Evaluation of Polycyclic Aromatic Hydrocarbons in the Activation of Early Growth Response-1 and Peroxisome Proliferator Activated Receptors

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1 TOXIOLOGIAL SIENES 85, (5) oi:.9/toxsi/kfi8 Avan Ass puliation Fruary 6, 5 Evaluation of Polyyli Aromati Hyroarons in th Ativation of Early Growth Rspons- an Proxisom Prolifrator Ativat Rptors Jong-Ho Kim,* Kiyoshi Yamaguhi,* Song-Ho L,* Patriia K. Tithof,* Gary S. Saylr, Joo-Hon Yoon, an Sung Joon ak*, *Dpartmnt of Pathoiology, an Dpartmnt of Miroiology, Univrsity of Tnnss, Knoxvill, Tnnss 7996, an Dpartmnt of Otorhinolaryngology, Yonsi Univrsity ollg of Miin, Soul, South Kora Riv Dmr 6, 4; apt Fruary 9, 5 Polyyli aromati hyroarons (PAHs) ar uiquitous nvironmntal an foo ontaminants with known or suspt arinogni proprtis. In this stuy, w hav valuat whthr PAHs ativat th arly growth rspons (EGR-) gn an in to proxisom prolifrator-ativat rptor alpha (PPARa) an lta (PPAR/) in ll ultur systms. Luifras rportr systms wr mploy an svral PAHs wr valuat for thir aility to ativat EGR- an PPARs. Som PAHs nhan EGR- xprssion an ativat PPARa an PPAR. Among thm, nz(a)anthran was foun to at as a rlativly potnt ativator of PPARa an PPAR/, an to signifiantly nhan EGR- transription. Ths in vitro assays wr onfirm y Wstrn lot analysis, using ll lysats of tissu sampls from mous trapp at a highly ontaminat Suprfun sit in th hattanooga rk flooplain in hattanooga, Tnnss. W hav foun that a PPAR targt gn, glyogn synthas kinas- (GSK-), was own-rgulat an EGR- was up-rgulat in th mous sampls of hattanooga rk. In aition, slt PAHs rprss GSK- an inu YP4A in FaO rat hpatoma lls. In onlusion, PAHs ativat PPARa an PPAR/, an up-rgulat EGR- xprssion in vitro as wll as in vivo. Ths ata may provi a ivrsity of PAH ativity in svral iologial pathways. Ky Wors: PAHs; PPARa; PPAR; EGR-; GSK-. Polyyli aromati hyroarons (PAHs) ar toxi an uiquitous nvironmntal pollutants. Thy ar gnrally form an mitt into th nvironmnt as a rsult of inomplt omustion of fossil fuls, woo, an othr organi matrials an from inustrial prosss. Humans an animals ar xpos to PAHs from air, watr, itary, an oupational sours, an also from igartt smok. Thir vry long iologial half-livs in omination with iologial ffts at To whom orrsponn shoul arss at Dpartmnt of Pathoiology, ollg of Vtrinary Miin, Univrsity of Tnnss, 47 Rivr Driv, Knoxvill, TN Fax: (865) sak@utk.u. vry low onntrations hav aus halth onrns aout arinogniity (Ruin, ), tratogniity (outur t al., 99; Inarona t al., 4), an ariovasular isas (Gustavsson t al., 996, ; Thirman t al., 994). Although many rports fous on luiating th molular mhanism of PAHs in svral isass, pathways othr than aryl hyroaron rptor (AhR) ativation hav riv littl attntion. Many hroni isass, inluing anr an ariovasular isass hav n link to hrity an/or th nvironmnt, whih an ithr nhan or inhiit th isas pross. On suh molular link twn isas an th nvironmnt is arly growth rspons- (EGR-, also known as NGFI-A, Zif68, Krox4, an Tis8). EGR- is th prototypial mmr of a family of zin fingr transription fators that inlus at last thr othr mmrs, EGR-, -, an -4. EGR- is spially inu y a rang of physiologial an nvironmntal stimuli inluing growth fators, ytokins, ultraviolt light, ionizing raiation, an mhanial injury (Gashlr an Sukhatm, 995; Khahigian an ollins, 998). EGR- appars to ritially involv in svral isass inluing angiognsis