Quinone compounds regulate the level of ROS production by the NADPH oxidase Nox4

Quinone

compounds

regulate

the

level

of

ROS

production

by

the

NADPH

oxidase

Nox4

Minh

Vu

Chuong

Nguyen

a,1,

*

,

Bernard

Lardy

a,d,1

,

Francis

Rousset

a

,

Florence

Hazane-Puch

e

_,

_Leilei

_Zhang

a

_,

_Candice

_Trocme´

d

_,

_Lena

_Serrander

b

_,

Karl-Heinz

Krause

c

,

Franc¸oise

Morel

a a

Universite´ JosephFourier,GREPIAGIMFRE3405,CNRS,EPHE,Grenoble,France

b

DepartmentofClinicalMicrobiology,UniversityHospital,Linkoping,Sweden

c

DepartmentofPathologyandImmunology,MedicalFacultyandUniversityHospital,Geneva,Switzerland

d

LaboratoiredeBiochimieEnzymeetProte´ine-DBTP,InstitutdeBiologieetdePathologie,UniversityHospitalCHU-Grenoble,Grenoble,France

e

LaboratoiredeBiochimieHormonaleetNutritionnelle-DBTP,InstitutdeBiologieetdePathologie,UniversityHospitalCHU-Grenoble,Grenoble,France

1. Introduction

Quinone derivative compounds inducea broad spectrum of effects in humans and representa class of toxicological inter-mediates,whichmayinduceacutecytotoxicity,immunotoxicity,

andcarcinogenesis,andleadtocellulardamage[1].Theycanbe formedfrombenzeneorpolycyclicaromatichydrocarbonsandare toxicologically important components of air pollution [2] and cigarettesmoke[3].Amongtheenzymesthatmodulatequinone toxicity,theNAD(P)Hquinoneoxidoreductasetype1(NQO1;EC 1.6.99.2) is one of the most important enzymes. NQO1 is a homodimeric flavoenzyme that catalyses the obligatory two-electron reduction of quinones to hydroquinones [4]. NQO1 activity can protect animal cells from the deleterious and carcinogeniceffects ofquinonesbypreventingtheone-electron reductionof quinonesbyotherreductases; however,prolonged exposure toenvironmental contamination, which contains qui-nones,damagespulmonarytissueandleadstoairway inflamma-tionandpathologiesthroughanoxidativestressmechanism[5,6]. NQO1ismainlycytosolicbutithasbeendescribedtobeexpressed atplasmamembrane[7].Semiquinoneradicalsofinhaledairborne particulate matter are believed to cause oxidative stress by generatingreactiveoxygenspecies(ROS),asreportedinthelung [8].Furthermore,dieselexhaustparticlescomposedofpolycyclic

ARTICLE INFO

Articlehistory:

Received3February2013 Accepted29March2013 Availableonline9April2013

Keywords:

NADPHoxidaseNox4

NAD(P)H:quinoneoxidoreductaseNQO1 Quinones

RedoxregulationofNox Reactiveoxygenspecies(ROS)

ABSTRACT

NADPHoxidaseNox4isexpressedinawiderangeoftissuesandplaysaroleincellularsignalingby

providingreactiveoxygenspecies(ROS)asintracellularmessengers.Nox4oxidaseactivityisthoughtto

beconstitutiveandregulatedatthetranscriptionallevel;however,wechallengethispointofviewand

suggestthatspecificquinone derivativescould modulate thisactivity. Infact,we demonstrated a

significant stimulation of Nox4 activity by 4 quinone derivatives (AA-861, tBuBHQ, tBuBQ, and

duroquinone)observedin3differentcellularmodels,HEK293E,T-RExTM_{,andchondrocytecelllines.Our}

resultsindicate thattheeffectisspecifictowardNox4versusNox2.Furthermore,we showedthat

NAD(P)H:quinone oxidoreductase (NQO1) may participate in this stimulation. Interestingly, Nox4

activityisalsostimulatedbyreducingagentsthatpossiblyactbyreducingthedisulfidebridge(Cys226,

Cys270)locatedintheextracellularE-loopofNox4.SuchmodelofNox4activityregulationcouldprovide

newinsightintotheunderstandingofthemolecularmechanismoftheelectrontransferthroughthe

enzyme,i.e.,itspotentialredoxregulation,andcouldalsodefinenewtherapeutictargetsindiseasesin

whichquinonesandNox4areimplicated.

ß2013ElsevierInc.Allrightsreserved.

Abbreviations:AA-861, 2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-p-ben-zoquinone; BQ, benzoquinone; CGD, chronic granulomatous disease; CHX, cycloheximide; DPI, diphenyliodonium; Duroquinone, 2,3,5,6-tetramethyl-1,4-benzoquinone; HQ, hydroquinone; tMetBQ, 2,5-dimethyl-1,4-benzoquinone; LDH, lactate dehydrogenase; 5-LO, 5-lipoxygenase; NQO1, NAD(P)H:quinone oxidoreductase1;PMA,phorbol12-myristate13-acetate;PMN, polymorphonu-clearcells;RLU,relativelightunit;ROS,reactiveoxygenspecies;tBuBHQ, 2,5-di-tert-butyl-1,4-benzo-hydroquinone; and tBuBQ, 2,5-di-tert-butyl-1,4-benzoqui-none.

*Corresponding authorat:GREPI AGIM FRE3405, CNRS,Universite´ Joseph Fourier,EPHE,CHUAlbertMichallon,BP217,38043Grenoble,France.

Tel.:+33476765752;fax:+33476766251. E-mailaddress:mvchuong@yahoo.fr(M.V.C.Nguyen).

1

Equalcontributors.

ContentslistsavailableatSciVerseScienceDirect

Biochemical

Pharmacology

j our na l ho me p a ge : w ww . e l se v i e r . com / l oc a te / b i och e mph a rm

aromatichydrocarbonsandquinonesareinvolvedinacellularROS productionassociatedwithlungfunctionimpairmentviaaNox4 redox-dependentmechanism[9].

Nox4belongstotheNADPHoxidase family thatcontains7 members[10–12].Thefunctionofthoseenzymesisexclusively dedicatedtotheproductionofROS,whichareessentialsignaling molecules.Primarydiscoveredinkidneytissue[13],Nox4appears tobeubiquitouslydistributed.Itsdysfunctionhasbeenlinkedto several pathologiesincludinghypertension [14],diabetes[15], atherosclerosis[16],cancer[17],osteoarthritis[18]and inflam-mation[19],andNox4representapotentialtherapeutic target [20].Despiteitswidedistribution,itsactivationmechanismsat themolecularlevelareunclear.Itis of therapeuticinterest to elucidatethemechanismofNox4NADPHoxidaseactivity.While activityofNox1,Nox2,andNox3largelydependsonthepresence of cytosolic activator or organizer subunits, no well-known NADPHoxidasepartners,besidep22phox_{,havebeenidentifiedor} showntoactivateNox4[21–23].However,twopartnersofNox4 havebeen described recently: theprotein disulfideisomerase (PDI)andthepolymeraseDNAdirected-delta-interactingprotein (Poldip2).Nox4isuniqueamongotherNoxisoenzymesinthatits activityisconstitutiveandmaydependonaspecificconformation ofthedehydrogenaseDHdomainthatshouldallowaspontaneous transferofelectronsfromNADPHtoFADandtothehemes[24,25]. DatafromvariousstudiesindicatedclearlythatNox4activityis regulatedatthemRNAlevel,implyingthatanincreaseordecrease of ROS production by Nox4 is correlated to an up regulation [9,16,26–33]ortoadecline[34]ofNox4transcripts.Althoughit hasnotbeenreportedyet,post-translationalregulationofNox4 oxidaseactivitycannotbeexcluded.

