Journal of Cell Science Gdf10/Bmp3b ieyi atb oneuaigBPsgaigadRn2activity. Runx2 hypertrophy and chondrocyte signaling represses BMP BMP Jab1 of that downregulating inhibitor demonstrates by study novel part our a Together, in likely treatment. likely is BMP Jab1 exogenous to that response reveals enhanced study our Notably, hypertrophy. chondrocyte of chondrocytes regulator in key signaling were a markers ossification chondrocyte Runx2, endochondral hypertrophic of via of activity developed expression elements increased skeletal and columns. arrest, the chondrocyte cycle all disorganized embryos, severely mutant with small the extremely In dwarfism. severe with chondrodysplasia o:10.1242/jcs.113795 doi: 234–243 126, Science Cell of Journal 2012 November 11 Accepted hnrct ifrnito,adotols differentiation proliferation, skeletal osteoblast of chondrocyte and regulator coordinating center master differentiation, ossification a cartilage. by chondrocyte primary is the a Ihh development of forming 2003). center matrix, and (Kronenberg, perichondrium the bone adjacent in from secrete here apoptosis recruited are undergo Osteoblasts model to cartilaginous the begin Runx2, within occurs chondrocytes regulators ossification hypertrophic endochondral when key chondrocytes and Subsequently, hypertrophy under (Ihh). overt the undergo collagen Hedgehog with Indian prehypertrophy X and start Runx3 type and stop of cycle, then cell expression Chondrocytes the columns. exit cartilage- orderly rapid dividing, of and master form first collagen secretion to II under type robust proliferation including precursors lineage the process matrix extracellular by chondrocyte specific followed mesenchymal differentiation to Sox9, commit regulator The well-controlled and condense 2003). and undergo chondrocytes (Kronenberg, elaborate which within involves anlagen an It cartilage bones. of long initial and most an ribs vertebrae, forms including skeleton, ossification the endochondral development, During Introduction words: Key the utilized we study, the this Strikingly, with In formation. interact cartilage hypertrophy. by can in chondrocyte Jab1 apoptosis signaling promote Although to and BMP pathways. loop repress proliferation, signaling feedback to various differentiation, Smad5 of cell effector output in downstream the skeletogenesis (BMP) regulating roles protein and critical factors morphogenetic plays diverse bone of Jab1 activity cofactor the transcriptional modulating conserved evolutionarily The Summary Chi of Republic People’s Province, Jilin Changchun, University, Jilin Hospital, Union § China-Japan Orthopaedics, of ` Department address: *Present 4 3 2 1 Chen Dongxing for crucial differentiation is chondrocyte Jab1 co-regulator transcriptional The 234 un Zhou Guang rsn drs:Oclg eerhLbrtre,DiciSny o,Ld. oy,Japan Tokyo, Ltd.., Co., Sankyo Daiichi Laboratories, Research Oncology address: Present uhrfrcrepnec ( correspondence for Author aeCmrhnieCne etr aeWsenRsreUiest,190Eci vne lvln,O 40,USA 44106, OH Cleveland, Avenue, USA Euclid 44106, 10900 OH University, Cleveland, Reserve Avenue, Italy Western Euclid Milan, Case 10900 Raffaele, Center, USA University, San Cancer 44106, Reserve Institute Comprehensive OH Western Scientific Case Cleveland, Case and Avenue, Genetics, Medicine Euclid of of 10900 Department School University, University, Reserve Raffaele Western San Case Orthopaedics, of Department 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. a1Cn,Codoypai,BP ux,CP signalosome COP9 Runx2, BMP, Chondrodysplasia, Jab1/Csn5, n fBPtresdrn hnrct yetoh uhas such hypertrophy chondrocyte during targets BMP of and nvivo in 1,3,4,§ 1 ida .Bashur A. Lindsay , [email protected] ssilpol nesod sakyrgltro kltgnss M inln euae h rtclIhh-Pthrp critical the regulates signaling BMP skeletogenesis, of regulator key a As understood. poorly still is nvivo in .In Jab1 Jab1 ) hnrct-pcfcknockout chondrocyte-specific K hnrcts hr a egtndepeso fBPsgaigcmoet including components signaling BMP of expression heightened was there chondrocytes, cKO 1 oinLiang Bojian , 1, ,MriaPanattoni Martina *, Jab1 nvivo in protein- eaiefebc opcnrl h egho proliferating of length 2003). (Kronenberg, the growth Ihh/Pthrp cartilage This during controls chondrocyte Ihh. columns the of poorly loop keep production still to the negative-feedback receptor mechanism inhibits its and a of on proliferating activation under acts the Pthrp Pthrp to understood. leads including that Patched-1 genes cascade receptor target a early its triggers and to which binds chondrocytes (Ptc-1), Ihh prehypertrophic chondrocytes. development, by hypertrophic synthesized bone is endochondral Ihh During 2003). (Kronenberg, ulnFbxpoen SF,amjrgopo ulnrn E3 cycles Repeated covalently ring NEDD8. is protein cullin ubiquitin-like cullin the component of with core modified 19S group SCF (CRL). the major Skip1– ligase complex a of ubiquitin of (SCF), lid stability has proteins the the CSN Cullin–F-box regulate 2003). to to Deng, activity composition, and is deneddylase (Wei and CSN particle (Csn5) 2009). size regulatory al., proteasome in signalosome et Olma both 2002; COP9 similar, al., et regulator (Bech-Otschir complex proteolysis evolutionarily the of subunit fifth conserved the also is Jab1 2010). Claret, (Shackleford and processes differentiation cell and pathways signaling Jab1 K hnrctsehbtdicesdaotss 2paecell phase G2 apoptosis, increased exhibited chondrocytes cKO h nrclua atrJb (c- Jab1 factor intracellular The Col10a1 flox/flox )itrcswt ueospoen orglt diverse regulate to proteins numerous with interacts 1) Ihh ; Furthermore, . loxP/Cre Col2a1-Cre and Runx2 2 ek Tamai Keiko , ytmt eiet h pcfcrl fJab1 of role specific the delineate to system nvitro in a1cnas nii h transcriptional the inhibit also can Jab1 . cO uat xiie entllethal neonatal exhibited mutants (cKO) Jab1 h oeo a1i BMP-mediated in Jab1 of role the , K hnrctsehbtdan exhibited chondrocytes cKO Jun 3, ` ciaindomain- activation ugr Pardi Ruggero , na eerhArticle Research binding nvivo in 2 and , Journal of Cell Science 02.Tu,Jb ly seta oe ohi general in both skeletogenesis in Jab1 and roles of organs vivo function in specific essential specific of the al., differentiation However, plays et tissues. the Sitte in 2011; Jab1 and