an tumor formation. Altration in xprssion oul ontriut to th ltrious ffts of PAH xposur. Rntly, Martinz t al. rport that halognat aromati hyroaron,,,7,8-ttrahloroinzo-p-ioxin (TDD), inus EGR- xprssion in human lung arinoma lls (Martinz t al., 4). Howvr, xprssion of EGR- in nonnoplasti lls unrlis ivrs pathophysiologial rsponss suh as survival rsponss to amaging irraiation (Huang t al., 999), vlopmnt of vasular olusion in artrioslrosis (Silvrman an ollins, 999), an formation of svr pulmonary mphysma (Zhang t al., ). In aition to ths ivrs ations, xprssion of EGR- is ommonly own-rgulat in tumor lls in ontrast with thir normal tissu ountrparts (alogro t al., 4; Hao t al., ; Huang t al., 997; Lvin t al., 995; Shozu t al., 4). Howvr, EGR- is xprss at a highr lvl an promots ll growth in prostat anr whn ompar with normal tissus (Ei t al., 998; Thigpn t al., 996). Thus, Toxiologial Sins vol. 85 no. Ó Th Author 5. Pulish y Oxfor Univrsity Prss on half of th Soity of Toxiology. All rights rsrv. For Prmissions, plas mail: journals.prmissions@oupjournals.org Downloa from

2 586 KIM ET AL. EGR- has multipl funtions in tumorignsis, an th xat iologial funtion of EGR- may pnnt on ll ontxt as wll as tissu typs (ak t al., 4). Nonthlss, th EGR- s rol in vasular isas has n firmly stalish y th fat that EGR- ontrols th xprssion of svral gns impliat in th pathognsis of athroslrosis an rstnosis (rslow, 996; Harja t al., 4; Maffry t al., ; Silvrman an ollins, 999). Proxisom prolifrator ativat rptors (PPARs) ar anothr molular link twn hroni isas an th nvironmnt. PPARs ar mmrs of th nular rptor suprfamily an xist as thr sutyps signat a, (or ), an. Among thos, PPARa ativation is rsponsil for th pliotropi ffts of proxisom prolifrator suh as nzym inution, proxisom prolifration, livr nlargmnt, an tumors (Klaunig t al., ). PPARa also plays a ritial rol in rgulation of llular uptak an -oxiation of fatty ais (rgr an Mollr, ; Marx t al., 4). In ontrast, PPAR (also known as PPAR) is wily xprss with rlativly highr lvls in rain, olon, an skin. Although thr hav n xtnsiv stuis on PPARa, muh lss is known aout th funtion of PPAR. Nonthlss, rnt stuis suggst that PPAR plays a rol in olon anr (Gupta t al., 4; H t al., 999; Wang t al., 4), an praipoyt prolifration (Hansn t al., ). W hypothsiz that transription fators, EGR- an PPARs may link nvironmntal toxi ompouns to human isass. Th aim of this stuy is to trmin whthr PAHs afft EGR- an PPARs ativity an to intify th iffrnt ativity of PAHs in rportr systm. In this stuy, fiftn PAHs, whih ar ommonly foun in th nvironmnt, wr xamin as potntial ativators of PPARa or PPAR/, an inur of EGR- gn xprssion in A549 human lung anoarinoma lls an HT-6 human olortal ano arinoma lls. Th luifras rportr gns wr us to masur th ativity of PPARs an transativation of th EGR- promotr. W hav monstrat that som PAHs may ativat PPARa an PPAR/, an transativat EGR- promotr ativity. Among thos, nz(a)anthran (aa) inus EGR- an PPAR ativation in ultur systms. Furthrmor fral mi (Pramysus gossypinus) trapp along th flooplain of a Suprfun sit with high lvls of PAH ontamination monstrat signifiant up-rgulation of EGR- an own-rgulation of GSK-, a PPAR targt gn. Ths ata provi vin for ivrs ffts of PAHs that may important in isass link to nvironmntal pollution suh as ariovasular isas an arinognsis. MATERIALS AND METHODS ll ultur an hmials. A549 human lung anoarinoma ll lins an HT-6 