Novel n-3 Fatty Acid Oxidation Products Activate Nrf2 by Destabilizing the Association between ...

13 Pages·2007·0.46 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRYVOL.282,NO.4,pp.2529–2537,January26,2007
©2007byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A.
Novel n-3 Fatty Acid Oxidation Products Activate Nrf2 by
Destabilizing the Association between Keap1 and Cullin3*□S
Receivedforpublication,August10,2006,andinrevisedform,October23,2006 Published,JBCPapersinPress,November25,2006,DOI10.1074/jbc.M607622200
LingGao‡1,JiakunWang§¶1,KonjetiR.Sekhar§¶,HuiyongYin‡,NicholasF.Yared§¶,ScottN.Schneider(cid:1),
SoumyaSasi§¶,TimothyP.Dalton(cid:1),MarkE.Anderson**2,JeffersonY.Chan‡‡,JasonD.Morrow‡3,
andMichaelL.Freeman§¶4
Fromthe‡DepartmentsofMedicineandPharmacology,DivisionofClinicalPharmacology,§DepartmentofRadiationOncology,
¶Vanderbilt-IngramCancerCenter,VanderbiltUniversitySchoolofMedicine,Nashville,Tennessee37232,the(cid:1)Departmentof
EnvironmentalHealthandCenterforEnvironmentalGenetics,UniversityofCincinnatiMedicalCenter,Cincinnati,
Ohio45267,the**DepartmentofMedicine,CarverCollegeofMedicine,UniversityofIowa,IowaCity,Iowa52242,
andthe‡‡DepartmentofPathology,UniversityofCalifornia,Irvine,California92697
Consumptionofeicosapentaenoicacid(EPA)anddocosa- oil. Epidemiological studies and randomized controlled trials
hexaenoic acid (DHA) can mitigate the progression of dis- have demonstrated that n-3 fatty acid supplementation can
easesinwhichoxidativestressrepresentsacommonunder- reducesuddencardiacdeath,nonfatalstroke,anddecreasepro-
lying biochemical process. Nrf2-regulated gene expression gression of arteriosclerosis (1–3). Dietary supplementation
regulatesdetoxificationofreactiveoxygenspecies.EPAand withfishoilalsoshowspromiseforsuppressingprogressionof
DHAweresubjectedtoaninvitrofreeradicaloxidationproc- neurodegenerativediseases(4),neuropsychiatricdisorders(5),
ess that models in vivo conditions. Oxidized n-3 fatty acids humanimmunodeficiencyvirus(6),anddiseasesoftheretina
reacted directly with the negative regulator of Nrf2, Keap1, (7).Thus,identifyingthemolecularmechanism(s)responsible
initiatingKeap1dissociationwithCullin3,therebyinducing forn-3fattyacidmitigationofthesediversediseasesrepresents
Nrf2-directedgeneexpression.Liquidchromatography-tan- animportantandintriguingquestion.
dem mass spectrometry analyses of oxidized EPA demon- Oxidativestressisacommonfactorintheetiologyofthedis-
strated the presence of novel cyclopentenone-containing easesimpactedbyDHAandEPA(1,8–17).Thesen-3fattyacids
molecules termed J -isoprostanes in vitro and in vivo and are very susceptible to free radical oxidation, exceeding that of
3
wereshowntoinduceNrf2-directedgeneexpression.These arachidonic acid (AA) (18–20). Yet, recent studies have shown
experiments provide a biochemical basis for the hypothesis thatEPAandDHAsupplementationreducedurinaryF -isopro-
2
that formation of J-ring compounds generated from oxida- tanelevels,amarkerforoxidativestress,aswellasenhancedcel-
tionofEPAandDHAinvivocanreachconcentrationshigh lularantioxidantdefensesystems(21–24).Althoughareduction
enoughtoinduceNrf2-basedcellulardefensesystems. ofF -isoprostanelevelscanbeattributed,inpart,toadecreasein
2
membraneAAcontent(20,25),therelationshipbetweenEPA/
DHAoxidationversusEPA/DHA-mediatedinductionofantioxi-
Eicosapentaenoic acid (EPA, C20:5n-3)5 and docosahexae- dantresponsesisnotwellunderstood.
noicacid(DHA,C22:6n-3)aretwomajorcomponentsoffish Non-enzymaticfree-radicalperoxidationofAAresultsinthe
formation of multiple stereoisomers of prostaglandin H -like
2
*ThisworkwassupportedinpartbyNCI,NationalInstitutesofHealth(NIH) bicyclicendoperoxidesthatcanundergothiol-mediatedreduc-
GrantsCA38079,CA104590,T32CA093240,DK26657,ES012463,DK48831, tiontoformprostaglandin-likecompoundstermedF -isopros-
CA77839, GM15431, and ES13125 and by NHLBI, NIH Grants HL070250, 2
tanes (F -IsoPs) or rearrangement and reduction to form
HL62494,andHL046681.Thecostsofpublicationofthisarticleweredefrayed 2
inpartbythepaymentofpagecharges.Thisarticlemustthereforebehereby E -IsoPs, D -IsoPs, and isothromboxanes. E /D -IsoPs can
2 2 2 2
marked“advertisement”inaccordancewith18U.S.C.Section1734solelyto then dehydrate to highly reactive A /J -isoprostanes contain-
indicatethisfact. 2 2
□S Theon-lineversionofthisarticle(availableathttp://www.jbc.org)contains ing cyclopentenone rings. These compounds readily adduct
supplemental“ExperimentalProcedures”andFigs.S1andS2. thiolgroupsonproteinsandhavebeenshowntoinduceNrf2/
1Bothauthorscontributedequallytothiswork. ARE-directedgeneexpression(26).
2AnEstablishedInvestigatoroftheAmericanHeartAssociation.
NF-E2-relatedfactor2,Nrf2,isamastertranscriptionfactor
3Towhomcorrespondencemaybeaddressed:DivisionofClinicalPharmacol-
showntoregulateexpressionofmorethan200genes,including
ogy,Dept.ofMedicineandPharmacology,VanderbiltUniversitySchoolof
Medicine,526RRB,23rdandPierceAves.,Nashville,TN37232.Tel.:615-322- thoseinvolvedinPhaseIIdetoxificationandantioxidantgene
4785;Fax:[phone];E-mail:[email protected]. expression(27).RegulationofNrf2activityrepresentsacritical
4Towhomcorrespondencemaybeaddressed:B902TVCRadiationOncology,
stepininitiatingacellularantioxidantresponsetoreactiveoxy-
VanderbiltUniversitySchoolofMedicine,Nashville,TN37232.Tel.:615-322-
3606;Fax:[phone];E-mail:[email protected]. genspecies.Nrf2-dependentgeneexpressionisregulatedbya
5Theabbreviationsusedare:EPA,eicosapentaenoicacid;LC/MS/MS,liq- cis-element located in the proximal promoter region and is
uidchromatography-tandemmassspectrometry;DHA,docosahexae-
noic acid; AA, arachidonic acid; IsoP, isoprostane; ARE, antioxidant
response element; MeOAMVN, 2,2(cid:1)-azobis(4-methoxy)-2,4-dimethyl-
valeronitrile; AAPH, 2,2(cid:1)-azobis(2-amidinopropane) hydrochloride; 5,5(cid:1)-dithiobis(2-nitrobenzoicacid);siRNA,smallinterferenceRNA;Nrf2,
PPh3,triphenylphosphine;GFP,greenfluorescentprotein;HPLC,high- NF-E2-relatedfactor2;E3,ubiquitin-proteinisopeptideligase;CID,col-
performanceliquidchromatography;GC,gaschromatography;DTNB, lision-induceddissociation.
