Nuclearreceptors核受體專題知識專家講座

INTRACELLULARRECEPTORS第1頁Theintracellular(nuclear)receptorsuperfamilySteroidhormones,thyroidhormones,retinoidsandvitaminD

第2頁IntracellularreceptorHYDROPHOBIC:Non-polarmoleculesGasesSteroids第3頁RegulationoftranscriptionactivityRegulatorymechanismsvaryHeterodimericreceptors-exclusivelynuclear;withoutligand,represstranscriptionbybindingtotheircognatesitesinDNAHomodimericreceptors-mostlycytoplasmic(withoutligands)&hormonebindingleadstonucleartranslocationofreceptorsWithoutligand-aggregationofreceptorwithinhibitorproteins(egHsp90)SteroidhormonesareoftenrequiredtodimerizewithapartnertoactivategenetranscriptionReceptorsforvitamin

D,retinoicacidandthyroidhormonebindtoresponsiveelementsasheterodimersSecondcomponentoftheheterodimerisRXRmonomer(i.e,RXR-RAR;RXR-VDR)Specificitiesofsomereceptors…第4頁Intracellularsignalmolecules

small,lipid-solublemoleculessuchassteroidhormones,retinoids,thyroidhormones,VitaminD.(madefromcholesterol)Thesemoleculesdiffusethroughplasmaandnuclearmembranesandinteractdirectlywiththetranscriptionfactorstheycontrol.第5頁6LipophilicHormonesCirculationinthebloodboundtotransportproteinsDissociationfromcarrierattargetcellsActioninthecell -Passthroughthecellmembraneand bindtoanintracellularreceptor,eitherin thecytoplasmorthenucleus -Hormone-receptorcomplexbindsto hormoneresponseelementsinDNA

-Regulategeneexpression第6頁7ReceptorBloodplasmaProteinLipophilichormonesmRNADNAHormoneresponseelement1.Hormonepassesthroughplasmamembrane2.Insidetargetcellthehormonebindstoareceptorproteininthecytoplasmornucleus3.Hormone-receptorcomplexbindstohormoneresponseelementonDNA,regulatinggenetranscription4.Proteinsynthesis5.ChangeinproteinsynthesisiscellularresponseCytoplasmPlasmamembraneNucleusCopyright?TheMcGraw-HillCompanies,Inc.Permissionrequiredforreproductionordisplay.第7頁SteroidHormonesSTEROIDHORMONES: -sexsteroids(estrogen,progesterone,testosterone) -corticosteroids(glucocorticoidsandmineralcorticoids)OTHERHORMONES

Thyroidhormone,vitaminD3,andretinoicacidhavedifferentstructureandfunctionbutsharethesamemechanismofactionwiththeothersteroids.第8頁第9頁BIOSYNTHESISOFSTEROIDS第10頁EndocrinedisruptionInterferenceofxenobioticswithnormalfunctionofhormonalsystemPossibleconsequences:Disruptionofhomeostasis,reproduction,development,and/orbehavior(andotherhormone-controlledprocesses).Shiftinsexratio,defectivesexualdevelopmentLowfecundity/fertilityHypo-immunity,carcinogenesisMalformations第11頁ToxicantsinteractwithhormonalsystematdifferentlevelsSynthesisTransportMetabolizationInteractionwithreceptorsStimulationSuppression第12頁biosynthesisandreleaseofhormonesbindingtoplasmatictransportproteinsbindingtonuclearhormonalreceptor(HR)activationofHR(dissociationofassociatedheatshockproteins,formationofhomodimers)bindingoftheactivatedreceptorcomplextospecificDNAmotifs-HREschromatinrearrangementandtranscriptionofestrogen-induciblegeneseffectsatthecellular,tissue,organ,organism,and/orpopulationlevele.g.modulationofCYP11Aand/orCYP19activitiese.g.down-regulationofreceptorlevelse.g.modulationofothernuclearreceptors(PPAR/RXR,RXR/TR)STEROIDOGENESIS第13頁Mechanismsof

steroidhormonessignallingdisruption-Nonphysiologicalactivationofhormonereceptor(HR)BindingtoHRwithoutactivationDecreaseofHRcellularlevelsDisruptionofthe?master“hormones(FSH/LH)Changesinhormonemetabolism第14頁Endocrinedisruptersintheenvironment?EDCs...PersistentOrganicCompounds

(POPsandtheirmetabolites)steroidhormonesandtheir

derivativesfromcontraceptionpillsalkylphenolsorganometallics(butyltins)pharmaceuticalsPesticides+numberofunknowns…第15頁

