LAMININ-5 SIRPA Function of the γ2 chain in epithelial cell adhesion and SALO migration, and expression in epithelial cells and Department of Biochemistry carcinomas 1999 SIRPA SALO LAMININ-5 Function of the γ2 chain in epithelial cell adhesion and migration, and expression in epithelial cells and carcinomas Academic Dissertation to be presented with the assent of the Faculty of Science, University of Oulu, for public discussion in Raahensali (Auditorium L 10), Linnamaa, on Octobet 2nd, 1999, at 12 noon. OULUN YLIOPISTO, OULU 1999 Copyright © 1999 Oulu University Library, 1999 Manuscript received 10.8.1999 Accepted 31.8.1999 Communicated by Professor Nils Brünner Professor Jyrki Heino ISBN 951-42-5342-6 (URL: http://herkules.oulu.fi/isbn9514253426/) ALSO AVAILABLE IN PRINTED FORMAT ISBN 951-42-5334-5 ISSN 0355-3221 OULU UNIVERSITY LIBRARY OULU 1999 SaloSirpa,Laminin-5:Functionoftheγ2 chaininepithelialcelladhesionand migration,andexpressioninepithelialcellsandcarcinomas BiocenterOuluandDepartmentofBiochemistry,UniversityofOulu,FIN-90570Oulu,Finland 1999 Oulu,Finland (Manuscriptreceived10.8.1999) Abstract Lamininsarebasementmembraneglycoproteinsconsistingofthreepolypeptidechainsα, β andγ. Untilnow12membersoftheproteinfamilyhavebeencharacterizedandallisoformshaveanαβγ chaincomposition,buttheyassembleinvaryingcombinationsofchainvariants.Thefunctional propertiesoflamininsincludecelladhesion,proliferation,differentiation,growthandmigration. Laminin-5hasachaincompositionofα3β3γ2withthedistributionmainlyrestrictedtoepithelial basementmembranes,whereitsbiologicalfunctionsinvolveanchorageandlocomotionofcells. Theimportanceofthisproteinfortheattachmentofbasalkeratinocytesisclearlydemonstratedby thefactthatallgenesencodingitschainshavebeenshowntobemutatedinthesevereskin blisteringdiseaseEpidermolysisbullosajunctionalis. Thepresentstudyfocusedoninvestigationsoftheroleofthelaminin-5isoformandparticularly itsγ2chainincelladhesionandmigration.Theroleoftheshortarmofthelamininγ2chaininthe processofepithelialcellattachmentistoserveasakindofabridgingmoleculetotheextracellular environment,becauseitdoesnothaveanycellbindingactivitybyitself.Itwasalsoshownthatthe newlysynthesizedγ2chainparticipatesinthecomplexprocessofcellmigration,probablyasoneof thefirstattachmentcomponentsformovingcells.Thus,asamigrationanddifferentiation- associatedmolecule,laminin-5wasconsideredapotentialmarkerfordetectionofmalignant processeswherecellmovementplaysarole.Subsequentlyitwasshownthattheγ2chainis expressednotonlyinarestrictedmannerinhumanepithelialtissues,butalsoinanumberofhuman epithelium-derivedcancers.Insomecarcinomas,expressionoftheγ2chainappearedtobea characteristicofcancercellswithinvasiveproperties.Examinationofover50dysplasiasand cervicaltumorsrevealedthatγ2chainantibodieswereabletodistinguishbetweenlesionswithor withoutinvasivecapacity.Thisisthefirstsystematicstudyofepithelialcancerswhereγ2chain antibodieshavebeenshowntobeausefulmarkerinthehistopathologicaldiagnostics.Inaddition, thisstudyshowedinamousetumormodelthattheγ2chainoflaminin-5hasapotentialforbeingof useforinvivotumorimaging. Keywords:Basementmembrane,cellanchorage,immunohistology,tumorimaging Acknowledgements ThepresentworkwascarriedoutattheBiocenterOulu,andtheDepartmentof Biochemistry,UniversityofOulu.Iwouldliketothankmysupervisor,ProfessorKarl Tryggvason,forintroducingmetothescientificworld,andforhissupportduringthis work.Ithasbeenaprivilegetoworkinhisinternationalresearchgroupwithexcellent facilities.Ialsowishtothankallformerandpresentmembersofthebasementmembrane groupbothinOuluandinStockholm.Especially,Iowemythankstopeoplewhostill workinOulu,toMaaritKangasandTomiAirenneformanycheerfulmoments,andto OlgaBeltcheva,PaulaMartin,VesaRuotsalainen,TuireSalonurmi,SirpaKontusaari, PaulaReponen,PaulaVäisänen,andMaireJarvaforcreatinganiceandrelaxed atmosphereatwork.IalsothankAriTuuttilaforsendingmestrange,butfunnyemail attachmentsfromStockholm.Iwishtoexpressmygratitudetoallco-authorsand collaborators.Particularly,ithasbeenapleasuretoworkwithDr.BarbroSkyldbergand Dr.GertAuerfromtheKarolinskaInstitute.MyspecialthanksgotoHeliHaakanafor collaborationwhenshewasstillworkinginourresearchgroup,andforherfriendship duringthattimeandafterwards.IamgratefultoProfessorNilsBrünnerandProfessor JyrkiHeinoforvaluablecommentsonthemanuscriptofmythesis,andtoSidneyHigley forcarefulandrapidrevisionofthelanguage. ProfessorKalervoHiltunen,aswellasotherprofessorsandgroupleaders,are acknowledgedforestablishinggreatfacilitiesandamodernattitudetowardsresearchwork attheDepartmentofBiochemistry.Iwouldalsoliketothankallcolleaguesandotherstaff attheDepartmentforhelpingmeinmanyways. Last,butnotleast,myveryspecialthanksgotopeoplewhohavetakencareofme outsidethelaboratoryand,whatisalsoimportantforaresearcher,helpedmetoforget worknowandthen.First,IamdeeplygratefultoIldikóVeressformanykindsof