This is an open access article under the CC BY license.
JANUARY26,2007•VOLUME282•NUMBER4 JOURNALOFBIOLOGICALCHEMISTRY 2529
n-3FattyAcidOxidationandNrf2
termedanantioxidantresponseelement(ARE)(28).Nrf2binds ried out as described (33). Immunoprecipitation was carried
to AREs as a heterodimer and regulates ARE-directed gene outasdescribedpreviously(34).Cardiactissuewasobtainedas
expression(29).Nrf2isnegativelyregulatedbyassociationwith describedinapreviousstudy(35).Slicesofflashfrozencardiac
theubiquitinligaseadaptorproteinKeap1(30).BindingofKeap1 tissueweresonicatedat4°Cfor10sinnuclearisolationbuffer
to Nrf2 directs the ubiquitination and proteasome-dependent (33).Nucleiwerepelletedfollowingcentrifugationat12,000(cid:3)
degradationofNrf2.InhibitionofNrf2ubiquitinationresultsin g,washedtwiceinnuclearisolationbuffer,andthensolubilized
Nrf2accumulation,increasedARE-directedgeneexpression,and in5(cid:3)Laemmlibuffer.
anenhancedabilitytorespondtooxidantstress(31). Preparation for Flow Cytometric Assay—Washed, trypsinized
Non-enzymatic free-radical peroxidation of n-3 fatty acids HepG2ARE-GFPreportercellswereassayedusingaBDLSRII
are predicted to generate reactive species analogous to those bytheVanderbiltInstitutionalflowcytometrycore.
formedfromAA.Itwouldbeofsignificantinteresttoidentify Transfection—COS-7cells,inoculatedinT25flasks,wereco-
those analogous species and elucidate their bioactivity. We transfected with a plasmid expressing human Keap1 (2
reporthereonnovelstudiesthatdemonstratetheformationof (cid:1)g/flask)andofaplasmidexpressinghumanV5-taggedCul3(2
cyclopentenone-containing J-ring isoprostanes following oxi- (cid:1)g/flask)usingDMRIE-C(Invitrogen).TheKeap1vectorhas
dationofn-3fattyacids.Furthermore,thesestudiesshowthat been described previously (36). A cDNA expressing human
J -IsoPs stabilize Nrf2 levels and induce Nrf2-directed gene Cul3wasobtainedfromtheI.M.A.G.EConsortium(Lawrence
3
expression. Whereas, AA-derived 15-deoxy-(cid:2)12,14-prostag- LivermoreNationalLaboratory)cDNAClonesandsubcloned
landin J was hypothesized to activate Nrf2-directed gene intopcDNA6/V5-His(Invitrogen).48haftertransfection,cells
2
expressionviadissociationofNrf2fromadductedKeap1(26), wereexposedto50(cid:1)MEPA .
ox
ourdatasupportanalternativemechanism:dissociationofCul- PurificationofActiveComponentinOxidizedEPA—EPAwas
lin3fromoxidizedKeap1. oxidized by AAPH, and the oxidation mixture was extracted
usingaC-18cartridge.Theeluatewasevaporatedtodryness,
EXPERIMENTALPROCEDURES
andtheresiduewasresuspendedinisopropanolandpurifiedby
Materials—Eicosapentaenoic acid, docosahexaenoic acid, normal phase high-pressure liquid chromatography (HPLC)
15-E -isoprostane, and 15-F -isoprostane were purchased usingaPhenomenexLuna10(cid:1)(10mm(cid:3)25cm)Silicasemi-
2 2
fromCaymanChemicalCo.15-A -isoprostaneand15-J -iso- preparationcolumnwithamobilephaseofhexane/isopropa-
2 2
prostane were obtained by total synthesis (38, 39). The free- nol/aceticacid(100/3/0.1,v/v)at9ml/minflowrate.Thefrac-
radicalinitiator2,2(cid:1)-azobis(4-methoxy)-2,4-dimethylvaleroni- tionwascollectedeveryminute.HepG2cellswereexposedto
trile (MeOAMVN) was generously donated by Wako theHPLCfractions,andnuclearNrf2wasmeasuredbyimmu-
Chemicals and 2,2(cid:1)-azobis(2-amidinopropane) hydrochloride noblotting.TheHPLCfractionthatmaximallyinducednuclear
(AAPH) was obtained from Kodak. Triphenylphosphine Nrf2accumulationwasfurtheranalyzedbyLC/MS/MS.
(PPh3) and diethyl maleate were purchased from Aldrich- PurificationandAnalysesofA /J -IsoPsfromTissue—Liver
3 3
Chemical.SulforaphanewasobtainedfromICN.Primaryanti- tissues were collected from mice fed with 6% fish oil (Dyets,
bodiestoNrf2,HO1,andactinwerefromSantaCruzBiotech- Inc.)for8weeks.Phospholipidsintheliverswereextractedby
nology,StressGen,andSigma,respectively,whereasGCLCwas FolchsolutionandhydrolyzedbyphospholipaseA torelease
2
purchasedfromLabVision. freeA /J -IsoPs.TheA /J -IsoPsinthesampleswerethenana-
3 3 3 3
Cell Culture—Human HepG2 cells stably expressing GFP lyzedbyLC/MS/MS.
undercontrolofamurineGSTA1ARE/thymidinekinasereporter Structural Characterization Using Tandem Mass Spec-
wereagraciousgiftfromDr.W.E.Fahl(ProCertusBioPharm). trometry—Theinvitroandinvivosampleswereanalyzedusinga
HepG2andCos-7cellsweremaintainedinDulbecco’smodified ThermoFinniganTSQQuantum1.0SR1massspectrometerin
Eagle’smediumsupplementedwith10%fetalbovineserum. negativeionmode.FortandemMSexperiments,collisionenergies
AnimalCare—MicewerefedanAIN-76Adietandwaterad wereoptimizedbetween10and30eVunder1.5millitorrofargon.
libitum and housed under controlled conditions (12-h light/ Spectrathatareshownwereobtainedat15eV.Dataacquisition
dark periods). All experimental protocols conducted on the andanalysiswereperformedusingXcalibersoftware,version1.3.
micewereperformedinaccordancewiththestandardsestab- For electrospray ionization-MS, the capillary temperature was
lishedbytheU.S.AnimalWelfareActs,assetforthinNational 300°C.Thevaporizertemperatureis350°C.Thesheathandaux
InstitutesofHealthguidelinesandthePolicyandProcedures gaspressurewere31and17p.s.i.Thesprayvoltagewas3.0kV,and
and the Vanderbilt University Institutional Animal Care and thetubelensvoltagewas(cid:4)102V.Sampleswereintroducedby
UseCommittee. reversed-phase HPLC with a Phenomenex Luna(cid:2)ODS 5(cid:1)(2.1
PreparationofOxidizedFattyAcid—EPAandDHAwereoxi- mm(cid:3)25cm)column.Themobilephasewasagradientstarting
dizedbyeitherAAPHinphosphate-bufferedsalinebuffer(20)or with80%solventA(2mMNH Ac:CH CN/MEOH(95/5)(cid:5)95:5)
4 3
MeOAMVNinbenzene(32).BothAAPHandMeOAMVNare and20%solventB(MeOH:CH CN(cid:5)5:95),holdingfor1minand
3
free radical initiators. The concentrations denoted in Figs. 1–3 increasingto50%Bin18minandholdingfor2min.Theflowrate
and 6 represent the initial free acid concentrations of EPA or was0.2ml/min.
