96-well mirotiter plte ontining humn MMP-9 ntiody-oted wells nd llowed to inute t room temperture for 2 h. Following the inution, the medi ws spirted nd eh well wshed 5 times with wsh uffer. One hundred μl Biotinylted nti-mmp-9 (iotin onjugte) solution ws dded to eh well, nd the plte ws inuted for 1 h t room temperture. Eh well ws wshed seond time with wsh uffer, nd 1 μl of streptvidin-hrp working solution ws dded nd the plte ws llowed to inute t room temperture for 3 minutes. After third wsh, 1 μl of stilized hromogen ws dded to eh well, nd the plte ws inuted t room temperture for 3 minutes in the drk, fter whih time 1 μl of stop solution ws dded nd thesorneredt45nm Cell Migrtion Assy. Either ontrol or knokdown humn pnreti ner ells were grown to onfluene in 1 m tissue ulture pltes nd then pretreted with TNFα with or without GW51516 for 24 h, s ove. Cell migrtion ssys were performed using the CytoSelet 96-well ell invsion ssy (sement memrne, fluorometri formt) ording to the mnufturer s instrutions. Briefly, the sement memrne ws llowed to reh room temperture for 3 minutes nd rehydrted using wrm, serum-free DMEM. Humn pnreti ner ells were then seeded into eh well t density of ells/ml in serumfree medi. Norml ell medi (DMEM ontining 1% FBS, long with TNFα with or without GW51516) ws dded tothefeedertry,ndtheentirepprtuswspledin n inutor t 37 C ontining 5% CO 2 for 24 h. CyQunt GR dye/lysis uffer solution ws dded to the invding ells following ompletion of the ssy, nd the resulting mixture ws inuted t room temperture for 2 minutes. Invding ells were quntified y reding the fluoresene t 48 nm/52 nm. All mesurements were performed in triplite Lentivirl shrna Infetion. HEK-293 ells were grown to onflueny in 1 m tissue ulture pltes under the onditions desried ove. The ells were then trnsiently trnsfeted with 4.6μg of either nontrgeting shrna or shrnas trgeted ginst humn PPARβ/δ, BCL-6, or MMP-9,swells2.4μg eh of ppackh1 pkging plsmids, using Lipofetmine 2. Cells were trnsfeted for 6 h nd llowed to reover overnight in norml medi. Fresh medi ws dded the following morning, nd pseudovirl superntnt ws generted for 72 h. Superntnt ws then hrvested nd pssed through.4μm filter under sterile onditions. Polyrene (Millipore, Billeri, MA) ws then dded to finl onentrtion of 5 μg per ml, nd the pseudovirl superntnt ws then dded diretlytotrgetellsfor6h.infetedellswerellowedto reover overnight following the ddition of 6 ml omplete medi, nd knokdown of trget genes ws ssessed y RT- PCR 48 h postinfetion Sttistil Anlysis. Quntittive dt re presented s men ± SEM. ANOVA with P-vlue <.5 ws used to determine whether differenes mong vriles were signifint. Normlity ws heked using Anderson-Drling test nd the generl liner model, followed y the Tukey post ho test to nlyze differenes etween tretments. All dt nlyses were performed y MiniTAB Ver.14 (MiniTAB, Stte College, PA) or JMP (SAS Institute, Cry, NC), nd dt were plotted y Prism 5.1 (GrphPd Softwre, Sn Diego, CA). 