Molecular Pharmacology Fast Forward. Published on June 1, 2010 as DOI: /mol

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1 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol Moleculr Phrmcology This rticle hs not Fst been Forwrd. copyedited nd Published formtted. The on finl June version 1, 2010 my differ s doi: /mol from this version. DK> eddde Cellulr nd Phrmcologicl Selectivity of the PPARβ/δ Antgonist GSK3787 Prjkt S. Plkr, Michel G. Borlnd, Simone Nruhn, Christin H. Ferry, Christin Lee, Ugir H. Sk, Arun K. Shrm, Shntu Amin, Iin A. Murry, Cherie R. Anderson, Gry H. Perdew, Frnk J. Gonzlez, Rolf Müller, nd Jeffrey M. Peters Deprtment of Veterinry nd Biomedicl Sciences nd The Center for Moleculr Toxicology nd Crcinogenesis, The Pennsylvni Stte University, University Prk, PA (P.S.P., M.G.B., C.H.F., C.L., I.A.M., C.R.A., G.H.P., J.M.P.); Institute of Moleculr Biology nd Tumor Reserch, Philipps University, Mrburg, Germny (S.N., R.M.); Deprtment of Phrmcology, Penn Stte Hershey Cncer Institute, The Pennsylvni Stte University, Milton S. Hershey Medicl Center, Hershey, Pennsylvni (A.K.S., U.H.S., S.A.); Lbortory of Metbolism, Ntionl Cncer Institute, Bethesd, Mrylnd (F.J.G) d Copyright 2010 by the Americn Society for Phrmcology nd Experimentl Therpeutics.

2 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol Running title: Biovilble PPARβ/δ ntgonist Address correspondence to: Dr. Jeffrey M. Peters, Deprtment of Veterinry nd Biomedicl Sciences nd The Center for Moleculr Toxicology nd Crcinogenesis, The Pennsylvni Stte University, University Prk, PA Emil: jmp21@psu.edu Text Pges: 43 Tbles: 1 Figures: Supplementl References: 35 Abstrct : 239 Introduction: 512 Discussion: 1648 ABBREVIATIONS: Adrp, dipocyte differentition-relted protein; ANOVA, nlysis of vrince; Angptl4, ngiopoietin-like protein 4; ChIP, chromtin immunoprecipittion; DMSO, dimethyl sulfoxide; DMEM, Dulbecco s Miniml Essentil Medium; FCS, fetl clf serum; GAPDH, glycerldehyde 3-phosphte dehydrogense; LDH, lctte dehydrogense; PCR, polymerse chin rection; PPAR, peroxisome prolifertorctivted receptor; PBS, phosphte buffered sline. d

3 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol ABSTRACT The vilbility of high ffinity gonists for peroxisome prolifertor-ctivted receptor-β/δ (PPARβ/δ) hs led to significnt dvnces in our understnding of the functionl role of PPARβ/δ. In this study, new PPARβ/δ ntgonist, GSK3787, ws chrcterized using in vivo nd in vitro models. Orlly dministrted GSK3787 cused ntgonism of GW0742-induced up-regultion of Angptl4 nd Adrp mrna expression in wild-type mouse colon, but not in Pprβ/δ-null mouse colon. Chromtin immunoprecipittion (ChIP) nlysis indictes tht this correlted with reduced promoter occupncy of PPARβ/δ on the Angptl4 nd Adrp genes. Reporter ssys demonstrted ntgonism of PPARβ/δ ctivity, nd wek ntgonism nd gonism of PPARγ ctivity, but no effect on PPARα ctivity. Time resolved fluorescence resonnce energy trnsfer ssys confirmed the bility of GSK3787 to modulte ssocition of both PPARβ/δ nd PPARγ co-regultor peptides in response to lignd ctivtion, consistent with reporter ssys. In vivo nd in vitro nlysis indictes tht the efficcy of GSK3787 to modulte PPARγ ctivity is mrkedly lower thn the efficcy of GSK3787 to ct s PPARβ/δ ntgonist. GSK3787 ntgonized GW0742-induced expression of Angptl4 in mouse fibroblsts, mouse kertinocytes, nd humn cncer cell lines. Cell prolifertion ws unchnged in response to either GW0742 or GSK3787 in humn cncer cell lines. Results from these studies demonstrte tht GSK3787 cn ntgonize PPARβ/δ in vivo thus providing new strtegy to delinete the functionl role of receptor with gret potentil s therpeutic trget for the tretment nd prevention of disese. d

4 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol Introduction There is considerble interest in trgeting nucler receptors for the tretment nd prevention of diseses due to their bility to specificlly modulte trnscription of regultory pthwys tht influence the etiology of diseses rnging from metbolic syndrome to cncer. This is due in prt to the successful development nd ppliction of nucler receptor gonists s therpeutic drugs. For exmple, the fibrte clss of hypolipidemic drugs ctivte peroxisome prolifertor-ctivted receptor-α (PPARα) cusing up-regultion of trget genes tht increse ftty cid ctbolism cusing decresed serum lipids nd incresed insulin sensitivity (Stels et l., 1998). Similrly, rosiglitzone (Avndi) nd pioglitzone (Actos) both ctivte PPARγ nd effectively enhnce insulin sensitivity nd decrese serum glucose, which is the bsis for their use in the tretment of type II dibetes (Gross nd Stels, 2007). There is evidence supporting the development of PPARβ/δ gonists for the tretment of metbolic syndrome, dibetes nd obesity, since ctivting PPARβ/δ increses ftty cid ctbolism, meliortes insulin resistnce nd decreses serum glucose (Billin, 2008). However, trgeting PPARβ/δ hs been met with significnt issues relted to clinicl sfety due to controversil reports surrounding the role of PPARβ/δ in cncer, with some suggesting tht ctivting PPARβ/δ potentites tumorigenesis while others suggest tht ctivting PPARβ/δ ttenutes tumorigenesis or hs no effect (Peters nd Gonzlez, 2009; Peters et l., 2008). A number of tools hve been developed in the lst ten yers tht hve significntly dvnced our understnding of the role of PPARβ/δ, in prticulr the genertion of Pprβ/δ-null mouse models (Brk et l., 2002; Ndr et l., 2006; Peters d