DHAthatweresubjectedtooxidation. Gaschromatography(GC)/negativeionchemicalionization
Preparation of Nuclear Extracts, Immunoprecipitation, and MSwasperformedusinganAgilenttechnologies6890Nnet-
Immunoblot Analysis—Preparation of nuclear extracts from workGCsystem.Methanewasusedasthecarriergasataflow
cellsandimmunoblottingofNrf2,HO1,andGCLCwerecar- rateof2ml/min.Ionsourcetemperaturewas250°C,electron
2530 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER4•JANUARY26,2007
n-3FattyAcidOxidationandNrf2
resentsnumberofaminoacidresi-
dues; and l represents cell path
lengthincentimeters.
hKeap1 was dissolved at a con-
centrationof0.1mg/mlin100mM
Tris buffer (pH 8.0). For the CD
measurements, hKeapl was incu-
batedwith10(cid:1)M15-J -IsoPor100
2
(cid:1)M oxidized EPA at 37°C for 1 h.
Percentage of secondary structure
was calculated using the K2d pro-
gram (37). Structural changes in
(cid:3)-helix, (cid:4)-sheet, and random coil
were expressed as the increase/de-
creaseofthepercentageofstructure
for 15-J -IsoP or oxidized EPA-
2
treated hKeap1 compared with
non-treatedhKeap1.
FIGURE1.DHA andEPA induceARE-directedGFPreporteractivityandincreaseexpressionofendog- RESULTS
ox ox
enousNrf2,HO1,andGCLC.DHA(A,C,andE)andEPA(BandD),atinitialconcentrationsdenotedinthe
Oxidationofn-3polyunsaturated
figure,wereoxidizedinvitrobyfreeradicalinitiationandthenaddedtoHepG2.RelativefluorescenceofaGFP
reporterstablyexpressedinHepG2cellsandundercontrolofamurineGsta1AREfusedtoathymidinekinase fattyacidsisrequiredforinduction
minimalpromoterisshowninAandB.ThefreeacidsofDHAandEPAwereaddeddirectlytocells.After16hof ofARE-directedGFPreportergene
exposure,GFPfluorescencewasmeasured.Cellsexposedto25or50(cid:1)Msulforaphanewereusedasapositive
expression. HepG2 cells stably
control.Theconcentrationsdenotedrefertotheinitialstartingconcentrationsthatweresubjecttooxidation.
HepG2cellswerealsoexposedtotheoxidizedn-3fattyacidsfortheindicatedtimespriortoimmunoblotting transfected with a plasmid that
(CandD).
expressedGFPundercontrolofthe
murineGsta1ARE(38)wereusedto
energywas70eV,andfilamentcurrentwas0.25mA.Foranal- determineifn-3fattyacidswerecapableofinducingNrf2-di-
ysis,samplesweredissolvedin20(cid:1)lofundecane. rectedgeneexpression.ThedatapresentedinFig.1(AandB)
ReactivityofKeap1Sulfhydryls—Thereactivityofsulfhydryl showthatawellcharacterizedNrf2activatorsuchassulfora-
groups in Keap1 was determined in a competition reaction phane(31)producedastatisticallysignificantincreaseinARE-
occurring between oxidized EPA and 5,5(cid:1)-dithiobis(2-nitro- reporterdirectedGFPfluorescence(p(cid:6)0.05,analysisofvari-
benzoic acid) (DTNB). Recombinant Keap1 (0.2 mg/ml) was ance). However, exposure to DHA or EPA free acids, did not
reactedat30°Cwith100(cid:1)MDTNBinargon-saturated100mM affectGFPfluorescencecomparedwithcontrol(p(cid:7)0.05,anal-
Tris,pH8.0,supplementedwitheithercarriercontrolsolvent ysisofvariance).
or100(cid:1)MoxidizedEPA.Theinitialrelativeabsorbanceofthe BecauseEPAandDHAreadilyundergoauto-oxidationinvivo
two reactions was set to zero and then monitored simulta- (39),itwasofinteresttodetermineifoxidizedn-3fattyacidsexhib-
neouslyatA usingadual-beamVarianDMS80spectropho- itedtheabilitytoinduceNrf2-regulatedactivity.Thus,EPAand
412
tometer. Competition between oxidized EPA and DTNB for DHAwereoxidizedinvitrobyfreeradicalinitiation(see“Experi-
Keap1sulfhydrylswasrecordedasadecreaseinabsorbanceat mental Procedures”). HepG2 cells were exposed to the EPA or
A ,asafunctionoftimerelativetotheabsorbanceproduced DHAoxidationproducts.Adose-dependentincreaseinARE-re-
412
in the reference cell by reaction of DTNB and Keap1 in the porter-directedGFPfluorescencewasobserved(Fig.1,Aand1B,
absence of oxidized EPA. Control experiments demonstrated p(cid:6)0.05,analysisofvariance).ThesedatademonstratethatEPA
thatoxidizedEPAdidnotreactwithDTNB.DTNBreactivity andDHAoxidationproductsinduceARE-directedreportergene
with Keap1, measured as an increase in absorbance at A activity.
412
demonstrated that all 27 His -Keap1 cysteine residues were Oxidized n-3 fatty acids stabilize Nrf2 and increase the
6
availablefortitration. endogenous expression of Nrf2-regulated genes. Nrf2 is a
CDSpectroscopy—CDspectrawereacquiredonaJascoJ-810 short-lived protein due to its association with Keap1, which
spectropolarimeterequippedwithaPeltier-controlledtemper- directstheubiquitinationofNrf2andsubsequentproteasome-
atureblockusinga1-mmpathlengthquartzcuvette.Thedata dependentdegradation(40).Underbasalconditions,verylittle
werecollectedat1-nmincrementsfrom200to260nmusinga Nrf2accumulatesinthenucleus(30).AgentsthatactivateNrf2
response time of 2 s and a scan speed of 10 nm/min. Spectra functiondosobyinhibitingKeap1-directedubiquitinationof
represent an average 2 scans with the background corrected Nrf2, allowing Nrf2-regulated gene expression to increase.
againstabufferblankorabuffertreatmentcompound.Dataare Inhibition of ubiquitination and stabilization of Nrf2 can be
reportedasmeanresidueellipticity,[(cid:2)](deg(cid:3)cm2(cid:3)dmol(cid:4)1), followedbymeasuringnuclearaccumulationofNrf2(31).