human olortal arinoma ll lins wr otain from th Amrian Typ ultur olltion (AT) (Manassas, VA). A549 lls wr ultur in RPMI-64 mium supplmnt with % FS an lg/ml gntamyin. HT-6 lls wr ultur in Moifi Moy 5A mium supplmnt with % FS an lg/ml gntamyin. FaO rat hpatoma ll lins wr gnrously provi y Dr. Song-Jin Kim (National anr Institut, thsa, MD) an wr ultur in Dulo s moifi Eagl s mium supplmnt with % FS an lg/ml gntamyin. All th PAHs wr purhas from Sigma-Alrih (St. Louis, MO) an ompltly issolv in DMSO. Plasmis. EGR- promotr (ÿ6 to þ5) link to th luifras gn (pegr6-lu) was sri prviously (ak t al., ). Th plasmis us for stuying PPARa an PPAR/ ativators wr a rportr gn ontaining four opis of a Gal4 ining sit (MH4-TK-Lu) an himri rptors (pmx-gal-mppara-ld for PPARa an pmx-galmppar-ld for PPAR). In this systm, whn a ompoun ins to th ligan ining omain (LD) from PPARa or PPAR of th himri rptor (pmx-gal4-mppara-ld or pmx-gal-mppar-ld), thn th DNA ining omain of th yast Gal4 (not as Gal) ins to o-transft Gal4 ining sit an initiats transription of th firfly luifras (Lu). A rportr plasmi ontaining thr opis of th PPAR rspons lmnt (PPRE-TK- Lu) an a mous PPARa DNA (pdna-mppara) wr prviously sri (Nixon t al., ). This systm irtly masurs ativation of PPARa via transriptional ativation of th luifras rportr gn as a rsult of PPARa ining to th PPAR rspons lmnt (PPRE). All th PPAR an PPAR rportr plasmis wr gnrously provi y Dr. Ronal M. Evans (Howar Hughs Mial Institut, A). Transint transftions an luifras rportr assays. lls ( 5 lls/wll) wr ultur in twlv-wll plats in ultur mium ontaining % FS. Aftr growth for 6 h, th intrnal ontrol,.5 lg prl-null (Promga, WI) an.5 lg of th othr plasmis wr transft using LipoftAMINE (Lif Thnologis, In.) aoring to th manufaturr s protool. Aftr 4 h, th mia wr hang an th lls wr trat with th various PAHs issolv in DMSO. Th final onntration of DMSO i not x.% (v/v) in any of th sampls. Tratmnts with PAHs wr prform unr srum-fr onitions. Aftr 4 h tratmnt, th lls wr wash with PS an harvst in X luifras lysis uffr. Th luifras ativity was masur y a ual luifras assay kit (Promga, WI), an normaliz to th intrnal ontrol, prl-null (rnilla luifras) ativity. Animal stuis. Fral mi (Pramysus gossypinus) wr trapp along th flooplain of th hattanooga Suprfun sit an in a ontrol sit two mils upstram from th ontaminat stion of rk. All animal prours wr in omplian with th National Institut of Halth guilins on animal us an wr approv y th Univrsity of Tnnss Institutional Animal ar an Us ommitt. Traps wr st ah vning an hk ah morning. Trapp animals wr transport to th laoratory an ansthtiz with O. Aftr th hst avitis wr opn, th mi wr xsanguinat y aria puntur an th istal aorta, hart an lungs prfus with phosphat uffr salin (PS) to rmov lott loo. Stions of hart, lung, an olon wr rmov an snap-frozn in liqui nitrogn for Wstrn lot analysis. Wstrn lot analysis. Th lvl of protin xprssion was valuat y Wstrn lot analysis. FaO lls wr grown to 6 8% onfluny in 6 m plats, follow y 4 h tratmnt of slt PAHs in th asn of srum. Total ll lysats wr isolat using RIPA uffr ( phosphat-uffr salin, % Nonit P-4,.5% soium oxyholat, an.% SDS) ontaining protinas inhiitor, an th solul protin onntrations wr trmin y A protin assay kit (Pir, Rokfor, IL). For fral mous tissus, frozn sampls wr lys in i-ol RIPA uffr as sri aov. All lysat protins wr sparat y SDS-PAGE an transfrr for h onto nitrollulos mmran (Osmonis In., MN). Th lots wr lok for h with 5% skim milk in Tris-uffr salin an Twn.5% an pro with GSK- (ll Signaling, MA), EGR- (Santa ruz ioth., A), YP4A (Affinity ioragnt, Goln, O), or Atin (Santa ruz ioth., A) antioy at 4 ovrnight. Aftr washing with Tris-uffr salin an Twn Downloa from