5 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol et l., 2000) nd high ffinity lignds tht re more selective for PPARβ/δ (Sherer nd Hoekstr, 2003). Coupling null mouse models with high ffinity lignds is n excellent pproch for delineting the biologicl function of PPARβ/δ, but there re considerble differences in responses in the different models found reported in the literture. Thus, there is distinct need to develop lterntive pproches to begin to ddress mny of the reported disprities. Towrds this gol, the recent identifiction of GSK0660 nd SR13904 s PPARβ/δ ntgonists ws step in the right direction (Sherer et l., 2008; Zveri et l., 2009). Unfortuntely, these ntgonists hve limited ppliction since GSK0660 is not biovilble nd the biovilbility of SR13904 hs not been evluted (Sherer et l., 2008; Zveri et l., 2009). In contrst, the recently described PPARβ/δ ntgonist GSK3787 (Fig. 1) exhibited more suitble phrmcokinetic properties s mximl concentrtion (C mx ) of 2.2 ± 0.4 µm with hlf-life of 2.5 ± 1.1 h ws ttinble in mouse serum fter orl dministrtion (10 mg/kg) (Sherer et l., 2010). Moreover, GSK3787 is n irreversible ntgonist of PPARβ/δ becuse it forms covlent bond with cysteine residue in the lignd binding domin of PPARβ/δ (Sherer et l., 2010). The present study provides further chrcteriztion of this new PPARβ/δ ntgonist by ssessing the bility of GSK3787 to ntgonize PPARβ/δ function in vivo, exmining the specificity of GSK3787 to ntgonize PPARβ/δ using null mouse models, nd by determining the effect of GSK3787 on PPARβ/δ function nd cell growth in pnel of humn cncer cell lines. d

6 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol Mterils nd Methods Mterils. GW0742 (Sznidmn et l., 2003), GSK0660 (Sherer et l., 2008) nd GSK3787 (Sherer et l., 2010) were synthesized by GlxoSmithKline (Reserch Tringle Prk, NC). GW501516, GW1929, GW7647 were purchsed from Sigm- Aldrich (Steinheim, Germny). Rosiglitzone ws purchsed from Biomol Interntionl (Plymouth Meeting, PA). Animls nd tretments. Animl experiments were pproved by the Institutionl Animl Cre nd Use Committee t Pennsylvni Stte University, which conforms to the Guide for the Cre nd Use of Lbortory Animls published by the Ntionl Institutes of Helth. For RNA nd DNA nlysis, mle wild-type nd Pprβ/δ-null mice (Peters et l., 2000) were dministered vehicle (corn oil), GW0742 (10 mg/kg), GSK3787 (10 mg/kg), or GW0742 nd GSK3787 by orl gvge 3 h prior to euthnsi. After euthnsi, colons were crefully dissected. To isolte colon epithelium, colons were flushed with phosphte buffered sline (PBS) nd epithelil cells were scrped from mucos using rzor blde. The isolted tissues were used for RNA isoltion. For glucose tolernce tests, mle wild-type nd Pprβ/δ-null mice were dministered vehicle (corn oil), GW0742 (10 mg/kg), GSK3787 (10 mg/kg) or rosiglitzone (20 mg/kg) by orl gvge 1X/dy for 2 weeks. RNA nlysis. Colon smples were immeditely homogenized in TRIzol Regent (Invitrogen, Crlsbd, CA), nd totl RNA ws prepred ccording to the e

7 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol mnufcturer s recommended protocol. The mrna encoding ngiopoietin-like protein 4 (Angptl4), dipose differentition-relted protein (Adrp), nd glycerldehyde 3- phosphte dehydrogense (Gpdh) ws mesured by quntittive rel-time polymerse chin rection (qpcr) nlysis. cdna ws generted from 2.5 µg of totl RNA using MultiScribe Reverse Trnsciptse kit (Applied Biosystems, Foster City, CA). The reltime primers for Angptl4, Adrp, nd Gpdh hve been previously described (Hollingshed et l., 2008). qpcr rections were crried out using SYBR green PCR mster mix (Qunt BioSciences, Githersburg, MD ) in the icycler nd detected using the MyiQ Rel-Time PCR Detection System (Bio-Rd Lbortories, Hercules, CA). The following rection conditions were used for PCR: 95 C for 15 s, 60 C for 30 s, 72 C for 30 s, repeted for 45 cycles. Ech PCR included no templte rection to control for contmintion, nd ll PCR rections hd greter thn 85% efficiency. The reltive mrna vlue for ech gene ws normlized to the reltive mrna vlue for Gpdh nd nlyzed for sttisticl significnce using two-wy nlysis of vrince with Bonferroni s multiple comprison test (Prism 5.0, GrphPd Softwre Inc., L Joll, CA). Chromtin immunoprecipittion (ChIP). Mle wild-type nd Pprβ/δ-null mice were treted with vehicle, GW0742, GSK3787, or GW0742 nd GSK3787 by orl gvge 3 h prior to euthnsi s described bove nd colon nd liver crefully dissected. Colon epithelium smples from five mice per group were individully snp frozen, pooled, nd then pulverized using mortr nd pestle. Cross-linking ws performed using 1% formldehyde sline solution with smple rottion for 10 min, fter which the cross-linking ws quenched by the ddition of glycine to finl e

8 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol concentrtion of 125 mm nd smples rotted for 10 min. Cells were wshed twice with PBS before the ddition of lysis buffer (50 mm Tris-HCl ph 8, 1% SDS, 10 mm EDTA, nd protese inhibitor cocktil). The lystes from ech tretment group were pooled, nd the DNA ws shered to obtin shered chromtin in the rnge of bp with the Digenode Bioruptor (Digenode, Sprt, NJ). The shered chromtin ws preclered by the ddition of protein A grose (Snt Cruz Biotechnology, Snt Cruz, CA) for 1 h tht ws previously blocked with bovine serum lbumin (BSA)/slmon sperm DNA (Invitrogen, Crlsbd, CA). The preclered chromtin ws immunoprecipitted by gentle gittion with specific ntibodies for either nti-pparβ/δ ntibody (Girroir et l., 2008b), nti-cetylted histone H4 (Upstte Biotechnology, Lke Plcid, NY) s positive control, or rbbit IgG (Snt Cruz Biotechnology, Snt Cruz, CA) s negtive control. After 4 h, the immune complexes were cptured by the ddition of preblocked protein A grose (Snt Cruz Biotechnology, Snt Cruz, CA) nd overnight incubtion. The beds were wshed three times with low slt wsh buffer (20 mm Tris-HCl ph 8, 2 mm EDTA, 0.1% sodium deoxycholte, 1% Triton-X, 150 mm NCl, nd protese inhibitor cocktil) nd once with high slt wsh buffer (20 mm Tris-HCl ph 8, 2 mm EDTA, 0.1% sodium deoxycholte, 1% Triton-X, 500 mm NCl, nd protese inhibitor cocktil). The beds were wshed once with TE8 (10 mm Tris- HCl ph 8, 1 mm EDTA) nd the immune complexes were relesed by the ddition of elution buffer (100 mm NHCO 3, 1% SDS). The formldehyde cross-links were reversed by overnight incubtion t 65 C. Immunoprecipitted DNA ws purified by phenol/chloroform/isomyllcohol (25:24:1) extrction nd subject to rel-time qpcr nlysis for occupncy in the Adrp or Angptl4 peroxisome prolifertor response e