whichwascalculatedas[(cid:2)](cid:5)100(signal)/(C(cid:3)n(cid:3)l),where HepG2 cells were exposed to oxidized DHA (DHA ) for
ox
degrepresentsdegrees;signalrepresentsrawoutputinmillide- up to 30 h, and nuclear protein was isolated and immuno-
grees;Crepresentsproteinconcentrationinmillimolar;nrep- blottedforthepresenceofNrf2(Fig.1C).NuclearNrf2levels
JANUARY26,2007•VOLUME282•NUMBER4 JOURNALOFBIOLOGICALCHEMISTRY 2531
n-3FattyAcidOxidationandNrf2
increasedwithin2hofexposuretoDHA .Similarly,HepG2 acidformofEPAdidnotstabilizeNrf2(Fig.1D).Moreover,
ox
cells exposed to oxidized EPA (EPA ) showed significant stabilization of Nrf2 was followed by the increased expres-
ox
increasesinnuclearNrf2levelswithin4h,althoughthefree sion of two well characterized endogenous Nrf2-regulated
genes,hemeoxygenase-1(HO1)(41)andGclc(42)(Fig.1,C
andE).Theseresultsareconsistentwiththedatapresented
in Fig. 1 (A and B) and suggest that oxidation products of
DHA and EPA result in Nrf2 stabilization, allowing
increasedNrf2-regulatedgeneexpression.
A siRNA approach was used to determine if oxidized n-3
fatty acid-mediated expression of HO1 was dependent upon
Nrf2. NMuMG cells were transiently transfected with either
controlsiRNAorsiRNAdirectedagainstNrf2.48haftertrans-
fection,cellswereexposedtoDHA for4h,andthennuclear
ox
extractswerepreparedforimmunoblotting.Theimmunoblot
was probed with antibodies directed against Nrf2, HO1, and
actin(Fig.2A).ExposuretoDHA producedarobustincrease
ox
in Nrf2 expression in cells transfected with control siRNA.
Gene silencing with siRNA directed against Nrf2 suppressed
DHA -mediatedexpressionofNrf2by50%(Fig.2B).Incells
ox
transfectedwithcontrolsiRNA,DHA alsoproducedarobust
ox
increase in the HO1 expression. The intensity of the HO1
immunoreactivebandwasmeasuredandexpressedrelativeto
the expression of actin (Fig. 2B). Transfection of siRNA
directedagainstNrf2suppressedDHA -mediatedexpression
ox
ofHO1by50%(Fig.2,AandB).Theseexperimentsdemon-
FIGURE2.TheincreaseinendogenousHO1mediatedbyDHA requires
ox strate a direct correlation between Nrf2 and HO1 expression
expressionofendogenousNrf2.A,NMuMGcellsweretransfectedwitheither
control siRNA or siRNA directed against Nrf2. After transfection, cells were andsupportthecontentionthatoxidizedn-3fattyacids-medi-
exposedtoDHAoxfor4h.B,imageanalysisquantitationofNrf2,HO1relativeto atedexpressionofHO1isNrf2-dependent.
theexpressionofactin.TheintensitiesofimmunoreactiveNrf2andHO1bands
Oxidizedn-3FattyAcidsReactwithKeap1Sulfhydryls,Alter-
weremeasuredbyimageanalysissoftwareandexpressedrelativetoactin.The
valuesobtainedfromcontrolcellswerearbitraryassignedavalueof1.0. ing Keap1 Secondary Structure That Is Followed by a Loss of
Association between Keap1 and
Cullin3—It was previously con-
cluded that adduction of Keap1
by 15-deoxy-(cid:2)12,14-prostaglandin
J2-initiatedreleaseofNrf2,allowing
forthelatter’sstabilization(26).To
determine if a similar mechanism
prevailedforn-3omegafattyacids,
experiments were undertaken to
determine if EPA could reacted
ox
directly with Keap1 cysteine resi-
dues. The reaction of cyclopen-
tenone isoprostanes with protein
sulfhydrylscanbemonitoredusing
DTNBtitration(43).Thisassaywas
modifiedtoincreasesensitivitysuch
thatwecouldmonitorthereactivity
of a small number of sulfhydryls.
The ability of EPA to compete
ox
with DTNB for reaction with
recombinantKeap1sulfhydrylswas
monitored as described under
“ExperimentalProcedures,”andthe
data are presented in Fig. 3A. The
decreaseinabsorbancemeasuredat
FIGURE3.EPA reactswithKeap1sulfhydryls,altersKeap1secondarystructure,andinitiatesdisassociation A can be attributed to EPA
ofKeap1fromoxCullin3.A,competitionbetweenEPA andDTNBresultedindifferentialtitrationofrecombinant 412 ox
1K5e-aJp2-1IsSoHPg(1r0ou(cid:1)pMs),fmore1ashuraetd37at°CA.4C12,.EBP,ACoDxisntiutidayteosfdriescaooxssmobciiantaionntKoefaKpe1ap(01.1frmomg/Cmull)lirne3a.cSteeedtwexitthfo1r0d0e(cid:1)taMilEs.PAoxor mgrooduipfsic,arteinodnerionfgt4hemKeuanpa1vailtahbiolel
2532 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER4•JANUARY26,2007
n-3FattyAcidOxidationandNrf2
fortitrationwithDTNB.Takentogether,theLC-MS-MS(see spectrometry (Fig. 4B), suggesting the presence of different
below)andDTNBtitrationdataareconsistentwiththemodel regioisomers in this fraction. We subsequently purified the
proposed by (44) in which an inducer transiently reacts with A /J -IsoPregioisomersshowninFig.4B,exposedHepG2cells
3 3
Keap1cysteineresiduesthatultimatelyundergodisulfidebond tothemfor16handmeasuredNrf2/ARE-directedGFPfluo-
formation. rescence.AsshowninFig.5,A /J -IsoPregioisomerswereable
3 3
CDwasusedtoprobeforsecondarystructuralchangespro- toproduceasignificantincreaseinGFPfluorescencecompared
duced by EPA interaction with Keap1 residues (Fig. 3B). withcontrol(p(cid:6)0.05).
ox
Basedonworkdescribedbelow,wealsointerrogatedsecondary We then performed collision-induced dissociation (CID)
structuralchangesinKeap1followingreactionwith15-J -IsoP. analysisonthem/z331iontogetmorestructuralinforma-
2
TheanalysisindicatedthatKeap1underwentanisoprostane-me- tion(Fig.4C).TheCIDoftheparentalionat331resultedin
diated loss of (cid:3)-helix content; coincident with an increase in the formation of a number of relevant product ions that
(cid:4)-sheetcontentandrandomcoilstructure.TheEPA -mediated would be predicted to be common to all of the A /J -IsoP
ox 3 3
lossin(cid:3)-helixconformationmaybeconsequenceofachangein regioisomers (Fig. 4C), including m/z 313[M-H O](cid:4), m/z
2
theBTBdomain(cid:3)-helixregion(45),becausethekelchdomain 295[M-2H O](cid:4), m/z 287[M-CO ](cid:4), and m/z 269[M-H O-
2 2 2
doesnotcontain(cid:3)-helixfolding(46).Currently,thereisnoinfor- CO ](cid:4). Otherprominentproductionswerepresentthatmight
2
mationconcerningthestructureoftheinterveningdomain. result from fragmentation of different A /J -IsoP regioisomers.