Inthisstudy,weprovidethefirstevidencethatNox4activity can be modulated independently of both transcriptional and translational processes. We showed a dual effect of quinone compounds, as being activators or inhibitors of Nox4 ROS production dependingon their redoxpotential (E).We suggest thatquinonesderivativescouldmodulateNox4activitybyaredox regulation pathway. Moreover, NQO1 is introduced as an intermediatebetweenquinonesandNox4activation,suggesting a functional interaction between Nox4 and NQO1. The results argueinfavorofamodulationofNox4activitybyanewfamilyof chemical compounds and suggest a potential role of Nox4 in pathologiesimplyingquinonetoxicity.

2. Materialsandmethods 2.1. Materials

Polyclonalantibodyagainst5-lipoxygenase(5-LO)(Cayman,Ann Arbor,USA)wasagenerousgiftfromF.Stanke(Grenoble,France). pEF6V5/HisBvector,penicillin,streptomycin,L-glutamine,

Dulbec-co’smodifiedEagle’smedium(DMEM),fetalbovineserum(FBS), trypsin,Earles’sbalancedsalts(EBSS),geneticin,Fluo-3/AM,BAPTA/ AM,andTRIzol1

werepurchasedfromInvitrogen(CergyPontoise, France).BlasticidinwasfromFunakoshiCo.(Japan);AMVreverse transcriptase was from QBiogene (Illkirch, France). Effectene transfection reagent was from Qiagen (Courtaboeuf, France). Luminol,ionomycin,horseradishperoxidase,thapsigargin, diphe-nyleneiodonium chloride (DPI), rotenone, N

v

-nitro-L-arginine

methylester hydrochloride(L-NAME),tiron,xanthine,

benzoqui-none (BQ), hydroquinone (HQ), 2,5-dimethyl-1,4-benzoquinone (tMetBQ),duroquinone,2,5-di-tert-butyl-1,4-benzo-hydroquinone (tBuBHQ), 2,5-di-tert-butyl-1,4-benzoquinone (tBuBQ), 2-(12-hydroxydodeca-5,10-diynil)-3,5,6-trimethyl-p-benzoquinone (AA-861),3,30_{-methylene-bis-4-hydroxycoumarin(Dicoumarol),}

cyclo-heximide (CHX) compounds were purchasedfrom Sigma(Saint QuentinFallavier,France).

b

-Mercaptoethanolwaspurchasedfrom

CarloErba(ValdeReuil,France).LightCyclerFastStartDNAMaster plus SYBR Green Ikit, protease inhibitorscocktail tablet, tosyl-lysine-chloromethyl ketone (TLCK), NADPH, NADH, xanthine oxidase, and lactate dehydrogenase (LDH) optimized kits were purchasedfromRoche(Meylan,France).Leupeptin,pepstatin,and ECLreagents werefromGEHealthcare(Orsay,France).UNI-ZAP humankidneylambdacDNAlibrarywaspurchasedfromStratagene (LaJolla,CA).HousekeepinggeneGAPDHwaspurchasedfromBD Bioscience (Pont de Claix, France). SuperScriptIII first-strand synthesiswasobtainedfromLifeTechnologies(SaintAubin,France). 2.2. Cellculture

Nox4 T-RExTM _{cells were a generous gift from K.H. Krause} (DepartmentofPathologyandImmunology,Gene`ve,Suisse)and weregeneratedfromHEK293cell(Invitrogen).TheHEK293Ecell linewaspurchasedfromInvitrogen(CergyPontoise,France). PLB-985humanmyeloidcelllines(wildtypePLB-985WT)orknockout forNox2(PLB-985KO-Nox2)wereagenerousgiftfromM.Dinauer (Department of Pediatrics, St. Louis, USA). PLB-985 cells were cultured at 378C under a 5% CO2 atmosphere in RPMI 1640 medium containing 2mM L-glutamine supplemented with 10%

fetal bovine serum and with 1% penicillin/streptomycin. The HEK293EcelllinewasmaintainedinDMEMsupplementedwith 10% (v/v) fetal bovine serum (FBS), 100units/mL penicillin, 100

m

g/mLstreptomycin,2mML-glutamineat378Cina5%CO2 humidifiedatmosphere.Five

m

g/mLBlasticidinwasaddedtothe culture medium of cells transfected with the mammalian expressionplasmidpEF6V5/HisB.

The T-RExTM _{system was used for Tetracycline-inducible} expressionofthegenesofinterest.T-RExTMcellsstablyexpressing Tetrepressorwereselectedby5

m

g/mLBlasticidinandmaintained inDMEMsupplementedwith10%(v/v)FBS,2mML-glutamineat

378Cina5%CO2humidifiedatmosphere.T-RExTMNox4cellsas comparedtoT-RExTM_{WTareabletoinduceNox4expression.Both} cellswereselectedwith5

m

g/mLblasticidinand400

m

g/mLG418. Nox4 expression was induced by the addition of 1

m

g/mL tetracyclineintheculturemedium.Experimentswereperformed aftertheincubationatsettimes.

2.3. Isolationofhumanneutrophils

Aspreviouslyreported[35],humanneutrophilswereisolated fromcitratedvenousbloodofhealthyvolunteers.Bloodsamples were dilutedtwice in PBS(137mMNaCl, 2.7mM KCl, 1.5mM KH2PO4,8mMNa2HPO4 pH7.3)containing1%(w/v)tri-sodium citrate,usinga33%(v/v)Hypaque–Ficollgradient.After20minof centrifugationat800gat208C,thepelletwassubmittedtoa hypotoniclysisfor5–15mininice.After5minofcentrifugationat 350g at48C,theneutrophilpelletwascollectedand washed onceinPBS.NeutrophilsweresuspendedinPBScontaining0.2% (w/v)BSAand0.5mMCaCl2ataconcentrationof107cells/mLand used for superoxide measurement by chemiluminescence and proteinextraction.

2.4. GenerationofplasmidconstructsforNox4andNQO1isoforms expression

and NQO1C were cloned into pEF6V5/HisB for mammalian expressionasdescribed[18].