al., embryogenesis et Thus, Deng type- 2008; increased cell 2012). al., and et whereas defects (Panattoni differentiation 2010), cell apoptosis postnatal apoptosis al., severe to et accelerated lead Tian of and 2004; deletions specific al., proliferation et impaired (Tomoda with E8.5 yetoh Mnn ta. 02 etn ta. 2009). al., et chondrocyte Retting promote 2002; of the al., to loss combined increases et the and Furthermore, directly (Minina and increase signaling to proliferation hypertrophy signaling chondrocytes prehypertrophic BMP prehypertrophic by BMP in chondrocyte 2011). Ihh chondrocytes of al., key levels proliferating expression within et in higher some Interestingly, than (Shu at of and chondrocytes 2009). hypertrophic is survival, expression al., proliferation, components the et chondrocyte cartilage, (Retting feedback promote Ihh-Pthrp hypertrophy critical BMP to various the that loop reveal regulate studies 2006; components genetic al., Mouse et signaling Shore 2010). 2003; al., Chang, (Asai- et and progressiva Ye Serra anomalies 2009; al., ossificans oculo-skeletal et Coakwell and fibrodysplasia chondrodysplasia, brachydactyly, (FOP), as such a1Cn steol uui ossigactltcmtlbinding metal catalytic signalosome, a possessing COP9 subunit whole only the the is Jab1/Csn5 Within I 2009). including Chamovitz, the regulators p27,2008; regulate transcriptional as can such and proteins CSN cell-cycle p53 activity, numerous of SCF removal activity regulating (Wei and stability By NEDD8 activity 2008). ligase al., and ubiquitin et E3 (neddylation) SCF maintain conjugation (deneddylation) NEDD8 of uain nBPsgaigcmoet,icuigtp I type including 2009). components, ligands Derynck, signaling and receptors BMP BMP (Wharton in in development Mutations diversity during context-dependent of cell large precise variety control the a exert wide of to produces outputs a levels Hill, transcriptional This and the and proteins. Smads, at (Ross Smad-interacting regulated antagonists, genes exquisitely receptors, target is ligands, of signaling BMP expression nucleus, 2008). the the to modulate signaling translocate then Smad4, and complex, BMP coactivator receptor the upon BMP canonical with the phosphorylated dimerize to are The binding 8 ligands BMP and 2004). various 5, kinase al., Smad1, effectors serine/threonine et intracellular with (Chen TGF- receptors activity the of and heterodimeric Wan members 2004; activate are Lyons, BMPs and 2005). Yoon 2004; endochondral Cao, al., during et differentiation (Chen and ossification growth skeletal of steps an suggesting constitutive The cancers, 2010). 2010). of Claret, the various deletion and Claret, in (Shackleford of role implicated and progression oncogenic been and has Shackleford various initiation Kato overexpression 2009; of 2009; developmental Jab1 Chamovitz, activity Furthermore, of 2008; Yoneda-Kato, and al., array et stability and vast (Wei the factors a regulating transcription and by repair, processes DNA apoptosis, Thus, 2010). activity. Claret, signalosome COP9 and for (Shackleford required SCF is Jab1 of from subunit NEDD8 removing cullin for the required is that motif metalloprotease a1i novdi inln rndcin elccecontrol, cycle cell transduction, signaling in involved is Jab1 oemrhgntcpoen(M)sgaigcodntsall coordinates signaling (BMP) protein morphogenetic Bone a opeeyukonpirt hsstudy. this to prior unknown completely was GDF3 Jab1 and BMPR1B nmc eut neryebyncltaiyby lethality embryonic early in results mice in GDF6 Jab1 edt kltldvlpetldefects developmental skeletal to lead , and nTcl,Bcl,o yli el all cells myeloid or B-cell, T-cell, in ACVR1 md,5 Smad1, antagonist , b k and B- uefml that superfamily a NOGGIN 8 Wie al., et (Wei specifically and , euae l h ao ee xrse yotolssi tissue in roles osteoblasts 1997). by Runx2 al., essential expressed et factor genes (Ducy play culture major transcription the the Runx2 domain all which regulates Runt and in 2003). Sox9 network (Kronenberg, transcriptional factors complex for transcription a dispensable on impinges mostly 2009). chondrocyte is al., of et (Retting Smad8 regulators formation cartilage redundant whereas and and Smad1 differentiation, positive that reveals are analysis Further Smad5 largely 1/5/8. formation Smad cartilage on embryonic depends in signaling BMP of effect specific reduced with chondrodysplasia severe Col10a1 in results chondrocytes in uatmice mutant ta. 01 eae l,20) ovrey chondrocyte Conversely, 2001). al., et Ueta chondrocyte 2001; osteoblast for (Takeda al., of in hypertrophy important the et role expression accelerated chondrocytes essential also proliferating Continuous its is Besides maturation. Runx2 1999). 1997; al., al., et differentiation, Mundlos 1997; et al., with et Zhou dysplasia (Lee defects skeletal bone inherited generalized dominantly a (CCD), in al., et dysplasia mutations Otto 1997; Moreover, al., et 1997). (Komori differentiation osteoblast of lack auainwsdlydi oeseea lmnsin elements skeletal some in delayed was maturation eudn oedrn hnrct hypertrophy. chondrocyte during role redundant iha niJb nioy h eut hwta a1is Jab1 performed that we show results cartilage, The sections antibody. study embryonic in anti-Jab1 To mouse embryogenesis 2004). an E18.5 expression al., mouse with on et during protein Carrabino immunohistochemistry 2000; broadly al., Jab1 expressed et (Bounpheng is Jab1 of chondrodysplasia Lethal 2003). al., et Zheng 2004; al., et hypertrophic Moreover, and (Yoshida prehypertrophic in chondrocytes activity binds directly their Runx2 to 1999). contributes in al., cis-elements et conserved Kim the 1999; to al., et (Inada mice Runx3 Results hypertrophy. chondrocyte of formation regulators cartilage key two Jab1 in the Jab1 signaling, ablated of most role we critical vivo the study, the the using unveiled mice this during and in is Jab1 in chondrocytes of in Hence, specifically role differentiation formation. the determine cartilage chondrocyte to and approach signaling straightforward during BMP on effect of its Runx2 of deletion analysis conditional cartilage the and during The chondrocytes activity unknown. potential and were expression the Runx2 been formation and on have interaction Jab1 of far signaling effect so Jab1-BMP studies of these relevance al., of et (Kim all performed degradation almost Jab1 Runx3 2006). induce However, Aigner, and 2009). and Runx3 (Haag bind signaling also act BMP can might of Jab1 inhibitor that an suggesting as culture, transcriptional chondrocyte BMP-dependent in of of responses attenuation overexpression an and in resulted immunoprecipitation, Jab1 in 2004). Smad5 al., effector et (Yoshida limbs embryonic the reduced in and size proliferation cell chondrocyte reduced with hypertrophy h feto M inln uigseeoeei ultimately skeletogenesis during signaling BMP of effect The oal,Jb a ietyitrc ihBPdownstream BMP with interact directly can Jab1 Notably, n h niioyefc fJb nRn2adBMP and Runx2 on Jab1 of effect inhibitory the and 2 / M receptors BMP 2 a1adcodoyedfeetain235 differentiation chondrocyte and Jab1 xrsin hntp eysmlrt chondrocyte- to similar very phenotype a expression, nvitro in ieso h opeeasneo chondrocyte of absence complete the show mice Runx2 ri elclue hrfr,tephysiological the Therefore, culture. cell in or n related and ulmc Rtige l,20) hs the Thus, 2009). al., et (Retting mice null Runx2 Jab1 Col10a1 RUNX2 Runx3 ulmc ipa complete a display mice null flox/flox lya seta and essential an play and euti cleidocranial in result ; Col2a1-Cre Runx2 Ihh loxP/Cre rmtr,and promoters, nmouse in Runx2 Runx2 Jab1 system 2 2 / 2 / in in 2 ; Journal of Cell Science itlgclaayi hwdthat the showed confirmed immunostaining analysis in The protein histological Jab1 1C). of than (Fig. deletion thinner and controls shorter much and the were vertebrae, elements, limbs, developed ribs, craniofacial that as some elements such skeletal ossification, the alizarin endochondral and all via blue that alcian showed by staining Skeletal embryos red 1B). mutant E18.5 (Fig. of as tongues featured well preparation protruding as mutants limbs, and short abdomens and The trunks, prominent generalized short The 1B,C). and snouts, short (Fig. 1B,C). severe heads, (Fig. round respiratory E18.5 very cages a from at rib displayed chondrodysplasia birth restricted embryos the at mutant to cKO died due mice likely distress mutant (cKO) knockout omladwr etl,all fertile, were and normal ih lyarl nrgltn hnrct hypertrophy in chondrocyte lethality crossed embryonic regulating we early 2010), in the role circumvent constitutive Jab1 that To a suggests progression. cartilage play in the pattern Thus, might 2006). hypertrophic expression Aigner, also the Jab1 and (Haag in distinct report cartilage expression fetal human previous JAB1 in of weaker zone in A much than expression 1A). a lower in (Fig. described much Jab1 chondrocytes including be to proliferating Interestingly, tissues, appeared chondrocytes musculoskeletal 1A). hypertrophic (Fig. in chondrocytes expressed widely 236 xrsinseiial tahg ee odifferentiating al., While to et (Retting 2009). factors applied Smad1/5/8 transcription including level been various ossification, of has endochondral in function high and the study 2000) to a al., successfully et at (Ovchinnikov chondrocytes specifically expression The chondrocytes. Col2a1-Cre uat a uhsalrcriaeeeet oprdwith controls wild-type compared While 2B). elements (Fig. E18.5 cartilage at littermates smaller type wild much had mutants ora fCl cec 2 (1) 126 Science Cell of Journal Jab1 rngncmc odelete to mice transgenic Jab1 dfcetmc Tmd ta. 04 ine al., et Tian 2004; al., et (Tomoda mice -deficient Jab1 flox/+ Col2a1-Cre flox/flox ; Jab1 Col2a1-Cre Jab1 ie(aatn ta. 08 with 2008) al., et (Panattoni mice flox/flox iedie r recombinase Cre drives line K atlg Fg A.The 2A). (Fig. cartilage cKO ; Jab1 Col2a1-Cre ieapae grossly appeared mice Jab1 flox/flox pcfclyin specifically ; conditionally Col2a1-Cre uaieJb agt Fg A SakeodadCae,2010). Claret, various and of IkB- (Shackleford stability of expression 4A) protein the (Fig. Interestingly, the of targets on effect Jab1 chondrocytes the putative in determine Jab1 to the of chondrocytes in loss primary than from rates proteins higher at phase G2 3D). (Fig. the controls primary at of of accumulated analysis chondrocytes cycle also cell that major cytometry specific showed flow (Panattoni modulate chondrocytes a differentiation Our cell 2008). can T caspase-3/7, al., in Jab1 et progression cycle 3C). of cell (Fig. of steps activity apoptosis Moreover, of higher 3B). (Fig. facilitator had E18.5 in at chondrocytes rate littermates apoptosis cytometry the control of in flow chondrocytes increase rib apoptosis one-fold primary V-based a confirmed Annexin analysis 3A). (Fig. in E18.5 proliferation apoptotic of overall TUNEL in staining However, positive cells for more 2008). the showed essential al., with experiments et labeling not consistent (Panattoni no is is cells This differentiating Jab1 revealed 2C,D). that (Fig. labeling notion controls BrdU and between E18.5 mutants via at differences analysis proliferation significant proliferation Cell in progression cycle cell Jab1 altered and apoptosis Increased iognzdcodoye Fg B.Tu,Jb sesnilfor essential is severely ossification Jab1 endochondral had Thus, proper 2B). mutants (Fig. cKO chondrocytes the disorganized chondrocytes, hypertrophic prehypertrophic, proliferating, and of structures columnar orderly had itntdwsra agt ndfeetcl ye during types component cell key another different Csn3, of in expression have The targets to not development. likely was is downstream 2008), Jab1 al., Thus, distinct mutants. et the (Panattoni in differentiation changed cell significantly T in Jab1 of et epromdtewsenbo nlsso extracted of analysis blot western the performed we Next, flox/flox Jab1 ; Col2a1-Cre K uatlmsta ncnrlltemtsat littermates control in than limbs mutant cKO mutants Jab1 uat;W,wl-yelittermates. wild-type WT, mutants; Col2a1-Cre hnrdslsai E18.5 in chondrodysplasia generalized and bone severe of show staining red alizarin of and staining cartilage blue alcian with preparations ( E18.5. at embryos of deletion Chondrocyte-specific 1. Fig. mount ( chondrocytes. hypertrophic HC, PC, chondrocytes; femur. proliferating E18.5 murine in expression ( chondrodysplasia. lethal generalized A muotiigrvassrn Jab1 strong reveals Immunostaining ) Jab1 nvivo in K uat oprdwith compared mutants cKO a Jab1 ao ontemtarget downstream major a , nmc ed osvr and severe to leads mice in flox/flox uat.MUT, mutants. . ; Jab1 