3 3
Keap1BTBandinterveningdomainsareessentialforassoci- Onthebasisofourpreviousworkandstudiesbyothergroups(50,
ation with the E3 ligase scaffold protein Cullin3 (47). Loss of 54–56), these ions can be tentatively assigned as follows, m/z
Keap1-Cullin3 association inhibits Nrf2 ubiquitination, 229[M-CH CH CH (OH)-CO ](cid:4) (18-series), m/z 215[M-
3 2 2 2
thereby stabilizing Nrf2 and initiating Nrf2-directed gene CH CH CH(cid:5)CHCH CH (OH)-H O](cid:4)(15-series),m/z189[M-
3 2 2 2 2
expression(47,48).COS-7cells,transientlyco-transfectedwith CH CH CH(cid:5)CHCH CH (OH)-CO ](cid:4) (15-series), and m/z
3 2 2 2 2
plasmids expressing Keap1 and V5-tagged Cullin3, were 175[M-CH CH CH(cid:5)CHCH CH(cid:5)CHCH CH (OH)-H O](cid:4)
3 2 2 2 2 2
exposed to solvent control or 50 (cid:1)M EPA for 2 h (Fig. 3C). (12-series). The structure was further confirmed by GC/MS
ox
ImmunoprecipitationwithV5antibodyfollowedbyimmuno- after trimethylsilylation derivatization (data not shown). The
blottingwithantibodytoKeap1demonstratedthatKeap1and molecular weight, CID results (Fig. 4C), and GC/MS analysis
V5-Cullin3 associated in a complex under control conditions areconsistentwiththepresenceofA /J -IsoPs.
3 3
whereNrf2immunoreactiveproteinwasbarelydetected(Fig. Although we have only demonstrated formation of A /J -
3 3
1D). Exposure to EPA , which resulted in Nrf2 stabilization IsoPs from oxidation of EPA, DHA should undergo a similar
ox
(Fig. 1D), disrupted the association between Keap1 and oxidationscheme.Tosupportthiscontention,EPAandDHA
V5-Cullin3(Fig.3C).Takenalltogether,thedatapresentedin were subjected to the oxidation process, and then PPh3 was
Fig. 3 indicate that EPA modification of Keap1 sulfhydryls added before applying the lipids to HepG2 cells. Addition of
ox
was followed by a change in secondary structure that was PPh3toEPA andDHA resultedintheformationofF-ring
ox ox
accompaniedbyalossofassociationwithCullin3andprovidea finalproductsratherthanAorJcyclopentenones.Asshownin
mechanismforNrf2stabilizationobservedinFig.1. supplementalFig.S1,theproductofDHA reductionbyPPh3
ox
A Series of Novel Cyclopentenone-containing Molecules wasnotabletoactivatetheARE-directedGFPreporter(p(cid:7)0.05,
TermedJ -isoprostanesGeneratedfromNon-enzymaticPeroxi- analysisofvariance).Similarly,PPh3reductionofEPA resulted
3 ox
dationofEPAArePotentActivatorsofNrf2—Majorfreeradi- inalossofinducingactivity(datanotshown).Thesedataarecon-
cal-catalyzed oxidation products of AA, including F -IsoPs, sistentwiththehypothesisthatA/J-ringcompoundsarerequired
2
D /E -IsoPs, and A /J -IsoPs, have been characterized previ- fortheactivationofARE-directedreporteractivity.
2 2 2 2
ously(49).EPAandDHAalsoundergoanalogousfreeradical- In addition, 15-F -IsoP, 15-E -IsoP, 15-A -IsoP, and 15-J -
2 2 2 2
induced oxidation, however significantly more regioisomers IsoPwereusedassurrogatecompoundstofurtherdefinethe
result (20, 50). In the presence of the free radical initiators, structure(s) responsible for activation of Nrf2. HepG2 cells
AAPHorMeOAMVN,EPAandDHAareoxidizedtobicyclic wereexposedtothesurrogatecompoundsfor16h,andNrf2/
endoperoxideswithdozensofregioisomersanddiastereomers. ARE-directed GFP fluorescence was measured (supplemental
ThesebicyclicendoperoxidescanbereducedtoF-ringisopros- Fig.S2A).Exposureto15-J -IsoPproducedarobustincreasein
2
tanes by GSH (in vivo) (51) and PPh3 (in vitro) (52) or rear- GFPfluorescencecomparedwithcontrol(p(cid:6)0.05,Student’st
ranged to D/E-ring isoprostanes in aqueous solution (53). To test).Moreover,nuclearNrf2levelsincreasedfollowingexpo-
identifytheactivecomponent(s)inoxidizedn-3fattyacids,we sureto15-J -IsoPfor4h(supplementalFig.S2B).Theseresults
2
oxidized EPA in phosphate-buffered saline buffer, separated aresupportedbythedatashowninFig.3Bandsuggestedthat
theoxidationmixturebynormalphaseHPLC,andcollected80 J-ringisoprostanestructureiscapableofNrf2activation.
fractions.WethenexaminedtheabilityofeachHPLCfraction A /J -isoprostanes Are Detected in Vivo—For these studies,
3 3
toinducenuclearaccumulationofNrf2,asmeasuredbyimmu- micewerefedadietsupplementedwith0.56%EPAfor8weeks.
noblotting (data not shown). The oxidation products in the We did not observe any significant formation of IsoPs in the
HPLC fraction that maximally induced Nrf2 accumulation in chow,whichwaschangeddaily,duringthe8weeksoffeeding
the nucleus were further identified by LC/MS/MS. The mass (datanotshown).AA,EPA,F -,andF -isoprostanelevelswere
2 3
spectrometryanalysisofthisfractionshowedthemajorionin quantitatedpergramofcardiactissue.Innon-stressedcondi-
this fraction was m/z 331 (Fig. 4A). A series of peaks can be tions, cardiac tissue obtained from mice fed a control diet
observed when m/z 331 was monitored by second ion mass contained0.35((cid:8)0.07)pgofF -IsoPspernmolofAA.Nei-
2
JANUARY26,2007•VOLUME282•NUMBER4 JOURNALOFBIOLOGICALCHEMISTRY 2533
n-3FattyAcidOxidationandNrf2
FIGURE4.StructurecharacterizationofA /J -IsoPinvitroandinvivo.A,MSspectrumshowingm/z331,themolecularweightofA /J -isoprostane,isthe
3 3 3 3
majorcomponentinHPLCfractionthatexhibitedthemaximalabilitytoinduceNrf2accumulationinnucleiofHepG2cells.B,secondionmassspectrometry
analysisillustratesthepresenceofA /J -IsoPregioisomersintheactiveHPLCfraction.C,CIDanalysisresultoftheactiveHPLCfraction.D,CIDanalysisofmouse
3 3
liver.Micewerefedadietsupplementedwith0.56%EPAfor8weeks.
