Col3a1 in procollagenIprocessingduringmusculoskeletaldevelopment. bone andmusculotendinoustissues.ADAMTS3inducedprocollagenIprocessingindermatosparacticfibroblasts,suggesting arole Adamts3 mouse embryogenesis,demonstrateddistincttissue-specific,overlappingexpressionpatternsoftheproteaseandsubstrategenes. the aorticwallappearednormal.Although specific geneexpression,andprovideinsightsintocollagenbiosynthesisthepathobiologyofdermatosparaxis. show howevolutionarilyrelatedproteaseswithsimilarsubstratepreferencesmayhavedistinctbiologicalrolesowingto tissue Adamts2 Adamts2 ␣ Adamts2 the samedegree,whichsuggestscompensationbyADAMTS2homologsADAMTS3andADAMTS14.Insituhybridizationof and asubtypeoftheEhlers-Danlossyndrome(dermatosparactictypeorVIIC)inhumans.Notallcollagen-richtissuesareaffect Mutations inADAMTS2,aprocollagenamino-propeptidase,causesevereskinfragility, designatedasdermatosparaxisinanimals, and SuneelS.Apte Carine LeGoff biosynthesis anddermatosparaxis homologous ADAMTSproteases:insightsoncollagen during mouseembryogenesisbyspecializationof Regulation ofprocollagenamino-propeptideprocessing Development 133,1587-1596(2006)doi:10.1242/dev.02308 DEVELOPMENT ANDDISEASE Accepted 2February 2006 3 1 one (Niederreither etal.,1995).CollagenIcontainstwo with collagenIatmany locations,withtheexception ofbone form heterotypicfibrilsanditisco-expressed invarying proportions Paepe andMalfait, 2004).Itcanco-polymerizewithcollagenIto as thevascular wall, dermis,lung,heartandgastrointestinaltract(De 1999). CollagenIIIisacomponentofmany stretchabletissues,such and inthenucleuspulposusofintervertebral disc(Zhuetal., collagen ofcartilage,but itisalsopresentinthevitreous oftheeye ligaments, andthedermisofskin.CollagenIIismajor major extracellular matrix(ECM)proteinofbone, tendons, supramolecular aggregates (Birketal.,1991).CollagenIisthe and byassemblyofthecollagentriple-helixintoavariety of 1991). They formorganotypic matricesbytissue-specificexpression quantitatively majorfibril-forming(fibrillar)collagens(Olsen, types I,IIandIIIareevolutionarily related,andcomprisethe elements ofalmostalltissuesoutsidethenervous system.Collagen Collagens aretriple-helicalmoleculesthatformthemajorstructural INTRODUCTION KEY WORDS:ADAMTS,Metalloprotease, Procollagen, Dermatosparaxis,Ehlers-Danlossyndrome *Author forcorrespondence (e-mail:[email protected]) USA. 44195, USA. Research Institute,ClevelandClinicFoundation,9500EuclidAvenue, Cleveland,OH Integrative Genoproteomics, University of Liège, 4000 Sart Tilman, Belgium. Integrative Genoproteomics, UniversityofLiège,4000SartTilman, Department ofCellBiology, ThomasJefferson University, Philadelphia,PA 19107, Department ofBiomedicalEngineeringandOrthopaedicResearch Center, Lerner 1(II) and ␣ 2(I) chain,whereascollagensIIandIIIarehomotrimersof in manytissuesincludingthelungsandaorta , butnot , –/– in maturedermis,whichpossiblyexplainsthepresenceofsomeprocessedskinprocollagendermatosparaxis.Thedata Adamts3 2 Laboratory of Connective Tissues Biology,Laboratory ofConnectiveTissues CenterofBiomedical ␣ mice hadabnormallungs,characterizedbyadecreasedparenchymaldensity. However, theaortaandcollagenfibrilsin 1(III) chains,respectively (Olsen,1991). Structural 1 Adamts2 , RobertP. T. Somerville and 1, Adamts14 * or Adamts14 andofthegenesencodingmajorfibrillarcollagens, , , wasco-expressedwith Adamts14 1 , Frederic Kesteloot ␣ 1(I) chainsand , and lacked developmentaltissue-specificexpression,itwasco-expressedwith Adamts2 Col2a1 Adamts2 –/– dermatosparactic type)inhumans(Coligeetal., 2004;Coligeetal., and theEhlers-Danlossyndrome-type VIIC(EDS-VIICorthe clinical syndrome–dermatosparaxis (fragilityofskin)inanimals procollagen chains.Instead, broader effects than structuralmutationsinvolving individual pathway, itwas expected thatdefectsinADAMTS2 would have supramolecular aggregates. Becauseofacommonbiosynthetic collagen monomersassembleintofibrilsandtissue-specific (Tuderman andProckop, 1982;Wang etal.,2003).Theprocessed 2) canexcise theN-propeptideofallthreemajorfibrillarcollagens And MetalloproteasedomainwithThrombospondintype1motifs protease (Kessler etal.,1996).ADAMTS2 (forADisintegrin-like Olsen, 1991).TheC-propeptideisexcised byBMP1,anastacin-like propeptide eitherpriortoorfollowing secretion(Cantyetal.,2004; procollagen moleculesundergo proteolyticexcision ofeach mechanisms. Following intracellularassembly ofthetriplehelix, three majorfibrillarprocollagenssharesimilarbiosynthetic end byglobular non-collagenouspropeptides(Olsen, 1991).The consist ofalongtriple-helical(collagenous)domainflanked ateach aneurysms andskinfragility(DePaepe andMalfait, 2004). Danlos syndrome(typeIV),whichischaracterizedbyaortic collagen IIImutationscausethevascular subtypeoftheEhlers- al., 1995;Cole,1997;SneadandYates, 1999;Tiller etal.,1995),and as Sticklersyndrome,arecausedbycollagenIImutations(Chanet 2004). Chondrodysplasias,sometimeswithocularanomalies,such mutations causeosteogenesisimperfecta(RauchandGlorieux, where singlecollagentypesaremajorconstituents.CollagenI mutations incollagengeneshave seriousconsequences fortissues mice showedwidespreaddefectsinprocollagenIIIprocessing. 2 Fibrillar collagensaremadeasaprecursor(procollagen)and , KimerlyPowell ncriaetruhu eeomn,andwith in cartilagethroughoutdevelopment, , butnot Adamts3 1 Col1a1 , DavidE.Birk or ADAMTS2 Adamts14 , Col2a1 RESEARCH ARTICLE , wasco-expressedwith mutations leadtoalimited and 3 , AlainC.Colige Col3a1 , during Col1a1 ed to - 1587 in 2