ESTROGENRECEPTOR–ER

themoststudiedtargetofEDCs第16頁

Estrogens:

playakeyroleinfemalehormoneregulationandsignallingareresponsibleformetabolic,behaviouralandmorphologicchanges

occurringduringstagesofreproductionareinvolvedinthegrowth,developmentandhomeostasisofanumberof

tissuescontroltheboneformation,regulationofhomeostasis,

cardiovascularsystemandbehaviourregulateproduction,transportandconcentrationoftesticularliquidand

anabolicactivityofandrogensinmalesSynthesisinovariesDISRUPTION->investigatedinaquaticbiota&laboratoryorganisms(seenotesonEDCs)

第17頁ESTROGENRECEPTORS-ER-&ER-:

subtype:ER-(inbreast,ovary,brain,liver,boneandcardiovascularsystem,

adrenals,testisandurogenitaltract)ER-(inkidneys,prostateandgastrointestinaltract) (ER-infish)第18頁NaturalproductsgenisteinnaringenincoumestrolzearalenoneEnvironmentalpollutantDDTkeponePCBs/OH-PCBsPAHsanddioxinsIndustrialchemicalsBisphenolANonionicsurfactantsPthalateestersendosulfanPharmaceuticalsEthinylestradiolDiethylstilbestrolgestodenenorgestrelDEHPEnvironmentalestrogens(xenoestrogens,exoestrogens)adiversegroupofsubstancesthat

donotnecessarilysharestructural

similaritytotheprototypicalestrogen(17-estradiol)MayactasAGONISTSand/orANTAGONISTS第19頁Exoestrogens-RelativePotenciestobindtoERa(REPs)第20頁Toxicityassessment

numberofinvivoandinvitromethodsJano?ek,J.,Hilscherová,K.,Bláha,L.,andHoloubek,I.(2023).Environmentalxenobioticsandnuclearreceptors-Interactions,effectsandinvitroassessment.ToxicologyinVitro

20,18-37.第21頁InvitroassaysINTERACTION(BINDING)tothereceptorcompetitiveligandbindingassayEffectunknown(?Activation/suppression/noeffect?)Testingtheeffectatcellularlevel(interferencewith

receptorbiologicalactivity)cellproliferationassayendogenousproteinexpression(orenzymeactivity)assayreportergeneassay第22頁InvitroER-mediatedeffects

luciferasereporterassay

第23頁ER-mediatedeffectsluciferasereporterassay96microwellplatecultivationoftransgeniccelllinesER:breastcarcinomaMVLNcellsSIMILARDESIGNFOR

OTHERRECEPTORS

(discussedbelow):AhR(H4IIE.luccells)AR(MDAcells)RAR/RXR(P19cells)Celllysis->extractionofinducedluciferaseExposure(6–24h)standards/samplesLuminoLuminescencedetermination(microplateluminescencereader)第24頁Invivoassays

uterotropicassayvaginalcornificationassaystandardtestproceduresforreproductiveanddevelopmentaltoxicity(e.g.FETAX)productionofestrogen-inducibleproteins(e.g.vittelogeninandzonaradiataprotein)第25頁Kidd,K.A.etal.2023.Collapseofafishpopulationfollowingexposuretoasyntheticestrogen.ProceedingsoftheNationalAcademyofSciences104(21):8897-8901Controls+Ethinylestradiol5ng/L(!)7years第26頁ANDROGENRECEPTOR(AR)

effectsknownbutlessexploredthanER第27頁AndrogensRoleinmalessimilartotheofestrogensinfemales -developmentofmalesexualcharacteristics

-stimulatingproteinsynthesis,growthofbones

-celldifferenciation,spermatogenesis -maletypeofbehaviour第28頁AndrogensEndogenousligands–androgenhormonestestosterone(T)dihydrotestosterone(DHT)

androstanedioldehydroepiandrosteroneandrostenedione

T:synthesisintestis(Leydigcells)inlesserextentinadrenalsDHT:FormedextratesticularyfromTInseveraltissues(seminalvesicles,prostate,skin)

higheraffinitytoandrogenreceptorthanTDailyproduction5-10%oftestosteroneTestosterone第29頁Mechanismsofandrogensignallingdisruption1)BindingtoARMostlycompetitiveinhibitionxenobioticsmostlyDONOTactivateAR-dependenttranscriptionOnlyfewcompoundsareabletoactivateARintheabsence ofandrogenhormones,andthesearealsoanti-androgenicin thepresenceofT/DHT (metabolitesoffungicide