ther EPA nor F -IsoPs were detected in cardiac tissue extracted and utilized for A /J -IsoPs analysis by LC/MS/
3 3 3
obtainedfromthecontrolmice.Innon-stressedconditions, MS. In the CID analysis of the liver, all of the product ions
cardiac tissue obtained from mice fed a diet supplemented that would be predicted to be common to all of the A /J -
3 3
with0.56%EPAcontained25((cid:8)5)pgofF -IsoPspernmolof IsoP regioisomers were detected (Fig. 4D), including m/z
3
EPAand0.40((cid:8)0.08)pgofF -IsoPspernmolofAA.These 313[M-H O](cid:4),m/z295[M-2H O](cid:4),m/z287[M-CO ](cid:4),and
2 2 2 2
measurementsdemonstratethatEPAissignificantlymoresus- m/z269[M-H O-CO ](cid:4).Inaddition,12-,15-,and18-series
2 2
ceptibletooxidationthanAAandprovidesupportforthecon- regioisomericcharacteristicfragmentswereformed,includ-
ceptthatoxidationofEPAwouldresultinsignificantgenera- ing m/z 229[M-CH CH CH (OH)-CO ](cid:4) (18-series), m/z
3 2 2 2
tionofoxidant-mediatedA /J -IsoPs. 189[M-CH CH CH(cid:5)CHCH CH (OH)-CO ](cid:4) (15-series),
3 3 3 2 2 2 2
AnalogoustoLC/ESI/MS/MSstudiesperformedinvitro, and m/z 175[M-CH CH CH(cid:5)CHCH CH(cid:5)CHCH CH (OH)-
3 2 2 2 2
experiments were then carried out to obtain evidence that H O](cid:4)(12-series).AsisevidentinFig.4D,theCIDpatternis
2
A /J -IsoPs are detected in vivo. Liver and heart were verysimilartothatobtainedfromtheinvitrostudies,although
3 3
2534 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER4•JANUARY26,2007
n-3FattyAcidOxidationandNrf2
formationofF -,F -,andF -isoprostanes(19,20,60).Oxida-
2 3 4
tive stress-mediated formation of isoprostanes represents an
important biochemical parameter, because the different iso-
prostanescanexhibitvariousbioactivefunctions:F -IsoPscan
2
increaseexpressionofthecytokineinterleukin-8(61)andstim-
ulate the thromboxane receptor (62), whereas 15-J -IsoP can
2
activate the peroxisome proliferator-activated receptor (cid:5)
nuclear receptor (63), as well as ARE-directed Phase II gene
expression(26).
ThetranscriptionfactorNF-E2-relatedfactor2(Nrf2)plays
an important role in regulating the expression of genes that
encodeproteinsresponsiblefordecreasingoxidativestressand
inflammationinvivo(64–67).Nrf2isnegativelyregulatedby
associationwithKeap1,aCullin3ubiquitinligaseadaptorpro-
tein(47,48).Keap1function,inturn,requiresassociationwith
FIGURE5.Them/z331fractionshowninFig.4BinducesARE-directedGFP
Cullin3, a scaffold protein that positions Keap1 and its sub-
reporteractivityinHepG2cellsstablyexpressingtheGFPreporter.Cells
wereexposedtovehiclecontrol(Me2SO(DMSO)),50(cid:1)Msulforaphane,or2(cid:1)M strateinproximitytotheE3ligaseRbx1,allowingthesubstrate
ofpurifiedm/z331for16h. tobepolyubiquitinated.Cullin3isregulatedbyneddylationand
deneddylation(34,68).HumanKeap1contains27cysteineres-
idues hypothesized to function as sensors of oxidative stress
(31,69).However,adductionofKeap1cysteineresiduesdoes
not induce release of Nrf2 (70). Nor does every reagent that
adducts Keap1 induce Nrf2-mediated gene expression (33).
Thus,therate-limitingstepsresponsibleforactivationofNrf2
arenotwellunderstood.
Levonen, et al. (26) found that 15-deoxy-(cid:2)12,14-prostag-
landin J adducted Keap1and hypothesized that activation of
2
Nrf2 by15-deoxy-(cid:2)12,14-prostaglandin J was a consequence
2
ofdissociationofNrf2fromKeap1.However,subsequentwork
hasshownthatadductionofKeap1by15-deoxy-(cid:2)12,14-pros-
FIGURE6.n-3fattyacidsstabilizeNrf2invivo.Immunoblotillustrating taglandinJ doesnotcausedisassociationfromNrf2(70).Thus,
increasedexpressionofendogenousNrf2incardiactissuefollowingintrap- 2
the mechanism by which cyclopentenone-containing IsoPs
eritonealinjectionofDHA .6-week-oldfemaleNrf2wild-typeC57BL/6Jmice
were injected intraperitoonxeally with either ETOH (lanes 1–4) or DHA (2 activateNrf2hasremainedobscure.Ourstudiesdemonstratea
ox
mg/animal,lanes5–10).6hafterinjection,themiceweresacrificed.Hearts requirement for free radical-mediated oxidation of DHA and
wereremovedandwashed.Nucleiisolatedfromhearttissueandnuclear
EPA for the process of Nrf2 stabilization, ARE-directed
proteinwasimmunoblottedforthepresenceofNrf2oractin.
reportergeneexpression,aswellasforexpressionofendoge-
therelativeabundanceofthedifferentproductionsareslightly nousHO1andGCLC.Wehavepresentednoveldatademon-
different(comparewithFig.4C).Asimilarresultwasobtained stratingthatfreeradical-mediatedoxidationproductsreacted
fromhearttissue(datanotshown).Takentogether,theseexperi- withKeap1sulfhydryls,alteringKeap1structure,aconforma-
mentsprovideevidencethatA /J -IsoPsarepresentinvivo. tionalchangethatwasassociatedwithlossofbindingtoCul-
3 3
n-3FattyAcidsStabilizeNrf2inVivo—AsshowninFig.6,a lin3.ThesedataidentifytheassociationofKeap1withCullin3
bolusintraperitonealinjectionofDHA resultedinstabiliza- asakeyregulatorystepinNrf2biologywithrespecttoactiva-
ox
tion of Nrf2 in heart tissue. Similar results were observed if tionbyJ -IsoPsand,byextension,othercyclopentenone-con-
3
animalswereinjectedwithfreeacidDHA,consistentwiththe tainingIsoPs.
occurrenceofinvivooxidation(datanotshown). LC-MS-MS analysis of the oxidized EPA identified novel
cyclopentenone-containing J -IsoPs that induced Nrf2-medi-
DISCUSSION 3
atedgeneexpression.Ouranalysisprovidedevidencethatthese
Oxidativestressrepresentsacommonunderlyingbiochem- novelIsoPsarepresentinvivo.Moreover,ourstudiessuggest
ical process that contributes to the progression of many dis- thatthebioactivityofJ -IsoPsmaycomefromthemixtureof
3
easesmitigatedbydietarysupplementationwithDHAandEPA different of regioisomers and suggest that J-ring IsoPs are far
(1, 8–14). Although EPA and DHA are more easily oxidized morepotentthanA-ringIsoPsinactivationofNrf2.Takenall
comparedwitharachidonicacid,DHA/EPAsupplementation togetherthedatasuggestthatJ-ringstructureiscrucialforNrf2
hasbeenassociatedwiththeinductionofcellularantioxidant activation.
responses (57–59). However, the biochemical pathways InvivoDHAandEPAaremoresusceptibletoauto-oxida-
responsiblefortheseeffectsarecurrentlynotwellunderstood. tionthanAA,becausetheypossessmoredoublebondsthan
Thegenerationofisoprostanesreflectingthenon-enzymatic AA.WefoundthatthelevelofF-ringIsoPsgeneratedfrom
processoflipidperoxidationofarachidonicacid,EPA,orDHA EPA and DHA is at least one order higher than that gener-
is a major consequence of oxidative stress, as exemplified by ated from AA after mice were supplemented with fish oil.