3 THE EXPRESSION OF EGR- AND ATIVATION OF PPARs Y PAHs 587.5%, th lots wr trat with horsraish proxias-onjugat sonary antioy for h an wash svral tims. Th signals wr tt y th nhan hmiluminsn systm (Amrsham iosins, Arlington Hight, IL). Th signal intnsitis wr masur y NIH Imag program (Sion orp., MD). Statistial analysis. For luifras ativitis of transint transftion xprimnts, ata wr xprss as man ± SD for at last thr inpnnt rpats. For quantitativ analyss, analysis of varian (ANOVA) with Tuky s multipl omparison tst or t-tst was us to ompar man valus. SAS for Winows (9.) (SAS Institut In., ary, N) statistial analysis softwar was us. A p-valu of lss than.5 was onsir signifiant. TALE haratristis of Polyyli Aromati Hyroarons Us in This Stuy ompoun Strutur Molular wight Gnotoxiity arinogniity Anaphthn 54 (?) (?) Anaphthyln 5 (?) AhR ativity Anthran 78 RESULTS W hav slt fiftn PAHs, ontaining two, thr, four, or fiv aromati rings. Ths PAHs wr foun to in th highst onntrations in th Suprfun sit in hattanooga, Tnnss (Elgayyar t al., manusript in prparation). Among ths, nz(a)anthran (aa), nzo(a)pyrn (ap), nzo()fluoranthn (F), nzo(k)fluoranthn (kf), an hrysn (HR) ar onsir to arinogni, whras anaphthn (ANY), anthran (ANT), naphthaln (NAP), pyrn (PYR), phnanthrn (PA), an triphnyln (TP) ar not onsir to arinogni aoring to WHO (Tal ). A limit numr of rnt stuis hav shown that fluoranthn (FA) is an xprimntal arinogn (Hht t al., 995). Effts of PAHs on EGR- Ativation Svral PAHs wr xamin for th inution of EGR- gn in A549 human lung arinoma ll lins, whih hav n shown to inu EGR- xprssion (Martinz t al., 4). Th pegr6-lu onstrut was transintly transft into A549 lls an th luifras ativity was masur in th prsn of PAHs. Th pegr6-lu onstrut (Fig. A) ontains. k of th EGR- promotr (ak t al., 5). All th PAHs tst hr show a signifiant inras in luifras ativity xpt ap an TP (Fig. ). PA, aa, an PA monstrat highr luifras ativity, an PA also inras EGR- ativity in lm onntration (Fig. ). As a positiv ontrol, sulina sulfi ( lm) was us in th promotr assay (ak t al., 5), showing a 4-fol inution of luifras ativity ompar to vhil trat sampl. Effts of PAHs on PPARa ativation In aition to ytohrom p45 as iomarkrs, proxisom prolifrators hav n us for a pollution iomarkr in aquati organism (ajaravill t al., ). Among thos, PPARa has riv muh attntion from many rsarhrs sin PPARa xprssion is tightly rgulat y various hormons. In aition, PPARa plays a pivotal rol in tumorignsis an athrognsis (Klaunig t al., ; Marx t al., 4). Thrfor, w hav xamin whthr PAH ompouns nz(a)anthran 8 þ þ þþ nzo(a)pyrn 5 þ þ þþ nzo()fluoranthn 5 þ þ þþþ nzo(k)fluoranthn 5 þ þ þþþ iphnyln 5 hrysn 8 þ þ þþþ Fluoranthn þ (þ) þ -Mthylanthran 9 Naphthaln 8 (?) Phnanthrn 78 (?) (?) Pyrn (?) (?) þ Triphnyln 8 þ (ÿ) Not. Gnotoxiity an arinignity aoring to WHO, 998: þ, positiv; ÿ, ngativ;?, qustional; parnthss, rsult riv from small ataas (WHO, 998). AhR-miat ativity (Mahala t al., ) valu for nzo(a)pyrn (an PAHs with similar ffts) was aritrarily st as þþ; valus þþþ an þ iniat PAHs with mor or lss signifiant ffts than nzo(a)pyrn; valu of ÿ iniats no fft. ativat PPARa. Th luifras rportr onstrut an xprssion vtor (Fig. A) wr o-transft into HT-6 lls that hav n shown a high transftion ffiiny (ak t al., ). Th lls wr trat with lm of various PAHs for 4 h an luifras ativity was xamin. Th Downloa from