9 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol elements (PPRE). The Adrp PPRE (Chwl et l., 2003) nd primer set spnning this region hve been previously described (Hollingshed et l., 2008). The primer set for Angptl4 ws designed bsed on previous identifiction of PPREs in intron 3 of the mouse Angtpl4 gene (Heinniemi et l., 2007). The primers for Angptl4 were 5 - CTAGCCAAGTAGAGGAAAGTTCAGAGC-3 (forwrd) nd 5 - CCAATCCCTCGGGCAGCTAGC-3 (reverse). qpcr rections were crried out s described bove. The specific vlues were normlized to tretment inputs nd verified to be greter thn rbbit IgG controls. Promoter occupncy ws determined bsed on fold ccumultion to normlized vehicle vlues. Reporter ssys. The LexA-mPPARβ/δ, LexA-mPPARα, LexA-mPPARγ, 7L- TATA inititor module (TATAi) nd PPRE-TATAi plsmids hve been described previously (Futi et l., 2005; Jerome nd Muller, 1998; Nruhn et l., 2010). Trnsfections were performed with polyethylenimine (verge MW 25,000; Sigm- Aldrich). NIH-3T3 cells were trnsfected on 6 well pltes t 70-80% confluence in Dulbecco s modified Egle medium (DMEM) plus 2% fetl clf serum (FCS) with 5 µg of plsmid DNA nd 5 µl of polyethylenimine (1:1000 dilution, djusted to ph 7.0 nd preincubted for 15 min in 200 µl phosphte- buffered sline for complex formtion). Four h fter trnsfection, the medium ws chnged nd cells were incubted in norml growth medium for 24 h with nd without the presence of the PPARα lignd GW7647 (0.3 µm), the PPARβ/δ lignd GW (0.3 µm) the PPARγ lignd GW1929 (0.3 µm) nd/or GSK3787 (1.0 µm). Luciferse ssys were performed s described (Gehrke et l., 2003). Vlues from three independent experiments were combined to clculte verges nd stndrd devitions. e

10 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol Time resolved fluorescence resonnce energy trnsfer (TR-FRET) ssys in vitro. The interction of coregultor peptides with PPARs in vitro ws determined by TR-FRET (Stfslien et l., 2007) using the Lnthscreen TR-FRET PPARα, PPARβ/δ nd PPARγ coregultor ssys ccording to the mnufcturer s (Invitrogen, Crlsbd, CA) instructions with the following peptides: co-ctivtor peptide C33, HVEMHPLLMGLLMESQWGA; co-ctivtor peptide thyroid hormone receptorssocited protein 220/vitmin D receptor intercting protein-1 (TRAP220/DRIP-1), KVSQNPILTSLLQITGNGG; co-repressor silencing meditor for retinoid nd thyroid hormone receptors interction domin 2 (SMRT-ID2), HASTNMGLEAIIRKALMGKYDQW; nd nucler receptor co-repressor interction domin 2 (NCoR-ID2), DPASNLGLEDIIRKALMGSFDDK. Incubtion times were min for ll ssys shown in this study. The ssy buffer contined 100 mm KCl, 20 mm Tris ph 7.9, 0.01% Triton X100 nd 1 µg/µl bovine serum lbumin. All ssys were vlidted for their robustness by determining the respective Z -fctors (Zhng et l., 1999). Mesurements were performed on VICTOR3V Multilbel Counter (WALLAC 1420; PerkinElmer, Wlthm, MA) with instrument settings s described in the mnufcturer s instructions for LnthScreen ssys. Cell culture. The humn heptocrcinom cell lines HepG2 nd Huh7, lung denocrcinom cell lines A549 nd H1838, squmous crcinom cell line A431, nd the brest cncer cell line MCF7 were obtined from Americn Type Culture Collection (Mnsss, VA). Cells were cultured ccording to the recommended procedures: HepG2, Huh7, A431, nd MCF7 cells were cultured in DMEM; A549 were cultured in Hm s F12K medium, nd H1838 cells were cultured in RPMI-1640 medium dd

11 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol supplemented with 10% fetl bovine serum (FBS) nd 1% penicillin-streptomycin. For mrna nlysis, cells were plted in 12-well tissue culture pltes, nd cultured until 80% confluency t which time they were treted with either DMSO, GW0742, GSK3787 or GW0742 nd GSK3787 for 24 h. The concentrtions of GW0742 ( µm) were used becuse they hve been previously shown to specificlly ctivte PPARβ/δ (Sherer nd Hoekstr, 2003). The concentrtions of GSK3787 ( µm) were used becuse they hve been previously shown to ntgonize PPARβ/δ in other cell bsed models nd re likely in the rnge of concentrtions tht could be chieved in vivo (Sherer et l., 2010). After this tretment, mrna ws isolted nd used for qpcr s described bove. Importntly, ll of the cell lines hve been shown to respond to lignd ctivtion of PPARβ/δ nd express PPARβ/δ mrna (Girroir et l., 2008; He et l., 2008; Hollingshed et l., 2007); nd dt not shown). However, reltive expression of PPARβ/δ hs been noted to be lower in these cncer cell lines s compred to norml cells/tissue (dt not shown). Isoltion of mouse kertinocytes nd fibroblsts. Kertinocytes nd fibroblsts were isolted from newborn mouse skin nd cultured s previously described (Dlugosz et l., 1995). Cell prolifertion ssys. Cell prolifertion ws exmined using rel-time monitoring of cell prolifertion of dherent cells using the xcelligence System (Roche Applied Science, Indinpolis, IN). Briefly, the optiml number of cells required to obtin exponentil growth in single well of n E-Plte 16 ws determined by monitoring cell prolifertion in rel time using incresing number of cells/well (Supplementl Fig. 1). The number of cells seeded per well to obtin exponentil growth curves nd the length dd

12 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol of time exmined for ech cell line is shown in Supplementl Fig. 1. Cell prolifertion ws monitored every 15 min using the RTCES system for up to 120 h. Cellsensor impednce is expressed s n rbitrry unit clled the Cell Index. The Cell Index t ech time point is defined s (Rn-Rb)/15, where Rn is the cell-electrode impednce of the well when it contins cells nd Rb is the bckground impednce of the well with the medi lone. Strt nd end times were selected during the log growth phse (Supplementl Fig. 1) nd used to clculte doubling time with RTCA Softwre v 1.2 (ACEA Biosciences, Inc, Sn Diego, CA) from independent triplicte wells per tretment. For exmintion of the effect of GW0742 nd/or GSK3787 on cell prolifertion, the humn squmous cell crcinom cell line A431, humn liver cncer cell lines HepG2, Huh7, humn lung cncer cell lines A549, H1838 or the humn brest cncer cell line MCF7 were seeded s described (Supplementl Fig. 1) nd treted with GW0742 (0.1, 1.0 or 10 µm) or GSK3787 (0.1, 1.0 or 10 µm). Quntittive Western Blotting. Protein smples were prepred from fibroblsts nd kertinocytes using lysis buffer contining protese inhibitors. Seventy-five microgrm of protein per smple ws resolved using sodium dodecyl sulfte 10% polycrylmide gels. Proteins were trnsferred onto polyvinylidene fluoride membrne. The membrne ws blocked with 5% non-ft milk or 0.5% geltin in Tris buffered sline Tween (TBST) nd incubted overnight with primry ntibodies ginst PPARβ/δ (Girroir et l., 2008b) or lctte dehydrogense (LDH). Membrnes were wshed nd incubted with biotinylted secondry ntibody (Jckson ImmunoReserch Lbortories, West Grove, PA) followed by incubtion with 125 I-lbeled streptvidin. Membrnes were exposed to phosphorimger pltes nd the level of rdioctivity dd

13 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol quntified with Pckrd phosphorimger. Hybridiztion signls for PPARβ/δ were normlized to the hybridiztion signls for the loding control LDH. Three independent smples were nlyzed for ech group. 3T3-L1 predipocyte cell culture nd differentition. Mouse 3T3-L1 predipocytes were cultured in DMEM with 10% FCS nd 1% penicillin/streptomycin. Cells were cultured to confluence nd then treted with differentition medium. The differentition medium ws DMEM with 10% FBS, 10 µg/ml insulin, 200 µm 3-isobutyl- 1-methylxnthine (IBMX) nd 250 µm dexmethsone. One dy fter tretment with the differentition medium, cells were treted with µm rosiglitzone, µm GSK3787, or both rosiglitzone nd GSK3787. For nlysis of PPARγ-dependent gene expression, RNA ws isolted s described bove from cells 24 h fter tretment for nlysis of the PPARγ trget gene p2 by qpcr s previously described by others (Rockwell et l., 2006). For nlysis of dipocyte differentition, cells were cultured for four dys in mintennce medium contining DMEM with 10% FBS, 1% penicillin/streptomycin nd insulin (10 µg/ml) beginning 24 h fter tretment with rosiglitzone, GSK3787 or both. Cells were fixed in 10% formlin nd stined with Oil Red O (0.2% in 60% isopropnol) for 15 min. After wshing with 60% isopropnol, Oil Red O stin ws extrcted with 4% NP-40 in isopropnol. The intensity of stining ws determined by mesuring bsorbnce t 570 nm. Glucose tolernce test. Mle wild-type nd Pprβ/δ-null mice were dministered GW0742, GSK3787 or rosiglitzone for 2 weeks s described bove. After six h fst, mice were injected with glucose (1.5 mg/g body weight) by intrperitonel injection. After this injection, blood ws collected from the mndibulr vein every 30 min dd

14 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol for 2 h nd used for nlysis of blood glucose using Accu-Chek Active Glucometer (Roche Dignostics, Indinpolis, IN). dd

15 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol Results GSK3787 ntgonizes lignd-induced PPARβ/δ-dependent gene expression in vivo. Preliminry chrcteriztion of GSK3787 indicted tht this ntgonist inhibits both bsl nd lignd-induced expression of pyruvte dehydrogense kinse 4 (PDK4) nd crnitine plmitoyl trnsferse 1 (CPT1) in humn skeletl muscle cells t concentrtions up to 1 µm (Sherer et l., 2010). Additionlly, this study lso demonstrted tht orl dministrtion of GSK3787 (10 mg/kg) led to serum C mx of 2.2 ± 0.4 µm in C57BL/6 mle mice (Sherer et l., 2010). To more definitively exmine the effect of GSK3787 in vivo, qpcr nlysis of PPARβ/δ trget genes nd ChIP ssys were performed using tissue collected from wild-type nd Pprβ/δ-null mice. Orl dministrtion of GW0742 cused n increse in expression of Angptl4 nd Adrp mrna (known PPARβ/δ trget genes) in wild-type mouse colon epithelium, nd this effect ws not found in Pprβ/δ-null mouse colon epithelium (Fig. 2A). Orl dministrtion of GSK3787 hd no effect on expression of Angptl4 nd Adrp mrna in mouse colon epithelium in either genotype (Fig. 2A). Co-dministrtion of GSK3787 with GW0742 effectively prevented the lignd-induced expression of both Angptl4 nd Adrp mrna in wild-type mouse colon epithelium nd this effect ws not found in Pprβ/δ-null mouse colon epithelium (Fig. 2A). GSK3787 did not modulte PPARβ/δdependent gene expression or ntgonize lignd-induced PPARβ/δ-dependent gene expression in liver (dt not shown). Since the ntgonism of PPARβ/δ by GSK3787 ws more evident in colon epithelium, consistent with high expression of PPARβ/δ in this tissue (Girroir et l., 2008b), ChIP ssys were performed using colon epithelil dd

16 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol DNA obtined from mice treted with either GW0742, GSK3787 or both compounds. Lignd ctivtion of PPARβ/δ with GW0742 cused n increse in cetylted histone H4 (AcH4) ssocited with the PPRE region of both the Angptl4 nd Adrp genes in wildtype mouse colon epithelium nd this effect ws not found in similrly treted Pprβ/δnull mice (Fig. 2B), consistent with pst results (Hollingshed et l., 2008). Acetyltion of histone H4 is importnt for chromtin remodeling nd recruitment of the trnscription initition complex. While orl dministrtion of GSK3787 hd essentilly no effect on promoter occupncy of AcH4 in the PPRE region of both the Angptl4 nd Adrp genes, co-dministrtion of GSK3787 with GW0742 resulted in mrkedly less ccumultion of AcH4 in the PPRE region of both the Angptl4 nd Adrp genes in wild-type mouse colon epithelium (Fig. 2B). Lignd ctivtion of PPARβ/δ with GW0742 cused n increse in promoter occupncy of PPARβ/δ in the PPRE region of both the Angptl4 nd Adrp genes in wild-type mouse colon epithelium nd this effect ws not found in similrly treted Pprβ/δ-null mice (Fig. 2B). Orl dministrtion of GSK3787 cused modest increse in promoter occupncy of PPARβ/δ in the PPRE region of both the Angptl4 nd Adrp genes, but co-dministrtion of GSK3787 with GW0742 resulted in mrkedly less ccumultion of PPARβ/δ in the PPRE region of both the Angptl4 nd Adrp genes in wild-type mouse colon epithelium (Fig. 2B). These chnges observed with codministrtion were not found in similrly treted Pprβ/δ-null mouse colon epithelium. While promoter occupncy of AcH4 on the Angptl4 gene ws modestly lower in Pprβ/δ-null mouse colon epithelium following co-tretment with GW0742 nd GSK3787, there ws no chnge in promoter occupncy of AcH4 on the Adrp gene in Pprβ/δ-null mouse colon epithelium following co-tretment with GW0742 nd de