DEVELOPMENT (forward) and5 unique C-terminal domain),5 generated bysubcloninga600basepair(bp) The following primerswereusedtoamplify IMAGE cloneAA832579intopBluescriptII(Stratagene,LaJolla,CA). 3 terminal domainofmouse embryo cDNA. cDNA fragmentsbyRT-PCR ofmouse 17.5dayspost-coitum(dpc) Naomi Fukai(Harvard MedicalSchool).The The mousepro- by E.Vuorio (University ofTurku, Finland)(Niederreitheretal.,1995). MATERIALS ANDMETHODS dermatosparaxis. in mice,withpossiblyimportantimplicationsforhuman perspective. ADAMTS2 isessentialforprocollagenIIIprocessing procollagen IandIIaminopropeptidasefromadevelopmental in cartilage,ADAMTS3, andnotADAMTS2, maybethemajor with itsprominentexpression insomeprocollagenIrich tissuesand demonstrate thatADAMTS3 canprocessprocollagenI.Together procollagen substratesduringmouseembryogenesis.We developmental expression oftwo oftheseenzymesandtheir encoding procollagenI,IIandIII,thatthereishighlycoordinated ADAMTS2, ADAMTS3 andADAMTS14, aswellofthat through thetemporalandspatialanalysisofmRNA encoding Fernandes etal.,2001;Wang etal.,2003).Here,wedemonstrate, their catalyticactivities arevery similar(Coligeetal.,2002; proteases. This,andprevious experimental evidence, suggestedthat (HETGHVLGMEHD) differs fromthatoftheotherADAMTS any otherenzyme.Theactive sitesequence intheircatalyticdomain domain structureandauniquemodularcompositionnotpresentin procollagen amino-propeptidases(PNPs).They have anidentical evolutionarily relatedclusterofADAMTS proteases,the collagenous matricesarelaiddown, hasnotbeenachieved. biosynthesis invivo, particularlyduringembryogenesis,asthefirst understanding ofhow theseenzymesparticipateincollagen III (Wang etal.,2003).Nevertheless, acomprehensive (Colige etal.,2002),andthatADAMTS2 couldprocessprocollagen homologous enzyme,ADAMTS14, couldprocessprocollagenI (Fernandes etal.,2001).Subsequently, itwas shown thatanother ADAMTS3, couldprocesstheamino-propeptideofprocollagenII expected site(Coligeetal.,2002).We previously foundthat dermatosparaxis, theresidualprocessedprocollageniscleaved atthe Nusgens, 1993);thisissupportedbytheobservation thatinbovine may processprocollagenIintissuesotherthanskin(Lapiereand or notatall(Fernandesetal.,2001;Li2001). dermatosparactic calves andmiceisonlyminimallycompromised, 1993). ProcessingofprocollagenIIinthecartilage rich tissues,suchasbone,tendonandmuscle(LapiereNusgens, (dermal) collagen,thereisonlyamodesteffect on othercollagenI- 1992). Althoughthisdisorderislargely causedbyanomaliesinskin and ofrelatively uniformdiameter(Lietal.,2001;Nusgens branched and‘hieroglyphic’oncross-section,insteadofbeinground the dermisofdermatosparacticcalves, humansandmicearethin, 1993; Smithetal.,1992;Wertelecki etal.,1992).Collagenfibrilsin 1999; LapiereandNusgens,1993;Nusgensetal.,1992;Petty 1588 collagen ( cDNA clonessuitableforinvitrotranscriptionofmousepro- Riboprobes Ј (forward) and5 Adamts14 Adamts3 ADAMTS2, ADAMTS3 andADAMTS14 constitutean It was hypothesizedthatenzymescloselyrelatedtoADAMTS2 RESEARCH ARTICLE Col1a1 (amplifying a528bpfragmentcorresponding totheuniqueC- (producing aPCRproductof310 bp thatcorrespondstothe Ј -AGGCTGCAGATCTGCACCATG-3 ) andmousepro- ␣ Ј 1(II) collagen( -TGGACTTGTCACCCAAACATG-3 Adamts3 Ј -AGGTGGGTGACAGAAGAGTGG-3 ␣ ), 5 Col2a1 1(III) collagen( Ј -CTGAAGAGCTGGTCTCAGTG- ) cDNA was provided by Dr Not Adamts3 Col3a1 I- Adamts2 Pst Ј (reverse). Ј I fragmentfrom (reverse); and ) wereprovided and probe was Adamts14 ␣ 1(I) Ј described (Birkand Trelstad, 1986).Thicksections (1 reconstruction ofthe images.SamplesforTEMwere prepared aspreviously by micro-computerizedtomography (mCT),followed bythree-dimensional mice werekilledbyCO Genotype analysiswas byPCRaspreviously described.Two-month-old (Tulane University) andwereusedunderapproved institutionalprotocols. [ vitro transcriptionusingT3,T7orSP6RNA polymerasesinthepresenceof WI). Radioactive senseandantisensecRNA probesweregeneratedbyin peptide coupledtoNHS-sulfolink(Pierce). International, Houston,TX).Theantiserumwas affinity-purified againstthe COOH), coupledtokeyhole-limpet hemocyanin (AlphaDiagnostics terminal peptidefromhumanADAMTS3 (CKKDGKIIDNRRPTRSSTLER- A polyclonalantibodywas generatedin rabbitsbyimmunizationwithaC- ADAMTS3 antibodies hybridized to[ embedded inparaffin wax, andsectionedat6 at 7.5dpcand9.5dpc,werefixed overnight in4%paraformaldehyde, gestational age7.5to17.5dpc.Embryos,includingthesurroundinguterus In situhybridizationwas performedontissuesectionsofmouseembryos In situhybridization Adamts2 Adamts2 Histology, transmission electron microscopy (TEM)andmCTof react withothercollagentypes(datanotshown). antisera, usedata1:2000dilution,werehighlyspecificanddidnotcross- guinea pigwithbovine collagenIorIII,respectively. These ECL. Theseantiserawereproducedbyrepeatedimmunizationofrabbit and western blottingusingtypeIorIIIcollagen-specificantiserum and DTT. Thecharacterization ofcollagenpolypeptideswas performed by separately anddenaturedinLaemmlisamplebuffer withorwithout0.1M G418. After24hours,theculturemediumandcelllayerwerecollected supplemented with2%FCSandascorbicacid(50 et al.,2002).Co-cultureexperiments wereperformedinDMEM expressing eitherADAMTS3 orADAMTS2 (asapositive control)(Colige empty plasmidvector (anegative control),orwiththeplasmidvector cultured withequivalent numbersofHEK293cellstransfectedwiththe al., 2003).Dermatosparacticcalffibroblastswereculturedaloneorco- conditioned mediumwas carriedoutas previously described(Somervilleet glycosidase F(PNGaseF, New EnglandBiolabs,Beverly, MA)treatmentof levels ofADAMTS3 wereusedinsubsequentstudies.PeptideN- polyclonal