Col2a1-Cre C Skeletal ) K mutants cKO Jab1 Jab1 Jab1 B Jab1 Whole- ) mutant flox/flox cKO cKO cKO ; Journal of Cell Science rgeso ihsgiiatG hs retin arrest phase G2 significant with progression hnsbetdt aps-l37asyfrqatfcto fcsae37atvt saotssidx ( index. apoptosis as activity caspase-3/7 of quantification for assay Caspase-Clo3/7 to subjected then ( tiigo ppoi npoieaigcodoye.Tergtpnlsosteelre mg ftebxdae ntemdl ae,wt seik in asterisks with panel, middle the in area boxed the of image ( enlarged mutant. the the shows in panel apoptosis right of The staining chondrocytes. positive proliferating in apoptosis of staining (40 i.3 nrae ppoi n bomlcl yl rgeso in progression cycle cell abnormal and apoptosis Increased 3. Fig. in decreased both were The Skip1 holocomplex. COP9 and of p27 integrity of the expressions for essential suggesting is downregulated, Jab1 be that to appeared signalosome, COP9 of C nrae aps-/ ciiyin activity caspase-3/7 Increased ) 6 .Sgiiataotsswsdtce ntemtn,weesteewsn eetbeTNLsann ntewl-yecnrl rospitt h posi the to point Arrows control. wild-type the in staining TUNEL detectable no was there whereas mutant, the in detected was apoptosis Significant ). Jab1 K rmr hnrcts 1. rmr hnrctswr rae iheooiefr2 or oidc ppoi and apoptosis induce to hours 24 for etoposide with treated were chondrocytes primary E18.5 chondrocytes. primary cKO B uniiaino nei tie el hwdicesdaotssin apoptosis increased showed cells stained V annexin of Quantification ) Jab K hnrcts MUT, chondrocytes. cKO 1 Jab1 Jab1 cKO flox/flox Jab1 ; Col2a1-Cre soitdpoen sa dpo ewe ulnrn E3 ring cullin inhibitor kinase- between kinase S-phase adaptor cyclin a an Skip1, cell is 2010). key protein, Claret, a associated and is (Shackleford p27 chondrocytes. K uat.W,wl-yelittermates. wild-type WT, mutants. cKO a1adcodoyedfeetain237 differentiation chondrocyte and Jab1 mutants. D uniiaino lwctmtyidctsatrdcl cycle cell altered indicates cytometry flow of Quantification ) ( A UE tiigo ppoi nE85dsa femur distal E18.5 in apoptosis of staining TUNEL ) MUT, E16.5. at proliferation chondrocyte normal tibia, showing proximal E16.5 of incorporation yetohcchondrocytes. hypertrophic HC, chondrocytes; prehypertrophic Pre, chondrocytes; proliferating Pro, littermates. (10 seik) u o in not but asterisks), by (indicated E18.5 chondrocytes in wild-type expression Jab1 strong shows Col2a1-Cre E16.5 ( oi tiigo rxmltbao E18.5 of tibia Jab1 proximal of staining eosin Cre hnrct oun in columns chondrocyte disorganized Severely 2. Fig. C rUlbln ftepoia ii of tibia proximal the of labeling BrdU ) 6 Jab1 uat (40 mutants flox/flox .( ). Jab1 Jab1 D n K rmr i chondrocytes. rib primary cKO uniiaino BrdU of Quantification ) 5 n ; 5 flox/flox o ahgroup. each for 3 Col2a1-Cre K uat.W,wild-type WT, mutants. cKO mutants. niiulmc e group. per mice individual 4 6 ; .( ). Col2a1-Cre B Jab1 ( eaoyi and Hematoxylin ) A cO mutants. (cKO) Immunostaining ) flox/flox Jab1 iaigthe dicating flox/flox mutants ; Col2a1- ; tive Journal of Cell Science Fg C.Teeoe a1cudb eaiergltro Runx2 of regulator negative a be could Jab1 Therefore, 4C). (Fig. al., Runx2- et the of one significantly, Zhou reporter the co-transfection of 1995; activated Karsenty, alone presence Runx2 and While the 2006). (Ducy on element OSE2 dependent binding partly this is of activity The fragment 2006). al., et (Zhou gene Cre el htd o xrs n uxpoen Dc ta. 1999). al., et (Ducy proteins osteocalcin Runx any express OSC-114x2-luc not do Runx2 that on cells an effect with direct chondrocyte co-transfected any were revealed has of Jab1 activity, also whether transcriptional test analysis regulator To 4A). in blot (Fig. expression positive Runx2 Western increased slightly 4B). a (Fig. chondrocytes hypertrophy large Runx2, hypertrophic abnormally to the adjacent expressed in of chondrocytes some chondrocytes of Furthermore, clusters of 4B). clusters (Fig. mutants large of already abnormally layers were and packed mice tightly in wild-type and missing in distinct chondrocytes the prehypertrophic E16.5, at Strikingly, in hypertrophy chondrocyte Increased might Jab1 differentiation. signaling chondrocyte that BMP during suggests canonical Smad1/5-mediated This regulate E18.5 4A). negatively (Fig. in expression, chondrocytes Smad1/5 mutant total in no blot change but cycle expression, drastic western cell phospho-Smad1/5 Importantly, altered increased decreased revealed with apoptosis. analysis The correlated increased 2010). Skip1 and and Claret, progression p27 and of (Shackleford expression survival cycle cell affects cell and and signalosome COP9 and ligases ubiquitin 238 uatmice mutant ora fCl cec 2 (1) 126 Science Cell of Journal Jab1 nacrfamn ue othe to fused fragment enhancer K uat.Ised hr eedisorganized were there Instead, mutants. cKO otistotne eet fa114-bp a of repeats tandem two contains Jab1 Jab1 erae t ciiyb lot50% almost by activity its decreased OSC-114x2-luc and Runx2 Jab1 Jab1 osteocalcin xrsinplasmids expression eotrit COS7 into reporter K chondrocytes cKO luciferase flox/flox Jab1 Jab1 ; enhancer Col2a1- reporter cKO cKO a1o M ontemtresi nohnrlossification, endochondral in in targets expression downstream BMP protein on Gdf10 Jab1 increased showed Jab1 also analysis hne nteegnssgetdicesdchondrocyte increased suggested genes The these 5). in (Fig. hypertrophy fold in one by al., upregulated et changes was expression (Zheng its marker and chondrocyte of 2003) hypertrophic expression chondrocytes. most-specific The mutant the 5). in 40% (Fig. by upregulated genes was dysregulated Runx2 severely most the as Our such genes analysis. hypertrophy-associated the markers to chondrocyte and proliferating subjected while rib that and showed Primary results collected performance. were E18.5 PCR littermates from real-time with PCR chondrocytes capability microarray Osteogenesis profiling the we combining an of technology a is using level, Array E18.5 PCR chondrocytes, Array. transcriptional in littermate analysis the wild-type RT-PCR real-time at performed chondrocytes 2009). al., differentiating et (Kim culture cell in transcriptional Runx3 the related context- repress highly be also of 4D). can activity to a (Fig. Jab1 Interestingly, likely that investigated. showed is transfection report further recent