2.5. Stabletransfectionofmammalianexpressionplasmids

HEK293Ecellsweretrypsinizedandcounted;4105_HEK293E cellswereseededin6-wellplatesandallowedtogrowfor24hto reach a 60% confluencein 2mL of culturemedium. Cells were transfected with0.4

m

g of vectors containing Nox4A or Nox4B accordingtothemanufacturingprotocol(Effectenetransfection reagent,Qiagen).After24hofculture,stabletransfectedcellswere selectedby5

m

g/mLblasticidinfor3weeksbeforeanalysis. 2.6. RealtimeRT-PCR

TotalRNAwasextractedusingaTRIzol1

reagentkitorRNeasy MiniKit (Qiagen) according tothemanufacturer’s instructions. TotalRNAwastreatedwithRNase-freeDNaseI(Qiagen).Oneor 5

m

gofRNAwereconvertedtocDNAbyreversetranscriptionwith 20U ofAMV reverse transcriptaseor SuperScriptIIIfirst-strand synthesis(LifeTechnologies).RealtimePCRswereperformedwith theLightCyclerFastStartDNAMasterplusSYBRGreenIkit(Roche) orQuantiTectSYBRGreenRT-PCRkit(Qiagen)asdescribed[18]. RealtimeRT-PCRwasconductedusingtheLightCycler1

Carousel-Based System (Roche) or a Stratagene Mx3005P (Stratagene). Briefly, the expression levels of human Nox4, NQO1 and housekeepingGAPDH,RPL27andRPL32mRNAsweredetermined usingspecificprimerschosentoincludeintronspanning(Table1). PCRs were carried out for each sample in triplicate. Gene expressionwasquantifiedusingthecomparativethresholdcycle (Ct) method. The amount of target gene, normalized to three endogenous reference genes (RPL27, RPL32 and GAPDH) was expressedrelativetothecontrolcells,asindicatedinFigures.The specificityoftheproductswasconfirmedforeachfragmentbya meltingcurveanalysisandgelelectrophoresis.

2.7. Proteinextraction

Twomethodswereused.First,HEK293Ecellswereharvested andlysedonicein1%(p/v)TritonX-100buffer(20mMTris–HCl,

150mMNaCl,1mMEDTA,pH7.6),containingaproteaseinhibitor cocktailcontaining2

m

Mleupeptin,2

m

Mpepstatin,and10

m

M TLCK l, for 20min at 48C. The lysate wasthen centrifuged at 10,000g for 10min at 48C and supernatant was used for Westernblottingexperiments.Second,humanneutrophilswere suspendedataconcentrationof5108_{cells/mLinPBScontaining} the protease inhibitor cocktail. The cells were sonicated for 310sat48Cand40WusingaBransonsonifier.Thehomogenate wascentrifugedat1000gfor15minat48Ctoremoveunbroken cellsandnuclei.Thepost-nuclearsupernatantwascentrifugedat 200,000g for 1h at 48C. The pellet consisting of crude membraneswassuspendedinthesamebuffer.

2.8. SDS-PAGEandWesternblot

TheTritonX-100solubleextract,membraneorcytosolfractions wereloadedona7%or10%SDS-PAGEandelectro-transferredto nitrocellulose, as previously described. Immunodetection was performed usingprimary polyclonal antibodiesagainst 5-lipox-ygenase (dilution 1:2000) following by a secondary antibody combined with horseradish peroxidase. The bound peroxidase activitywasmeasuredusingECLreagents.

2.9. DeterminationofROSproductionbychemiluminescence ROSproductionwasmeasuredaspreviouslydescribed [18]. CellswerewashedtwicewithPBS,detachedwithtrypsin,and collected by centrifugation (250g, 5min at 208C). The cell viabilitywassuperiorto90%,asdeterminedbythetrypanblue exclusion method. For measurement of Nox4 activity that is constitutive,5105 living cellsper well (96-wellplate) were resuspended in 250

m

l of PBS containing 20

m

M luminol and 10U/mLhorseradishperoxidase.Thephagocyteoxidase(Nox2) activitywasinitiatedby0.13

m

MPMAorby 0.15

m

MfMLPin medium containing 0.9mM CaCl2, 0.5mM MgCl2, and 20mM glucose.Relativeluminescenceunit(RLU)countswererecorded every minute for a total of 60min using a Luminoscan1 luminometer (Labsystems, Helsinki, Finland). Insome experi-ments,chemicalcompounds(quinones)wereaddedjustbefore luminescence measurement (Nox4) or before the addition of

Table1

PrimersusedinthisstudyforRT-PCRexperiments.tot,totalandexo,exogenous.

Name Primers(50_...30_{) Gene TM}

RealtimeRT-PCR

Nox4Forward CTGAATGCAGCAAGATACCGAGAT Nox4 668C

Nox4Reverse CTGGCTTATTGCTCCGGA Nox4 668C

NQO1Forwarda GAAGAGCACTGATCGTACTGGC NQO1tot 608C

NQO1Reversea GGATACTGAAAGTTCGCAGGG NQO1tot 608C

NQO1Forwardb GCAAGTCCATCCCAACTGACA NQO1exo 608C

NQO1Reverseb CTAGAAGGCACAGTCGAGGC NQO1exo 608C

RPL27Forward TGATGGCACCTCAGATCGC RPL27 608C

RPL27Reverse AGAGTACCTTGTGGGCATTAGG RPL27 608C

RPL32Forward TTAAGCGTAACTGGCGGAAAC RPL32 608C

RPL32Reverse GAGCGATCTCGGCACAGTAA RPL32 608C

GAPDHForwarda CATGAGAAGTATGACAACAGCCT GAPDH 608C

GAPDHReversea AGTCCTTCCACGATACCAAAGT GAPDH 608C

GAPDHForwardb GTGGTGGACCTGACCTGC GAPDH 708C

GAPDHReverseb CCCTGTTGCTGTAGCCAAATTCG GAPDH 708C

Semi-quantitativeRT-PCR

GAPDHForward BDBioscience GAPDH 558C

GAPDHReverse BDBioscience GAPDH 558C

ActinForward Clontech Actin 558C

ActinReverse Clontech Actin 558C

NQO1Forward GTTGGTACCATGGTCGGCAGAAGAGC NQO1 558C

NQO1Reverse GTTACTAGTTCATTTTCTAGCTTTGATCTGG NQO1 558C

stimulating agents (Nox2). For the xanthine-xanthine oxidase assay,ROSproductionwasmeasuredaspreviouslyinamedium containing luminol and horse radish peroxidase plus 1mM Xanthineand0.75mUIXanthineoxidaseinthepresenceornotof quinones.

2.10. IntracellularfreeCa2+_measurement

Intracellular free Ca2+ _{levels were measured by flow} cytometry (FACSCalibur, Becton Dickinson cytometer) using Fluo-3/AM. The fluorescence intensity of Fluo-3/AM dye was detectedin thegatedcellpopulationat526nm(FL1channel), whichisproportionaltotheintracellularfreecalciumlevel.Cells were harvested andthen counted. 107_{cells/mL,suspended in} Tyrode’s«free»buffer(10mMHEPES,145mMNaCl,2.5mMKCl, 10mMglucose,1.2mMMgCl2,pH7.3)wereincubatedinalight freeenvironmentwith10

m

MofFluo-3/AMfor30minat378C before analysis. After washing cells with the Tyrode’s ‘‘free’’ buffer containing 1.5mM CaCl2 or 1mM EGTA, basal fluores-cencewasmeasuredat526nmfor1min.Chemicalcompounds wereaddedtothetubescontaining5106_cellsper500

_m

_l,and measurement was continued for 5 more minutes. The data analysiswasperformedasfollow:gates(5s)werecreatedalong thetimeaxisofthedotplotswindowsatdefinedtimepoints,and the mean fluorescence intensity wasstatistically analyzed for everygate(WinMDI2.8software).