antibodyata1:1000dilution,andclonesexpressing thehighest Culture mediawereanalyzedbywesternblottingwithADAMTS3 pSHTS3 wereisolatedaspreviously described(Fernandesetal.,2001). serum). Clonesstablyexpressing the medium (DMEMsupplementedwithantibioticsand10%fetalbovine HEK 293Fcells(Invitrogen, Carslbad,CA)weremaintainedingrowth dermatosparactic fibroblasts Cloning ofcellsexpressing ADAMTS3andco-culture with hind limbsofthe stain, Hart’s stainforelastinandtheMallory trichromestain.Theskullsand al., 2005),followed byroutinehistology, includingHematoxylinandEosin fixed in10%formalinunder inflationaspreviously described(Oblanderet histological analysisandfortransmission electronmicroscopy. Lungswere pseudocolor andsuperimposedonnuclearfluorescence(blue). MD). Silver grainsvisualizedondarkfieldmicroscopy weregiven ared viewed withImageProPlussoftware (MediaCybernetics,Silver Spring, crystal colorfilterslider(QImaging,Burnaby, BC,Canada).Imageswere GmbH), withaRetigaExiFast 1394cooledCCDcamerawithRGBliquid captured onaLeicaDMRuprightmicroscope(LeicaMicrosystems,Wetzlar signal) andfluorescencemicroscopy (nuclearstaining)imageswere with Hoechst33258(Sigma,StLouis,MO).Dark-field(autoradiographic Rochester, NY)andexposed at4°Cfor2to10days.Nucleiwerestained (Somerville etal.,2003).SlidesweredippedinNTB-2emulsion(Kodak, 35 S]-UTP (Perkin-Elmer). The PCRproductswereclonedintopGEMT-Easy (Promega, Madison, –/– -deficient ( mice inthe129Svstrainwerekindlyprovided byD.Prockop 35 Adamts2 S]-UTP labeledriboprobes,aspreviously described Adamts2 2 inhalation, andtissuesorgans wereisolatedfor –/– mice andwild-typelittermateswere analyzed –/– ) mice ADAMTS3 ␮ ␮ Development 133(8) m. Thesectionswere g/ml) intheabsenceof expression plasmid ␮ m) werecutand

DEVELOPMENT mesenchymal condensations thatbegin to express procollagenII and endochondral ossification.Cartilage isfirstformedaround12dpcin Most oftheskeleton isformed fromcartilagemodelsthatundergo expressed with Adamts3 inclusion ofasignificantamount ofmaternaltissue. previously describednorthernanalysisfindingsresultedfromthe uterus thatoccursduringpregnancy. Thesedatasuggestthatthe these biosyntheticenzymesreflectstheintenseremodelingof the with The expression overlapped significantlyinthedecidualreaction(Fig.1). expression was seeninthesetissues. reaction andthemyometriumofuterus.Prominent strong expression inmaternaltissues,specificallythedecidual there was noexpression intheembryoitself,whereastherewas (Fernandes etal.,2001).However, insituhybridizationclarifiedthat peritoneal liningandthewall ofvisceralorgans. expression was prominentinthevascular wall, lungmesenchyme, Col2a1 tendons andtheadventitia (outermostlayer)ofblood vessels. primarily expressed inbone,skin,perichondrium,periosteum, previous reports(Niederreitheretal.,1995).Thus, The distribution ofthefibrillarcollagenmRNA was consistentwith RESULTS Individual organs wereisolatedfrom Procollagen processing in 2K Ultrascandigitalcamera. Tecnai 12transmissionelectronmicroscopeequippedwithaGatanUS1000 lead citrate.Sectionswereexamined andphotographedat80kVusinga The aortawas stainedwithaqueous2%(w/v)uranyl acetatefollowed by (w/v) ethanolicuranyl acetatefollowed by0.04%(w/v)bismuthsubnitrate. UCT ultramicrotomeandadiamondknife.Thelungwas stainedwith2.5% stained withmethyleneblue-azureB.ThinsectionswerecutusingaLeica Procollagen amino-propeptidases rotated for2hoursat4°Candcentrifuged(20000 suspended in0.15Mextraction buffer [0.15MNaCl,50mMTris (pH7.5)], discarded andpelletswashed againasdescribedabove. Pelletswerethen buffer (aorta).Aftercentrifugation(20000 PMSF, 50mMTris (pH7.5)]ordirectlysolubilizedinLaemmlisample washing buffer [0.25Msucrose,20mMEDTA, 2.5mMNEM,0.5 littermates. Thesewereeithergroundinliquidnitrogenandsuspended highest expression ofboth Northern analysisofcommercialembryoRNA blotshadshown the tissuesatday7.5ofgestation maternal Adamts2 above backgroundlevels. probes forany ofthecollagenorADAMTS genesgave asignal expressed with blotted withanti-collagenIIIasdescribedabove. extracted inLaemmlisamplebuffer, separatedbySDS-PAGE andwestern sponges wereground,washed asdescribed above, andthenproteinswere tissue containingfibroblastsandbloodvessels (datanotshown). The collected. Histologydemonstratedthepresenceofabundant granulation hydrated inPBS.After4weeks,miceweresacrificedandsponges PVA spongesgrade3,M-PACT Worldwide Management,Eudora,KS)re- ofcircularPVA sponges(10mmdiameter, clinical subcutaneous insertion and analyzedbySDS-PAGE. Spongegranulomaswereinducedby the lyophilized, denaturedinLaemmlisamplebuffer withorwithout0.1DTT, the various sampleswerethendialyzedat4°Cin0.1Maceticacid, M NaCl,50mMTris (pH7.5)]and0.1Maceticacid.Adequateamountsof were collectedandpelletssequentiallyextracted in1Mextraction buffer [1 Adamts2 Col1a1 was expressed almostexclusively incartilage. , butnot and (Fig. 1).Theprominentco-expression of expression patternwas different, but alsooverlapped Col1a1 Adamts3 Col2a1 Adamts2 in skin,but notinbone.Noneofthesense Adamts2 are expressed inthe in cartilage Adamts2 –/– or Adamts2 g mice , 10minutes),supernatantswere Adamts14 and Col1a1 –/– g , 10minutes).Extracts Adamts3 mice andwild-type Col3a1 and , isco- Col1a1 Col1a1 at 7dpc Adamts3 was co- Col1a1 Col3a1 with was patients (DeCoster etal.,2003;Malfait et al.,2004).Incisorsfrom the secondarydentitionhave beendocumentedinEDS-VIIC from thatofwild-typelittermates (Fig.4A).Severe abnormalitiesof and thedimensionsofitscomponent bones,appearednodifferent of theskull,aswellsurface featuressuchasvascular