Nuclearreceptors核受体培训课件

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1、Nuclearreceptors核核受体受体The intracellular(nuclear)receptor superfamilySteroid hormones,thyroid hormones,retinoids and vitamin D 2Nuclearreceptors核受体核受体Intracellular receptorHYDROPHOBIC:-Non-polar molecules-Gases-Steroids3Nuclearreceptors核受体核受体Regulation of transcription activityRegulatory mechanisms v

2、aryHeterodimeric receptors-exclusively nuclear;without ligand,repress transcription by binding to their cognate sites in DNAHomodimeric receptors-mostly cytoplasmic(without ligands)&hormone binding leads to nuclear translocation of receptorsWithout ligand-aggregation of receptor with inhibitor prote

3、ins(eg Hsp90)Steroid hormones are often required to dimerize with a partner to activate gene transcriptionReceptors for vitamin D,retinoic acid and thyroid hormone bind to responsive elements as heterodimersSecond component of the heterodimer is RXR monomer(i.e,RXR-RAR;RXR-VDR)Specificities of some

4、receptors 4Nuclearreceptors核受体核受体Intracellular signal molecules small,lipid-soluble molecules such as steroid hormones,retinoids,thyroid hormones,Vitamin D.(made from cholesterol)These molecules diffuse through plasma and nuclear membranes and interact directly with the transcription factors they co

5、ntrol.5Nuclearreceptors核受体核受体6 6Lipophilic HormonesCirculation in the blood bound to transport proteinsDissociation from carrier at target cellsAction in the cell-Pass through the cell membrane and bind to an intracellular receptor,either in the cytoplasm or the nucleus-Hormone-receptor complex bind

6、s to hormone response elements in DNA -Regulate gene expression6Nuclearreceptors核受体核受体7 7ReceptorBloodplasmaProteinLipophilic hormonesmRNADNAHormone response element1.Hormone passes through plasma membrane2.Inside target cell the hormone binds to a receptor protein in the cytoplasm or nucleus3.Hormo

7、ne-receptor complex binds to hormone response element on DNA,regulating gene transcription4.Protein synthesis5.Change in protein synthesis is cellular responseCytoplasmPlasma membraneNucleusCopyright The McGraw-Hill Companies,Inc.Permission required for reproduction or display.7Nuclearreceptors核受体核受

8、体Steroid HormonesSTEROID HORMONES:-sex steroids(estrogen,progesterone,testosterone)-corticosteroids (glucocorticoids and mineralcorticoids)OTHER HORMONESThyroid hormone,vitamin D3,and retinoic acid have different structure and function but share the same mechanism of action with the other steroids.8

9、Nuclearreceptors核受体核受体9Nuclearreceptors核受体核受体BIOSYNTHESIS OF STEROIDS10Nuclearreceptors核受体核受体Endocrine disruptionInterference of xenobiotics with normal function of hormonal system Possible consequences:Disruption of homeostasis,reproduction,development,and/or behavior(and other hormone-controlled p

10、rocesses).Shift in sex ratio,defective sexual developmentLow fecundity/fertilityHypo-immunity,carcinogenesisMalformations11Nuclearreceptors核受体核受体Toxicants interact with hormonal system at different levelsSynthesis TransportMetabolizationInteraction with receptorsStimulationSuppression12Nuclearrecept

11、ors核受体核受体biosynthesis and release of hormonesbinding to plasmatic transport proteinsbinding to nuclear hormonal receptor(HR)activation of HR(dissociation of associated heat shock proteins,formation of homodimers)binding of the activated receptor complex to specific DNA motifs-HREschromatin rearrange

12、ment and transcription of estrogen-inducible geneseffects at the cellular,tissue,organ,organism,and/or population levele.g.modulation of CYP11A and/or CYP19 activitiese.g.down-regulation of receptor levelse.g.modulation of other nuclear receptors(PPAR/RXR,RXR/TR)STEROIDOGENESIS13Nuclearreceptors核受体核

13、受体Mechanisms of steroid hormones signalling disruption-Nonphysiological activation of hormone receptor(HR)-Binding to HR without activation-Decrease of HR cellular levels-Disruption of the master“hormones(FSH/LH)-Changes in hormone metabolism14Nuclearreceptors核受体核受体Endocrine disrupters in the enviro

14、nment?EDCs.EDCs.Persistent Organic Compounds Persistent Organic Compounds(POPs and their metabolites)(POPs and their metabolites)steroid hormones and their steroid hormones and their derivatives from contraception pillsderivatives from contraception pills alkylphenolsalkylphenols organometallics(but