Runx2 be affect to on transient remains Jab1 and not of dependent in effect did the stability expression Therefore, protein Jab1 increased Runx2 However, activity. M inln opnnswsurgltdin upregulated Bmpr1a was including some components chondrocytes, of expression signaling the that revealed BMP also analysis array PCR Our in components signaling Jab BMP of expression Upregulated oivsiaetecneuneo h osof loss the of consequence the investigate To Sox9 flox/flox K hnrcts(i.4) oivsiaeteefc of effect the investigate To 4A). (Fig. chondrocytes cKO , xrsin eentsgiiatycagd chondrocyte changed, significantly not were expressions Smad1 ; Col2a1-Cre Jab1 , Smad4 K mutants. cKO uatchondrocytes mutant Bmp3b and lsi (0.25 expression plasmid Jab1 Flag-tagged of increasing amounts and plasmid expression Runx2 0.25 co- with were transfected cells COS7 transfection. transient ( ae n ujce owsenbo analysis antibodies. blot indicated western with to hours subjected 24 and collected later was extract cell Whole mut (0.25 amounts o ux (0.25 Runx2 for plasmids expression and OSC-p114x2-luc reporter osteocalcin Runx2-responsive vitro activity transcriptional Runx2 represses ( littermates. wild-type WT, ltaayi of analysis blot itltbao E16.5 of tibia distal in Runx2 of staining Immunohistochemical ( chondrocytes. rib primary tiigidctdb ros ntemutant. the MUT, in arrows) by indicated (brown staining expression Runx2 was There ectopic panels. strong left the of in images areas boxed enlarged the the are panels right The Cre i.4 a1i eaiergltrof regulator negative hypertrophy. a chondrocyte is Jab1 4. Fig. Jab1 D a1de o fetRn2saiiyin stability Runx2 affect not does Jab1 ) uat.Pe rhprrpi zone. prehypertrophic Pre, mutants. O7clswr rnfce ihthe with transfected were cells COS7 . Smad7 Jab1 K uat n wild-type and mutants cKO as named (also Col10a1 flox/flox m m Fg ) etr blot Western 5). (Fig. ,0.5 g, Jab1 ,0.5 g, m ; )adJb nincreasing in Jab1 and g) Col2a1-Cre , Jab1 flox/flox Runx2 m m m pestagged- Xpress g n 1.0 and g n 1.0 and g Gdf10 Jab1 flox/flox ; B C ( Col2a1-Cre ) A eeamong were Jab1 ) Jab1 Western ) Col10a1 ; mutants; ), K and cKO Col2a1- Jab1 m m Col2a1 g). Bmp5 g). cKO in in is , Journal of Cell Science eioae rmr i hnrctsfrom chondrocytes rib to primary isolated due of we is effect components autonomous signaling cell the BMP and markers hypertrophic during regulated survival. progression tightly cycle and cell be proper to ensure has to differentiation signaling chondrocyte BMP Thus, BMP7-treated material S2). (supplementary between Fig. chondrocytes cycle, primary cell wild-type LDN-193189- the treated inhibitor of BMP molecule phases small versus G2 chondrocytes cycle and the cell S in differences, Furthermore, G1, significant revealed cycle cytometry S3). cell flow by S1, the analysis of Figs arrest material G2 (supplementary and apoptosis of increased those exhibited significantly as they phenotypes BMP7, BMP cellular with similar repress treated were might when chondrocytes Jab1 Interestingly, wild-type differentiation. that chondrocyte during indicates signaling This hypertrophy chondrocytes. accelerated and in signaling BMP Thus, dysregulated 5). to (Fig. leads mutants the significantly in were upregulated three all of expressions the Indeed, hypertrophy. Cre Fg A.Tu h niioyefc fJb nchondrocyte autonomous. on cell Jab1 be to of likely is effect signaling inhibitory BMP and the hypertrophy Thus 6A). (Fig. md/ oiiit ontemeet.Ntby etr blot western Notably, phosphorylate events. receptors downstream BMP initiate ligands, to BMP Smad1/5 of binding Upon in response signaling BMP Enhanced genes target changes BMP the direct study in to RT-PCR quantitative real-time performed we ieR-C nlssrvae infcn nraein Real- increase (Ade-GFP). significant and GFP a revealed expressing analysis virus RT-PCR time control recombinase Cre a expressing or adenovirus (Ade-Cre) with infected were cells odtriewehrteices nchondrocyte in increase the whether determine To uatchondrocytes mutant Bmp3b/Gdf10 xrsini el netdwt Ade-Cre with infected cells in expression Ihh , Jab1 Id1 and nciaini chondrocytes, in inactivation ni 2 Snail Jab1 Jab1 Jab1 Jab1 uigchondrocyte during K el,namely cells, cKO flox/flox eiinylikely deficiency flox/flox ie The mice. ; Col10a1 Col2a1- 919cnptnl nii M inln epnesc as such response LDN- signaling TGF- kinases, not BMP receptor but I inhibit expression, phospho-Smad1/5 type potently BMP LDN-193189. can of inhibitor inhibitor 193189 molecule selective a small As the with chondrocytes in signaling indicates TGF- This panel). lower 6B, that (Fig. controls the versus mutants xgnu M7tetet(i.6,tppnl.Ti suggests This panel). top 6B, (Fig. that after phospho- treatment and BMP7 before of both exogenous controls, expression wild-type with upregulated compared Indeed, Smad1/5 had chondrocytes antibody. anti- mutant an (activated)-Smad1/5 with primary immunoblotting The to respectively. phospho subjected minutes, cultured then 45 were stimulated extracts and then cell chondrocytes, 15 for hours, BMP-7 12 mutant recombinant signaling for by BMP serum-starved cKO the were chondrocytes analyze chondrocytes in further primary To response 4A). from E18.5 (Fig. in mutants proteins expression cKO phospho-Smad1/5 extracted increased revealed of analysis odatcpopoSa23epeso hnei the in change was expression there phospho-Smad2/3 expression, drastic phospho-Smad1/5 no altered the to contrast In TGF- key of the effector antibody, downstream an (activated)-Smad2/3 with phospho TGF- immunoblotting against to antibody subjected with were then treated and respectively, hours, 12 serum-starved were for the chondrocytes TGF- determine primary related cultured TGF- To closely mutants, 2004). of cKO of al., response et modulators the Kim of key 2002; status al., two et Smad7, (Wan signaling inhibitory and Smad4 response. signaling BMP canonical increased odtrieteudryn ehns fehne BMP enhanced of mechanism underlying the determine To a1wspeiul eotdt neatwt coactivator with interact to reported previously was Jab1 Jab1 b Jab1 inln epnei culture. in response signaling a1adcodoyedfeetain239 differentiation chondrocyte and Jab1 flox/flox K uatcodoye aen rs hnein change gross no have chondrocytes mutant cKO Jab1 ; Col2a1-Cre K hnrcts etetdprimary treated