3. Results 3.1. Tissue Smples from Humn Pnreti Dutl Crinoms Show Signifintly Inresed Levels of MMP-9 mrna. It is well known tht the mtrix metlloproteinses re key regultors of ell prolifertion nd migrtion in humn pnreti ner ells [34]ndthtMMP-9 protein is inresed in the pnreti juie from ptients dignosed with pnreti dutl denorinoms [35]. Reently, MMP-9 hs een linked to PPARβ/δ nd the trnsriptionl repressor BCL-6; inpparβ/δ / mrophges, for exmple, there ws lower MMP-9 expression ompred with wild-type ells [15]. Tissue smples from ptients dignosed with hroni pnretitis or pnreti ner were otined, nd we set out to ssess the differenes in expression of severl genes involved in inflmmtion nd metstsis. Indeed, there ws 1-fold inrese in MMP-9 gene expression in dutl rinoms ompred with smples from ptients dignosed with hroni pnretitis (Figure 1). Interestingly, PPARβ/δ expression ws lso elevted while mrna expression of the trnsriptionl repressor BCL-6 ws lmost 3-fold lower in tumor smples ompred with those from hroni pnretitis ptients. Despite the low expression of BCL-6 in dutl rinoms, the reltive expression of two BCL-6 trget genes, VCAM-1 [36] ndmcp-1, wsnotsignifintlyelevtedin tumor smples. A PPARβ/δ trget gene, ADRP,wslsonot different etween tumor, pnretitis, nd other pnreti tissue smples (dt not shown) Regultion of MMP-9 Expression y PPARβ/δ nd BCL6 in Pnreti Cner Cells. PPARβ/δ tivtion negtively influenesmmp-9 gene expression in IL-1β-stimulted vsulr smooth musle ells [23]. To study the mehnism of this response nd to determine its ppliility to nother ell type, Mip-2 ells were trnsiently infeted with nontrgeting ontrol, hbcl-6, hpparβ/δ, or hmmp9 lentivirl shrnas (Figure2()). Cells trnsiently infeted with nontrgeting ontrol shrna showed no ltertions in either BCL-6, PPARβ/δ, or MMP9 mrna expression. Mip ells infeted with n shrna trgeted ginst BCL-6

4 4 PPAR Reserh E-seletin ICAM-1 VCAM-1 IL-1β MCP-1 MMP-9 Bl-6 PPARβ/δ 15 Reltive expression 1 5, Pnretitis Dutl rino. Pnretitis Dutl rino. Pnretitis Dutl rino. Pnretitis Dutl rino. Pnretitis Dutl rino. Pnretitis Dutl rino. Pnretitis Dutl rino. Pnretitis Dutl rino. Figure 1: Reltive mrna expression in humn pnreti tissues t vrying stges of rinogenesis from hroni pnretitis to pnreti ner. Totl mrna ws isolted using stndrd Tri-Regent protool nd reverse-trnsried. Gene expression ws determined using qrt- PCR nd expressed s fold indution fter normliztion to β-tin. P <.5. showed pproximtely 5% redution in BCL-6 mrna levels, nd ells infeted with n shrna trgeting PPARβ/δ or MMP9 showed pproximtely 7% redution in orresponding mrna levels (Figure 2()). To determine if gene expression is ltered fter lentivirl-medited BCL-6 or PPARβ/δ repression, the PPAR trget gene ADRP ws exmined upon tretmentwiththreeisoform-speifippar gonists (iprofirte, PPARα; GW51516, PPARβ/δ; rosiglitzone, PPARγ). Control ells nd those trnsiently expressing the indited shrnas were treted with either 2 μmiprofirte,5nm GW51516, or 1 μm rosiglitzone. Mip-2 ells ontin funtionl PPARs s indited y the lignd-indued expression of ADRP, with eh tretment resulting in threefold inrese in trnsript levels (Figure 2()). Cells expressing PPARβ/δ-speifi shrna did not indue expression of ADRP in response to GW51516 t onentrtion tht tivtes only PPARβ/δ [37], while ells expressing BCL-6- trgeting shrna retined induile expression of ADRP y ll three isoform-speifi lignds. Following BCL-6, PPARβ/δ or MMP-9 knokdown, ellsweretretedwith1 ng/mltnfα with or without 5 nm GW51516 for 24 hours, nd MMP-9 protein levels were ssessed y ELISA. MMP-9 protein levels were signifintly elevted in BCL-6 knokdown Mip- 2 ells following