vinclozoline,somePAHs)2)FSH/LH(gonadotropins)signallingdisruption–lessexploredFSH/LHexpression-regulationvianegativefeedbackbytestosteroneSuppressionleadstoalterationsofspermatogenesis第30頁Mechanismsofandrogensignallingdisruption3)AlterationsoftestosteronesynthesisInhibitionofP450sccneededforsidechain cleavageofcholesterol(fungicideketoconazol)Inhibitionof17--hydroxylaseandotherCYPs-– enzymesneededfortestosteronesynthesis (ketoconazol)4)TestosteronemetabolicclearanceInductionofUDP-glucuronosyltransferaseor monooxygenasesCYP1A,1BinvolvedinandrogencatabolismPesticidesendosulfan,mirex,o-p′-DDT第31頁EffectsofmaleexposuretoantiandrogensExposureduringprenataldevelopment:malformationsofthereproductivetract -reducedanogenitaldistance -hypospadias

(abnormalpositionoftheurethral openingonthepenis) -vaginadevelopment

-undescendentectopictestes -atrophyofseminalvesiclesandprostategland

Exposureinprepubertalage:delayedpuberty

reducedseminalvesicles

reducedprostateExposureinadultage:

oligospermiaazoospermialibidodiminution

第32頁Antiandrogeniccompoundtris-(4-chlorophenyl)-methanol -Ubiquitouscontaminantofuncertainorigin -ProbablemetaboliteofDDT-mixturecontaminant -Levelsinhumanbloodserumcca.50nM -EC50–cca.200nM

第33頁AR-binding-potencies

(Ref:DHTEC50~0.1uM)CompoundIC50

(μM)Benz[a]anthracene3.2Benzo[a]pyrene3.9Dimethylbenz[a]anthracene10.4Chrysene10.3Dibenzo[a,h]anthraceneactivationinrange0.1-10μMBisphenolA5vinclozolinmetabolites9.7hydroxyflutamide5Aroclortypicalvalues0.25-1.11IndividualPCBstypicalvalues64-87tris-(4-chlorophenyl)-methanol0.2第34頁(Anti)androgenicityassessmentInvivoHershbergerassay

-castratedratstreatedwithexaminedsubstance -Endpoint–after4-7days–seminalvesiclesand ventralprostateweightInvivomeasurementoftestosteronebloodlevelsInvitrocellproliferationassays –celllineswithandrogen-dependentgrowth

-mammarycarcinomacelllines -prostaticcarcinomacelllines¨Receptor-reporterassaysGeneforluciferase

(orGFP)undercontrolofARAR-CALUX(humanbreastcarcinomaT47D)PALM(humanprostaticcarcinomaPC-3)CHO515(ChinesehamsterovaryCHO)Yeasttransfectedcells beta-galactosidasereporterTreatment:

testedchemicalonly ->androgenicityCotreatmentwithDHT ->antiandrogenicity第35頁Thyroidhormones第36頁Thyroid

hormonesRegulationofmetabolism- increasingoxygenconsumptionmodulatinglevelsofotherhormones(insulin,glucagon,somatotropin,adrenalin)importantincelldifferenciationcrucialroleindevelopmentofCNS,gonadsandbones

Playcrucialrolesinstimulatingmetabolism,developmentandmaturationHYPOTHYROIDISMHYPERTHYROIDISM第37頁Thyroid

hormonesT4–prohormone5′-deiodinationleadstoactiveform,T3Thyroxine(T4)AlsocalledtetraiodothyronineContains4iodideionsTriiodothyronine(T3)Contains3iodideionsMostT3produced

bydeiodination

intargettissues(deiodinases)第38頁EnzymesinvolvedinthyroidmetabolismThyroidperoxidases

iodinationoftyrosylresiduescouplingofiodinatedtyrosylresiduesThyroiddeiodinasesD1,D2-activationofT4intoT3viadeiodinationon?outer“ringD3-deactivationintorT3viadeiodinationon?inner“ringEDCs->mayaffectmetabolismofthesekeyenzymes?outer“?inner“第39頁ThyroidhormonesaretransportedinthebloodbythyroidbindingproteinsRegulatingfreeT4andT3levelsinblood3types:

Thyroid-bindingprealbunin(transthyretin)(20-25%)Albumin(5-10%)Thyroidbindingglobulin(75%)NUMBEROFENVIRONMENTALTOXICANTSactattransportproteinsOH-PCBs,brominatedandchlorinatedflameretardants,DDT,dieldrinOH-PCBs–equalaffinitytoTBPasT4andT3(!!!)MoreoffreeT4inbloodnegativefeedbacktoTSHrelease