we chondrocytes, cKO akr npiaycodoye rmE18.5 from Jab1 chondrocytes primary in markers differentiation chondrocyte signaling and BMP components for assay RT-PCR qualitative ttsial infcn ihlreS,likely SD, large not with were significant cases statistically some in gene changes The expression littermates. wild-type WT, mutants; K uatsmlsanalyzed. of samples numbers mutant limited cKO the also embryo and mouse each sample of heterogeneity the to due ersn h mean the represent Col2a1-Cre hnrct ifrnito in altered and differentiation signaling chondrocyte BMP Increased 5. Fig. eoye MUT, genotype. flox/flox b inln Rs n il 2008). Hill, and (Ross signaling uatcodoye aean have chondrocytes mutant ; b Col2a1-Cre uatchondrocytes. mutant o 5ad4 minutes, 45 and 15 for 1 Jab1 6 flox/flox b ..fo – ieper mice 3–5 from s.d. b uat.Tedata The mutants. inln response signaling inln in signaling ; Col2a1-Cre Jab1 Jab1 Jab1 Real-time flox/flox Jab1 Jab1 Jab1 cKO cKO ; b Journal of Cell Science md/ rti xrsinwsurgltdi h ogbones long the phospho- in upregulated that was of showed expression protein phospho-Smad1/5 Smad1/5 of immunostaining M iadsiuain ned ohbfr n fe BMP7 after and before the both Indeed, to stimulation. ligand adjacent BMP of clusters size whether the large in 6D). especially abnormally (Fig. zone intense, hypertrophic of more be chondrocytes to (Retting phospho- appeared reported In and cartilage, previously 2009). proliferating al., et as wild-type to restricted chondrocytes the mainly prehypertrophic was In expression Smad1/5 6D). (Fig. littermates activity. signaling BMP upregulated to utemr,LN138 eue h aps-/ ciiyin activity caspase-3/7 the activity. primary receptor reduced BMP LDN-193189 enhanced Furthermore, and/or ligands BMP increased in signaling xrsigCercmiae(d-r) u o in not but (Ade-Cre), recombinase Cre expressing F)(upeetr aeilFg 3.Tu,increased Thus, S3). Fig. material in apoptosis (Ade- (supplementary GFP expressing virus GFP) control a with infected chondrocytes of including increased light components of In signaling Gdf10/Bmp3b BMP 6C). the of (Fig. expression model manner enhanced the reverse dose-dependent a mouse in partially chondrocytes 2008). in al., a can expression et phospho-Smad1/5 LDN-193189 (Yu in can (FOP) LDN-193189 Indeed, progressiva Moreover, ossification ossificans 2008). fibrodysplasia al., heterotopic et (Yu reduce culture cell in 240 aty epromdra-ieR-C nlsst determine to analysis RT-PCR real-time performed we Lastly, above the with Consistent Jab1 Jab1 Jab1 ora fCl cec 2 (1) 126 Science Cell of Journal K mro tE75cmae ihwild-type with compared E17.5 at embryos cKO Jab1 Jab1 , K hnrctshv oerbs epneto response robust more a have chondrocytes cKO flox/flox Bmp5 Jab1 dfcetcodoye ih edei part in due be might chondrocytes -deficient K iei ieyt ei atdeto due part in be to likely is mice cKO K uat,popoSa15expression phospho-Smad1/5 mutants, cKO and hnrctsifce ihadenovirus with infected chondrocytes Bmpr1a Jab1 xvivo ex Fg ) h peuae BMP upregulated the 5), (Fig. flox/flox utr experiments, culture ; Col2a1-Cre Jab1 mutant flox/flox nrae xrsino h akro prehypertrophic of marker the of chondrocytes, component expression signaling BMP increased of expression increased oe niio fcnnclBPsgaigi chondrocytes in profiles expression signaling Gene (1) BMP observations: following canonical the on of BMP based canonical inhibitor the novel on a Jab1 one of first effect the signaling specific is the study investigate Our growth. ensure to to and development formation be during cartilage to hypertrophy controlled proper has tightly signaling chondrocyte and BMP tuned that very represses finely reconfirming inhibitory 2011), A signaling, al., an and 2009). et Smad6, BMP (Estrada al., that survival, of et revealed study Song Smad proliferation, genetic 2009; mouse chondrocyte al., recent feedback et promote of Ihh-Pthrp (Retting hypertrophy activation critical to subsequent the loop the regulates and targets, its downstream proteins of phosphorylation Smad1/5 the through effectors 1, signaling, (Figs BMP columns Canonical chondrocyte 2). disorganized formation and deletion dwarfism cartilage chondrocyte-specific severe the in that activity here of report Runx2 We and effect 7B). novel its (Fig. signaling and especially essential BMP differentiation, the chondrocyte on demonstrate in to Jab1 one of first roles the is study Our BMP Discussion 7B). canonical (Fig. activity inhibiting Runx2 downregulating specifically possibly by and chondrocyte signaling part regulates in negatively Jab1 hypertrophy that suggest upregulating chondrocyte collectively by promoting part in in Runx2 hypertrophy and signaling BMP canonical cultured stimulation, Jab1 nmc e onoaa ehlcodoypai with chondrodysplasia lethal neonatal to led mice in nvivo in Ihh u eut eosrt htJb slkl obe to likely is Jab1 that demonstrate results Our . Fg A.I ih fteetbihdrl of role established the of light In 7A). (Fig. Jab1 K uatpiaycodoye had chondrocytes primary mutant cKO nagdiaeo h oe rai the in MUT, area panel. boxed middle the of image enlarged Jab1 TGF- not but signaling, Col2a1-Cre Cre 1. rxmltbao the of tibia proximal E17.5 in phospho-Smad1/5 of immunostaining to subjected ( being immunoblotting. before hours 24 LDN-193189 for of amounts indicated and FBS 10% containing medium DMEM in cultured were chondrocytes Primary manner. Col2a1-Cre nrae M inln in signaling BMP the increased block partially can LDN-193189 indicated. ( as then immunoblotting and to times, subjected indicated the for panel) TGF- or panel) ng/ (top (100 ml) stimulation BMP7 before hours 12 for serum-starved were chondrocytes oprsnwt hs netdwt Ade- ( with GFP. infected those with in comparison Ade-Cre with infected chondrocytes ieR-C nlsssoe increased Runx2 showed analysis RT-PCR time in signaling Jab1- BMP Upregulated 6. Fig. xrsinin expression C h ml oeueBPinhibitor BMP molecule small The ) uat;W,wl-yelittermates. wild-type WT, mutants; flox/flox eiin chondrocytes. deficient , B Col10a1 nrae epnet BMP to response Increased ) ; Ihh Col2a1-Cre uat(20 mutant uat nadose-dependent a in mutants Jab1 xrsin u studies our expression, and D flox/flox Increased ) Jab1 Bmp3b/Gdf10 6 b b uat.Primary mutants. .Bto panel, Bottom ). 