TNFα hllenge ompred with ontrol ells, while PPARβ/δ knokdown Mip-2 ells showed signifintredution intnfα-indued MMP-9 protein levels (Figure 2()), onsistent with previous reports in PPARβ/δ / mrophges. While GW51516 otretment signifintly suppressed TNFα-indued MMP-9 protein levels in ontrol (nontrgeting) Mip-2 ells, this effet ws not oserved in either of the BCL-6 or PPARβ/δ knokdown ells. Not unexpetedly, lentivirl shrna trgeted ginst MMP-9 signifintly redued oth mrna nd protein expression in Mip-2 ells, nd GW51516 tivtion of PPARβ/δ did not further redue MMP-9 protein levels in these ells MMP-9 Knokdown Redues Mip-2 Cell Invsion. Beuse MMP-9 is key regultor of humn pnreti ner ell invsion nd metstsis, we further exmined the effet of lentivirl shrna-medited MMP-9 knokdown on the sl ility of Mip-2 ells to invde sement memrne. Mip-2 ells treted with non-trgeting ontrol or MMP-9-trgeting lentivirl shrnas were seeded into 96-well invsion plte nd llowed to migrte ross memrne for 24 hours. Using the migrtion ssy desried, we found tht MMP-9 knok-down signifintly redued the sl migrtion of Mip-2 ells (Figure 2(d)) PPARβ/δ Ativtion Dereses TNFα-Indued Expression of Proinflmmtory nd Cell Adhesion Genes in Humn Pnreti Cner Cells. PPARβ/δ ssoites with the trnsriptionl repressor BCL-6 whih, upon PPARβ/δ tivtion, is relesed nd dereses expression of trget genes. To determine if the PPARβ/δ/BCL-6 pthwyistivein humn pnreti ner ells, we used shrna knok-down of PPARβ/δ nd BCL-6 in onjuntion with PPARβ/δ-speifi tivtion y GW51516 to nlyze the gene expression hnges. Mip-2 ells trnsiently expressing non-trgeting ontrol shrna, or shrnas trgeted ginst PPARβ/δ or BCL-6, were stimulted with 1 ng/ml TNFα with or without GW51516 for 24 hours. In ontrol Mip-2 ells, TNFα stimultion indued the roust expression of the ell dhesion moleules E-seletin, ICAM-1 nd VCAM-1, theproinflmmtory genes IL-1β nd MCP-1, nd the promigrtory gene MMP-9, while otretment with 5 nm GW51516 signifintly suppressed their expression t the mrna level (Figure 3). Tretment of Mip-2 ells with BCL-6- trgeting shrna ttenuted the GW51516 inhiitory effet on the genes tested, inditing role for BCL-6 in GW medited repression. Consistent with the findings of Lee et l. in PPARβ/δ / RAW264.7 mrophge ells, PPARβ/δ knok-down Mip-2 ells displyed signifintly lower levels of these genes when hllenged with TNFα lone, nd PPARβ/δ tivtion with GW51516 hd no further signifint repressive effet. Of note is the ft tht lthough BCL- 6 repression resulted in inresed MMP-9 protein levels in

5 PPAR Reserh Normlized mrna expression 1.5 Normlized ADRP expression Nontrgeting shrna hbl6 shrna hpparβ/δ shrna hmmp-9 shrna hbl-6 mrna hpparβ/δ mrna hmmp-9 mrna Bl6 Ctrl Ctrl Cipro Bl6 Cipro PPARβ/δ Cipro Bl6 Ctrl Bl6 GW shrnai, Trt Bl6 Ctrl Ctrl Rosi Bl6 Rosi PPARβ/δ Rosi () () Humn MMP-9 (pg/ml) ,,, d, d d Perent invding ells Nontrgeting shrna hbl6 shrna hpparβ/δ shrna hmmp-9 shrna Nontrgeting shrna hmmp-9 shrna TNFα TNFαGW51516 () (d) Figure 2: Effet ofpparβ/δ tivtion on MMP-9 expression. () Mip-2 ells were trnsiently infeted with nontrgeting ontrol, hbcl- 6, hpparβ/δ, or hmmp9 lentivirl shrnas for 48 hours. Totl mrna ws isolted nd gene-speifi knokdown ws ssessed using qrt- PCR. P <.5. () Mip-2 ells ontin funtionl PPARs. Mip-2 ells were trnsiently