CCN Proteins: Methods and Protocols PDF
Preview CCN Proteins: Methods and Protocols
Methods in Molecular Biology 2582 Masaharu Takigawa Editor CCN Proteins Methods and Protocols Second Edition M M B ETHODS IN OLECULAR IO LO GY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire, UK Forfurther volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologiesinthecriticallyacclaimedMethodsinMolecularBiologyseries.Theserieswas thefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandardinall biomedicalprotocolpublishing.Eachprotocolisprovidedinreadily-reproduciblestep-by- step fashion, opening with an introductory overview, a list of the materials and reagents neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitutethekeyingredientineachandeveryvolumeoftheMethodsinMolecularBiology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexedinPubMed. CCN Proteins Methods and Protocols Second Edition Edited by Masaharu Takigawa Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan Editor MasaharuTakigawa AdvancedResearchCenterforOraland CraniofacialSciences OkayamaUniversityDentalSchool/GraduateSchool ofMedicine,DentistryandPharmaceuticalSciences Okayama,Japan ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-2743-3 ISBN978-1-0716-2744-0 (eBook) https://doi.org/10.1007/978-1-0716-2744-0 ©TheEditor(s)(ifapplicable)andTheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,part ofSpringerNature2023 Thisworkissubjecttocopyright.AllrightsaresolelyandexclusivelylicensedbythePublisher,whetherthewholeorpart of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,reproductionon microfilmsorinanyotherphysicalway,andtransmissionorinformation storageand retrieval,electronicadaptation, computersoftware,orbysimilar ordissimilar methodologynow knownorhereafter developed. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations andthereforefreeforgeneraluse. Thepublisher,theauthors,andtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressedorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeen made.Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations. ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partofSpringer Nature. Theregisteredcompanyaddressis:1NewYorkPlaza,NewYork,NY10004,U.S.A. Preface The acronym CCN previously stood for Cyr61/CCN1, CTGF/CCN2, and Nov/CCN3, which are the three original members of this family. With the addition of three more members in WISP1–3 (CCN4–6), this family now consists of six distinctive members. All members are cysteine-rich secreted proteins composed of four distinct modules connected intandem:IGFbindingprotein-like,vonWillebrandtypeC,thrombospondintype1repeat, and C-terminalmodules—exceptforCCN5, whichlackstheCTmodule.Theyareknown to play multiple roles in fundamental biological processes as intercellular signaling regula- tors, which are considered as a kind of matricellular proteins and were once referred to as “signal conductors.” This multifunctionality is thought to be due to these modules inter- acting physically with various growth factors and cytokines, extracellular matrices, and cell membraneproteins,includingreceptorsinvariousmicroenvironments.Recently,becauseof these characteristics of CCN proteins, as a result, the abbreviation CCN was redefined as “CellularCommunicationNetwork”andtheirgenenamesCCN1–6wereofficiallyadopted by the HUGO Gene Nomenclature Committee (HGNC) in 2018. The abnormal regula- tionoftheseproteins,consequentlydisturbingthecellularcommunicationnetwork,isalso involved in various diseases, such as fibrosis and renal, vascular, muscle, and metabolic diseases, as well as malignancies. Furthermore, the recent discovery of novel functions of these proteins, such as their intracellular functions, functions as circulating factors, and involvement in the biological defense, which stretch beyond the scope of intercellular signaling regulators, is expected to attract even more attention from basic scientists, espe- cially molecular and cellular biologists, and medical/dental scientists, as well as clinical scientists,inthefuture. ThisvolumewillbevaluableforthoseinterestedinCCNproteinsandserveasamanual depictingcutting-edgemethodologiesanddiscussingpracticaltipstohelpovercomeobsta- cles associated with experimentation pertaining to chemistry, biology, physiology, pathol- ogy,medical/dental