4 588 KIM ET AL. A pegr6-lu Lu Rlativ Luifras Unit 4 VEH ** *** µm PA 5µM PA Rlativ Luifras Unit 5 4 a VEH SS ANT aa ap F kf HR FA PA PYR TP ANY FIG.. PAH ffts on EGR- xprssion. (A) Shmati iagram of pegr6-lu onstrut. () A549 lls wr xpos to PAHs aftr transftion with a perg6-lu onstrut. lls wr trat with 5 lm PA an lm of all othr PAHs for 4 h. Sulina sulfi was us as a positiv ontrol for EGR- ativator (ak t al., 4, 5). Transftion ffiiny for luifras ativity was normaliz to th Rnilla luifras (prl-null vtor) ativity. Th y-axis shows rlativ luifras unit (firfly luifras unit/rnilla luifras unit) y th fol inras in PAH tratmnt ovr vhil tratmnt. Th rsults ar xprss as man ± SD of thr inpnnt transftions an iffrnt lttrs iniat signifiant iffrn (Tuky s multipl omparision tst, p<.5). VEH, vhil; SS, sulina sulfi; ANT, anthran; aa, nz(a)anthran; ap, nzo(a)pyrn; F, nzo()fluoranthn; kf, nzo(k)fluoranthn; HR, hrysn; FA, fluoranthn; PA, phnanthrn; PYR, pyrn TP, triphnyln; ANY, anaphthyln. () A549 lls wr transft an xpos to lm, 5 lm of PA. Th luifras ativity was masur, an rlativ luifras unit was shown at y-axis. t-tst, **p <.; ***p <., ompar to vhil. luifras ativity to PAH tratmnt was ompar to that of vhil tratmnt. aa was stimat as a strong ativator among PAHs tst hr. Othr PAHs show slight inrass or littl hangs in luifras ativity (Fig. ). As a positiv ontrol, Wy464 was us in this systm an w foun aroun 55-fol inution of luifras ativity, ompar to vhil trat sampls (ata not shown). Transft lls wr also xpos to aa an luifras ativity was signifiantly inras in a os pnnt mannr (Fig. ). It is important to not that th rsults shown in Figur rflt solly ativator ativity with xognous transftants. Thrfor, to xamin th transativation of PPARa, A549 lls wr transintly otransft with a pppre-tk-lu an pdna-mppara xprssion vtor (Fig. A), an thn trat with vhil, an or lm of aa. As shown in Figur, w osrv a signifiant inution of luifras ativity in a os pnnt mannr, iniating that aa ins to PPARa an transativats th gn ontaining PPRE sits in th promotr. Effts of PAHs on PPAR/ ativation It has n suggst that PPAR/ may play an important rol in tumorignsis an athrognsis (Gupta t al., 4; H t al., 999; Marx t al., 4; Tong t al., ). PAH ompouns wr tst for PPAR/ ativity in HT-6 lls transintly transft with pmh4-tk-lu an pmx- Gal4-mPPAR-LD onstruts (Fig. 4A). Th PPAR/ ativity of ah PAH was ompar with vhil. Intrstingly, tratmnt with aa also inu strong luifras ativity among PAHs tst hr. Howvr, othr PAHs also slightly inras luifras ativity, whih is signifiant (Fig. 4). aa also inras luifras ativity in a os pnnt mannr (Fig. 4). GSK- an EGR- Exprssion in th Prsn of PAHs YP4A is inu y PPARa ativation in livr tissu (Johnson t al., 996). GSK- is rprss whn FaO rat hpatoma lls ar xpos to PPARa an PPAR/ ligans (Vann Huvl t al., ). W hav shown that som PAHs ar al to in th PPARa an PPAR/ as an ativator. To trmin whthr PAHs affts PPARa ownstram in FaO lls, w prform Wstrn lot analysis using ll lysats prpar from FaO lls trat with aa, ap, or PA. As shown in Figur 5A, all th PAHs tst inras YP4A xprssion an supprss GSK- xprssion rlativ to vhil-trat lls. aa was th rlativly strong supprssor of GSK- xprssion, whras ap or PA was th wak supprssor. Ths ata ar onsistnt with prvious ata showing that aa is th rlativly strongst PPARa an PPAR/ ativator, whras ap an PA ar rlativly wak (Figs. an 4). Nxt, w xamin GSK- xprssion in vivo Downloa from