infeted with the indited shrnas nd treted with the indited PPAR isoform-speifi gonists. Indution of the PPAR-trget gene ADRP ws determined using qrt-pcr. P<.5. () Mip-2 ells were stimulted with humn TNFα with or without GW51516 for 24 hours following trnsient infetion with the indited shrnas. Humn MMP-9 protein expression ws quntified using MMP-9-speifi ELISA (Invitrogen). P <.5. (d) Mip- 2 ells with redued MMP-9 expression re less invsive thn ontrol Mip-2 ells. Following infetion with humn MMP-9-trgeting shrna, Mip-2 ells were seeded in 96-well invsion pltes (Cell Biols, In.) nd llowed to invde the sement memrne overnight. Reltive ell invsion ws quntified using the CytoSelet 96-well ell invsion ssy with fluorometri redings t 48 nm/52 nm. P<.5. medi, it did not onordntly inrese the mrna expression of this gene PPARβ/δ Ativtion Inhiits Humn Pnreti Cner Cell Migrtion. To exmine if PPARβ/δ tivtion y GW51516 nd susequent repression of pro-inflmmtory nd pro-migrtorygenes vibcl-6 influened their ility to invde sement memrne, Mip-2 (COX-2 negtive, Figure 4()) nd BxP-3(COX-2 positive, Figure 4()) were tretedwithnon-,pparβ/δ- or BCL-6-trgeting shrna, nd the effets of GW51516 on ell migrtion were exmined. In

6 6 PPAR Reserh Normlized mrna expression , E-seletin ICAM-1 VCAM-1 IL-1β MCP-1,, Bl6 Ctrl Bl6 GW,, Bl6 Ctrl Bl6 GW,, Bl6 Ctrl Bl6 GW, Bl6 Ctrl Bl6 GW,, Bl6 Ctrl Bl6 GW MMP-9, Bl6 Ctrl Bl6 GW shrnai, Trt Figure 3: Effets of PPARβ/δ nd BCL-6 knokdown on Mip-2 gene expression. Mip-2 ells were trnsiently infeted with nontrgeting ontrol, hbcl-6 or hpparβ/δ-speifi shrnas nd then stimulted with humn TNFα with or without GW51516 for 24 hours. Totl mrna ws isolted nd gene expression ws determined using qrt-pcr. Dt is normlized to β-tin nd indited s fold hnge. ontrol ells, GW51516 tretment negtively influened the ility of either Mip-2 or BxP-3 ells to migrte ross memrne (5% redution). Lentivirl-medited knokdown of BCL-6, however, inresed ell migrtion in oth ell lines ompred with ontrol ells. GW51516 tretment of BCL-6 repressed ells did not hve n effet on Mip (Figure 4()) utdidhveneffetonomprlebxp-3 ells (Figure 4()). Interestingly, Mip-2 nd BxP-3 ells trnsiently expressing n shrna trgeted ginst PPARβ/δ showed signifintly redued ell migrtion ompred with ontrol ells with or without GW51516 tretment. These results suggested tht the trnsriptionl repressor BCL-6 medites the ntimigrtory tions of GW51516 in humn pnreti ner ells ut does so in PPARβ/δ-dependent mnner. 4. Disussion The PPAR nuler reeptors re regultors of inflmmtion nd prolifertion in humn pnreti ells [16, 38, 39]. Although severl studies implite PPARγ tivtion in inhiition of pnreti ner ell growth, little is known out the role of PPARβ/δ, sve for its role in suppressing inflmmtion vi BCL-6 [16]. Generlly, the role of PPARβ/δ in ner ell growth nd tumorigenesis remins ontroversil. In oloretl ner ells, nonsteroidl ntiinflmmtory drugs inhiit tumorigenesis through inhiition of PPARβ/δ [27], nd PPARβ/δ promotes intestinl rinogenesis [4]. Studies in the PPARβ/δ null mouse, however, show tht tivtion of PPARβ/δ indues terminl differentition [41], nd PPARβ/δ-speifi lignds inhiit the growth of kertinoytes in vivo [42, 43] ndin vitro [32]. Furthermore, PPARβ/δ tivtion is linked to inhiition of IL-1β-stimulted prolifertion nd migrtion