foramina appendicular, axialorcraniofacial skeleton. Theoverall morphology attained, demonstratednoconsistent anomaliesineitherthe mCT analysisoftwo-month-old miceinwhomskeletal maturitywas with both typesofosteogenicprocesses, osteoid, whichissubsequentlymineralizedtoformbone.During membrane ossification,osteoblastsdepositcollagenIinthe During endochondralossification,aswellduringintra- musculotendinous development expressed with Adamts3, stage examined. detectable byinsituhybridizationcartilageatany developmental perichondrium (Fig.2B-G).Neither around cartilage(Zhuetal.,1999);The cartilaginous mesenchymaltissueislocatedintheperichondrium chondrocytes becamehypertrophic(Fig.2C,E-G).After 12dpc,pre- and temporalassociationof RT-PCR (Fernandesetal.,2001).However, inthatstudy, thespatial higher levels than procollagen IIinvitro,andthat aggrecan. We have previously shown thatADAMTS3 processed processed. Interestingly, collagen, typeX(LuValle etal.,1989),whoseN-propeptidesarenot collagen IIproductionandproduceanabundance ofashort-chain into hypertrophicchondrocytes and,asthey doso,they suppress (Fig. 2A-G).Chondrocytes indeveloping growth platesdifferentiate models, whetherintheappendicular, axialorcraniofacial skeleton subsequently, throughoutskeletal development inallcartilage expression fromtheinitiationofchondrogenesis(Fig. 2A)and, Scale bar:100 overlap. Asteriskindicatestheembryo,whichdidnotshowexpression. Col1a1 the 7.5dpcuterus. Fig. 1. Adamts2 musculotendinous tissueduringembryonicdevelopment. were detectablebyinsituhybridizationbone or with extended beyond thelimitsof determined. 17.5 dpc(Fig.2D,E,Fig.3B-D).Neither expressed inthesestructuresatmany locations intheskeleton at synthesize abundant collagenIlateindevelopment; Musculotendinous structuresdevelop around theskeleton and hypertrophic zoneofcartilage(Fig.2C,Fig.3A,C). Col1a1 Col1a1 , whereas Adamts2 –/– Adamts3 but not (Fig. 3A,B).Inaddition, ␮ mice haveanormalskeletonandteeth m. in perichondrium,includingthataroundthe Adamts2 and ADAMTS2 Note that Adamts3 Col1a1 RAwas associatedwith mRNA Adamts2 expression isdifferent buthaspartial Adamts3 Adamts3 mRNA inculturedchondrocytes by during boneand expression overlapwith ADAMTS3 Col2a1 Adamts3 RESEARCH ARTICLE or Adamts2 expression issimilartothatof Adamts3 Adamts3 Adamts3 Adamts14 expression domainintothe was downregulated as Adamts2 and mRNA was presentat nor expression domain was co-expressed Col2a1 was co-expressed Adamts14 Col2a1 nor isco- , Adamts3 Col1a1 Adamts14 was not mRNA 1589 were was in

DEVELOPMENT 1590 Adamts2 Adamts2 Adamts3 Adamts2 compared withthewild-typelittermates(Fig.4A).Although dermatosparactic calfskinfibroblasts(DS)inculture consistsofpro linked carbohydrate(Fig. 4C).Type Icollagen producedby PNGase Fasexpected, since ADAMTS3 ispredictedtohave aN- antibodies. Thereactive bandmigratedfaster afterdigestionwith protein thatwas reactive withtheaffinity-purified ADAMTS3 plasmid, but notvector-transfected cells,producedan~130-150kDa HEK293F cellsstablytransfected withanADAMTS3 expression defect indermatosparacticskinfibroblasts ADAMTS3 rescues aprocollagen Iprocessing papilla andatlower levels inameloblasts(Fig.4B). RESEARCH ARTICLE –/– –/– , but not expression was notdetectableduringtoothdevelopment, mice showed asubtlelossofsurface contourwhen mice appearednormal,althoughmolarteethfrom Adamts14 was highlyexpressed inthedental , pN ␣ processed atthe Nterminus[ cell layer. Intheconditionedmedium,onlycollagenmoleculesfully and asignificantamountofpC control. Whenco-culturedwith DS, fullyprocessed HEK293F cellsexpressing ADAMTS2 wereusedasapositive confluence conditionsobserved inco-culturesthanmonocultures. ADAMTS14 byDS,enzymesthatmaybemoreactive inthehigh- the synthesisoflow levels ofBMP1,ADAMTS3 and/or carboxy-procollagen peptidaseactivity. Thisisprobablyrelatedto medium. Thesedataillustratethepresenceoflow amino-and and pN are alsothemostabundant products,but pC with controlHEK293Ftransfectedcells(Ct),pro type Iaminoprocollagenpeptidaseactivity (Fig.4D).Inco-culture 1(I) andpro ␣ 1(I) inthemedium,demonstratingabsenceofsignificant ␣ 2(I) arealsodetected,especiallyintheconditioned expression of dens (d)ofthesecondcervicalvertebra.Notestrong Sagittalsectionthrough thebasi-occiput(BO)and (G) oval indicatesaspurioussignalfrom erythrocytes. manubrium sterni. Note manubrium sterni. expression inthehypertrophic chondrocytes (HC)ofthe hypertrophic chondrocytes. (E)Absenceof elements, demonstratingthat chondrocytes (asterisk)thatdonotexpress process. Theribelementscontainhypertrophic intercostal muscle(arrow). (F)Ribsandvertebrallateral Scale bar:100 (asterisk). Expression isseenintheperichondrium(arrows). chondrocytes andabsencefrom thehypertrophic zone small chondrocytes andtheseexpress contrast, thelateralprocess ofathoracicvertebra(v)has primary ossificationcenter(B). Fig. 2.Co-expression of but not arrow indicatesatendoninsertionsitewhere perichondrium (arrow) andbone.(D)Proximal femur. The Adamts3 Hypertrophic zonechondrocytes (HZ)donotexpress that expression isrestricted totheproliferating zone(PZ). (C) Expression intheproximal humerusgrowth plate.Note cartilage oftheanklejoint.T, tibia;Ts, Talus; C,calcaneus. developing ribs(arrows). (B)Expression inprimordial (A) Expression ininitialsitesofchondrogenesis inthe from whichthesectionwastakenisshownonright. right formorphologicalcorrelation; theageofembryo Hoechst 33258stainedimagesare includedonthefar used toshowexpression of musculoskeletal tissues. include hybridizationof cartilage from 12.5dpcto17.5dpc.InA-D,images ␣ 2(I) inthecelllayer, andpro Col2a1 . Col2a1 Adamts3 ␮ is expressed. (E-G)17.5dpcskeletal m. is notexpressed intheboneof ␣ ␣ 1(I), werefoundassociatedwith the 1(I), in regions containingsmall Col2a1 Adamts3 Adamts3 ␣ ( A-G Adamts3 2(I), pC Adamts3 Adamts3 to adjacentserialsections. Insituhybridizationis ) expression indeveloping ␣ Development 133(8) ␣ and 1(I), pN ␣ in developing Adamts3 1(I), pro ␣ 1(I) andpC is notexpressed by 1(I) andpro is expressed inthe Col2a1 ␣ Adamts3 Adamts3 1(I) and Adamts3 ␣ . Theblack 1(I), ␣ in 2(I) and ␣ . By ␣ ␣ ␣ 2(I)] 2(I), 1(I) , 2(I)