5 THE EXPRESSION OF EGR- AND ATIVATION OF PPARs Y PAHs 589 A pmx-gal4-mppar -LD MX pmhx4-tk-lu 4XGal Gal4 DD TK mppar LD Lu Rlativ Luifras Unit VEH ** µm aa *** µm aa Rlativ Luifras Unit 4 a VEH ANT ANP aa ap F kf P HR FA -MA NAP PA PYR TP FIG.. PAH ffts ozn PPARa ining affinity. (A) Shmati iagram of pmx-gal4-mppara-ld an pmhx4-tk-lu vtor. () HT-6 lls wr transft with th vtors shown in Figur A, an th lls wr trat with lm of various PAHs iniat for 4 h. Transftion ffiiny for luifras ativity was normaliz to Rnilla luifras (prl-null vtor) ativity. Th y-axis shows rlativ luifras unit (firfly luifras unit/rnilla luifras unit) y th fol inras in PAH tratmnt ovr vhil tratmnt. Th rsults ar xprss as man ± SD of four inpnnt transftions an iffrnt lttrs iniat signifiant iffrn (Tuky s multipl omparison tst, p<.5). ANP, anaphthn; P, iphnyln; -MA, -mthylanthran; NAP, naphthaln. Othr arviations ar list in Figur. () Th transft lls wr trat with th iniat onntration of aa for 4 h an luifras ativity was masur. t-tst, **p <.; ***p <. ompar to vhil. A Rlativ Luifras Unit 4 pdna-mppar ppprex-tk-lu MV mppar TK Lu Vhil * PPREx µm nz(a)anthran µm nz(a)anthran FIG.. PAH ffts on PPARa transativation in th promotr ontaining PPRE sits in A549 lls. (A) Shmati iagram of pdna-mppara xprssion vtor an ppprex-tk-lu rportr vtor. () A549 lls wr transft an th lls wr trat with ithr vhil or iniat aa for 4 h. *p <.5 ompar to vhil. Luifras ativity was masur as sri aov. * using fral mi (Pramysu gossypinus) trapp along th flooplain of a highly ontaminat ara of th hattanooga rk an also along a nonontaminat ara. W valuat GSK- xprssion in olon, hart, an lung tissus of mi from oth aras. As shown in Figur 5, GSK- was ownrgulat in hart ut not lung from mi trapp in th ontaminat ara of th hattanooga rk whn ompar to mi trapp in th ontrol ara. Howvr, GSK- was not tt in olon tissu from oth mi trapp from ithr sit. W also masur EGR- xprssion an foun that EGR- was inu in olon sampls from mi trapp in th Suprfun sit whn ompar to ontrol. Howvr, no EGR- was tt in hart or lung sampls from ithr sit. EGR- inution in th in vivo stuy is onsistnt with in vitro ata, showing that PAHs inras EGR- xprssion as assss y a rportr systm (Fig. ). Takn togthr, ths rsults suggst that som PAHs, partiularly aa ativats PPARa an PPAR/, an altrs th PPAR targt gn xprssion suh as YP4A an GSK-. DISUSSION Humans an othr living organisms ar onstantly xpos to a larg numr of potntially toxi nvironmntal Downloa from

6 59 KIM ET AL. A pmx-gal4-mpparβ/δ-ld ** MX Gal4 DD mppar β/δ LD pmhx4-tk-lu 4XGal TK Lu Rlativr Luifras Unit ** VEH µm aa 5µM aa a Rlativ Luifras Unit VEH ANT ANP aa ap F kf P FIG. 4. PAHs ffts on PPAR/ ativation using luifras assay. (A) A shmati iagram of th pmx-gal4-mppar-ld himri gn an th pmhx4-tk-lu rportr vtor us in this stuy. () HT-6 lls wr transft with vtors shown in A, an th lls wr trat with lm of various PAHs for 4 h. Transftion ffiiny for luifras ativity was normaliz to th Rnilla luifras (prl-null vtor) ativity. Th y-axis shows rlativ luifras unit (firfly luifras unit/rnilla luifras unit). Th rsults ar xprss as man ± SD of thr inpnnt transftions an iffrnt lttrs iniat signifiant iffrn (Tuky s