JANUARY26,2007•VOLUME282•NUMBER4 JOURNALOFBIOLOGICALCHEMISTRY 2535
n-3FattyAcidOxidationandNrf2
OurdatasupportthehypothesisthattheformationofJ-ring 31. Zhang,D.D.,andHannink,M.(2003)Mol.Cell.Biol.23,8137–8151
compounds generated from oxidation of EPA and DHA in 32. Yin,H.,Musiek,E.S.,Gao,L.,Porter,N.A.,andMorrow,J.D.(2005)
J.Biol.Chem.280,26600–26611
vivocanreachconcentrationshighenoughtoinduceNrf2-
33. Hong,F.,Sekhar,K.R.,Freeman,M.L.,andLiebler,D.C.(2005)J.Biol.
based antioxidant and Phase II detoxification defense
Chem.280,31768–31775
systems.
34. Lo,S.C.,andHannink,M.(2006)Mol.Cell.Biol.26,1235–1244
35. Zhang,R.,Khoo,M.S.,Wu,Y.,Yang,Y.,Grueter,C.E.,Ni,G.,Price,
REFERENCES
E. E., Jr., Thiel, W., Guatimosim, S., Song, L. S., Madu, E. C., Shah,
1. Chen,J.,andMehta,J.L.(2004)IndianHeartJ.56,163–173 A.N.,Vishnivetskaya,T.A.,Atkinson,J.B.,Gurevich,V.V.,Salama,
2. Barnham,K.J.,Masters,C.L.,andBush,A.I.(2004)Nat.Rev.DrugDiscov. G.,Lederer,W.J.,Colbran,R.J.,andAnderson,M.E.(2005)Nat.Med.
3,205–214 11,409–417
3. Harrison,N.,andAbhyankar,B.(2005)Curr.Med.Res.Opin.21,95–100 36. Sekhar,K.R.,Crooks,P.A.,Sonar,V.N.,Friedman,D.B.,Chan,J.Y.,
4. Lukiw, W. J., Cui, J. G., Marcheselli, V. L., Bodker, M., Botkjaer, A., Meredith,M.J.,Starnes,J.H.,Kelton,K.R.,Summar,S.R.,Sasi,S.,and
Gotlinger,K.,Serhan,C.N.,andBazan,N.G.(2005)J.Clin.Invest.115, Freeman,M.L.(2003)CancerRes.63,5636–5645
2774–2783 37. Andrade,M.A.,Chacon,P.,Merelo,J.J.,andMoran,F.(1993)ProteinEng.
5. Young,G.,andConquer,J.(2005)Reprod.Nutr.Dev.45,1–28 6,383–390
6. Woods,M.N.(2005)Nutr.Clin.Care8,24–30 38. Zhu,M.,andFahl,W.E.(2001)Biochem.Biophys.Res.Commun.289,
7. SanGiovanni,J.P.,andChew,E.Y.(2005)Prog.Retin.EyeRes.24,87–138 212–219
8. Cross,C.E.,Halliwell,B.,Borish,E.T.,Pryor,W.A.,Ames,B.N.,Saul, 39. Porter,N.A.,Caldwell,S.E.,andMills,K.A.(1995)Lipids30,277–290
R. L., McCord, J. M., and Harman, D. (1987) Ann. Intern. Med. 107, 40. Itoh, K., Wakabayashi, N., Katoh, Y., Ishii, T., O’Connor, T., and
526–545 Yamamoto,M.(2003)GenesCells8,379–391
9. Kehrer,J.P.(1993)Crit.Rev.Toxicol.23,21–48 41. Alam,J.,Stewart,D.,Touchard,C.,Boinapally,S.,Choi,A.M.,andCook,
10. Lee,J.M.,Li,J.,Johnson,D.A.,Stein,T.D.,Kraft,A.D.,Calkins,M.J., J.L.(1999)J.Biol.Chem.274,26071–26078
Jakel,R.J.,andJohnson,J.A.(2005)FASEBJ.19,1061–1066 42. Chan,J.Y.,andKwong,M.(2000)Biochim.Biophys.Acta1517,19–26
11. Soccio,M.,Toniato,E.,Evangelista,V.,Carluccio,M.,andDeCaterina,R. 43. Shibata,T.,Yamada,T.,Ishii,T.,Kumazawa,S.,Nakamura,H.,Masutani,
(2005)EurJ.Clin.Invest.35,305–314 H.,Yodoi,J.,andUchida,K.(2003)J.Biol.Chem.278,26046–26054
12. Moreira,P.I.,Smith,M.A.,Zhu,X.,Honda,K.,Lee,H.G.,Aliev,G.,and 44. Wakabayashi,N.,Dinkova-Kostova,A.T.,Holtzclaw,W.D.,Kang,M.I.,
Perry,G.(2005)DrugNewsPerspect.18,13–19 Kobayashi,A.,Yamamoto,M.,Kensler,T.W.,andTalalay,P.(2004)Proc.
13. Sacktor,N.,Haughey,N.,Cutler,R.,Tamara,A.,Turchan,J.,Pardo,C., Natl.Acad.Sci.U.S.A.101,2040–2045
Vargas,D.,andNath,A.(2004)J.Neuroimmunol.157,176–184 45. Li,X.,Lopez-Guisa,J.M.,Ninan,N.,Weiner,E.J.,Rauscher,F.J.,3rd,and
14. Zarbin,M.A.(2004)Arch.Ophthalmol.122,598–614 Marmorstein,R.(1997)J.Biol.Chem.272,27324–27329
15. Palozza, P., Sgarlata, E., Luberto, C., Piccioni, E., Anti, M., Marra, G., 46. Li,X.,Zhang,D.,Hannink,M.,andBeamer,L.J.(2004)ActaCrystallogr.