2.11. Lactatedehydrogenase(LDH)activitymeasurement

TheactivityofLDHwasmeasuredintheincubationmedium asanindexofplasmamembrane integrity.Cellswerewashed twicewithPBS,detachedwithtrypsin,collectedby centrifuga-tion (250g, 5min at 208C), and counted. In a 96-well plate, 5105 _{cells per well were resuspended in 250}

_m

_{l of PBS.} Cells were incubated with quinone compounds at different concentrationsfor60min.Atthe endof thetreatmentperiod, supernatantswerecollectedandassessedforLDHactivityona Hitachi/MODULAR automated analyzer using the Roche opti-mizedkit.Thecellviabilitywasexpressedasthepercentageof LDH released into the incubation medium versus total cell activitymeasuredaftercell lysiswith1%(v/v)Triton-X100. 2.12. Statisticalanalysis

DatawereexpressedasthemeanSD.Statisticalanalysiswas performedusingMannandWhitneyorKruskalandWallistests.The results are reported when significantly different (p<0.05) from control.

3. Results

3.1. OverexpressionofNox4inHEK293Ecelllineandconstitutive NADPHoxidaseactivity

Nox4wasidentifiedforthefirsttimeintubularcellsofadult kidneyanditsoxidaseactivitywasshowntobeconstitutive[13]. We,therefore,performedthefunctionalcharacterizationofNox4 inHEK293E(humanembryonickidney)celllinewhichexpresses Nox4 mRNA but not Nox1 and Nox2, (unpublished data). The oxidaseactivityofthesecellswasverylowandwasnotsensitiveto PMA(unpublisheddata).Westablyoverexpressedtwoisoformsof Nox4,Nox4A theactive isoform(GenbankTM_{accessionnumber:} AF254621), anda splicing variantNox4B(GenbankTM _accession number: AY288918)that is unable toproduce ROSdue tothe absenceofoneNADPHbindingsites[36].Theresultsshoweda significant increase of mRNA expression encoding both Nox4 isoformscomparedtoWTcells(Fig.1A).Whiletheoverexpression of Nox4B failed to produce ROS, Nox4A demonstrated a spontaneous ROS generation compared to WT-HEK293E cells (Fig.1B)andthatcorrespondedtoaNADPHoxidaseactivitysinceit wassolelyinhibitedbyDPI(inhibitorofflavoproteins)andTiron (superoxide scavenger) but not by rotenone (mitochondrial respiratory chain inhibitor) and L-NAME (competitive inhibitor

ofNOsynthase)(Fig.1C).

3.2. StimulationofNox4activitybytwoquinonederivatives Next,weevaluatedtheeffectoftwoquinonecompounds, AA-861 and tBuBHQ on Nox4 constitutive activity. Incubation of Nox4A-HEK293EcellswithAA-861ortBuBHQledtoanincreaseof ROS production (Fig. 2A). The stimulated activity reached its optimum level at 10min or 20min after AA-861 or tBuBHQ incubation respectively.Theincubation withWT-HEK293Ecells didnotleadtoahighlevelofROSproduction.Infact,onlysmall increase of ROS wasobserved reflecting thestimulation of the endogenous Nox4 by those two quinones (Fig. 2A, inset). The specificityofaNADPHoxidaseactivitywasconfirmedbyusingDPI whichhadaninhibitoryeffectwhereasrotenoneandL-NAMEdid

not(Fig.2A).

ToconfirmthattheobservedeffectofAA-861andtBuBHQwas specifically related to Nox4 proteins, we used two other characterizedcelllinesthatover-expressedNox4:human chon-drocyteC-20/A4[18]andNox4T-RExTMinwhichNox4expression couldbetemporallyinducedbytheadditionoftetracycline[27]. Consistently, theconstitutive ROS production in Nox4 C-20/A4 cellsorin4h-inducedNox4T-RExcells wasenhancedafterthe additionofAA-861ortBuBHQ(Fig.2BandC).Itisnoticeablethat

Fig.1.CharacterizationofNox4expressionandconstitutiveactivityinHEK293Etransfectedcells.(A)Real-timeRT-PCRanalysisofNox4mRNA.TotalRNAwasextractedfrom non-transfectedWT-,Nox4A-,orNox4B-HEK293Etransfectedcells.(B)MeasurementofconstitutiveROSproductionbyHEK293Ecells.TheROSproductionwasmeasuredby chemiluminescenceon5105

intactWT-,Nox4A-,orNox4B-HEK293Etransfectedcells.(C)ThespecificityofconstitutiveNADPHoxidaseactivityofHEK293Ecells overexpressingNox4Awasconfirmedbyusingseveralinhibitors:DPI(10mM),rotenone(Rot,1mM),L-NAME(L-N,100mM),tiron(0.5mM).Resultsareexpressedasthesum

theROS production of uninduced T-RExTM _{cells, which do not} expressNox4,wasnotaffectedbyAA-861orbytBuBHQindicating thatthosetwoquinonesdonotproduceROSspontaneouslyinthe presenceofcells(Fig.2C).Consideringtheshorttimecourseof AA-861andtBuBHQstimulation,itisunlikelythattheaugmentation of Nox4 activity observed was correlated toan increase of its protein expression. However, to rule out this eventuality, we inhibitedNox4proteinsynthesisbyusingcycloheximide(CHX),an inhibitorofproteintranslation.ThetimecourseoftheNox4mRNA expressioninducedbytetracyclineindicatedthatthemaximum levelwas reached at4hand thenremained constant until8h (Fig.2D).After2hofNox4inductionbytetracycline,timepoint whentheNox4mRNAsynthesisisstillincreasing,CHXwasadded inthemediumofNox4T-RExTM_{celllineandoxidaseactivitywas} measuredaftertwoadditionalhoursofincubation.Asexpected, CHX-treatedcellsexhibitedalowerconstitutiveoxidaseactivity (66%), indicating a lower amount of induced Nox4 proteins, comparedtonon-CHX-treatedcells(Fig.2E,whitebars).Although anincreaseofNox4proteinwasabolishedunderCHXtreatment, Nox4activitywasstillstimulatedbyAA-861incubation(Fig.2E, blackbarscomparedtowhite).Theseresultssupportourabove

consideration that AA-861 specifically increases Nox4 activity independentlyofitsproteinlevel.

3.3. MolecularmechanismandsignificanceofNox4activity stimulationbyquinonecompoundsAA-861andtBuBHQ: 5-lipoxygenaseandcalciumfluxarenotinvolvedintheactivationof Nox4

Since AA-861 was described as a 5-lipoxygenase (5-LO) inhibitor,we investigatedwhether 5-LO couldbeimplicatedin thestimulationofNox4activity.5-LOwasoriginallyreportedtobe expressedinleucocytesandsomeepithelialcells[37],butnodata concerning5-LOexpressionintheHEK293Ecelllineareavailable. Wefoundthatneither5-LOmRNAnor5-LOproteinwasexpressed inHEK293Ecells(Fig.3AandB),whichexcludestheinvolvement of 5-LO in Nox4 activation by AA-861. Furthermore, MK-886, another5-LO inhibitordidnotmodulateNox4 activity (unpub-lishedobservations).