sciences, and pharmacologyofCCN proteins.Wealso believethat, in conjunction with the first edition of CCN Proteins: Methods and Protocols, published in 2017, this volume will provide a comprehensive guide to the methods and protocols necessary for CCN research, from fundamental to state-of-the-art techniques, and be a valuable resource for a wide audience ranging from experienced CCN researchers looking fornewapproachestojuniorgraduatestudentstakingtheirfirststepsintothefieldofCCN research. Okayama,Japan MasaharuTakigawa v Contents Preface ..................................................................... v Contributors................................................................. xi 1 CCNProteins(CellularCommunicationNetworkFactors):Expanding TheirRepertoireTowardaNewConcept.... ....... ........ ....... ........ 1 MasaharuTakigawa PART I BASIC METHODS AND PROTOCOLS 2 OptimalCCN4ImmunofluorescenceforTissueMicroarray.......... ........ 13 Pierre-OlivierGaudreau,IsabelleCousineau,andJohnStagg 3 CellularFluorescenceImagingfor theEvaluationofBioactivity ofCCNFamilyProteins .... ........ ....... ....... ........ ....... ........ 23 HarumiKawaki,SatoshiKubota,andMasaharuTakigawa 4 ImagingofMolecularInteractionBetweenCCNProteinandItsBinding Partners:AnInSituProximityLigationAssayofInteractionBetween CCN2andRab14inChondrocytes ......... ...... ....... .. ... .... ........ 31 MitsuhiroHoshijima,ErikoAoyama,HiroshiKamioka, andMasaharuTakigawa 5 WesternBlotProtocolsforAnalysisofCCNProteinsandFragments inExosomes,Vesicle-FreeFractions,andCells....... ........ ....... ........ 39 KishoOno,YukaOkusha,ManhTienTran, KokiUmemori,andTakanoriEguchi 6 ComprehensiveMethodforExosomeIsolationandProteome AnalysisforDetectionofCCNFactorsin/onExosomes...... ....... ........ 59 TakanoriEguchi,YukaOkusha,YanyinLu, KishoOno,EmanA.Taha,andShiroFukuoka 7 EvaluationoftheMolecularInteractionBetweenCCNProteinandIts BindingPartners:ASolid-PhaseBindingAssayandSurface PlasmonResonance ........ ........ ....... ....... .... .... ...... ......... 77 ErikoAoyamaandMasaharuTakigawa 8 ProtocolsforScreeningPeptidesBindingtoCCNFamilyProteins andTheirExtendedUtility.......... ....... ....... ........ ....... ........ 87 SatoshiKubota,HarumiKawaki,andMasaharuTakigawa 9 Transfection,Spinfection,Exofection,andLuciferaseAssaysforAnalysis ofCCNGenesExpressionMechanism....... ....... ........ ....... ........ 103 TakanoriEguchi,YanyinLu,EmanA.Taha,andYukaOkusha 10 AnalysesofthePosttranscriptionalRegulationofCCNGenes:Approach toMultipleStepsofCCN2GeneExpression........ ..... ... ....... ........ 127 SeijiKondo,SatoshiKubota,andMasaharuTakigawa vii viii Contents 11 ProtocolforCRISPR/CasGenomeEditingforInvestigatingCell CommunicationNetwork........... ....... ....... ........ ....... ........ 157 YukaOkushaandTakanoriEguchi 12 UtilizingPublicMolecularBiologicalDatabasesforCCN FamilyResearch .... ....... ........ ....... ....... ........ ....... ........ 169 SatoshiKubota PART II FUNCTIONAL ANALYSIS 13 EvaluationoftheEffectsofCCN4onPancreaticBeta CellProliferation .......... ... ..... ...... ....... ...... ... ....... ........ 191 RebecaFerna´ndez-RuizandRosaGasa 14 AnalysisofCCNFunctionsinLiverRegenerationAfter PartialHepatectomy ....... ........ ....... ....... ........ ....... ........ 209 NaiyuanCheng,Ki-HyunKim,andLesterF.Lau 15 TheEvaluationofMeniscusRegenerativeEffectsofLIPUS-Induced CCNProteins:InductionbyLIPUSofCCN2andMeniscus-Related GenesinCulturedMeniscusCellsandMeniscusTissues...... ....... ........ 223 YusukeKamatsuki,ErikoAoyama,TakayukiFurumatsu, ToshifumiOzaki,andMasaharuTakigawa 16 AnalysisofChemotacticPropertyofCCN2/CTGFinIntramembranous Osteogenesis...... ....... ..... .... ....... ....... ........ ....... ........ 237 NobuoTakeshitaandTerukoTakano-Yamamoto 17 NovelCellBiologicalAssaysforMeasuringBoneRemodeling ActivitiesofCCNProteins.......... ....... ....... ........ ....... ........ 255 TakashiNishida,SatoshiKubota,andMasaharuTakigawa 18 ImagingAnalysisofNeurotrophicEffectsbyCCN2ProteininNeuronal PrecursorCellsDerivedfromHuman-InducedPluripotentStemCells........ 269 MasakiShojiandTakashiKuzuhara 19 GeneExpressionAnalysisofCCNsAlongwithOdontoblastic DifferentiationInVivo .. ... ...... ......... ....... ........ ....... ........ 