DEVELOPMENT Adamts2 minimal toothinvolvementin development, bonestructure and ( processing intheskeleton. has arole inprocollagen I Fig. 4. molars from the poorlydefinedtopographyof morphology from littermates.Note lower panelsshowtooth any craniofacialelements.The size, dimensionsororganizationof differences inoverallpatterning, type (+/+)littermatesdonotshow similar. ( although theoverallmorphologyis of confluencein the monoculture. Alternatively, HEK-293 cellsmaysecrete factors activatingthesynthesisofprocollagen. Scal amount ofcollagensynthesizedby DScultured alone,ascompared toco-cultures, wasrepeatedly observedandmayberelated to by ADAMTS2andADAMTS3inDS. SamplesusedinDwereblottingusing ananti-collagenIIIantiserum.Th alsoanalyzedbywestern cell layerorrecovered from theconditionedculture mediumisindicated between thetwopanels.( antibody useddetectsboth ADAMTS3 (TS3).Aftertwodaysin culture,blottingofanon-reducing western SDS-PAGE gelwasused todetecttypeIcollagen.N identical numberofHEK-293cells stablytransfectedwithanemptyexpression vector(Ct),or with thesamevectorencodingADA procollagen Iprocessing defectbyADAMTS2andADAMTS3indermatosparacticcalf skin fibroblasts (DS).DSwere platedaloneorm F, ADAMTS3 migratesfaster. Untransfectedcellsshowednoprotein. Molecularmassmarkersare indicatedontheright.( Conditioned mediumfromblottingwith apolyclonalantibodytoADAMTS3.Notethatupontreatment cellswasanalyzedbywestern stably transfected293cells. Adamts2 Procollagen amino-propeptidases skulls of three-dimensional reconstruction of ( strongly intheameloblasts(arrow). (DP), andwasexpressed less development inthedentalpapilla strongly expressed duringtooth A C ) Expression ofADAMTS3in Normalcraniofacial ) Adamts3 B Adamts2 –/– ) or Adamts3 mice. mCTanalysisand Adamts14 Adamts2 , not –/– , butnot mice andwild- –/– Adamts2 , was mice, ␣ 1 and , ␣ 2 chains,butreacts more strongly withthe ␣ 1 chain.Theidentityofcollagenpolypeptides associatedwiththe area inmiddlepanel).Scalebar:100 shows theAchillestendonatahighermagnification(boxed the anklecartilage(C)andtendon(arrow); thatontheright 15.5 dpc.Theimageontheleftshows 2D.( Fig. arrow. Theleftpanelinthisfigure isthesameasthatin (TF), whichcontainstheobturatortendons,isindicatedby ( maxilla, aswellindevelopingmassetermuscle(mt). of Fig. 3.Co-expression of ( zone (HZ)are indicated;arrows indicatemRNAinmuscle. Chondrocytes intheproliferating zone(PZ)andhypertrophic hypertrophic zoneandtheperiosteum(around bone). with (proximal humerus).Notethat right-hand panel.( dpc embryos;Hoechst33258-stainedimagesare showninthe musculoskeletal system C B Expression intheproximal femur. Thetrochanteric fossa ) Expression inthecraniofacialskeleton.Noteco-expression ) Col1a1 Col1a1 D and ) Expression inthedevelopingAchillestendonat in bone(B)andtheperichondriumaround the E Adamts3 ) Rescueoftheprocollagen IIIprocessing defect A ) Expression intheappendicularskeleton in bone(B)themandibleand . Allimagesare from 15.5and17.5 Adamts3 RESEARCH ARTICLE Adamts3 D and ␮ ) Rescueofthe m. Adamts3 expression overlaps e bar:100 Col1a1 MTS2 (TS2)or ote thatthe the lowerlevel ixed withan e lower expression in with PNGase in the ␮ m. 1591

DEVELOPMENT (Fig. 5E). Adamts3 vitreous humor, anotherextraskeletal siteof (Fig. 5D).We have notinvestigated third andfourthventricles (Fig.5C),andindorsalroot ganglia subcommisural organ, aspecializedependymalzonebetweenthe cortex (Fig.5B),andat17.5dpcitwas stronglyexpressed inthe dpc, Col2a1 addition, one suchlocationinthehindbrainat12.5dpc(Fig.5A).In We found are notassociatedwithcartilageformation(Cheahetal.,1991). Col2a1 Adamts3 cultured withcellsexpressing ADAMTS3. Procollagen IIIprocessingwas alsodetectedwhenDSwereco- processed procollagen processing(Fig.4E).Asignificantamountoffully ADAMTS2. activity, althoughitislower thanthatobserved inpresenceof collagen patterndemonstratesahighaminoprocollagenpeptidase activity. InDS/ADAMTS3-HEK293F cellco-culture,analysisofthe were detected,illustratingahighaminoprocollagenpeptidase 1592 The sameco-culturesampleswereinvestigated fortypeIII Adamts3 RESEARCH ARTICLE , expression hasbeenpreviously reportedatlocationsthat Col1a1 Adamts3 expression was seeninthewall oftheurinary bladder Adamts3 ␣ is expressed atextraskeletallocations 1(III) was observed inthepresenceofADAMTS2. was highlyexpressed inthedeveloping cerebral or was highlyexpressed atotherlocationswhere Col3a1 to bepreciselyco-expressed with eeexpression was absent.At15.5 gene Adamts3 Col2a1 expression inthe expression. Col2a1 at whether procollagenIIIprocessingwas affected in because ADAMTS2 processesprocollagenIII,weinvestigated As only Adamts2 Procollagen IIIprocessing isdecreased in expressed with 5E, Fig.6F).With thisexception, overlapped withtheexpression ofboth expression of Adamts14 the wall oftheurinarybladder(Fig.6F)andskin(seeFig.8A). (Fig. 6D),theintestinalwall, peritoneumandmesentery(Fig.6E), also co-expressed with was highestinthelungmesenchyme(Fig.6B,C). arteries (datanotshown). Expressionofboth Col1a1 throughout mouseembryogenesis(Fig.6A,B).Bycontrast, arteries, suchastheaorta,carotidandpulmonary Adamts2 expressed with all cases,asubstantialproportionofprocollagenIIIwas The different typesofextraction gave essentiallysimilarresults.In western blottingwas usedtoevaluate procollagenIIIprocessing. mice. Collagenswereextracted fromvarious mouseorgans and Adamts2 was expressed onlyintheadventitial (outermost)layerof Adamts2 was co-expressed with , butnot –/– Scale bar:100 cerebral cortex.