15、yltins)organometallics(butyltins)pharmaceuticalspharmaceuticals PesticidesPesticides+number of unknowns+number of unknowns 15Nuclearreceptors核受体核受体ESTROGEN RECEPTOR ERthe most studied target of EDCs16Nuclearreceptors核受体核受体 Estrogens:Estrogens:play a key role in female hormone regulation and signalli

16、ng are responsible for metabolic,behavioural and morphologic changes occurring during stages of reproduction are involved in the growth,development and homeostasis of a number of tissues control the bone formation,regulation of homeostasis,cardiovascular system and behaviour regulate production,tran

17、sport and concentration of testicular liquid and anabolic activity of androgens in malesSynthesis in ovaries DISRUPTION-investigated in aquatic biota&laboratory organisms(see notes on EDCs)17Nuclearreceptors核受体核受体ESTROGEN RECEPTORS ESTROGEN RECEPTORS-ER-ER-&ER-&ER-:subtype:ER-(in breast,ovary,brain,

18、liver,bone and cardiovascular system,adrenals,testis and urogenital tract)ER-(in kidneys,prostate and gastrointestinal tract)(ER-in fish)18Nuclearreceptors核受体核受体Natural productsgenisteinnaringenincoumestrolzearalenoneEnvironmental pollutantDDTkeponePCBs/OH-PCBsPAHs and dioxinsIndustrial chemicalsBis

19、phenol ANonionic surfactantsPthalate estersendosulfanPharmaceuticalsEthinyl estradiolDiethylstilbestrolgestodenenorgestrelDEHPEnvironmental estrogens(xenoestrogens,exoestrogens)Environmental estrogens(xenoestrogens,exoestrogens)a diverse group of substances that do not necessarily share structural s

20、imilarity to the prototypical estrogen(17-estradiol)May act as AGONISTS and/or ANTAGONISTS19Nuclearreceptors核受体核受体Exoestrogens-Relative Potencies to bind to ERExoestrogens-Relative Potencies to bind to ER (REPs)(REPs)20Nuclearreceptors核受体核受体Toxicity assessment Toxicity assessment number of number of

21、 in vivo and in vitro methodsin vivo and in vitro methodsJanoek,J.,Hilscherov,K.,Blha,L.,and Holoubek,I.(2006).Environmental xenobiotics and nuclear receptors-Interactions,effects and in vitro assessment.Toxicology in Vitro 20,18-37.21Nuclearreceptors核受体核受体In vitro assaysIn vitro assaysINTERACTION(B

22、INDING)to the receptor competitive ligand binding assayEffect unknown(?Activation/suppression/no effect?)Testing the effect at cellular level(interference with receptor biological activity)cell proliferation assay endogenous protein expression(or enzyme activity)assay reporter gene assay22Nuclearrec

23、eptors核受体核受体In vitro ER-mediated effectsluciferase reporter assay 23Nuclearreceptors核受体核受体ER-mediated effectsluciferase reporter assay 96 microwell platecultivation of transgenic cell linesER:breast carcinoma MVLN cellsSIMILAR DESIGN FOROTHER RECEPTORS(discussed below):AhR(H4IIE.luc cells)AR(MDA cel

24、ls)RAR/RXR(P19 cells)Cell lysis-extraction of induced luciferaseExposure (6 24 h)standards/samplesLuminoLuminescence determination(microplate luminescence reader)24Nuclearreceptors核受体核受体In vivo assays uterotropic assay vaginal cornification assay standard test procedures for reproductive and develop

25、mental toxicity(e.g.FETAX)production of estrogen-inducible proteins (e.g.vittelogenin and zona radiata protein)25Nuclearreceptors核受体核受体Kidd,K.A.et al.2007.Collapse of a fish population following exposure to a synthetic estrogen.Proceedings of the National Academy of Sciences 104(21):8897-8901Control

26、s +Ethinylestradiol5 ng/L(!)7 years26Nuclearreceptors核受体核受体ANDROGEN RECEPTOR(AR)effects known but less explored than ER27Nuclearreceptors核受体核受体Androgens-Role in males similar to the of estrogens in females-development of male sexual characteristics-stimulating protein synthesis,growth of bones -cell

27、 differenciation,spermatogenesis-male type of behaviour 28Nuclearreceptors核受体核受体Androgens-Endogenous ligands androgen hormones-testosterone(T)-dihydrotestosterone(DHT)-androstanediol-dehydroepiandrosterone-androstenedione T:synthesis in testis(Leydig cells)in lesser extent in adrenalsDHT:Formed extr