of vsulr smooth musle ells [23] vi regultion of IL-1R nd TGF-β nd negtive regultion of MMP-9. Our results show tht PPARβ/δ tivtion y GW51516 suppresses expression of MMP-9 in humn pnreti ner ells vi BCL-6, with further inhiition on the ility of two ell lines, Mip-2 nd BxP-3, to invde sement memrne. Consistent with previous work [35], nlysis of the expression levels of severl genes in oth dutl rinoms nd hroni pnretitis showed elevted levels of the mtrixremodeling gene MMP-9. Severl studies link inresed MMP-9 levels to inresed invsiveness nd metstsis [44, 45]. MMP-9 is ritil plyer in the erly stges of tumor invsion y degrding sement memrne type IV ollgen [46], onsidered to e ruil step in tumor ell invsion [47]. MMP-9 lso prtiiptes in the degrdtion of the vrious omponents of the ECM [48]. Inhiition of MMP tivity y orlly ioville mtrix metlloproteinse inhiitors hs shown promise in deresing tumor metstsis in linil trils [46]. In the ell lines BxP-3 nd Mip-2, tretment with the neurotrnsmitter norepinephrine inresed ell invsiveness vi ugmented MMP- 2, MMP-9, ndvegf [49], while tretment with the βloker proprnolol inhiited these effets. Clerly the regultion of MMP tivity is importnt in ontrolling, nd possily treting, pnreti ner. The ssoition etween MMP-9, PPARβ/δ, nd BCL-6 ws estlished y Lee et l. [15], demonstrting tht MMP-9 expression in PPARβ/δ / mrophgesisrepressedompredtowildtype.ativtion of the reeptor signifintly deresed the expression of proinflmmtory mrkers, suggesting tht BCL-6 relesed from the PPARβ/δ omplexplysroleintheregultionofmmp- 9. A similr result ws otined in VSMCs [23]. In the present studies, PPARβ/δ mrna were inresed in dutl rinoms, while BCL-6 expression ws deresed.

7 PPAR Reserh 7 2 Mip-2 2 BxP-3 Perent invding ells , Perent invding ells Nontrgeting Bl-6 shrna PPARβ/δ Nontrgeting Bl-6 shrna PPARβ/δ TNFα TNFαGW51516 () () Figure 4: GW51516 tretment redues TNFα-stimulted Mip-2 nd BxP-3 ell invsion through sement memrne. Humn pnreti ner ells were trnsiently infeted withtheinditedshrnasndstimultedwithhumntnfα with or without GW Cells were llowed to invde sement memrne overnight, nd reltive invsion ws quntified usingthe CytoSelet 96-well ell invsion ssy with fluorometri redings t 48 nm/52 nm. GW51516 tretment inhiits the invsion of the COX-2 negtive pnreti ner ell line Mip-2 () nd the COX-2 positive pnreti ner ell line BxP-3 () through sement memrne. In oloretl ner tissue smples, PPARβ/δ expression inresed during multistge rinogenesis nd ws tightly ssoited with highly mlignnt morphology [5]. It is possile tht PPARβ/δ plys role in humn pnreti ells, ut whether PPARβ/δ ontriutes to pnreti ner ell metstsis or if its overexpression is the result of some ltered signling pthwy remins unler. Sine the regultory region of the PPARβ/δ ontins severl AP-1 response elements nd is ontrolled y vriety of inflmmtory signls [51], the inresed expression of this nuler reeptor my e inditive of stress response nd not uslly relted to the tumor phenotype. Inresed expression of PPARβ/δ in the intivted stte my sequester BCL-6, inresing the expression of genes normlly ontrolled y this trnsriptionl repressor. Of prtiulr note is the oservtion tht the reltive expression of BCL-6, proto-onogene known to suppress genes involved in ell yle progression, prtiulrly ylin D1 [52], nd