TherelationshipbetweencalciumionfluxandNox4activity hasnotbeenclearlydescribed.Pedruzzietal.[16]reportedthat Nox4 activity located in the endoplasmic reticulum was

Fig.2.AA-861andtBuBHQstimulateROSproductionbyNox4A-HEK293Ecellsandtwoothercellularmodels.(A)StimulationofROSgenerationbytheendogenousNox4 (inset)inWT-HEK293EcellsorNox4A-HEK293Ecellswith50mMtBuBHQor10mMAA-861.TheROSproductionwasmeasuredbychemiluminescenceon5105

intact cellsandresultsareexpressedasthesumofRLUmeasurementsfor90min.ThespecificityofNADPHoxidaseactivitystimulationbytBuBHQorAA-861wasdemonstratedby usingseveralinhibitors:DPI(10mM);rotenone(Rot,1mM)andL-NAME(L-N,100mM).ValuesrepresentthemeanSDoftriplicatedeterminationsofatleast3independent experiments,*p<0.05versuscellswithoutactivators.ROSproductionbyhumanchondrocytecelllineC-20/A4(B)orNox4T-RExTM

cells(C)[27]induced4hornotwith1mg/mL tetracycline(tet-induced)wasmeasuredaftertheadditionornotof10mMAA-861or50mMtBuBHQ.ResultsareexpressedasthesumofRLUmeasurementsfor90minforeach condition.ValuesrepresentthemeanSDoftriplicatedeterminationsandarerepresentativeofatleast3independentexperiments,*p<0.05versuscellswithoutactivators.(D) Real-timeRT-PCRanalysisofNox4mRNAtimecourseexpression.TotalRNAwasextractedfromNox4T-RExTM

exposedtotetracyclinefrom0to8h.(E)Inordertostopdenovo proteintranslation,tet-inducedNox4T-RExTM

cellsweretreatedwithcycloheximide(CHX).After2hoftetracyclineinduction,tet-inducedNox4T-RExTM

cellswereincubatedwith CHX(100mg/mL)ornotfor2morehoursandthenROSmeasurementsbychemiluminescenceof5105

intactcellswasperformed.ResultsareexpressedasthesumofRLU measurementsfor90minandareshownaspercentageofROSproducedbyNox4T-RExTM

concomitanttoCa2+_{oscillationafter exposureto} 7-ketocholes-terol.Howeverinapreviousstudy,weshowedthationomycin (calcium ionophore) had no effect on the Nox4 activity in chondrocytes C-20/A4cells over-expressingNox4A [18]. Since tBuBHQwasdescribedasaSERCApumpinhibitor,itisnecessary toevaluate thepotentialcontributionoftheCa2+_fluxonNox4 stimulation.Tothispurpose,we measuredthe calciumflux in Nox4-HEK293E cells by FACs using a fluorescent Fluo-3/AM probeandcomparedthecalciumchangestoNox4activation.As illustratedinthefigure3(Fig.3CandD),anincreaseofcytosolic calciumfromintracellularstoreswasobservedinthepresence of extracellular EGTA after adding ionomycin, thapsigargin or tBuBHQ whereas the addition of AA-861 or tBuBQ, two compoundsthatstimulateNox4activitywereunabletomodify

Ca2+ _{level. Furthermore, thapsigargin or ionomycin could not} modulate the constitutive activity of Nox4 in Nox4-HEK293E cells (Fig. 3E). Same experiments were performed in the presenceof1.5mMCaCl2intheextracellularmediumwithout EGTA andled tothe same results (unpublishedobservations). Thesedatastronglysuggestthatthereisnocorrelationbetween Ca2+_{fluxandNox4activity.}

3.4. QuinonestructuremayberesponsibleforthemodulationofNox4 activity

AA-861 and tBuBHQ share the same quinone moiety. To evaluate whether quinone structure was responsible for Nox4 activation, the ROS production by Nox4-HEK293E cells was

Fig.3.AA-861ortBuBHQNADPHoxidaseactivitystimulationisindependentof5-lipoxygenaseandofcalciumflux.(A)5-lipoxygenasemRNAexpressioninHEK293Ecells. TotalRNAwasextractedfromHEK293EandBlymphocytes(BL),asemi-quantitativeRT-PCRwasperformedusingspecificprimersfor5-lipoxygenase(5-LO).The housekeepingactingene(Act)wasusedasapositivecontrol.(B)5-lipoxygenaseproteinexpressioninHEK293Ecells.150mgoftotalproteinsfromHEK293EWT,and75mg ofcytosolic(Cyto)proteinfraction(positivecontrol)fromneutrophils(PMN)wereloadedona7%SDS-PAGEgel.5-lipoxygenaseproteinswerecharacterizedwithaspecific monoclonalantibodyagainst5-LO.(C)and(D)CalciumfluxmeasurementwasassessedbyafluorescentprobeinNox4A-HEK293Ecells.Intactcellswereincubatedwith 10mMFluo3/AMwithorwithout20mMBAPTA,during30minat378C,washedandresuspendedat106_{cells/mL.Calciumfluxvisualizationwasperformedbyflow}

cytometry.Thearrowindicatesthetimeoftheadditionofionomycin(Iono,10mM),AA-861(10mM),tBuBHQ(50mM),orthapsigargin(Thaps,10mM)inpresenceof1mM EGTA(C);orthatoftBuBQ(50–200mM)andtBuBHQ(50–200mM)(D).Resultsareexpressedasthemeanofthefluorescenceintensity(FL1change)measuredevery5s(E). 5105

measuredafterincubationwithvariousquinonederivativesthat differ with respect to their reduced or oxidized state and substitution levels.Theresults shownin Table 2 illustratetwo opposite effects. Duroquinone as well as tBuBHQ, tBuBQ (the oxidativeformoftBuBHQ),andAA-861,stimulatedNox4activity at a starting concentration of 1

m

M, whereas BQ (30

m

M), HQ (30

m

M) and tMetBQ(100

m

M)inhibitedthis activityin a dose dependentmanner.Wealsoconfirmedtheinhibitoryeffectofthe naphthoquinone,plumbagin,onNox4activityasreported[38,39]. To exclude a non-specific spontaneous ROS production by quinonecompoundsincontactwithmembranephospholipidsas describedfortBuBHQ[40],wemeasuredtheROSsynthesisinthe presenceof25

m

gphosphatidylcholine.Theresultsillustratedin Table3 showthat therewasno ROSenhancementbyquinones afteradditionofphospholipidsversuscontrol,exceptforaslight increase observed as expected with tBuBHQ, as previously reported[40].Furthermore,quinoneconcentrationsusedinthis studywerenotcytotoxicasexaminedbytheLDHactivity(Table3). 3.5. EffectofquinonesonNox2

WenextevaluatedwhetherthosequinonescouldaffectNox2 oxidaseactivityinisolatedhumanneutrophilswhichexpressonly