28、atesticulary from T-In several tissues(seminal vesicles,prostate,skin)higher affinity to androgen receptor than T-Daily production 5-10%of testosteroneTestosterone29Nuclearreceptors核受体核受体Mechanisms of androgen signalling disruption1)Binding to AR-Mostly competitive inhibition-xenobiotics mostly DO N

29、OT activate AR-dependent transcription-Only few compounds are able to activate AR in the absence of androgen hormones,and these are also anti-androgenic in the presence of T/DHT(metabolites of fungicide vinclozoline,some PAHs)2)FSH/LH(gonadotropins)signalling disruption less explored-FSH/LH expressi

30、on-regulation via negative feedback by testosterone-Suppression leads to alterations of spermatogenesis30Nuclearreceptors核受体核受体Mechanisms of androgen signalling disruption3)Alterations of testosterone synthesis-Inhibition of P450scc needed for side chain cleavage of cholesterol(fungicide ketoconazol

31、)-Inhibition of 17-hydroxylase and other CYPs-enzymes needed for testosterone synthesis(ketoconazol)4)Testosterone metabolic clearance-Induction of UDP-glucuronosyltransferase or monooxygenases CYP1A,1B involved in androgen catabolism-Pesticides endosulfan,mirex,o-p-DDT31Nuclearreceptors核受体核受体Effect

32、s of male exposure to antiandrogensExposure during prenatal development:-malformations of the reproductive tract-reduced anogenital distance-hypospadias(abnormal position of the urethral opening on the penis)-vagina development-undescendent ectopic testes-atrophy of seminal vesicles and prostate gla

33、nd Exposure in prepubertal age:-delayed puberty-reduced seminal vesicles-reduced prostateExposure in adult age:-oligospermia-azoospermia-libido diminution 32Nuclearreceptors核受体核受体Antiandrogenic compoundtris-(4-chlorophenyl)-methanol-Ubiquitous contaminant of uncertain origin-Probable metabolite of D

34、DT-mixture contaminant-Levels in human blood serum cca.50nM-EC50 cca.200nM 33Nuclearreceptors核受体核受体AR-binding-potencies(Ref:DHT EC50 0.1 uM)CompoundIC50(M)Benzaanthracene3.2Benzoapyrene3.9Dimethylbenzaanthracene10.4Chrysene10.3Dibenzoa,hanthraceneactivation in range 0.1-10MBisphenol A5vinclozolin me

35、tabolites9.7hydroxyflutamide5Aroclor typical values0.25-1.11Individual PCBs typical values64-87tris-(4-chlorophenyl)-methanol0.234Nuclearreceptors核受体核受体(Anti)androgenicity assessmentIn vivo Hershberger assay-castrated rats treated with examined substance-Endpoint after 4-7 days seminal vesicles and

36、ventral prostate weightIn vivo measurement of testosterone blood levelsIn vitro cell proliferation assays cell lines with androgen-dependent growth-mammary carcinoma cell lines-prostatic carcinoma cell linesReceptor-reporter assaysGene for luciferase(or GFP)under control of ARAR-CALUX(human breast c

37、arcinoma T47D)PALM(human prostatic carcinoma PC-3)CHO515(Chinese hamster ovary CHO)Yeast transfected cellsbeta-galactosidase reporterTreatment:tested chemical only-androgenicity Cotreatment with DHT-antiandrogenicity35Nuclearreceptors核受体核受体Thyroid hormones36Nuclearreceptors核受体核受体Thyroid hormonesRegu

38、lation of metabolism-increasing oxygen consumption-modulating levels of other hormones(insulin,glucagon,somatotropin,adrenalin)-important in cell differenciation-crucial role in development of CNS,gonads and bones Play crucial roles in stimulating metabolism,development and maturationHYPOTHYROIDISMH

39、YPERTHYROIDISM37Nuclearreceptors核受体核受体Thyroid hormones-T4 prohormone-5-deiodination leads to active form,T3Thyroxine(T4)Also called tetraiodothyronineContains 4 iodide ionsTriiodothyronine(T3)Contains 3 iodide ions-Most T3 produced by deiodination in target tissues (deiodinases)38Nuclearreceptors核受体

40、核受体Enzymes involved in thyroid metabolism-Thyroid peroxidases-iodination of tyrosyl residues-coupling of iodinated tyrosyl residues-Thyroid deiodinases-D1,D2-activation of T4 into T3 via deiodination on outer“ring-D3-deactivation into rT3 via deiodination on inner“ringEDCs-may affect metabolism of t