2n/l (lower ng/ml) (2 1 rmr rib primary inln,in signaling, flox/flox Jab1 Jab1 Gdf10 ( A ; flox/flox flox/flox Real- ) Col2a1- ; and ; Journal of Cell Science oe o h nerto fJb,Rn2adBPsgaigduring signaling BMP and Runx2 Jab1, differentiation. of chondrocyte integration the for model tre vrih,adte rae ihBP 10n/l o or before analysis. hours RT-PCR 7 for real-time ng/ml) to serum- (100 subjected BMP7 days, being with 2 treated then for and cultured overnight, were starved chondrocytes primary E18.5 chondrocytes. uatchondrocytes. mutant inr 06.Cnesl,orrslscnitnl hwdthat in showed upregulated consistently was chondrocyte signaling results BMP our and human (Haag Conversely, clear in 2006). entirely not Aigner, signaling was mechanism the BMP although culture, regulate Smad5 to negatively bind BMP directly exogenous to to reported to previously is was response Jab1 enhanced treatment. Smad1/5 an form) exhibit chondrocytes (activated ( in phospho- markers upregulated effector differentiation downstream chondrocyte and Snail2 targets ( ligands BMP BMP of expression heightened reveal in response signaling BMP Upregulated 7. Fig. el sdi h tde n a eylkl lorfetthe reflect also likely very TGF- may in and Jab1 different studies of the role the to complex due in Smad7 TGF- likely used are promotes cells results Jab1 enhances conflicting These of activity. and Overexpression Smad7 inhibitory 2004). with degradation al., constitutively et associate (Kim can inhibited Jab1 and hand, levels steady Smad4 TGF- of endogenous expression and Ectopic decreased Smad4 degradation. Jab1 binds for Jab1 ubiquitylation phospho-Smad1/5 Smads. its various induces TGF- enhanced with modulate interaction direct can LDN- reversed by Jab1 inhibitor 6C). partially BMP (Fig. expression molecule can small a 193189 since BMP enhanced activity and/or ligands receptor BMP and due of part expression complex increased in the be be to may to signaling BMP likely upregulated The is multi-layered. differentiation chondrocyte during auain(cree ta. 05.I u study, our In 2005). al., et (Schroeder maturation differentiation. type cell Jab1 and diverse Smads in various between interaction the study to important h ehns fJb-eitdBPsgaiginhibition signaling BMP Jab1-mediated of mechanism The rncito atrRn2i seta o chondrocyte for essential is Runx2 factor Transcription b )in idcdgn rncito Wne l,20) nteother the On 2002). al., et (Wan transcription gene -induced Jab1 Jab1 K hnrcts 2 h xrsino BMP of expression The (2) chondrocytes; cKO ( A K atlg;ad(3) and cartilage; cKO nrae M inln epnein response signaling BMP Increased ) b BPsgaig hs twl be will it Thus, signaling. /BMP Jab1 b mdae transcriptional -mediated n 5 K cartilage. cKO e ru.( group. per 3 Jab1 Jab1 Gdf10 flox/flox b BPsignaling /BMP K primary cKO , ; BMP5 Col2a1-Cre B Proposed ) Jab1 and ) Jab1 cKO Ihh , prvdb h ntttoa nmlCr n s omte ICC fCase of (IACUC) Committee Use and and reviewed Care been The Animal University. have Reserve Institutional Western protocols the animal by All approved conditions. animal University standard Reserve Western under Case the facility at housed and maintained were mice All genotyping and breeding Mouse Methods and Materials ramn flta hnrdslsa htmgtb soitdwith associated be might that dysregulated chondrodysplasias lethal of treatment Cre ahgnsso ua ehlcodoypais ihterapid the the of With study further chondrodysplasias. tools, genomics lethal in human advances of pathogenesis Cre ogenerate to N earmciey(un ta. 07.Frhroe the Furthermore, 2007). other al., and et of complex (Huang loss the machinery (9-1-1) with repair Rad9-Rad1-Hus1 interact DNA to repair reported DNA is key Jab1 since G2 defect The damage-repair 2008). al., in et (Panattoni arrest stage G2-M at progression cycle Interestingly, 2010). points Claret, Jab and multiple Shackleford the at 2008; when affects al., pathway et complex control (Wei CSN checkpoint Jab1-containing DNA-damage 2A). (Fig. mutants 3 following the floxed with GGCCTAAGAAGG-3 for USA) WI, pairs: Madison, (Promega, primer polymerase DNA Flexi GoTaq of were littermates controls All background. C57/BL6J on maintained were sig BMP in abnormalities an therapies osteoarthritis, during novel fractures, of signaling development treat the BMP to to of lead may regulation im which Jab1 skeletogenesis, be of will mechanism It underlying 7B). (Fig. signaling repair DNA the of status it the Thus, investigate 2010). in al., to double- apparatus et interesting (Tian DNA be cells those p53-dependent will in capability upregulated repair an break with associated ta. 08 eecosdwith crossed were 2008) al., et ee owr rmr5 primer forward gene, es npr,tesotradsalrcriaein cartilage smaller at and cause, might shorter defects the These part, 3). (Fig. in arrest least cycle cell chondrocyte G2 and differentiation. accelerated chondrocyte during interaction the Runx2 elucidate and that further Jab1 to critical between differentiation. be will it suggests and activity mediated, negative chondrocyte transcriptional in a This Runx2 hypertrophy during be repress might 4C). levels can (Fig. Runx2 Jab1 protein Jab1 the that Furthermore, of and indicating mRNA 5), regulator the Fig. both 4A, increased (Fig. at in expression resulted chondrocytes Runx2 differentiating in deficiency rcue,adfripoigtesceso pnlfsos u study endochondral Our proper for fusions. factor essential spinal ossification an of is Jab1 success that establishes the l here improving for for and worldwide fractures, patients million as all BMP2andBMP7havebeenusedforboneregenerationinoverone hea fracture for including important regeneration, is signaling Jab by skeletogenesis of regulation 5 9 9 -CATGTTTAGCTGGCCCAAATGT-3 Jab1 nsmay u td provides study our summary, In wl-yeall,30b;floxed bp; 350 allele, (wild-type nl el lodslydrdcdsria n eae cell delayed and survival reduced displayed also cells T -null uatmc a ev sa ex an as serve can mice mutant uatseea hntp a aiiaetedanssand diagnosis the facilitate can phenotype skeletal mutant Jab1 Jab1 K uatcodoye xii ceeae apoptosis accelerated exhibit chondrocytes mutant cKO a1adcodoyedfeetain241 differentiation chondrocyte and Jab1 Jab1 Jab1 Jab1 a odtoal nce u nTclsi ie the mice, in cells T in out knocked conditionally was nvivo in JAB1 nmueebyncclsadotoacm el is cells osteosarcoma and cells embryonic mouse in