( Note, inB-E, Adamts3 mainly hypertrophic chondrocytes. ( expression inthevertebralcartilage(v)thatcomprises the hindbrain(arrow). ( Fig. 5.Expression of ( of thesubcommissuralorgan.III,third ventricle. Adamts3 are shownintheright-handpanel.( musculoskeletal system. showed noco-expression with D ) Adamts3 mice Adamts3 Col3a1 was substantiallyco-expressed with in theinnermusculature oftheurinarybladder. and Col3a1 . Adamts3 in thewall oftheurinarybladderpartially Adamts3 in dorsalroot ganglia(drg).Notethesparse Col3a1 Col2a1 ␮ m. C ) Adamts3 in theflooroffourthventricle Adamts3 was notco-expressed with at othersites,suchasthepalate B Expression of ) Adamts3 or Col3a1 Hoechst 33258-stainedimages expression intheependyma Adamts14 Adamts2 outside the was nototherwiseco- E in thewall oflarge Development 133(8) A Col3a1 ) Expression of ) Co-expression of Col3a1 Adamts3 and , isco- Adamts2 and Col3a1 Col3a1 Adamts2 , although in the Adamts2 Col2a1 (Fig. , and was –/– .

DEVELOPMENT more dispersed.( bladder. Scalebar:100 mesentery ofthegut(G)andpancreas (Pa).( basisphenoid (BS),atwhichlocation (Fig. 6B,C).Lungsfromtwo-month-old ( granulomas inducedin whereas littleornoprocollagenIIIprocessingoccurredinthe induced inthegranulomasofwild-typemicewas processed, demonstrated thatmostofthenewly synthesized procollagenIII subcutaneous implantationofaPVA sponge.Theanalysis of The lungwas themostprominentsiteofdevelopmental expression were present. Spongegranulomasfrom two a consistentdecreaseinparenchymaldensityatboth2months(Fig. granulomas inducedin inflammation-induced fibrosis,proteinswereanalyzedfrom whether procollagenIIIprocessingby completely processedinwild-typemice(Fig.6G).To investigate unprocessed inthe Procollagen amino-propeptidases lung. ( expressed with Fig. 6.Co-expression of inflation tooptimizeparenchymaldistension. type littermateswereevaluated histologicallyafterfixationunder Adamts2 proportion offullyprocessed moleculesinthevarioustissues(lowerpanel).( collagen IIImolecularspeciesare indicatedontheleft.Scanningdensitometryofdifferent collagenIIIsignalswasused tissues with0.15MNaClisshown. Thetissueoriginofthesampleandrespective genotype isshownaboveeachlane;procol Adamts2 D ) Co-expression of Adamts2 C ) Expression of –/– mice. Protein wasextracted fromblottingusedtodefinethe molecularspeciesofprocollagen spongegranulomasandwestern III , whereitsdistribution correspondedwiththatof –/– Col3a1 mice havedistalairspacedistension G ) Defectiveprocollagen IIIprocessing intissuesfrom Adamts2 Adamts2 Col3a1 in thearterialwall,asshownattwodevelopmentalstages:12.5dpc carotid artery(A)and17.5dpcpulmonary(B).L, ␮ m. InD-F, Adamts2 Adamts2 Adamts2 –/– and and mice, whereasitwas extensively or –/– Adamts2 Col3a1 Adamts2 Col3a1 and mice (Fig.6H). –/– Col3a1 in themesenchymeofpalate(P). Adamts2 and wild-typemiceby is absent.( in lung.Notetheexpression inlungmesnchymebutnottheepitheliumofbronchial tubes(asterisk). F expression isweakerthan in lungandarteries, Adamts2 ) Co-expression of Adamts2 anddeficiencyofprocollagen IIIprocessing in , Adamts2 –/– –/– E mice andwild- ) Co-expression of mice andtheirwild-type littermatesare shown. Scalebar:100 occurred in –/– lungs had Col3a1 Adamts2 Adamts2 and Adamts2 Adamts2 procollagen IIIwereimpairedintheaortaandlungsfrom to beunaffected. from contrast, thehistology(Fig.7C)andelectronmicroscopy oftheaorta and surfactant-producing pneumocytes (datanot shown). By presence ofnormalbasementmembranes(Fig.7B),elasticfibers not reveal any abnormalitiesandtheultrastructureshowed the Histochemical detectionofelastinandcollagen(datanotshown) did was unaccompaniedbyinflammatorycellsorobvious fibrosis. 7A) and2weeks(datanotshown). Thisemphysema-like appearance Adamts2 the wild-typelittermates.Inbothlungandaorta(Fig.7D)of it isclearthatbothnewborn andadult data ontherelative proportionofthetwo collagensinthesetissues, type littermates(Fig.7E,F). significantly lessprocessingofbothcollagensthandidtheirwild- H ) Defectiveprocollagen IIIprocessing innewlysynthesizedcollagen We investigated whethertheprocessingofbothprocollagenIand Col3a1 –/– Adamts2 Adamts2 mice. Westernblotanalysisofprocollagen isolatedfrom mouse and –/– –/– within thesubepitheliallayerof developingurinary mice, theshapeanddiameterofcollagenfibrilsappeared mice. Althoughitisnotpossibletocommentfromthis Col3a1 is alsoexpressed intheperichondriumof –/– mice didnotshow consistentchangesfromthatof Col3a1 in theperitoneumliningliver(L,arrow), and Adamts2 expression isalsosignificantly weakerand –/– mice. RESEARCH ARTICLE Adamts2 ␮ ( m. A to calculatethe , B ) –/– Adamts2 mice demonstrated lagen IIIand is co- that 1593