17 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol GSK3787 (Fig. 2B). It remins possible tht the former chnge could reflect n offtrget effect of GSK3787 or limittion due to the use of technicl replictes for the ChIP ssy, which precluded ssessing mesures of vribility. Collectively, these results demonstrte tht GSK3787 cn effectively ntgonize lignd-induced effects on PPARβ/δ trget genes in vivo, nd tht these effects re due to receptor-dependent mechnisms since they re not found in Pprβ/δ-null mice. GSK3787 ntgonizes lignd-induced PPARβ/δ-dependent gene expression in vitro. While preliminry chrcteriztion of GSK3787 indictes tht this compound cn ntgonize both bsl nd lignd-induced expression of PDK4 nd CPT1 in humn skeletl muscle cells t concentrtions up to 1 µm (Sherer et l., 2010), the specificity of this effect on gene expression ws not exmined. For this reson, the effect of GSK3787 in cells expressing reltively low level of PPARβ/δ (fibroblsts) nd reltively high level of PPARβ/δ (kertinocytes) ws exmined using cells isolted from wild-type nd Pprβ/δ-null mice. Expression of PPARβ/δ protein is ~7-fold lower in fibroblsts s compred to kertinocytes (Fig. 3A). Kertinocytes re known to express PPARβ/δ t high level s compred to most other mouse tissues/cells (Girroir et l., 2008b). Despite the reltively low level of expression of PPARβ/δ observed in fibroblsts, tretment with 10 or 50 nm GW0742 cused up to ~10-fold increse of Angptl4 mrna compred to control; this effect ws not found in fibroblsts from Pprβ/δ-null mice (Fig. 3B). The increse in Angptl4 mrna observed in response to 10 nm GW0742 ws not found in wild-type fibroblsts tht were cultured with both 10 nm GW0742 nd 0.1 or 1.0 µm GSK3787 (Fig. 3B). The increse in Angptl4 mrna observed in response to 50 nm GW0742 ws mrkedly lower in wild-type fibroblsts de

18 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol tht were cultured with 50 nm GW0742 nd 0.1 µm GSK3787 nd essentilly bsent in wild-type fibroblsts tht were cultured with 50 nm GW0742 nd 1.0 µm GSK3787 (Fig. 3B). None of these effects were found in similrly treted fibroblsts from Pprβ/δ-null mice (Fig. 3B). Consistent with the difference in expression of PPARβ/δ protein (Fig. 3A), the chnge in expression of Angptl4 mrna in response to GW0742 ws greter in kertinocytes s compred to fibroblsts s 50 nm GW0742 cused greter thn 30- fold increse in Angptl4 mrna compred to control (Fig. 3C). The increse in Angptl4 mrna observed in response to 50 nm GW0742 ws mrkedly lower in wild-type kertinocytes tht were cultured with 50 nm GW0742 nd 0.1 µm GSK3787 nd not found in wild-type kertinocytes tht were cultured with 50 nm GW0742 nd 1.0 µm GSK3787 (Fig. 3C). None of these chnges were found in similrly treted kertinocytes from Pprβ/δ-null mice (Fig. 3C). Combined, these results estblish tht GSK3787 cn effectively ntgonize lignd-induced gene expression medited by PPARβ/δ in cultured fibroblsts nd kertinocytes using concentrtions rnging from µm in the presence of n gonist with ffinity for PPARβ/δ in the nm rnge. GSK3787 ntgonizes lignd-induced PPARβ/δ-dependent gene expression in humn cncer cell lines. There is considerble interest in the effect of PPARβ/δ in humn cncer (reviewed in Burdick et l., 2006; Peters nd Gonzlez, 2009; Peters et l., 2008). Thus, the reltive bility of GSK3787 to ntgonize PPARβ/δ ws exmined in pnel of humn cncer cell lines including those for skin (A431), liver (HepG2, Huh7), brest (MCF7) nd lung cncer (H1838, A549). Expression of ANGPTL4 nd ADRP mrnas in A431 cells were incresed by ~20-fold nd ~6-fold, respectively, by 50 nm GW0742 (Fig. 4). Co-tretment with 50 nm GW0742 nd 1.0 µm GSK3787 lrgely de

19 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol diminished these GW0742-induced responses in A431 cells (Fig. 4). The mgnitude of lignd-induced chnges in PPARβ/δ trget genes ws greter in A431 cells s compred to the other humn cncer lines. Tretment with 50 nm GW0742 cused n increse in expression of ANGPTL4 mrna in MCF7, Huh7, HepG2, H1838 nd A549 cells rnging from ~2-fold to 6-fold (Fig. 4). The mgnitude of chnge in GW0742- induced ANGPTL4 mrna expression in these cells ws mrkedly lower s compred to primry mouse kertinocytes, where >30-fold increses were noted (Fig. 3C). Cotretment of 50 nm GW0742 nd 1.0 µm GSK3787 ntgonized the GW0742-induced increse of ANGPTL4 mrna in MCF7, Huh7 nd HepG2 cells but not in H1838 or A549 cells (Fig. 4). ADRP mrna incresed following tretment with 50 nm GW0742 in Huh7, HepG2 nd H1838 cells but not in MCF7 or A549 cells (Fig. 4). Co-tretment of 50 nm GW0742 nd 1.0 µm GSK3787 ntgonized the GW0742-induced increse of ADRP mrna in Huh7 nd HepG2 cells but not in H1838 cells (Fig. 4). These dt show tht GSK3787 cn ntgonize lignd-induced chnges in gene expression in most, but not ll, humn cncer cell lines exmined in this study. This is in contrst to effective ntgonism of lignd-induced chnges in gene expression observed in mouse primry fibroblsts nd kertinocytes using the sme concentrtions of GW0742 nd GSK3787 (Fig. 3B, 3C). No decrese in bsl expression of either ANGPTL4 or ADRP mrna ws observed following tretment with GSK3787 suggesting tht GSK3787 does not ntgonize bsl expression of either of these two PPARβ/δ trget gene in A431, MCF7, Huh7, HepG2, H1838 or A549 humn cncer cell lines. Effect of lignd ctivtion of PPARβ/δ by GW0742 nd ntgonism of PPARβ/δ by GSK3787 on cell prolifertion. There is considerble controversy de