multipl omparision tst, p<.5). Th arviations ar sri in Figur. () Transft lls wr trat with iniat onntrations of aa for 4 h an luifras ativity was masur. t-tst,. **p <.; ***p <., ompar to vhil tratmnt. HR FA -MA NAP PA PYR TP ANY hmials, inluing th uiquitous polyyli aromati hyroarons (PAHs). A numr of PAHs hav n known to afft svral hroni isass, inluing anr an ariovasular isas, in xprimntal animals following oral, pulmonary, rmal, or suutanous aministration. Most of th iologial ffts of PAHs ar onsir to miat via aryl hyroaron rptor (AhR)-pnnt gn xprssion (Nrt t al., ). PAHs also aus oxiativ DNA amag an DNA aut formation, whih is th most wily apt mhanisms of PAH-inu tumorignsis an ariovasular isas (L an lair, ; Toraason t al., ; Wogan t al., 4). ompar to gnotoxiity or AhR-miat toxiitis of PAHs, knowlg aout th nongnotoxi ffts of PAHs rmains to luiat. Th major goal of our invstigation was to trmin th llular mhanisms that ar inu y PAHs in AhR inpnnt mannr. EGR- an PPARs ar th transription fators that ontrol many gns in th prsn of stimuli, inluing hmial ompouns. Th aility of slt PAHs to ativat th EGR- gn an to in to PPARa an PPAR/ was xamin in this stuy using svral PAHs ommonly foun in a Suprfun sit in hattanooga, Tnnss. W foun that som PAHs ativat EGR- gn an nhan PPARa an PPAR/ ativity in vitro. Th lattr rsult in th supprssion of GSK- xprssion, whih may play an important rol in tumorignsis. EGR- an inu xprssion of a st of vasulatur gns, suh as PDGF-A an hain, FGF, TGF-, TNF-a, an intrallular ahsion molul-. Exprssion of th EGR- is lvat in prostat anr an orrlats with tumor progrssion. Thus, EGR- is th ky miator in orhstrating th funtional haratristis of th vssl wall an tumorignsis. Howvr, EGR- an rlat to anti-tumorignsis an protumorignsis, pning on ll an tissu typs. Whil EGR- inus anti-tumorigni protins inluing p5, PTEN, an NAG- (ak t al., 5), EGR- is xprss at a highr lvl an promots ll growth in prostat anr (Ei t al., 998; Thigpn t al., 996). Thus, EGR- oul play a rol in oth ll prolifration an growth arrst. In ontrast to EGR- funtions in tumorignsis, xprimntal vin is mrging to link EGR- to hroni vasular an inflammatory strss in vivo. Th rol of EGR- in athroslrosis rlat to PAH xposur has not n xamin. In this stuy, EGR- was not up-rgulat in hart tissu from mi xpos naturally to nvironmntal ontaminants; howvr, vasular tissu was not xamin. Furthr stuis ar warrant to valuat th potntial rol of EGR- in athroslrosis progrssion rlat to athroslrosis an hart isas. Downloa from

7 THE EXPRESSION OF EGR- AND ATIVATION OF PPARs Y PAHs 59 A YP4A GSK- Atin DMSO aa ap PA GSK- olon S Hart S Lung S EGR- Rlativ Unit of GSK- /Atin DMSO aa ap PA SDS- PAGE FIG. 5. Rgulation of GSK-, YP4A, an EGR- y PAHs. (A) FaO rat hpatoma lls wr trat with lm of various PAHs for 4 h (DMSO, vhil ontrol; aa, nz(a)anthran; ap, nzo(a)pyrn; PA, phnanthrn). Wstrn lotting was prform using a GSK- antioy (ll Signaling, vrly, MA) an YP4A antioy (Affinity ioragnt, Goln, O). Equal loaing ( lg) was onfirm y trmining atin immunorativity. Th rlativ GSK- xprssion lvls normaliz y atin from thr inpnnt xprimnts ar shown on th ottom. () Mous tissu sampls from olon, hart, an lung wr isolat from mous tissu ollt from th ontrol sit () or from th Suprfun sit (S) in hattanooga, Tnnss. ll lysats ( lg) wr sujt to Wstrn lot analysis for GSK- an EGR- protin xprssion. Equal loaing was onfirm y io-saf oomassi staining (io-ra, A) of