DEVELOPMENT erythrocyte. ( exudate. ( expression of In embryonicskin,low-level mature mousedermis Fig. 7.Histologyandultrastructure oflungandaortain 1594 Adamts2 from procollagen III(above)andI(below)processing inthemouseaorta.Thevariousmolecularspeciesofcollagenare indicated. (below) processing inmouselungs.Samplesfromandadultmiceare wild-typeandmutantnewborn illustrated.( and shape,uniformityoffibrildiameter. Scalebar:100 elastin lamellae(arrow). ( However, intwo-week oldmice,both detected inthedermis,but not process procollagen IIIinvitro,but the invivo relevance was unclear 1982). Wang etal.recentlydemonstratedthatADAMTS2 could Halila etal.,1986;Peltonen al.,1985;Tuderman andProckop, enzyme thatprocessedprocollagen I(HalilaandPeltonen,1986; and thattheprocollagenIII-processing enzymewas notthesame significant proportionofprocollagen IIIintissueswas unprocessed function withADAMTS3. Previous literaturehadsuggested that a mice, but thatitappearstosharetheprocollagen Iprocessing ADAMTS2 isthemajorprocollagenIII amino-propeptidase in proteinases duringmouseembryogenesis.Theresultssuggest that We appliedthesecriteria toexploring therolesofprocollagenN- substrate shouldhave asignificantimpactonbiologicalsystem. should bereducedorabsent.Inaddition,failure tocleave the and thatintheabsenceofprotease,cleavage ofthesubstrate should beco-expressed, orthatthey shouldco-localize intissues, substrate interactioninvivo requiresthattheproteaseandsubstrate To rigorouslyestablish thephysiologicalsignificanceofaprotease- DISCUSSION Adamts2 RESEARCH ARTICLE B ) Ultrastructure ofanalveolarwall,demonstratingcomparablebasementmembranes(arrows) in C and –/– ) Methyleneblue-azure Bstainforelastinin Adamts3 and theirwild-type(+/+)littermates.Thedistalairspacesare muchlargerinthemutantmice,butthere isnoinflammatory Adamts14 D ) Electron micrograph ofcollagenfibrilsincross section(inarea markedbytheasterisk)demonstratescomparable size or Adamts2 Adamts14 Adamts3 are co-expressed in Adamts2 expression was noted,but no (Fig. 8B). was detected(Fig.8A). and Adamts14 Adamts2 ␮ m inA,C;500nmB,D.( Adamts2 were –/– and wild-typeaortasdemonstratescomparableaorticwallthickness –/– mice andwild-type(+/+)littermates. in Col3a1 (Wang etal.,2003).Theoverlapping expression of development inmice.Expression of contribution toprocollagenIprocessingduringmusculoskeletal induction ofstressontheaortic wall inappropriatevivo models. is possiblethatchangesmay be seeninoldermice,orbythe effect onhistology andcollagenultrastructureat2monthsofage.It the near-total lackof procollagenIIIprocessinghadnoapparent aorta, whichisseverely affected bystructuralcollagenIIImutations, there was nocompensatingprocessingenzyme.However, inthe Adamts3 pulmonary walls, similartoskinfragilityindermatosparaxis. Neither III inthelung.Lungabnormalitiesmayresultfromafragilityof the Adamts2 lungs andthenear-total lackoflungprocollagenIII processingin (Shiomi etal.,2003).Thepseudo-emphysematousappearanceof the mice, collagenIIIisacrucialstructuralcomponentofthe lung development, inaccordancewithaprevious studysuggestingthat,in hybridization revealed prominent skin, ascollagenIandIIIarebothpresentinthedermis.In situ contribute tothelossofmechanicalintegrity ofdermatosparactic procollagen III-processingenzyme. Adamts2 The datasuggestthatADAMTS3 probablymakes asignificant The roleofADAMTS2 inprocessingprocollagenIIImayfurther , andthesubstantialreductioninprocollagenIIIprocessing –/– nor E –/– mice stronglysuggestsavitalrequirementforcollagen ) Westernblotanalysisofprocollagen III(above)andI Adamts14 mice provides solidproofthatADAMTS2 isthemajor were expressed inthelungand,therefore, Adamts2 F Col3a1 ) Westernblotanalysisof ( A –/– Comparative lunghistology ) Adamts3 and wild-typemice.E, expression duringlung Development 133(8) in many Adamts2 Col1a1 and -

DEVELOPMENT procollagens? Although expression. overlapping riboprobesshowed no discernibledevelopmental RNA (datanotshown), insituhybridizationwiththreenon- Although wedetected Adamts3 Col1a1 chondrodysplasia orosteochondrodysplasia.Although enzyme, andpredictthat ADAMTS3 isprobablythemajorprocollagenII-processing complete procollagenIIprocessing,thedatasuggestthat analysis ofbovine dermatosparacticcartilagethatdemonstrated dermatosparaxis areinthedermis.Together withourprevious most severe deficienciesinprocollagenIprocessingmouse expressing tissues,suchasboneandtendon,mayexplain whythe Procollagen amino-propeptidases resistant tothe actionoftheseproteases(Zhu etal.,1999).By not excised duringdevelopment, suggestingperhapsthatitis Ultrastructural andbiochemical studiessuggestitsN-propeptideis perichondrium (RyanandSandell, 1990;Ryanetal.,1990). termed IIAisexpressed inpre-cartilaginousmesenchymeand appear tocompensatesignificantly. Asplicevariant ofcollagenII, do notcompensateforitsabsence. Thusotherenzymesdonot locations, neither loss ofprocessingprocollagenIIIinmany tissues.Atthese Adamts2 enzyme(s) closelyrelatedtoADAMTS2. Itisclearfromthe as innormalskin(Coligeetal.,2002),itsupportsprocessingby an processing indermatosparacticskinoccursatthesamecleavage site ancillary domainandthetriplehelix.Becauseresidualprocollagen suggesting aneedforspatiallystringentinteractionsbetween the efficiently processedbyADAMTS2 (Tuderman etal.,1978), binding totriple-helicalcollagens.Heat-denaturedcollagenis not from allotherproteasesandisprobablyhighlyspecialized for organization (except forthevariable C-terminalmodule)isdifferent processed byotherproteases?Thehighlyconserved PNP modular might alsobeaSCO-spondin-processingenzyme. spinal cord(Gobronetal.,1996).ItispossiblethatADAMTS3 processing priortopolymerizationintotheReissnerfiberof product isSCO-spondin,aglycoproteinthatundergoes proteolytic located attheentrancetoaqueductofSylvius.Itsmajorsecretory sub-commisural organ (SCO),aspecializedependymalorgan I, IIorIIImRNA. For instance, Adamts3 procollagen geneexpression, wenotedstrongexpression of Is itpossiblethatthePNPshave substratesother thanthefibrillar Can themajorfibrillarprocollagenamino-propeptidesbe expression aregenerallymutuallyexclusive intissues, –/– in thenervous systematlocationsthatlacked procollagen expression overlapped withthatofbothcollagengenes. mice thatabsenceofADAMTS2 leadsto a significant Adamts3 Adamts14 Adamts2 nor Adamts3 Adamts3 Adamts14 mRNA byRT-PCR oftotalembryo expression was associatedwith mutations willleadtoa was highlyexpressed inthe are expressed, and,thus, Col2a1 and 2003). individuals withEDS-VIIC,includinghypodontia(DeCosteretal., clinical studyemphasizedtheabnormalpermanentdentition of ADAMTS2 inthedeveloping mouseattheselocations.Arecent VIIC. Ourstudiesdemonstrateco-expression ofprocollagenIIIand intestinal perforationshave beenreportedinindividuals withEDS- monitoring ofEDS-VIICpatientsasthey age.Bladderruptureand noted inEDS-VIICpatients,ourfindingssuggestaneedfor the wall, andnoaorticorothervascular aneurysms have beenhitherto electron microscopy didnotreveal specificanomaliesintheaortic processing inthemouseaortawas defective. Althoughhistologyand tissues. We wereparticularlyconcernedthatprocollagenIII in theabsenceofADAMTS2 appearstobedramaticinmostmouse in thewalls ofbloodvessels. TheprocollagenIII-processingdefect in mice,thatitistheonlyPNPexpressed atsignificantly highlevels ADAMTS2 toprocessprocollagenIII,andourobservation, albeit patients withEDS-VIIC,canbepartlyattributed totheabilityof with avariety ofotheranomalies.Easybruising,auniversal signin of dermatosparacticskinchangeasaffected individuals getolder. change withageinhumans,perhapsexplaining how theproperties by of theirgenes.Theexpression of enzymes inhumansandmicethroughthetranscriptionalregulation compatible withthedifferent deployment offunctionally equivalent differences betweenEDS-VIICandmousedermatosparaxisare have nocraniofacial dysmorphism(Lietal.,2001).These have fragileskin,but they arenotsignificantlygrowth retardedand not aprominentfeatureinearlychildhood. somewhat amelioratedandthereisincreasingjointlaxity, whichis mice. Asindividuals withEDS-VIICage,theskinfragilityis changes anddecreasedgrowth; featuresthatareabsentin the PNPbond(Fukuietal.,2002). cleave thepropeptidefromcollagenIIAatnumeroussitesotherthan contrast, matrixmetalloproteinases(MMPs)have beenshown to diversification ofexpression patterns) rather than subfunctionalization (retention ofmolecularfunctionbut with and ADAMTS14, werehighlyconserved toprovide duplication, closelyrelatedgenes, suchasADAMTS2, ADAMTS3 al., 2005;Nicholsonet2005). We speculatedthat, following arose byduplicationoftheirprimitive ancestors(Huxley-Jones et evolution suggeststhatgeneswithinmammalianADAMTS clades 2005; Nicholsonetal.,2005).RecentanalysisofADAMTS gene phylogenetically distinctclades(Apte,2004;Huxley-Jones etal., In additiontoskinfragility, humanswithEDS-VIICmanifest ADAMTS2 Most ofthe19ADAMTS proteases can beassignedto Adamts3 Adamts2 in thedermisoftwo-week-oldskin.Scalebar:100 Adamts14 mRNA wasdetectedinthedermis.( dpc embryo. of eacto feiemsaddri.( demarcation ofepidermisanddermis. skin. Fig. 8.Procollagen N-propeptidase expression in may accountforthis. mutations inhumansleadtocharacteristiccraniofacial Col3a1 –/– D, dermis;thewhitedashedlineindicatesof mice have milddentalchangesandcompensation and mRNA, butnot Adamts2 Col1a1 , butnot in thedermisofskina17.5 ADAMTS3 RESEARCH ARTICLE Adamts3 Adamts14 Adamts2 and B mRNA, isdetectable ) Adamts2 A ADAMTS14 ) Co-expression or –/– Adamts3 Adamts2 mice also and ␮ m. 1595 may , –/–

DEVELOPMENT Colige, A.,Sieron, A.L.,Li,S.W., Schwarze,U.,Petty, E.,Wertelecki, W., Chan, D.,Rogers,J.F., Bateman,J.F. andCole,W. G. Apte, S. References We thankD.Prockop for exhibit thesamephenomenon. regulation, supportsthishypothesis.OtherADAMTS cladesmay but specializationinthisregard asaresultofdifferential gene et al.,2005).TheabilityofallthreePNPstoprocessprocollagenI, neofunctionalization (acquisitionofnew functions) (Huxley-Jones 1596 Halila, R.andPeltonen,L. Canty, E.G.,Lu,Y., Meadows,R.S.,Shaw, M.K.,Holmes,D.F. andKadler, Musculoskeletal Disorders award from NIH(AR050953). (7.4.561.03.F). HistologyandmCTwassupportedbyaCore Centerfor Recherche Scientifique(grant3.4562.03),andF.K. byaTélévie grant AR44745 toD.E.B.).A.C.C.issupportedbytheBelgianFondsNationaldela by grantsfrom theNationalInstitutesofHealth(AR49590toS.S.A.and microscopy; andCraigBennettsformCTscanning.Thestudieswere supported assistance withinsituhybridization;KaitlinPetrella forassistancewithelectron N. FukaiforcDNAprobes; AmandaAllamongforhistology;KatieJungers Fernandes, R.J.,Hirohata,Fernandes, S.,Engle,J.M.,Colige,A.,Cohn,D.H.,Eyre, D.R. De Paepe,A.andMalfait,F. Colige, A.,Vandenberghe, I.,Thiry, M.,Lambert,C.A.,Van Beeumen,J.,Li, Cole, W. G. Cheah, K.S.,Lau,E.T., Au,P. K.andTam, P. P. Huxley-Jones, J.,Apte,S.S.,Robertson, D.L.andBoot-Handford, R.P. Halila, R.,Steinmann,B.andPeltonen, L. Birk, D.E.,Silver, F. H.andTrelstad, R.L. Birk, D.E.andTrelstad, R.L. Fukui, N.,McAlinden,A.,Zhu,Y., Crouch, E.,Broekelmann, T. J.,Mecham,R. De Coster, P. J.,Malfait,F., Martens,L.C.andDePaepe,A. Colige, A.,Nuytinck,L.,Hausser, I.,Van Essen,A.J.,Thiry, M.,Herens, C., Kessler, E.,Takahara, K.,Biniaminov, L., Brusel,M.andGreenspan, D.S. Gobron, S.,Monnerie,H., Meiniel,R.,Creveaux, I.,Lehmann,W., Lamalle, in theprocollagen IN-proteinase gene. 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