entertainment,forinstance,fortakingmetoLaplandwithforeigners,andforimporting UnicumtoFinlandseveraltimes.MysincerethanksgotomyfriendsJariandJulie Pekkarinen,andtoKaisuAinassaari,formanypleasantmomentsduringtheseyears,and toMirkaPohjanrinne,forherniceandchallengingfriendship.IwishtothankAnu Leinonenforbeingmyfriend,forhernumerous5o’clockearly-morningphonecallsand, especially,forbeinganexcellenttravelguideduringourtrips.LahjaUittodeservesmy warmestthanksforsharinggoodandbaddays,andforherpatienceinlisteningtomy storiesthatcanbeabitstrangesometimes.Finally,IthankmydearparentsAnjaand 8 Armas, brother Jyrki, sister-in-law Anneli, and two lovely young ladies, my favorite girls, Linda and Erika, for their support and love. This work was granted by the Cancer Foundation of Northern Finland, the Finnish Cancer Foundation, the Pohjois-Pohjanmaa Branch of Finnish Cultural Foundation, and the University of Oulu. Oulu, August 1999 Abbreviations AP-1 activatorprotein-1 bp basepair cDNA complementaryDNA ECM extracellularmatrix EGF epidermalgrowthfactor EHS Engelbrecht-Holm-Swarm GBM glomerularbasementmembrane GST glutathioneS-transferase HD1 hemidesmosomalprotein-1 IF intermediatefilament IgG immunoglobulinG i.m. intramuscularly i.v. intravenously kb kilobase kDa kilodalton mRNA messengerribonucleicacid PBS phosphatebufferedsaline TGF transforminggrowthfactor uPA urokinaseplasminogenactivator uPAR urokinaseplasminogenactivatorreceptor Originalarticles Thepresentworkisbasedonthefollowingarticles: I PykeC,SaloS,RalfkiærE,RømerJ,DanøK&TryggvasonK(1995)Laminin-5 isamarkerofinvadingcancercellsinsomehumancarcinomasandis coexpressedwiththereceptorforurokinaseplasminogenactivatorinbudding cancercellsincolonadenocarcinomas.CancerRes.55:4132-4139 II SaloS,HaakanaH,KontusaariS,HujanenE,KallunkiT&TryggvasonK (1999)Laminin-5promotesadhesionandmigrationofepithelialcells: identificationofamigration-relatedelementintheγ2chaingene(LAMC2)with activityintransgenicmice.MatrixBiol.18:197-210. III SaloS,AirenneT,HeikkiläJ&TryggvasonK(1999)Intravenouslyinjected anti-lamininγ2chainantibodytargetstoepithelialtumors.Submittedfor publication. IV SkyldbergB,SaloS,ErikssonE,AspenbladU,MobergerB,TryggvasonK& AuerG(1999)Laminin-5isamarkerofinvasivenessincervicallesions.JNatl CancerInst,Inpress. Contents Abstract Acknowledgements Abbreviations Originalarticles 1.Introduction...................................................................................................................13 2.Reviewoftheliterature.................................................................................................15 2.1.BasementMembranes.............................................................................................15 2.2.Laminins.................................................................................................................16 2.2.1.Isoformsandchains.....................................................................................16 2.2.2.Structure,self-assemblyandpolymerization................................................19 2.2.3.Location,structureandregulationofgenes.................................................24 2.2.4.Tissuedistribution........................................................................................26 2.2.5.Interactionswithotherbasementmembraneligands....................................28 2.2.6.Interactionswithcellularreceptors..............................................................29 2.2.7.Examplesofbiologicalfunctions.................................................................32 2.2.7.1.Laminin-5asacomponentoftheepithelialanchoringsystem........32 2.2.7.2.Laminin-2/laminin-4andattachmentofmusclecells......................34 2.2.7.3.β2chainoflaminin-3inneuromuscularjunctionsandkidney........35 2.2.7.4.Importanceoftheα5andγ1 chainsinembryogenesis....................35 2.3.InteractionsoftumorcellswithECMandbasementmembrane.............................36 2.3.1.Generalaspectsoftumorprogression..........................................................36 2.3.2.Lamininsandcancer....................................................................................37 3.Outlinesofthepresentstudy.........................................................................................38 4.Materialsandmethods...................................................................................................39 4.1.Preparationandcharacterizationofpolyclonalantibodies.....................................39 4.2.Expressionofrecombinantlamininγ2chain..........................................................39 4.3.Immunohistochemistryandinsituhybridization....................................................40 4.4.Cells........................................................................................................................40