Nox2,theredoxcoreofphagocyteNADPHoxidase.Comparedto thewellknownactivatorPMA,noNADPHoxidaseactivationwas observedwhenneutrophilswereincubatedwithAA-861,tBuBQ, or duroquinone (Fig. 4A) as opposed to what was previously observedinNox4T-RExTMcells(Table2).However,slightoxidase activationwasobservedupontheadditionoftBuBHQ,probably duetoitseffectonthecalciumfluxsince thapsigargin,another SERCApumpinhibitor,orionomycingavesimilarresults (unpub-lisheddata).Onthecontrary,thequinonesBQ,HQ,tMetBQ,and plumbaginalsoinhibitedthePMAstimulatedneutrophiloxidase activity(Fig.4B).Byusinganinvitroxanthine/xanthineoxidase superoxide production system, we observed that the ROS productionwasabolishedin thepresenceof30

m

MBQ, 30

m

M HQ,and100

m

MtMetBQwhilenoeffectonthexanthine/xanthine oxidaseROSproductionwasnoticeablewith50

m

Mduroquinone, 50

m

M tBuBHQ, 50

m

M tBuBQ,or 10

m

MAA-861 (unpublished data).UsingdifferentiatedPLB-985asanothercellularmodel,we obtained similarresultsthan those observedwithhumanPMN (Fig.4C). We,next, evaluated whetherquinonescouldincrease Nox2activityalreadyactivatedbythephysiologicalagonists(the formylatedpeptidefMLP)indifferentiatedPLB-985cellsandfound that AA-861, tBuBQ, and duroquinone did not stimulate Nox2 activity(Fig.4D).

These results suggest that BQ, HQ, and tMetBQinhibit ROS production through an antioxidant property. On the contrary, duroquinone, tBuBHQ, tBuBQ, and AA-861 appear to stimulate specificallyNADPHoxidaseactivityofNox4.

3.6. Putativequinonebindingsite(Qsite)onNox4

Quinonescompoundsbindtoitstargetproteinsthroughwell describedubiquinonebindingsites[41].Basedoncomputational prediction,aconsensussequenceforapredictablequinonebinding site or Qsite was suggested: ‘‘aliphatic-(X)3-H-(X)2-3-(Leu/Thr/ Ser)’’[42].TheanalysisoftheNox4proteinsequenceshoweda similar putative motif in the fifth transmembrane domain (‘‘203LLTLHVS209’’) that is missing in Nox2 sequence (Fig. 5A). Therefore, quinones could regulate Nox4 activity by binding directlytotheproteinthroughthisputativedomain.Toinvestigate therelevanceofthispotentialQsiteinNox4protein,wemutated

Table2

Quinoneeffect on Nox4 ROS production of Nox4A-HEK293cells. Data were expressedasthemeanSD.

Effect Structure [C]mM ROSproduction None PBS 9.010.8 Activators AA-861 1 24.260.91* 5 34.401.16* 10 45.700.77* 50 37.202.14* tBuBHQ 1 14.200.76 10 36.010.68* 50 61.740.78* 100 38.901.82* tBuBQ 1 13.580.42 10 32.700.22* 50 36.500.63* 100 34.991.22* Duroquinone 1 35.691.65* 10 30.211.70* 50 29.301.80* Inhibitors Benzoquinone 0.01 7.880.36 0.5 8.090.37 1 7.690.51 3 3.630.15* 30 1.010.11* Hydroquinone 0.01 7.650.50 0.5 8.090.37 1 8.700.52 3 4.180.34* 30 0.120.07* tMetBQ 1 10.091.68 10 12.700.43 50 12.870.82 100 1.320.20* 200 0.380.01* Plumbagine 0.01 7.400.67 0.5 6.640.27 1 4.000.22* 5 1.930.29* 10 1.340.09* *

p<0.01versusPBStreatedcells.

Table3

CytotoxicityofquinonesandROSproductioninthepresenceof phosphatidylcho-line.

Structure [C]mM Toxicity%LDHa _{ROSproduction}b PBS PC – – 10.800.29 1.90.1 2.70.2 Benzoquinone 1 11.370.53 ND ND 30 10.720.53 2.00.1 2.30.2 Hydroquinone 1 10.730.53 ND ND 30 10.990.80 2.10.1 2.30.1 tMetBQ 50 12.790.97 2.30.3 2.40.1 200 12.661.00 ND ND tBuBQ 50 12.440.42 2.30.1 2.20.2 tBuBHQ 50 11.931.41 4.40.9 6.80.4 Duroquinone 50 11.031.13 2.30.1 2.80.2 AA-861 10 12.000.84 2.30.1 2.40.3 a

CytotoxicityevaluationofquinonecompoundsonHEK293Ecellline.5105

intactHEK293Ewereincubatedwithchemicalcompoundsandlactate dehydroge-nase(LDH)activity wasassessed60min laterintheculturemedium.Values representthemeanpercentageofLDHactivitycomparedtototalLDHenzymatic activity(cellslyzedbyatreatmentwith1%tritonX-100).

b

key amino acid residues of that sequence, i.e., substitutions of L203G and S209A (Fig. 5A). Our results showed that those mutationsdidnotmodifythestimulatoryeffectofthequinone tBuBHQ on Nox4 activity which remainedsimilar toWT Nox4 (Fig.5B).Sinceourdatashowedthatquinonesdidnotactivate Nox2 activity nor enhance its activity (Fig. 4), we therefore exchanged the sequence 201Y...Y215 of Nox4, which contains theputativeQbindingofNox4,withthehomologoussequenceof Nox2 216_F...G230_{. This Nox4/2 chimera protein remained still} stimulatedbytBuBHQ (Fig.5B). Thoseresultssuggestthat this putativeQsitedomainonNox4isnotinvolvedinthestimulation ofNox4activitybyquinonecompounds.

3.7. RedoxregulationofNox4activity

Arecentstudy[43]showedthatstructuralmodificationsofthe E-loop,bydeletionorexchangeofcysteine,hadanimpactonthe production rate and nature of ROS generated. The authors hypothesizedthat extracellularcysteinesC226 and C270might form a disulfide bridge that modulates Nox4 ROS production. Quinonescompounds arehighly reducing moleculescapableof breakingdisulfidebridges and, therefore,couldmodulateNox4 activitythrougha redoxregulation.Totest this hypothesis,we measuredtheeffectofknown reducingagentsonNox4 activity andfoundthatintet-inducedNox4T-RExTM_{cells,theadditionof} 80

m

M NADPH, NADH, or

b

-mercaptoethanol increased signifi-cantly the ROS production compared to non-induced and untreatedNox4T-RExTM_{cells(Fig.5C).Theseresultssuggestthat} reducingagentsstimulateNox4activityandpointoutapossible

mechanismby whichquinonesmight regulateNox4activityby reducingtheE-loopdisulfidebridge.