20 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol regrding the effects of PPARβ/δ on the prolifertion of cultured humn cncer cells s there is evidence tht PPARβ/δ either increses, decreses or hs no effect on cell growth (reviewed in Burdick et l., 2006; Peters nd Gonzlez, 2009; Peters et l., 2008). Thus, the effect of GW0742 nd/or GSK3787 on cell prolifertion ws exmined in the humn cncer cell lines A431, HepG2, Huh7, MCF7, H1838, nd A549. Neither GW0742 nor GSK3787 hd ny effect on cell prolifertion in MCF7, Huh7, HepG2, A431, A549 or H1838 humn cncer cell lines t concentrtions rnging from µm (Tble 1). GSK3787 selectively ntgonizes lignd-induced PPARβ/δ trnscription but lso modultes PPARγ ctivities. Reporter ssys were performed to determine if GSK3787 could ctivte the two other members of the PPAR fmily, PPARα nd PPARγ. GSK3787 did not modulte PPARα-dependent trns-ctivtion nd hd no effect on lignd-induced trns-ctivtion of PPARα by GW7647 (Fig. 5). In contrst, GW induced PPARβ/δ-dependent trns-ctivtion ws effectively ntgonized by GSK3787 (Fig. 5). Interestingly, GSK3787 ws ble to modestly increse PPARγdependent reporter ctivity s compred to the PPARγ gonist GW1929 (Fig. 5). Additionlly, GSK3787 lso ntgonized GW1929-induced PPARγ trns-ctivtion (Fig. 5). These dt reveled tht GSK3787 hd no influence on PPARα ctivity, is effective s PPARβ/δ ntgonist, nd wek PPARγ gonistic nd ntgonistic ctivities. To more closely exmine the bility of GSK3787 to ntgonize PPAR ctivity, the interction between PPARβ/δ, PPARγ nd co-ctivtor or co-repressor peptides ws determined using TR-FRET. In this ssy, the interction between the PPARβ/δ or PPARγ lignd binding domin (LBD; indirectly lbeled with terbium) with either, the co- dd

21 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol ctivtor peptides C33 or TRAP220/DRIP-1 (lbeled with fluorescein), or the corepressor peptides SMRT ID2 or NCoR ID2 (lbeled with fluorescein) ws determined. The in vitro TR-FRET ssy mesures the intensity of terbium-induced fluorescence emission of the fluorescein moiety of the lbeled peptides, expressed s the rtio of fluorescein- nd terbium-derived fluorescence. In the bsence of lignd (GW501516), GSK3787 decresed co-repressor peptide SMRT ID2 dissocition t higher concentrtions (0.75 nd 1.0 µm) nd dose-dependently decresed recruitment of coctivtor peptide C33 to the PPARβ/δ LBD (Fig. 6A, 6C). In the bsence of lignd, GSK0660 did not influence co-repressor peptide SMRT ID2 dissocition but did decrese recruitment of co-ctivtor peptide C33 to the PPARβ/δ LBD (Fig. 6A, 6C). Previous studies estblished tht mximl SMRT ID2 dissocition from, nd mximl C33 recruitment with, the LBD of PPARβ/δ occurs by 30 min post-lignd tretment (dt not shown). A dose dependent prevention of co-repressor peptide SMRT ID2 dissocition (Fig. 6B) nd inhibition of co-ctivtor peptide C33 recruitment (Fig. 6D) ws observed following co-tretment of 0.15 µm GW with GSK3787. Similr chnges in PPARβ/δ LBD/co-ctivtor/co-repressor interctions were not observed with co-tretment of GW with GSK0660, nother PPARβ/δ ntgonist (Fig. 6B, 6D). Since the reporter ssys (Fig. 5) indicted tht GSK3787 lso modultes PPARγ ctivity, TR-FRET ssys were performed to compre co-regultor peptide recruitment/dissocition between PPARγ nd PPARβ/δ. In the bsence of lignd (GW501516), GSK3787 cused dissocition of co-repressor peptides SMRT ID2 nd NCoR ID2 from the PPARβ/δ LBD but hd no effect on recruitment of the PPARγ coctivtor TRAP220/DRIP-1 to the PPARβ/δ LBD (Fig. 7A). GSK0660 hd no effect on dd

22 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol either dissocition of co-repressor peptides SMRT ID2 or NCoR ID2 from the PPARβ/δ LBD, or recruitment of the PPARγ co-ctivtor TRAP220/DRIP-1 to the PPARβ/δ LBD (Fig. 7A). In the bsence of lignd (GW1929), GSK3787 nd GSK0660 cused some dissocition of co-repressor peptides SMRT ID2 but not NCoR ID2 from the PPARγ LBD (Fig. 7B). In the bsence of lignd, GSK3787 enhnced recruitment of the PPARγ co-ctivtor TRAP220/DRIP-1 to the PPARγ LBD, while GSK0660 hd no effect on recruitment of TRAP220/DRIP-1 to the PPARγ LBD (Fig. 7B). GW induced recruitment of TRAP220/DRIP-1 to the PPARβ/δ LBD nd dissocition of SMRT ID2 from the PPARβ/δ LBD were both inhibited by GSK3787, nd GSK0660 did not influence either of these effects (Fig. 7C). Interestingly, GW1929-induced recruitment of TRAP220/DRIP-1 to the PPARγ LBD nd dissocition of SMRT ID2 from the PPARγ LBD were both inhibited by GSK3787 nd GSK0660 did not chnge either of these effects (Fig. 7D). Combined, these dt suggest tht while GSK3787 cn ntgonize PPARβ/δ, this compound cn lso cuse moleculr interctions between co-ctivtors nd co-repressor peptides with the LBD of PPARγ, similr to effects found with PPARγ gonists nd ntgonists, consistent with the reporter ssys (Fig. 5). To begin to determine the reltive efficcy of GSK3787 to modulte PPARγ ctivity, the effect of GSK3787 to regulte PPARγ-dependent gene expression ws exmined in n dipocyte cell-bsed model. 3T3-L1 predipocytes were cultured with dipocyte differentition medium to enhnce PPARγ ctivity nd then treted with either, PPARγ gonist (rosiglitzone), GSK3787 or rosiglitzone nd GSK3787. Expression of the PPARγ trget gene p2 mrna ws mrkedly incresed by tretment with 1 or 10 µm rosiglitzone (Fig. 8). GSK3787 (1 or 10 µm) hd no effect on dd

23 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol expression of p2 mrna in 3T3-L1 cells nd did not ntgonize rosiglitzone-induced expression of p2 mrna (Fig. 8). Similrly, rosiglitzone cused n increse in lipid ccumultion ssocited with dipocyte differentition s shown by Oil Red O stining, wheres GSK3787 did not significntly influence Oil Red O stining or modulte the increse in dipocyte differentition cused by rosiglitzone (Fig. 8C, Supplementl Fig. 2). These dt suggest tht the efficcy of GSK3787 to ctivte PPARγ is mrkedly lower s compred to rosiglitzone. These dt lso suggest tht the efficcy of GSK3787 to ntgonize PPARγ-dependent gene expression is mrkedly less s compred to its bility to ntgonize PPARβ/δ-dependent gene expression. Effect of GSK3787 on glucose tolernce. Lignd ctivtion of PPARβ/δ or PPARγ cn both improve insulin sensitivity nd glucose tolernce (reviewed in Quinn et l., 2008; Reilly nd Lee, 2008). Since GSK3787 my modulte both PPARβ/δ nd/or PPARγ ctivities, the effect of GSK3787 on glucose tolernce ws exmined. Higher blood glucose, consistent with glucose intolernce, ws observed in Pprβ/δ-null mice s compred to wild-type mice, similr with pst results (Lee et l., 2006). Tretment with GW0742 for 2 weeks mrkedly improved glucose tolernce in wild-type mice nd this effect ws not found in GW0742-treted Pprβ/δ-null mice (Fig. 9). In contrst, tretment with rosiglitzone for 2 weeks improved glucose tolernce in both wild-type nd Pprβ/δ-null mice (Fig. 9). Administrtion of GSK3787 hd no effect on glucose tolernce in either genotype (Fig. 9). dd