total protins shown in ottom. Th lot shown is a rprsntativ of two inpnnt xprimnts. PPARa is a ky ontriutor in th prosss of proxisom prolifration, hyprtrophy, ll prolifration, an hpatoarinognsis in vivo. Its ovr-xprssion is osrv in avan prostat anr (olltt t al., ), an ativation of PPARa promots ll prolifration in rast anr lls (Klaunig t al., ; Suhank t al., ). In aition, ativation of PPARa has n monstrat to moulat many aspts of lipoprotin mtaolism an inflammation in vitro, as wll as in animal an human stuis (Isralian-Konaraki an Ravn, 4). Thus, ativation of PPARa may play a rol in isas suh as tumorignsis an athrognsis. On th othr han, th ativation of PPAR/ plays an anti-apoptoti rol in kratinoyts via transriptional ontrol of th AKT signaling pathway (Di-Poi t al., ). Gnti isruption of PPAR/ also rass th tumorigniity of human olon anr lls transplant into mi (Park t al., ). Our rsults support th ontntion that som PAHs with known arinogni ativity ar rlativly strong PPARa an PPAR/ ativators as assss y rportr systm. Ths rsults support that som PAHs may inu hroni isas through PPAR ativation mhanism othr than AhR ativation. Ths rsults also suggst that minut iffrns in PAH strutur rsult in th ativation of two iffrnt PPARs. In this rport, w hav shown that GSK- is supprss in hart tissu from mi trapp in a highly ontaminat Suprfun sit as wll as in th FaO lls. GSK- is known to a ngativ rgulator of aria hyprtrophy (Hart an Saoshima, ), an w hav rntly rport that AKT/ GSK- plays an important rol in apoptosis (Yamaguhi t al., 4). Thrfor, th supprssion of GSK- y nvironmntal ontaminants suh as PAHs may important in th prosss y whih nvironmntal pollution alrats aria isorrs or tumorignsis suh as ariomyopathy or anr; howvr, furthr stuis may rquir to luiat th xat molular mhanism. In onlusion, our ata suggst that som PAHs, partiulary aa, ar al to ativat EGR- promotr an at as an ativator of PPARa an PPAR/ in vitro. aa an ativat targt gns of PPARa an PPAR/, thry rprssing th GSK- xprssion in vitro an in vivo an inuing th YP4A xprssion in vitro. Th rprssion of GSK- an ativation of EGR- y som PAHs may provi a novl approah to luiating th various ffts of PAHs on human hroni isas. AKNOWLEDGMENTS W thank Dr. Song-Jin Kim (National anr Institut, thsa, MD) for proviing FaO lls, Wi Guan for thnial assistan, an Jaa Husky for hr ritial raing of th manusript. This work was in part support y Downloa from

8 59 KIM ET AL. grant from th National Instituts of Halth (ES657) an y start-up funs from th Univrsity of Tnnss. Finanial support for J.H.K. was provi y Dpartmnt of Oupational an Environmntal Miin, Inj Univrsity Shool of Miin, Pusan, Kora. REFERENES ak, S. J., Horowitz, J. M., an Eling, T. E. (). Molular loning an haratrization of human nonstroial anti-inflammatory rug-ativat gn promotr. asal transription is miat y Sp an Sp. J. iol. hm. 76, ak, S. J., Kim, J. S., Moor, S. M., L, S. H., Martinz, J., an Eling, T. E. (5). ylooxygnas inhiitors inu th xprssion of th tumor supprssor gn EGR-, whih rsults in th up-rgulation of NAG-, an antitumorigni protin. Mol. Pharmaol. 67, ak, S. J., Kim, J. S., Nixon, J.., DiAugustin, R. P., an Eling, T. E. (4). Exprssion of NAG-, a transforming growth fator-ta suprfamily mmr, y troglitazon rquirs th arly growth rspons gn EGR-. J. iol. hm. 79, ak, S. J., Wilson, L.., Hsi, L.., an Eling, T. E. (). 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