41、hese key enzymesouter“inner“39Nuclearreceptors核受体核受体Thyroid hormones are transported in the blood by thyroid binding proteins-Regulating free T4 and T3 levels in blood-3 types:-Thyroid-binding prealbunin(transthyretin)(20-25%)-Albumin(5-10%)-Thyroid binding globulin(75%)-NUMBER OF ENVIRONMENTAL TOXI

42、CANTS act at transport proteins-OH-PCBs,brominated and chlorinated flame retardants,DDT,dieldrin-OH-PCBs equal affinity to TBP as T4 and T3(!)-More of free T4 in blood negative feedback to TSH release=increased depletion=increased weight,histological changes in thyroid glandObserved after exposure t

43、o POPs in mammals,birds,fish40Nuclearreceptors核受体核受体 Competitive binding to TR-Probably less important than binding to TBP-Chemicals that affect thyroid signalling in vivo mostly dont bind to TR(DDT,PCBs)or bind with much lesser affinity than T3(OH-PCBs 10000 x)Accelerated depletion of THUDP-glucuro

44、nosyltransferase detoxication enzyme(II.biotransformation phase)Induced by PCBs,dioxins Key enzyme in thyroid catabolismIncreased by disruption of TBP bindingOther possible effects of EDCs on Thyroid signalling41Nuclearreceptors核受体核受体Effects of thyroid disruptionDisruption during prenatal developmen

45、t-severe damage of CNS(cretenism,delayed eye opening,cognition)-Megalotestis-Histological changes in thyroid gland(goitre)-nervous system fails to develop normally-mental retardation-skeletal development42Nuclearreceptors核受体核受体Assessment of effects-In vivo approaches-TH serum levels simple,nondestru

46、ctive x variation within time of day,age,sensitive to other than biochemical stresses-Thyroid gland weight and folicular cells number-Developmental toxicity assays-delayed eye opening,abnormalities in brain development and cognition,increased testis weight and sperm counts-Perchlorate discharge test

47、(TH synthesis)-Hepatic UDP-glucuronosyltransferase activity(marker of enhanced TH clearance from serum)-In vitro-Enzyme inhibition assays(thyroid peroxidase,deiodinases)assessment of thyroid metabolism-Competitive binding assays with TBP-TH-dependent proliferation assay(pituitary tumor GH3,thyroid t

48、umors like FRTL-5 cell line)or TSH-dependent proliferation assay(thyroid tumors)-Receptor-reporter gene assays with luciferase(monkey kidney CV-1,chinese hamster ovary CHO or insect Sf9 cell lines)43Nuclearreceptors核受体核受体Retinoids Vitamin A and its derivativesToxicants affect retinoid action but eff

49、ects are much less explored44Nuclearreceptors核受体核受体Suppressive effects in cancer development RetinoidsNecessary for visionImportant for cell growth,apoptosis and differenciation Development of embryonic,epithelial cells(gastrointestinal tract,skin,bones)Antioxidative agentAffect nervous and immune f

50、unctionRegulation of development and homeostasis in tissues of vertebrates and invertebrates45Nuclearreceptors核受体核受体 Retinoids Retinol(vitamin A)Bond cleavageRetinoic Acid-karotenSources:from diet(dietary hormones)Retinyl esters animal sourcesPlant carotenoids46Nuclearreceptors核受体核受体RE:Retinol-Ester

51、R:RetinolRBP:Retinol BindingProtein(LMW)TTR:Transthyrethin(HMW)TRANSPORT OF RETINOIDS47Nuclearreceptors核受体核受体RAL-RetinalCRBP cellular retinol binding protein-binding of retinol,immediate decrease of retinol concentrationCRBAP cellular retinoic acid binding protein-Controlling ratio free retinol/free

52、 retinoic acidRetinoid binding proteins48Nuclearreceptors核受体核受体 Mode of action-Isoforms of RAR a RXR-Both have isoforms ,andg,each of them several subtypes-Formation of homo-and heterodimers-48 possible RAR-RXR heterodimers=sensitive regulation of gene expression-RXR heterodimers even with other rec

53、eptors like VDR,TR,PPAR-3 basic subtypes-all-trans-,9-cis-and 13-cis-retinoic acid-All-trans RA binds selectively to RAR-Cis RA bind to both receptor typesRetinoic acidGene expression49Nuclearreceptors核受体核受体Disruption of retinoid signalling by xenobiotics-Relatively little known-Possible modes of ac