24 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol Discussion These studies re the first to demonstrte tht GSK3787 cn effectively ntgonize lignd-induced effects on PPARβ/δ trget genes in vivo through receptordependent mechnisms since they re not found in Pprβ/δ-null mice. This conclusion is supported by qpcr nlysis demonstrting PPARβ/δ-dependent ntgonism of lignd-induced chnges in gene expression in colon epithelium, s well s ChIP ssys demonstrting PPARβ/δ-dependent ntgonism of lignd-induced promoter occupncy of PPARβ/δ on trget genes in colon epithelium. Further confirmtion of receptorspecificity for t lest some GSK3787 ctivity ws provided by results showing tht GSK3787 cn ntgonize GW0742-induced chnges in gene expression in wild-type mouse fibroblsts nd kertinocytes, but not in Pprβ/δ-null cells. It is noteworthy tht GSK3787 cused no overt signs of toxicity s ssessed by reltive cell prolifertion. The specificity of GSK3787 for PPARβ/δ could be due in prt to the fct tht it is n irreversible ntgonist tht forms covlent bond within the lignd binding domin of PPARβ/δ (Sherer et l., 2010). GSK3787 lso ntgonized lignd-induced chnges in gene expression in most, but not ll humn cncer cell lines exmined in this study. This is in contrst to PPARβ/δ-dependent ntgonism by GSK3787 of lignd-induced chnges in gene expression observed in mouse fibroblsts nd kertinocytes using the sme concentrtions of GW0742 nd GSK3787. Determining the mechnisms for the resistnce of the humn lung cncer cell lines H1838 nd A549 to PPARβ/δ ntgonism by GSK3787 requires further study. dd

25 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol It is worth noting tht potency nd efficcy of lignd ctivtion of PPARβ/δ on trget gene expression in fibroblsts nd kertinocytes were mrkedly greter s compred to MCF7, Huh7, HepG2, H1838 nd A549 cells. This suggests tht the bility of lignd to ctivte PPARβ/δ is reltively less in these humn cncer cell lines s compred to mouse fibroblsts nd kertinocytes. While the mechnism for this difference is not known, the lignd my be more rpidly degrded in the ltter cell lines s compred to fibroblsts nd kertinocytes. Further, since GSK3787 ntgonized lignd-induced gene expression in mouse fibroblsts nd kertinocytes tht express reltively low nd reltively high levels of PPARβ/δ protein, respectively, this demonstrtes tht GSK3787 is effective for in vitro models t concentrtions rnging from µm in cells with vrying levels of receptor expression. This is importnt to point out becuse these concentrtions re comprble to concentrtions chievble in vivo since the C mx in serum observed in mice dministered GSK3787 (10 mg/kg) is 2.2 ± 0.4 µm with hlf life of 2.5 ± 1.1 h (Sherer et l., 2010). In model systems tht lck the presence of high ffinity lignds, but re dependent on endogenous lower ffinity lignds (e.g. ftty cids, ftty cid derivtives), concentrtions of GSK3787 between µm should be cpble of ntgonizing constitutive PPARβ/δ function. Thus, it is surprising tht GSK3787 hs no effect on bsl expression of PPARβ/δ trget genes exmined in these studies in fibroblsts, kertinocytes or humn cncer cell lines. This is in contrst to reduced bsl expression of CPT1 nd PDK4 in humn skeletl muscle cells observed following tretment with GSK3787 (Sherer et l., 2010). This could be due to differences in regultory elements in the promoters between CPT1 or dd

26 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol PDK4 nd ANGPTL4 or ADRP. Future in vivo nd in vitro studies should consider this possibility. Despite demonstrting tht GSK3787 specificlly ntgonizes lignd-induced ctivity of PPARβ/δ bsed on nlysis of cells nd mice lcking expression of PPARβ/δ, evidence ws lso obtined demonstrting tht GSK3787 hs wek PPARγ gonist nd ntgonist ctivities. However, comprison of PPARγ function in 3T3-L1 cells demonstrted negligible PPARγ gonistic or ntgonistic ctivities by GSK3787 s shown by both the lck of chnge in expression of known PPARγ trget gene nd nlysis of dipocyte differentition. Since the concentrtions required to specificlly ntgonize PPARβ/δ re less thn or equl to 1 µm, nd this concentrtion of GSK3787 did not cuse significnt gonism or ntgonism of PPARγ ctivity in 3T3-L1 dipocytes, this suggests tht GSK3787 cn be used to study the effects of PPARβ/δ ntgonism in vitro without considering confounding chnges due to PPARγ-dependent ctivity; s suggested by the FRET nlysis nd reporter ssys tht my exhibit incresed sensitivity. In vivo exmintion of glucose tolernce lso demonstrted negligible PPARγ ctivity of GSK3787 s compred to known PPARγ gonist using dose of GSK3787 tht effectively ntgonized lignd-induced PPARβ/δ trnscriptionl ctivity in colonic epithelium. However, it is importnt to emphsize tht experiments should control for this potentil PPARγ gonism/ntgonism by GSK3787, s it remins possible tht other PPARγ-dependent ctivities could influence interprettion. Further, whether GSK3787 cn be used s n ntgonist to study lignd-induced improvement of insulin sensitivity medited by PPARβ/δ requires further exmintion. de