3.8. InvolvementofNQO1onNox4activationbyquinone

Quinonecompoundsaremetabolizedthroughdifferent path-waysand one of them involves theNAD(P)Hoxidoreductase 1 (NQO1). By RT-PCR and sequencing, we revealedthat Nox4 T-RExTM_{cellsexpressedtwotypesofmRNAencodingfor} NQO1:N-QO1A,theactiveformandNQO1C,theinactiveform,whichlacks2 essentialaminoacidresidues(Tyrosine-126and-128)necessary foritsquinonebindingcapacity[4].Toinvestigatethepotential role of NQO1 in the stimulation of Nox4 activity by quinone compounds,weuseddicoumaroltoinhibitNQO1.Pre-incubation of the tetracycline induced Nox4 T-RExTM _{cells with 100}

_m

_M dicoumarol inhibited Nox4 constitutive activity as well as its stimulationbytBuBHQ(Fig.6A).Tofurtherinvestigatetheroleof NQO1, we stably overexpressed NQO1A and NQO1C in Nox4-HEK293andconfirmedthepresenceoftheexogenousNQO1Aand NQO1C mRNAin thecorresponding transfected cells compared withtheemptyvectorcontroltransfectedcells(Fig.6B). Further-more,byrealtimeRT-PCRweshowedthemRNAlevelofNQO1A andNQO1Cincreased2and8fold,respectively,comparedtothe control Nox4-HEK293 cells (Fig.6B). We then investigated the consequencesoftheseoverexpressionsonNox4activity.Results showed that overexpression of NQO1A enhanced twice the stimulatoryeffectof tBuBHQon Nox4activitycomparedtothe controlemptyvectorcells.Thisisconsistentwiththeincreaselevel ofNQO1AmRNAobservedpreviously(Fig.6B).Onthecontrary,

Fig.4.EffectofquinonederivativesontheactivationofNox2oxidaseactivity.(A)Thereactiveoxygenspeciesproductionwasmeasuredbychemiluminescenceon5105

intactPMNcellsinthepresenceofanincreasingconcentration(1mM,10mM,or50mM)ofAA-861,tBuBQ,tBuBHQ,orduroquinone.ResultsareexpressedasthesumofRLU measurementsfor60minandareshownaspercentageofROSproducedbyPMNstimulatedbyPMA(0.13mM).ValuesrepresentthemeanSDoftriplicatedeterminationsof atleast3independentexperiments,*p<0.05versuscellswithoutPMAactivationandquinones.(B)PMN(5105_{cells)werestimulatedbyPMA(0.13mM)inthepresenceornotof}

BQ(1mM,3mM,and30mM),HQ(1mM,3mM,and30mM),tMetBQ(10mM,50mM,and100mM)orplumbagin(1mMand5mM).ResultsareexpressedasthesumofRLU measurementsfor90minandarereportedasapercentageofROSproducedbyPMNstimulatedbyPMA(0.13mM).ValuesrepresentthemeanSDoftriplicatedeterminationsofat least3independentexperiments,*p<0.05versuscellsactivatedbyPMAwithoutquinones.(C)Thereactiveoxygenspeciesproductionwasmeasuredbychemiluminescenceon 5105

intactdifferentiatedPLB-985cellsinthepresenceof30mMBQ,30mMHQ,50MetBQ,10mMAA-861,50mMtBuBQ,50mMtBuBHQ,or50mMduroquinone.Resultsare expressedasthesumofRLUmeasurementsfor60minandareshownaspercentageofROSproducedbydifferentiatedPLB-985cellsstimulatedbyfMLP(0.15mM).Valuesrepresent themeanSDoftriplicatedeterminationsofatleast3independentexperiments,*p<0.05versuscellswithoutfMLPactivationandquinones.(D)DifferentiatedPLB-985 (5105_{cells)werestimulatedbyfMLP(0.15mM)inthepresenceornotof30mMBQ,30mMHQ,50MetBQ,10mMAA-861,50mMtBuBQ,50mMtBuBHQ,or50mMduroquinone.}

although the overexpression of NQO1C is higher than that of NQOA,itdidnotmodifythestimulatoryeffectwhichiscomparable to that observed with the control empty plasmid (Fig. 6C). Together,thesedatasuggesttheimplicationofNQO1enzymesin quinone-stimulatedNox4activity.

4. Discussion

Our studyfocuseson thecapacityof quinonecompoundsto modulate the NADPH oxidase activity of Nox4. Quinones are importanttoxiccompoundspresentinairpollutionandabundant inallburntorganicmaterial,includingurbanairparticles,cigarette smoke,anddieselexhaustparticles(DEP)[44].DEPwerereported to increase Nox4 ROS production after 1h of incubation, suggestingthe implication of Nox4in the quinonemetabolism [9]. Considering the structure/function relationship of the qui-nonesusedinthisstudy(Table2),weobservedadualstimulation/ inhibitioneffect thatdepends on their substitutionstate: Nox4 activity was stimulated by AA-861, duroquinone, tBuBQ, and tBuBHQwhereasotherlesssubstitutedderivativessuchasBQ,HQ,

tMetBQ, and plumbagin, developed antioxidant properties (Table2).ThespecificityofAA-861,duroquinone,andtBuBQfor Nox4 over Nox2, suggests either a direct effect of quinone derivatives in the molecular dynamics of electron transfer mediated by Nox4, or an indirect role on a quinone sensitive functionalpartnerofNox4.Inthiswork,wesuggestanewaspect of Nox4 oxidase activity regulation that takes place not only atatranscriptionallevelasusuallydescribed[13,27,45]butalsoat a post-translational level as described above with quinone molecules.

Fig.5. Putative Q site and redox regulationof Nox4 activity.(A) Schematic representationofthealignmentofpartialsequencesofNox4,Nox2andNox4 mutants(Nox4L203G/S209A

andNox4/2).Grayboxrepresents thepredicted5th transmembranedomain.BoxedaminoacidsonNox4sequencecorrespondtothe putativeQsiteandboldlettersarekeyaminoacidsdescribedfortheQsite[42]. Underlinedaminoacidsrepresentaminoacidsthathavebeenexchangedfrom Nox2sequencetoproducethechimeraNox4/2.(B)MeasurementofROSgeneration bychemiluminescenceon5105_{intacttransfectedNox4-,Nox4}L203G/S209A_-and

Nox4/2-HEK293cellswithorwithout50mMtBuBHQ.(C)MeasurementofROS generationbychemiluminescenceon5105

intactcells.ROSproductionbyNox4 T-RExTM

cellinduced4hornot with1mg/mL tetracycline(tet-induced)was measuredaftertheadditionornotofNADPH,NADH,b-mercaptoethanol(b-ME) (80mMofeach),ortBuBHQ(50mM).ResultsareexpressedasthesumofRLU measurementsfor90minforeachcondition.ValuesrepresentthemeanSDof triplicatedeterminationsofatleast3independentexperiments,*p<0.05versuscells withoutredoxcompounds.