inflmmtion [53], ws lower in dutl rinoms ompred with pnretitis. BCL-6 is mutted in severl disorders [54 56] nd is implited in ell line immortliztion nd trnsformtion y overriding ellulr senesene downstrem of p53 [57]. Interestingly, DNAhip hyridiztion ssys identified oth BCL-6 nd BCL- 1 s novel ndidte genes in pnreti ner tht were overexpressed in pnreti ner ell lines nd primry tumor smples [58]. Contrry to dutl ells, BCL-6 is sent in pnreti et ells, potentilly explining the lk of nti-inflmmtory PPARβ/δ signling in this ell type [16]. Our results suggest tht the BCL-6 pthwy is disrupted s inflmed tissue trnsforms into tumor, with the possiility tht loss of BCL-6 expression leds to inresed ner invsion vi inresed MMP-9 expression. Ativtion of PPARβ/δ deresed the expression of MMP-9 t the protein level, while knokdown of BCL- 6 inresed MMP-9 protein prodution. Ativtion of PPARβ/δ in BCL-6 knok-down ells showed no signifint effet on reduing MMP-9 protein, suggesting key role for BCL-6 in the regultion of MMP-9. Knokingdown PPARβ/δ in humn pnreti ner ells redued MMP- 9 protein levels to those omprle to GW51516-treted ontrol ells despite PPARβ/δ tivtion. It is our hypothesis tht MMP-9 is n indiret PPARβ/δ trget gene nd tht PPARβ/δ tivtion releses BCL-6 whih my then relote to the MMP-9 promoter. Further studies, suh s hromtin immunopreipittion ssys, for exmple, re required to sustntite this point. Our results, however, re in greement with previous work inditing tht low levels of PPARβ/δ result in deresed MMP-9 expression [15], nd we elieve tht, in the sene of PPARβ/δ, BCL-6 is ville to repress trget genes, either through diret repression on trget gene promoters s in the se of VCAM-1 nd E-seletin, two genes lking PPREs, [36] or through intertions with other ell signling meditors, suh s NF-κB [59]. GW742 hs een found to inhiit inflmmtion y TNF-α while not preventing TNF-α-indued degrdtion of IκBα nd the trnslotion of NFκB. No derese in DNA inding tivity indites PPARβ/δ must interfere vi orepressors mehnism t the hromtin level [36]. The reltionship etween MMP-9 nd metstsis in pnreti ner is well doumented. Tretment of Mip- 2 ells with MMP-9 shrna redued protein levels y pproximtely 5% regrdless of GW51516 tretment, with orresponding inhiition on the ility of the ell line to invde sement memrne. Mip-2 ells re onsidered highlymetsttiellline[6], nd our results support

8 8 PPAR Reserh the ide tht regulting MMP-9 expression my effetively ontrol humn pnreti ner ell invsion nd metstsis. PPARβ/δ tivtion is ssoited with redued inflm- mtorynddhesionellmrkers.ourresultsinmip- 2 ells show tht the PPARβ/δ BCL-6 nti-inflmmtory pthwy is tive nd represses E-seletin, ICAM-1, VCAM-1, IL-1β, MCP-1, nd MMP-9.RepressionofVCAM-1, E-seletin, [36], nd MCP-1 [15] in prtiulr is dependent upon dissoition of BCL-6 from PPARβ/δ nd susequent relotion to the orresponding promoters. Our results demonstrte tht the prodhesion moleules E-seletin, ICAM-1, nd VCAM-1, the pro-inflmmtory IL-1β nd MCP-1, nd the prometstsis gene MMP-9 re BCL-6-regulted genes in humn pnreti ner ells. Tretment with BCL-6 shrna ttenuted the GW51516-medited inhiition of TNFαindued expression of these moleules, while tretment with PPARβ/δ shrna redued their expression t the mrna level regrdless of GW51516 tretment. E-seletin, ICAM- 