281 KazumiKawataandSatoshiKubota PART III PATHOPHYSIOLOGY 20 AngiogenesisAssaysfor theAnalysisofCCNProteins........ ....... ........ 295 TsuyoshiShimo,MariShimatani,AkihikoTanimura, andMasaharuTakigawa 21 UsingtheBleomycin-InducedModelofFibrosistoStudytheContribution ofCCNProteinstoSclerodermaFibrosis.... ....... ........ ....... ........ 309 AlexanderPeidl,JohnNguyen,PratyushaChitturi, BruceL.Riser,andAndrewLeask 22 AtomicForceMicroscopy-BasedMeasurementsofRetinalMicrovessel StiffnessinMicewithEndothelial-SpecificDeletionofCCN1........ ... .. ... 323 BrahimChaqour,MariaB.Grant,LesterF.Lau,BiranWang, MateuszM.Urbanski,andCarmenV.Melendez-Vasquez Contents ix 23 ImmunohistochemicalAnalysisofCCN2inExperimentalFracture HealingModels .... ....... ........ ....... ....... ........ ....... ........ 335 TsuyoshiShimo,HiroakiTakebe,SakiFujii,andAkihiroHosoya 24 MouseModelsofTumorBoneMetastasisandInvasionforStudying CCNProteins...... ....... ........ ....... ....... ........ ....... ........ 343 TsuyoshiShimo,TatsuoOkui,NaohiroHorie, KenjiYokozeki,MasaharuTakigawa,andAkiraSasaki 25 RegulationofCCN2andItsBioactivitybyAdvanced GlycationEndProducts .... ...... .. ... .... ....... ........ ....... ........ 355 XiaoyuWang,DanqingMin,andStephenM.Twigg 26 AnalysisofCCN4/WISP1EffectsonJointTissuesUsing Gain-andLoss-of-FunctionApproaches..... ....... ........ ....... ........ 369 MartijnH.J.vandenBoschandEsmeraldaN.BlaneyDavidson 27 GenerationofConditionalKOMiceofCCN2andItsFunction intheKidney....... ....... ........ ....... ....... ...... .. .... ... ........ 391 HidekiYokoi,Naohiro Toda,andMasashiMukoyama 28 AnalysisoftheFunctionofCCN2inTubularEpitheliumCells withaFocusonRenalFibrogenesis......... ....... ........ ....... ........ 411 HiroakiAmano,TsutomuInoue,TakeruKusano, andHirokazuOkada Index ...................................................................... 427 Contributors HIROAKIAMANO • DepartmentofNephrology,FacultyofMedicine,SaitamaMedical University,Saitama,Japan ERIKO AOYAMA • AdvancedResearchCenter forOralandCraniofacialSciences (ARCOCS),OkayamaUniversityDentalSchool/GraduateSchoolofMedicine,Dentistry, andPharmaceuticalSciences,Kitaku,Okayama,Japan ESMERALDAN.BLANEYDAVIDSON • ExperimentalRheumatology,Radbouduniversity medicalcenter,Nijmegen,TheNetherlands BRAHIMCHAQOUR • DepartmentofOphthalmology,UniversityofPennsylvania,Perelman SchoolofMedicine,Philadelphia,PA,USA NAIYUANCHENG • DepartmentofBiochemistryandMolecularGenetics,UniversityofIllinois atChicago,Chicago,IL,USA PRATYUSHACHITTURI • CollegeofDentistry,UniversityofSaskatchewan,Saskatoon,SK, Canada ISABELLECOUSINEAU • CentrederechercheduCentrehospitalierdel’Universite´deMontre´al, InstitutduCancerdeMontre´al,Montre´al,QC,Canada TAKANORIEGUCHI • DepartmentofDentalPharmacology,GraduateSchoolofMedicine, DentistryandPharmaceuticalSciences,OkayamaUniversity,Okayama,Japan REBECA FERNA´NDEZ-RUIZ • Institutd’InvestigacionsBiome`diquesAugustPiiSunyer (IDIBAPS),Barcelona,Spain;CentrodeInvestigaci(cid:1)onBiome´dicaenReddeDiabetesy EnfermedadesMetab(cid:1)olicasAsociadas(CIBERDEM),Barcelona,Spain SAKIFUJII • DivisionofReconstructiveSurgeryforOralandMaxillofacialRegion, DepartmentofHumanBiologyandPathophysiology,SchoolofDentistry,HealthSciences UniversityofHokkaido,Hokkaido,Japan SHIRO FUKUOKA • DepartmentofDentalPharmacology,GraduateSchoolofMedicine, DentistryandPharmaceuticalSciences,OkayamaUniversity,Okayama,Japan; DepartmentofOrthopaedicSurgery,OkayamaUniversityGraduateSchoolofMedicine, DentistryandPharmaceuticalSciences,Okayama,Japan TAKAYUKI FURUMATSU • DepartmentofOrthopedicSurgery,OkayamaUniversityGraduate SchoolofMedicine,Dentistry,andPharmaceuticalSciences,Kitaku,Okayama,Japan ROSAGASA • Institutd’InvestigacionsBiome`diquesAugustPiiSunyer(IDIBAPS), Barcelona,Spain;CentrodeInvestigaci(cid:1)onBiome´dicaenReddeDiabetesyEnfermedades Metab(cid:1)olicasAsociadas(CIBERDEM),Barcelona,Spain PIERRE-OLIVIER GAUDREAU • DepartmentofOncology,Queen’sUniversityandCanadian CancerTrialsGroup,Kingston,ON,Canada MARIAB.GRANT • DepartmentofOphthalmologyandVisualSciences,Universityof AlabamaatBirmingham(UAB),Birmingham,AL,USA NAOHIROHORIE • DivisionofReconstructiveSurgeryforOralandMaxillofacialRegion, DepartmentofHumanBiologyandPathophysiology,SchoolofDentistry,HealthSciences UniversityofHokkaido,Hokkaido,Japan xi
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