Fig.6.EffectofNQO1ontBuBHQactivationofROSproductionbyNox4.(A)Nox4 T-RExTM

cellswereinduced(ornot)with1mg/mLtetracyclinefor4h.Dicoumarol was added in the culture medium 2h before ROS measurements. Chemiluminescenceassaywasdoneon106

intactcellsperwell,activatedornot by50mMtBuBHQ.ResultsareexpressedasthesumofRLUmeasurementsfor 90min.ValuesrepresentthemeanSDoftriplicatedeterminationsofatleast3 independent experiments, *p<0.05 versus cells in same condition without dicoumarol.(B)Real-timeRT-PCRanalysisofexogenousandtotalNQO1mRNAin Nox4A-,Nox4A/NQO1C-andNox4A/NQO1A-HEK293cells.Leftpanel,realtimeRT-PCR showingthe presenceofexogenousNQO1mRNAinNox4A/NQO1C-and Nox4A/ NQO1A-HEK293cellsandnotinNox4A-HEK293transfectedwiththecontrolpEFb emptyplasmid.N.D.,notdetected.Rightpanel,realtimeRT-PCRshowingtheincrease ofNQO1mRNAlevelinNox4A/NQO1C-andNox4A/NQO1A-HEK293cellscomparedto Nox4A-HEK293transfectedwiththecontrolpEFbemptyplasmid.(C)Nox4A-HEK293E cellsweretransfectedwiththeemptyvectorpEFb(Control)orcontainingNQO1Aor NQO1B.Thereactiveoxygenspeciesproductionwasmeasuredbychemiluminescence on5105

Quinonesarebioreactivemoleculesthataresensitivetoredox mechanism.Ubiquinone,forexample,isamobileelectroncarrier ofmitochondriaandahighlyhydrophobicmoleculethatdiffuses into the core of innermembrane wherethe electron transfer proceedsthroughaQcycleoneithersideofmembrane[41,46]. Apredictablequinonebindingsitehasbeensuggested[42]and we identified a similar domain in Nox4 protein sequence ‘‘203_LLTLHVS209_{’’. However, the implication of such domain} was not confirmed since the ability of quinones to stimulate Nox4activitywasnot abolishedwhenappropriate residuesof thissequenceweremutated(Fig.5AandB).

Alternatively,Takacandhiscolleagues[43]haveshownthat disulfidebridgesformedbytwocysteinesC226andC270maybe involvedinthemaintenanceoftheextracellularE-loopintegrity. Indeed, the disruption of the bridge by mutagenesis modified notably Nox4 ROS production [43]. Disulfide bridges with a standardredoxpotential(E0)of180mV[47]couldbereducedby NADPH, NADHand

b

-mercaptoethanolthat possess a lowerE0 (324,320,and253mV,respectively)[48].Ourresultsusing those reducing agents indicated that Nox4 activity could be stimulatedbyextracellularredoxmodifications,possiblythrough thereductionofitsdisulfidebridge.Interestingly,duroquinone, whichenhancesNox4activity,hasalowE0(260mV)[49]and then could theoretically reduce disulfide bridge. Conversely, compoundssuch asBQ, HQ and tMetBQthat do not stimulate Nox4exhibitahigherE0,78mV,78mVand67mV,respectively, comparedtothatofthedisulfidebridge.Thus,itispossiblethat quinonesexhibitinganE0lowerthanthatofdisulfidebridgeare abletostimulateNox4activity.

NAD(P)H-quinone oxidoreductase-1 (NQO1) is a broadly distributedFAD-dependentflavoproteinthatcatalysesthe reduc-tionofawidevarietyofquinonecompounds.Itreducesquinones, bythetransferof2electrons,tothecorrespondinghydroquinones whicharetheelectrondonorstatecapabletoinitiatereductive reactions.WeshowedthatNQO1isexpressedinuninducedor tet-induced Nox4 T-RExTM _{cells; however, the stimulation of ROS} productionbyquinonesoccursonlywhenNox4expressionwas induced. Moreover, the increase of Nox4 ROS production by quinones was inhibited by a well known inhibitor of NQO1, dicoumarol.Noticeably,dicoumarolalsodecreasesNox4 constitu-tiveactivityinquinone-untreatedTet-inducedNox4T-RExTMcells (Fig.6A),possiblybyinhibitingtheendogenousNQO1enzymes. More specifically, only the functional NQO1 enzyme, NQO1A, enhanced the ROS production of Nox4 induced by quinone compounds(Fig.6C).Interestingly,NQO1 hasbeendescribedto partitionbetweencytosolandplasmamembrane[7]whereNox4 isalsolocatedinHEK293cells[50].Theseobservationssuggesta potentiallinkbetweenNQO1andNox4.Suchpotentiallinkcould be illustrated in pancreatic cancer. Pancreatic cancer is very aggressiveandunresponsivetotreatmentsduetoaresistanceof cellstoapoptosis.Vaqueroetal.[51]reportedthatROSproduced byNox4areprosurvivalandantiapoptoticforhumanpancreatic adenocarcinomacell lines,MIAPaCa-2and PANC-1.In theMIA PaCa-2cellline,Lewisetal.[52]showedthatinhibitionofNQO1by dicoumarol suppresses the malignant phenotype of pancreatic cancercellsandinducescellapoptosis.

Finally,ourstudydescribed4quinonesderivativesespecially AA-861andtBuBHQcapableofenhancingNox4ROSproduction. WeshowedthatNQO1maybeinvolvedinthestimulationofNox4 ROSproductioninducedbyquinones.We furtherobservedthat Nox4activityisalsostimulatedinthepresenceofreducingagents thatmaypossiblyactbyreducingthedisulfidebridgelocatedin theextracellularE-loopofNox4.Alltogether,wehypothesizethat inthepresenceof quinones,NQO1maygeneratehydroquinone forms,providing,therefore,potentialreducingagentsnearNox4 protein.Dependingonthequinoneredoxpotential,Nox4E-loop

disulfidebridgecouldbedisrupted consequentlyleadingtothe increaseofitsoxidaseactivity.However,thedirectimplicationof the E-loop disulfide bridge needs further investigations. The proposedmodelcouldprovidenewinsightintotheunderstanding ofmolecularmechanismoftheelectrontransferthroughNox4and alsodefinenewtherapeutictargetsindiseasesinwhichquinones andNox4areimplicated.

Acknowledgments

This workwassupported bygrants from: the‘Ministe`re de l‘Enseignementsupe´rieurdelarechercheetlatechnologie’,Paris, France; the ‘CNRSInstitute’; theAssociation pour la Recherche contre le Cancer (ARC), Paris, France; the Re´gion Rhoˆne-Alpes, programmeARCUS,France/Chine2007–2008,programme Emer-gence2003–2006;the‘CGDresearchTrust2006–2007’,UK;the ‘Groupement des Entreprises Franc¸aises de la Lutte contre le Cancer’,de´le´gationdeGrenoble;the‘UFRdeMe´decine,Universite´ Joseph Fourier, Grenoble’; and the ‘Direction Re´gionale de la Recherche Clinique, Center Hospitalier Universitaire, Grenoble’. WethankProf.Stanke(Grenoble,France)forkindlyprovidingthe 5-lipoxygenase polyclonal antibody, Prof. P.E. Cole, Drs M.H. HuynhandM.H.PacletforEnglishlanguagecorrections,S.Berthier for FACS measurements, A. Dariz-Soldini for real time RT-PCR experimentsandProf.A.Maitreand Prof.R.Brandesforhelpful readinganddiscussions.

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