Armelao,F.,Franceschelli,P.,andBartoli,G.M.(1996)Am.J.Clin.Nutr. Sect.DBiol.Crystallogr.60,2346–2348
64,297–304 47. Kobayashi,A.,Kang,M.I.,Okawa,H.,Ohtsuji,M.,Zenke,Y.,Chiba,T.,
16. Vericel,E.,Polette,A.,Bacot,S.,Calzada,C.,andLagarde,M.(2003)J. Igarashi,K.,andYamamoto,M.(2004)Mol.CellBiol.24,7130–7139
Thromb.Haemost.1,566–572 48. Cullinan,S.B.,Gordan,J.D.,Jin,J.,Harper,J.W.,andDiehl,J.A.(2004)
17. Grundt,H.,Nilsen,D.W.,Mansoor,M.A.,andNordoy,A.(2003)Eur. Mol.Cell.Biol.24,8477–8486
J.Clin.Nutr.57,793–800 49. Morrow,J.D.,andRoberts,L.J.(1997)Prog.LipidRes.36,1–21
18. Roberts,L.J.,2nd,Montine,T.J.,Markesbery,W.R.,Tapper,A.R.,Hardy, 50. Reich,E.E.,Zackert,W.E.,Brame,C.J.,Chen,Y.,Roberts,L.J.,2nd,
P.,Chemtob,S.,Dettbarn,W.D.,andMorrow,J.D.(1998)J.Biol.Chem. Hachey,D.L.,Montine,T.J.,andMorrow,J.D.(2000)Biochemistry39,
273,13605–13612 2376–2383
19. Fam,S.S.,Murphey,L.J.,Terry,E.S.,Zackert,W.E.,Chen,Y.,Gao,L., 51. Morrow,J.D.,Roberts,L.J.,Daniel,V.C.,Awad,J.A.,Mirochnitchenko,
Pandalai,S.,Milne,G.L.,Roberts,L.J.,Porter,N.A.,Montine,T.J.,and O., Swift, L. L., and Burk, R. F. (1998) Arch. Biochem. Biophys. 353,
Morrow,J.D.(2002)J.Biol.Chem.277,36076–36084 160–171
20. Gao,L.,Yin,H.,Milne,G.L.,Porter,N.A.,andMorrow,J.D.(2006)J.Biol. 52. Yin,H.,andPorter,N.A.(2003)Anal.Biochem.313,319–326
Chem.281,14092–14099 53. Roberts,L.J.,2nd,andFessel,J.P.(2004)Chem.Phys.Lipids128,173–186
21. Sarsilmaz,M.,Songur,A.,Ozyurt,H.,Kus,I.,Ozen,O.A.,Ozyurt,B., 54. Kerwin,J.L.,andTorvik,J.J.(1996)Anal.Biochem.237,56–64
Sogut,S.,andAkyol,O.(2003)ProstaglandinsLeukot.Essent.FattyAcids 55. Kerwin,J.L.,Wiens,A.M.,andEricsson,L.H.(1996)J.MassSpectrom.31,
69,253–259 184–192
22. Iraz,M.,Erdogan,H.,Ozyurt,B.,Ozugurlu,F.,Ozgocmen,S.,andFadil- 56. Waugh,R.J.,Morrow,J.D.,Roberts,L.J.,2nd,andMurphy,R.C.(1997)
lioglu,E.(2005)Ann.Clin.Lab.Sci.35,169–173 FreeRadic.Biol.Med.23,943–954
23. Mori,T.A.,Puddey,I.B.,Burke,V.,Croft,K.D.,Dunstan,D.W.,Rivera, 57. Choi-Kwon,S.,Park,K.A.,Lee,H.J.,Park,M.S.,Lee,J.H.,Jeon,S.E.,
J.H.,andBeilin,L.J.(2000)RedoxRep.5,45–46 Choe,M.A.,andPark,K.C.(2004)BrainRes.Dev.BrainRes.152,11–18
24. Nalsen, C., Vessby, B., Berglund, L., Uusitupa, M., Hermansen, K., 58. Galvez,J.,deSouzaGracioso,J.,Camuesco,D.,Vilegas,W.,Monteiro
Riccardi, G., Rivellese, A., Storlien, L., Erkkila, A., Yla-Herttuala, S., SouzaBrito,A.R.,andZarzuelo,A.(2006)Fitoterapia77,515–520
Tapsell,L.,andBasu,S.(2006)J.Nutr.136,1222–1228 59. Bhattacharya,A.,Sun,D.,Rahman,M.,andFernandes,G.(2007)J.Nutr.
25. Calviello,G.,Palozza,P.,Franceschelli,P.,Frattucci,A.,Piccioni,E., Biochem.18,23–30
Tessitore,L.,andBartoli,G.M.(1999)Nutr.Cancer34,206–212 60. Morrow,J.D.,Hill,K.E.,Burk,R.F.,Nammour,T.M.,Badr,K.F.,and
26. Levonen,A.L.,Landar,A.,Ramachandran,A.,Ceaser,E.K.,Dickinson, Roberts,L.J.,2nd.(1990)Proc.Natl.Acad.Sci.U.S.A.87,9383–9387
D.A.,Zanoni,G.,Morrow,J.D.,andDarley-Usmar,V.M.(2004)Biochem. 61. Scholz,H.,Yndestad,A.,Damas,J.K.,Waehre,T.,Tonstad,S.,Aukrust,P.,
J.378,373–382 andHalvorsen,B.(2003)Cardiovasc.Res.59,945–954
27. Kwak,M.K.,Wakabayashi,N.,Itoh,K.,Motohashi,H.,Yamamoto,M., 62. Kinsella,B.T.,O’Mahony,D.J.,andFitzgerald,G.A.(1997)J.Pharmacol.
andKensler,T.W.(2003)J.Biol.Chem.278,8135–8145 Exp.Ther.281,957–964
28. Favreau,L.V.,andPickett,C.B.(1991)J.Biol.Chem.266,4556–4561 63. Musiek,E.S.,Gao,L.,Milne,G.L.,Han,W.,Everhart,M.B.,Wang,D.,
29. Venugopal,R.,andJaiswal,A.K.(1996)Proc.Natl.Acad.Sci.U.S.A.93, Backlund,M.G.,DuBois,R.N.,Zanoni,G.,Vidari,G.,Blackwell,T.S.,and
14960–14965 Morrow,J.D.(2005)J.Biol.Chem.280,35562–35570
30. McMahon,M.,Itoh,K.,Yamamoto,M.,andHayes,J.D.(2003)J.Biol. 64. Hirayama,A.,Yoh,K.,Nagase,S.,Ueda,A.,Itoh,K.,Morito,N.,Hirayama,
Chem.278,21592–21600 K.,Takahashi,S.,Yamamoto,M.,andKoyama,A.(2003)FreeRadic.Biol.
2536 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER4•JANUARY26,2007
n-3FattyAcidOxidationandNrf2
Med.34,1236–1242 68. Wu,J.T.,Lin,H.C.,Hu,Y.C.,andChien,C.T.(2005)Nat.CellBiol.7,
65. Cho,H.Y.,Reddy,S.P.,andKleeberger,S.R.(2006)Antioxid.Redox. 1014–1020
Signal.8,76–87 69. Dinkova-Kostova, A. T., Holtzclaw, W. D., Cole, R. N., Itoh, K.,
66. Ma, Q., Battelli, L., and Hubbs, A. F. (2006) Am. J. Pathol. 168, Wakabayashi, N., Katoh, Y., Yamamoto, M., and Talalay, P. (2002)
1960–1974 Proc.Natl.Acad.Sci.U.S.A.99,11908–11913
67. Thimmulappa,R.K.,Lee,H.,Rangasamy,T.,Reddy,S.P.,Yamamoto,M., 70. Eggler,A.L.,Liu,G.,Pezzuto,J.M.,vanBreemen,R.B.,andMesecar,A.D.
Kensler,T.W.,andBiswal,S.(2006)J.Clin.Invest.116,984–995 (2005)Proc.Natl.Acad.Sci.U.S.A.102,10070–10075
JANUARY26,2007•VOLUME282•NUMBER4 JOURNALOFBIOLOGICALCHEMISTRY 2537