54、tion:-Metabolization of retinoids by detoxication enzymes-Disruption of binding retinoids to retinoid binding proteins-Retinoids as antioxidants may be consumed cause of oxidative stress caused by xenobiotics-Interference of chemicals(binding to RAR/RXR)50Nuclearreceptors核受体核受体Consequences of retino

55、id signalling disruptionDecreased retinoid levels in organisms-Downregulation of growth factors-Xerophtalmia,night blindness-Embryotoxicity,developmental abnormalitiesXIncreased ATRA concentration teratogenic effectChange may cause severe developmental anomalies(both excess and deficiency)51Nuclearr

56、eceptors核受体核受体Disruption of retinoid signalling by xenobioticsPolluted areas mostly decrease of retinoid levels in aquatic birds,mammals and fishDisruption of retinoid transport:PCBsEffects on retinoid receptors:-RAR,RXR binding and/or transactivation pesticides(chlordane,dieldrin,methoprene,tributy

57、ltin)-Effect on ATRA mediated response TCDD,PAHsDisruption of retinoid metabolism:PCDD/Fs,PAHs,PCBs,pesticides -changes of serum concentrations of retinol and RA-mobilization of hepatic storage forms-in kidney,concentration of all forms elevated52Nuclearreceptors核受体核受体How to assess retinoid signalli

58、ng disruption?In vivo-Mostly derived from classical toxicity tests,particularly of developmental toxicity-Direct measurements of various retinoid forms in living organisms(laboratory and wildlife)In vitro-Mostly epithelial cell lines(keratinocytes)-Mouse embryonic cell lines P19 pluripotent cells di

59、fferentiation dependent on circumstances,triggered by ATRA-reporter gene assay P19/A15-Other cell lines rainbow trout gonads,human salivary gland,breast or prostatic carcinomas etc.53Nuclearreceptors核受体核受体AhR(Arylhydrocarbon receptor)AhR structure54Nuclearreceptors核受体核受体AhR ligand-activated transcri

60、ption factor activation of different responsive elements(genes)important mediator of toxicity of POPs primary target of coplanar aromatic substances regulator of xenobiotic metabolism and activation of promutagens crossactivation/crosstalk with other receptors strongest known ligand TCDD55Nuclearrec

61、eptors核受体核受体AhR regulated genes:AhR regulated genes:contain xenobiotic response elements(XRE)or dioxin responsive elements(DRE)in their promoter region:phase I enzymes-CYP 1A1,CYP 1A2,CYP 1B1;phase II enzymes-UDP-glucuronosyltransferase,GST-Ya,NADP(H):oxidoreductase;other genes-Bax,p27Kip1,Jun B,TGF

62、-b b-regulation of cell cycle and apoptosis;57Nuclearreceptors核受体核受体6-formylindolo3,2-bcarbazole(FICZ)potent endogenous physiological(natural)ligand of AhR 58Nuclearreceptors核受体核受体Denison&Nagy,Annu.Rev.Pharmacol.Toxicol.43:30959Nuclearreceptors核受体核受体Non-classical“AhR ligandsNon-classical“AhR ligands

63、60Nuclearreceptors核受体核受体Physiological role for AhR not known(?)Physiological role for AhR not known(?)Effects in AhR-deficient mice:Effects in AhR-deficient mice:significant growth retardation;defective development of liver and immune system;retinoid accumulation in liver;abnormal kidney and hepatic

64、 vascular structures.resistant to BaP-induced carcinogenesis and TCDD-induced teratogenesis;no inducible expression of CYP 1A1 and 2.61Nuclearreceptors核受体核受体Schmidt&Bradfield,Annu.Rev.Cell Dev.Biol.12:55Biological responses&effects of TCDD(mostly related to AhR activation)62Nuclearreceptors核受体核受体Com

65、pounds having similar toxicological properties as TCDD(strongest AhR ligand)may be evaluated by TEF/TEQ conceptTEF=Toxic Equivalency Factor(characteristic of the Chemical)TEQ=Toxic Equivalent(sum of TEFs x concentrations)TEFs are consensus values based on REPs(relative potencies)across multiple spec

66、ies and/or endpoints.TEFs are based upon a number of endpoints,from chronic in vivo toxicity to in vitro toxicity with the former having the greatest importance in determining overall TEF.TEQs provide a simple,single number that is indicative of overall toxicity of a sample containing a mixture of dioxins and dioxin-like compounds.The total potency of a mixture can be expressed in TCDD TEQ concentration:Toxic equivalency factors(TEF)/TEQ concept:Toxic equivalency factors(TEF)/TEQ concept:63Nucle