1, nd VCAM-1 re importnt in pnreti ner, where they prtiipte in the dethment of ells from the primry tumor nd ontriute to ner spred [61], nd their overexpression in pnreti denorinoms is ssoited with stimultion in tumor growth, inresed metstti ility, nd potentilly shorter postopertive survivl following tumor resetion [62]. IL-1β indues MMP-9 expression in other ell lines [63 65] nd enhnes the invsiveness of humn pnreti ner ells [66]. Monoyte hemotti protein-1 is produed y pnreti ner ells in response to TNFα hllenge nd my ontriute to the umultion of tumorssoited mrophges [67] whih influene key events in the tumor invsion proess [68]. Tken together, these results suggest tht PPARβ/δ tivtionndsusequent suppression of prodhesion nd pro-migrtory genes vi BCL-6 might prove useful in the ontrol of pnreti ner. GW51516 tretment lso inhiited the TNFα-promoted invsion of sement memrne y the pnreti dutl ell lines Mip-2 nd BxP-3. Pnreti ner ells trnsiently expressing BCL-6 shrna were signifintly more invsive, while GW51516 tretment ttenuted their invsive potentil lose to ontrol levels. Conversely, ells trnsiently expressing PPARβ/δ shrna were signifintly less invsive thn ontrol ells, nd GW51516 tretment showed no signifint effet in further reduing invsion. We hypothesize tht ells expressing lower levels of the trnsriptionl repressor BCL-6 remoreinvsiveowing to lkofontrol over pro-migrtory gene regultion nd inresed protein levels of MMP-9, while knoking down PPARβ/δ vi shrna methods llow for greter popultion of unssoited BCL-6 whih is ville to repress pro-migrtory genes resulting in lower invsion. Although we present here firly simplified mehnism, it is possile tht more omplex signling sde is tking ple resulting in the inhiition of ell invsion. In VSMCs, repression of MMP-9 tivity ws effeted in TGF-β-dependent mnner following PPARβ/δ tivtion [23], nd indeed, TGF-β suppresses MMP-9 expression in monoytes through prostglndin E2- nd AMP-dependent mehnism [69]. TGF-β is PPARβ/δ trget gene in VSMCs [7], nd it is possile tht the PPARβ/δ-TGF-β pthwy ould in ft e tive in humn pnreti ner ells. Our results suggest, however, tht BCL-6 nd PPARβ/δ ply ritil roles in suppressing pro-migrtory gene expression t the mrna level nd in ultimtely ontrolling humn pnreti ner ell invsion. Our oservtions demonstrte tht tivtion of PPARβ/δ y speifi gonist redues the TNFα-indued mrna levels of genes known to e involved in the regultion of humn pnreti ner ell invsion nd metstsis, nd this negtive regultion is lso mnifested t the protein level. Furthermore, we show tht PPARβ/δ tivtion redues Mip-2 nd BxP-3 invsion through sement memrne, nd tht the trnsriptionl repressor BCL-6 plys ritil role in the pthwy(s) regulting humn pnreti ner ell invsion. 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12 MEDIATORS of INFLAMMATION The Sientifi World Journl Gstroenterology Reserh nd Prtie Journl of Dietes Reserh Interntionl Journl of Journl of Endorinology Immunology Reserh Disese Mrkers Sumit your mnusripts t BioMed Reserh Interntionl PPAR Reserh Journl of Oesity Journl of Ophthlmology Evidene-Bsed Complementry nd Alterntive Mediine Stem Cells Interntionl Journl of Onology Prkinson s Disese Computtionl nd Mthemtil Methods in Mediine AIDS Behviourl Neurology Reserh nd Tretment Oxidtive Mediine nd Cellulr Longevity