=>increaseddepletion =>increasedweight,histologicalchangesinthyroidgland

ObservedafterexposuretoPOPsinmammals,birds,fish第40頁

CompetitivebindingtoTRProbablylessimportantthanbindingtoTBPChemicalsthataffectthyroidsignallinginvivomostlydon′tbindtoTR(DDT,PCBs)orbindwithmuchlesseraffinitythanT3(OH-PCBs–10000x)AccelerateddepletionofTHUDP-glucuronosyltransferase–detoxicationenzyme(II.biotransformationphase)InducedbyPCBs,dioxinsKeyenzymeinthyroidcatabolismIncreasedbydisruptionofTBPbindingOtherpossibleeffectsofEDCs

onThyroidsignalling第41頁EffectsofthyroiddisruptionDisruptionduringprenataldevelopment- severedamageofCNS(cretenism,delayedeyeopening,cognition)MegalotestisHistologicalchangesinthyroidgland(goitre)nervoussystemfailstodevelopnormallymentalretardationskeletaldevelopment第42頁AssessmentofeffectsInvivoapproachesTHserumlevels–simple,nondestructivexvariationwithintimeofday,age,sensitivetootherthanbiochemicalstressesThyroidglandweightandfolicularcellsnumberDevelopmentaltoxicityassays-delayedeyeopening,abnormalitiesinbraindevelopmentandcognition,increasedtestisweightandspermcountsPerchloratedischargetest(THsynthesis)HepaticUDP-glucuronosyltransferaseactivity(markerofenhancedTHclearancefromserum)InvitroEnzymeinhibitionassays(thyroidperoxidase,deiodinases)–assessmentofthyroidmetabolismCompetitivebindingassayswithTBPTH-dependentproliferationassay(pituitarytumorGH3,thyroidtumorslikeFRTL-5cellline)orTSH-dependentproliferationassay(thyroidtumors)Receptor-reportergeneassayswithluciferase(monkeykidneyCV-1,chinesehamsterovaryCHOorinsectSf9celllines)第43頁Retinoids

VitaminAanditsderivatives

Toxicantsaffectretinoidactionbuteffectsaremuchlessexplored第44頁SuppressiveeffectsincancerdevelopmentRetinoidsNecessaryforvisionImportantfor

cellgrowth,apoptosisanddifferenciationDevelopmentofembryonic,epithelialcells(gastrointestinaltract,skin,bones)AntioxidativeagentAffectnervousandimmunefunctionRegulationofdevelopmentandhomeostasisintissuesofvertebratesandinvertebrates第45頁RetinoidsRetinol(vitaminA)BondcleavageRetinoicAcid-karotenSources:fromdiet(dietaryhormones)Retinylesters–animalsourcesPlantcarotenoids第46頁RE:Retinol-EsterR:RetinolRBP:RetinolBindingProtein(LMW)TTR:Transthyrethin(HMW)TRANSPORT

OFRETINOIDS第47頁RAL-RetinalCRBP–cellularretinolbindingprotein-bindingofretinol,immediatedecreaseofretinolconcentrationCRBAP–cellularretinoicacidbindingprotein-Controllingratiofreeretinol/freeretinoicacidRetinoidbindingproteins第48頁

Modeofaction

IsoformsofRARaRXRBothhaveisoformsa,

bandg, eachofthemseveralsubtypesFormationofhomo-andheterodimers48possibleRAR-RXRheterodimers=>sensitiveregulationofgeneexpressionRXR–heterodimersevenwithotherreceptorslikeVDR,TR,PPAR3basicsubtypesall-trans-,9-cis-and13-cis-retinoicacidAll-transRAbindsselectivelytoRARCisRAbindtobothreceptortypesRetinoicacidGeneexpression第49頁DisruptionofretinoidsignallingbyxenobioticsRelativelylittleknownPossiblemodesofaction:Metabolizationofretinoidsbydetoxication enzymesDisruptionofbindingretinoidstoretinoidbinding proteinsRetinoidsasantioxidantsmaybeconsumedcause ofoxidativestresscausedbyxenobioticsInterferenceofchemicals(bindingtoRAR/RXR)第50頁ConsequencesofretinoidsignallingdisruptionDecreasedretinoidlevelsinorganisms -Downregulationofgrowthfactors -Xerophtalmia,nightblindness -Embryotoxicity,developmentalabnormalitiesXIncreasedATRAconcentration–teratogeniceffectChangemaycauseseveredevelopmentalanomalies(bothexcessanddeficiency)第51頁DisruptionofretinoidsignallingbyxenobioticsPollutedareas–mostlydecreaseofretinoidlevelsinaquaticbirds,mammalsandfishDisruptionofretinoidtransport:

PCBsEffectsonretinoidreceptors:RAR,RXRbindingand/ortransactivation–pesticides(chlordane,dieldrin,methoprene,tributyltin…)EffectonATRAmediatedresponse–TCDD,PAHsDisruptionofretinoidmetabolism:

–PCDD/Fs,PAHs,PCBs,pesticides -changesofserumconcentrationsofretinolandRAmobilizationofhepaticstorageformsinkidney,concentrationofallformselevated第52頁Howtoassessretinoidsignallingdisruption?Invivo

Mostlyderivedfromclassicaltoxicitytests,particularlyofdevelopmentaltoxicityDirectmeasurementsofvariousretinoidformsinlivingorganisms(laboratoryandwildlife)Invitro-Mostlyepithelialcelllines(keratinocytes)-MouseembryoniccelllinesP19 pluripotentcells differentiationdependentoncircumstances,triggeredbyATRA-reportergeneassayP19/A15-Othercelllines–rainbowtroutgonads,humansalivarygland,breastorprostaticcarcinomasetc.

第53頁AhR(Arylhydrocarbonreceptor)AhRstructure第54頁AhRligand-activatedtranscriptionfactoractivationofdifferentresponsiveelements(genes)importantmediatoroftoxicityofPOPs–primarytargetofcoplanararomaticsubstancesregulatorofxenobioticmetabolismandactivationofpromutagenscrossactivation/crosstalkwithotherreceptorsstrongestknownligandTCDD第55頁AhRactivation:第56頁AhRregulatedgenes:containxenobioticresponseelements(XRE)ordioxinresponsiveelements(DRE)intheirpromoterregion:

phaseIenzymes-CYP1A1,CYP1A2,CYP1B1;

phaseIIenzymes-UDP-glucuronosyltransferase,GST-Ya,NADP(H):oxidoreductase;othergenes-Bax,p27Kip1,JunB,TGF-b-regulationofcellcycleandapoptosis;第57頁6-formylindolo[3,2-b]carbazole(FICZ)potentendogenousphysiological(natural)ligandofAhR第58頁Denison&Nagy,Annu.Rev.Pharmacol.Toxicol.43:309第59頁?Non-classical“AhRligands第60頁PhysiologicalroleforAhRnotknown(?)EffectsinAhR-deficientmice:

significantgrowthretardation;defectivedevelopmentofliverandimmunesystem;retinoidaccumulationinliver;abnormalkidneyandhepaticvascularstructures.resistanttoBaP-inducedcarcinogenesisandTCDD-inducedteratogenesis;noinducibleexpressionofCYP1A1and2.第61頁Schmidt&Bradfield,Annu.Rev.CellDev.Biol.12:55Biologicalresponses&effectsofTCDD

(mostlyrelatedtoAhRactivation)第62頁CompoundshavingsimilartoxicologicalpropertiesasTCDD(strongestAhRligand)maybeevaluatedbyTEF/TEQconcept TEF=ToxicEquivalencyFactor(characteristicoftheChemical) TEQ=ToxicEquivalent(sumofTEFsxconcentrations)TEFsareconsensusvaluesbasedonREPs(relativepotencies)acrossmultiplespeciesand/orendpoints.TEFsarebaseduponanumberofendpoints,fromchronicinvivotoxicitytoinvitrotoxicitywiththeformerhavingthegreatestimportanceindeterminingoverallTEF.TEQsprovideasimple,singlenumberthatisindicativeofoveralltoxicityofasamplecontainingamixtureofdioxinsanddioxin-likecompounds.ThetotalpotencyofamixturecanbeexpressedinTCDDTEQconcentration:Toxicequivalencyfactors(TEF)/TEQconcept:第63頁ToxicequivalencyfactorsforPCDDs,PCDFsandPCBs:Eljarrat&Barceló,TrendsAnal.Chem.22:655Finalconcentrationisexpressedas?EquivalentsofTCDD“

(e.g.ngTEQ/kg=ngTCDD/kg)第64頁Biomarkers/bioanalyticalmethodsforAhRtoxicity

invivo:liverenlargement,reductionofthymusweight,wastingsyndrome,reproductiveanddevelopmentaldisordersinvivobiomarkers:ERODactivity,CYP1A1and1B1expr