27 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol Results from these studies lso illustrte differences in functionl roles of PPARβ/δ in cell prolifertion. There is considerble controversy regrding the role of PPARβ/δ in cell prolifertion s some studies suggest tht ctivting this receptor increses cell prolifertion while others indicte tht ctivting PPARβ/δ hs either no effect, or inhibits cell prolifertion in ssocition with the induction of terminl differentition (reviewed in Peters nd Gonzlez, 2009; Peters et l., 2008). This is lrgely due to the lck of stringency in pproches used to exmine cell prolifertion, in prticulr the limited published time course nd concentrtion-dependent studies. Lignd ctivtion of PPARβ/δ with GW0742 hd no effect on cell prolifertion in A431, MCF7, Huh7, HepG2, H1838 or A549 humn cncer cell lines, results tht re consistent with some, but not ll studies, showing tht GW0742 or GW hve little influence on cell prolifertion t concentrtions less thn 10 µm (reviewed in Peters nd Gonzlez, 2009; Peters et l., 2008). The present studies exmined cell prolifertion by determining doubling time using rel-time nlysis, nd mny published studies typiclly limit nlysis of cell prolifertion to single timepoint (reviewed in Peters nd Gonzlez, 2009; Peters et l., 2008). Thus, it remins possible tht the pproch used to detect cell growth could impct these findings. Importntly, rel-time nlysis of cell prolifertion provides n outstnding pproch yielding ccurte ssessment of doubling time during the liner growth phse over brod concentrtion rnge of compound. Since lignd ctivtion of PPARβ/δ hd no influence on cell prolifertion, it is not surprising tht GSK3787 hd no effect on cell prolifertion in A431, MCF7, Huh7, HepG2, H1838 or A549 humn cncer cell lines, despite the fct tht GSK3787 ntgonized lignd-induced chnges in PPARβ/δ-dependent gene de

28 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol expression in these cells. This is consistent with recent study where GSK3787 hd no effect on cell prolifertion in SW480, HCT116, DLD1, RKO, A549 or HEK293 cncer cell lines with concentrtions of GSK3787 up to 10 µm (Sherer et l., 2010). This could be due to the fct tht expression of PPARβ/δ is low in tumors nd cncer cell lines s compred to norml cells. For exmple, expression of PPARβ/δ in the C20 mmmry glnd cncer cell line is less thn 25% of tht found in kertinocytes (Foremn et l., 2010). Further, while it hs been suggested tht PPARβ/δ expression is up-regulted by the APC/β-CATENIN signling pthwy tht is often enhnced in cncer, recent findings indicte tht this ide is incorrect (Foremn et l., 2009) nd mny studies show tht PPARβ/δ expression is either lower or unchnged in tumors s compred to control tissue (Berglund et l., 2008; Uhlen et l., 2005; reviewed in Peters nd Gonzlez, 2009; Peters et l., 2008). Thus, while ctivtion of PPARβ/δ with high ffinity lignd modultes chnges in gene expression in cncer cell lines, the effect of either n gonist or n ntgonist on cell prolifertion cn be negligible in humn cncer cell lines. Results from these studies re in contrst to recent report suggesting tht ntgonism of PPARβ/δ with 10 µm SR13904 inhibited cell prolifertion of A549 cells (Zveri et l., 2009). However, dt from the present study nd recent work by others (Sherer et l., 2010) show tht ntgonism of PPARβ/δ in the sme humn lung cncer cell line hs no effect on cell prolifertion t concentrtions tht specificlly ntgonize PPARβ/δ. It is importnt to note tht the study by Zveri et l hd limittions tht preclude definitive conclusions regrding the specificity of the response observed with SR13904 since they did not demonstrte n increse in A549 cell growth by lignd de

29 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol ctivtion of PPARβ/δ tht ws prevented by co-tretment with SR This is concern since no effect on cell prolifertion is found in response to lignd ctivtion of PPARβ/δ in A549 cells s shown by the present study nd nother recent report (He et l., 2008). Moreover, Zveri et l did not demonstrte specific ntgonism of ligndinduced PPARβ/δ trget gene(s) or ltered cell prolifertion using knockout or knockdown pproches. This rises the possibility tht the observed inhibition of cell prolifertion in A549 cells by high concentrtion SR13904 is due to off trget effects rther thn ntgonism of PPARβ/δ, in prticulr since SR13904 ws lso shown to ntgonize PPARγ (Zveri et l., 2009). While it hs been suggested tht ntgonism of PPARβ/δ my be useful pproch for chemoprevention (Zuo et l., 2009), this ide is not supported by results from the present study showing no influence on cell prolifertion of humn cncer cell lines nd the fct tht the effect of lignd ctivtion of PPARβ/δ on tumorigenesis is entirely uncler (reviewed in Peters nd Gonzlez, 2009; Peters et l., 2008). Further, since lignd ctivtion of PPARβ/δ improves insulin sensitivity, increses skeletl muscle ftty cid ctbolism, nd hs potent nti-inflmmtory ctivities, therpeutic ntgonism of PPARβ/δ could likely led to negtive effects on these essentil functions. Nevertheless, results from these studies demonstrte tht GSK3787 cn be used to ntgonize PPARβ/δ in vivo nd in vitro, providing new strtegy to delinete the functionl role of receptor with gret potentil s therpeutic trget for the tretment nd prevention of diseses including dyslipidemis, obesity nd cncer. Given the potentil for GSK3787 to interct with PPARγ, receptor specificity must be controlled for in future studies. de

30 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol Acknowledgements. The uthors grtefully cknowledge Andrew Billin nd Timothy Willson for providing GW0742, GSK0660 nd GSK3787. dd

31 Moleculr Phrmcology Fst Forwrd. Published on June 1, 2010 s DOI: /mol References Brk Y, Lio D, He W, Ong ES, Nelson MC, Olefsky JM, Bolnd R nd Evns RM (2002) Effects of peroxisome prolifertor-ctivted receptor δ on plcenttion, diposity, nd colorectl cncer. Proc Ntl Acd Sci U S A 99: Billin AN (2008) PPAR-β/δ gonists for Type 2 dibetes nd dyslipidemi: n dopted orphn still looking for home. Expert opinion on investigtionl drugs 17(10): Burdick AD, Kim DJ, Perz MA, Gonzlez FJ nd Peters JM (2006) The role of peroxisome prolifertor-ctivted receptor-β/δ in epithelil cell growth nd differentition. Cell Signl 18(1):9-20. Chwl A, Lee CH, Brk Y, He W, Rosenfeld J, Lio D, Hn J, Kng H nd Evns RM (2003) PPARδ is very low-density lipoprotein sensor in mcrophges. Proc Ntl Acd Sci U S A 100(3): Dlugosz AA, Glick AB, Tennenbum T, Weinberg WC nd Yusp SH (1995) Isoltion nd utiliztion of epiderml kertinocytes for oncogene reserch. Methods Enzymol 254:3-20. Futi T, Müller-Brüsselbch S, Kreutzer M, Rieck M, Meissner W, Rpp U, Schweer H, Kömhoff M nd Müller R (2005) Induction of PPARβ nd prostcyclin (PGI 2 ) synthesis by Rf signling: filure of PGI 2 to ctivte PPARβ. Febs J 273(1): Foremn JE, Shrm AK, Amin S, Gonzlez FJ nd Peters JM (2010) Lignd ctivtion of peroxisome prolifertor-ctivted receptor-β/δ (PPARβ/δ) inhibits cell growth in mouse mmmry glnd cncer cell line. Cncer Lett 288: dd