flox/flox Jab K uat.Muegntpn a efre yPRusing PCR by performed was genotyping Mouse mutants. cKO K el ih eascae ihaDNA a with associated be might cells cKO K uatcodoye ntefuture. the in chondrocytes mutant cKO 1 Jab1 ; 9 ees rmr5 primer reverse , xrsino activity. or expression Col2a1-Cre ieyi atb niiigcnnclBMP canonical inhibiting by part in likely , 9 -TGCAACGAGTGATGAGGTTCG-3 Jab1 K uat ih npr eRunx2- be part in might mutants cKO Col2a1-Cre aig Additionally, naling. lee owr rmr5 primer forward allele, Jab1 odtoa ncot(K)mtns l mice All mutants. (cKO) knockout conditional Jab1 odtoa allele conditional kltldsresascae with associated disorders skeletal d uigmuedvlpet BMP development. mouse during 1 9 9 . -GGCATGCATCACCATTTTCAGTAG- rngncmc Ocinkve l,2000) al., et (Ovchinnikov mice transgenic otn ofrhrudrtn the understand further to portant et fseea omto and formation skeletal of pects n-oennuin n acute and non-unions ong-bone igadatclrcriaerepair. cartilage articular and ling lee 5 p;for bp); 450 allele, oe nihsit h critical the into insights novel eln oe osuythe study to model cellent Jab1 Jab1 9 Jab1 -GGTCAGAAAGCTA- Cre flox/flox flox/flox flox/flox 9 Col2a1-Cre eaiewild-type negative ,reverseprimer ie(Panattoni mice Jab1 ; ; Col2a1- Col2a1- cKO trans- Journal of Cell Science rmr i hnrctso otaa a 3 day infection, al., postnatal L-glutamine, adenovirus of mM et For chondrocytes 2 extraction. (Lefebvre rib FBS, RNA 10% primary described before containing penicillin/streptomycin as medium 1% DMEM collected and in were cultured and chondrocytes 1994) rib primary Mouse RT-PCR quantitative Real-time at ethanol 90% from in fixed chondrocytes PBS, ice-cold rib 2 in al., primary washed were cultured et littermates control analysis, (Zhou and mutants described cycle as cell performed For were 1999). plasmids reporter plasmids (PI) expression OSC-p114x2-luc Runx2 iodide and and Jab1 propidium with cells with COS7 analysis in assays transfection cycle Transient cell and assay Transfection 500 BMP Subsequently, nM 200 indicated. or BMP7 when per ng/ml hours 100 25,000 24 of with density treated for a were at LDN-193189 cells plate day, inhibitor 24-well next a The in seeded well. were chondrocytes assay Primary MTT and assay Caspase-Glo3/7 the per cells V-FITC (Annexin apoptotic apoptotic scoring identify and by cytometry, determined to flow was by rib (PI) analyzed rate were Fluor apoptotic mouse iodide cells Alexa stained using The propidium Cultured stained instruction. manufacturer’s and and instruction. PBS, V cold manufacturer’s in 488-Annexin washed the harvested, were to (#V13241, chondrocytes Kit Assay according Apoptosis Vybrant staining a Invitrogen) using TUNEL performed and was staining staining V V Annexin annexin by analysis Apoptosis for was eosin activity peroxidase first H and 1 endogenous 3% were in The hematoxylin with minutes USA). sections quenched 10 with CA, for the Burlingame, stained steamer Laboratories, immunohistochemistry, a were in For heated al., sections et analyses. (Zhou described tissue as morphological bone of The staining Embryos alcian red age. with alizarin 2006). embryo’s made and was the cartilage preparation for of skeletal 0.5 staining Mouse E blue times. as indicated counted at was collected day were plugging the of immunohistochemistry noon and The labeling BrdU processing, Embryo 242 hog elsrie ue n nlzdb ASClbrfo yoee (BD cytometer Facility. flow Core Calibur Cytometry Flow FACS Case by at analyzed USA) CA, and Jose, tube, San Biosciences, strainer cell a through irsses ufl rv,I,UA sn ec plcto ut 1.3 Suite Application Leica Leica microscopes. (DM500, using IRB camera DM USA) and digital DM6000B a IL, Leica under with Grove, software, captured Buffalo kit were staining Microsystems, BrdU images formalin a 10% with All in incorporation fixed BrdU (Invitrogen). harvested, for were analyzed Carlsbad, and embryos sectioned, injection, (Invitrogen, overnight, embryos after reagent hours E16.5 labeling 4 with BrdU USA). CA, pregnant with Laboratories). mice injected the intraperitoneally (Vector were labeling, Medium (BrdU) Mounting and bromodeoxyuridine peroxidase Permanent Laboratories), For DAB (Vector VectaMount with solution with counterstaining incubation covered nuclear counterstained by QS were followed hematoxylin sections Afterwards, with temperature, Sections Laboratories). (Vector room temperature. reagent solution at room (ABC) substrate hour at complex 1 avidin-biotin-peroxidase hour for with 1 treated for subsequently antibody were secondary biotinylated with h T aeigraet(M44 nirgn A a de,adthe and added, was CA) reader. microplate a Invitrogen, with measured (#M6494, was were nm cells reagent 570 day, at were next absorbance culture, labeling The 48-hour spectrophotometric chondrocytes well. another MTT After per primary indicated. 25,000 when of the proliferation, LDN-193189 density or a and BMP7 at with plate treated viability 24-well a cell in seeded evaluate assay to Caspase-Glo3/7 WI). assay luminescent Madison, Twenty-four a (Promega, apoptosis. instructions with induce manufacturer’s measured the to to was added according apoptosis were MO) later, Louis, hours St (Sigma, etoposide .%BA n .%Tio -0 nPS o 0mnts h etoswr then were 4 serum, sections at The normal overnight minutes. 3% antibodies 20 primary for contained specific PBS, which in in X-100 incubated buffer, Triton 0.1% blocker and BSA, serum 0.1% normal in incubated tiigwspromdwt ppa lsPeroxidase Plus ApopTag with performed was staining 0mnts el eete hle niefr1 iue,floe yicbto in incubation by followed minutes, 10 for ice on chilled 50 then were Cells minutes. 30 elpae tadniyo 000cells/cm 50,000 of density a at plates well eoiaesbtaewste ple ntescin odvlpsignal. develop to temperature. with sections room mounted and the at Laboratories). green (Vector methyl on minutes Medium 30 with Mounting by applied counterstained Permanent for followed were VectaMount then slides sections, chamber the was the Afterwards humidified on substrate a applied Peroxidase was in conjugate incubation Anti-digoxigenin hour. 1 h etoswr rtetdi rsl iue rtiaeK(20 K proteinase diluted freshly in instruction. pretreated manufacturer’s were the to sections according USA) The MA, Billerica, (Chemicon, Kit yrgnprxd o iue tro eprtr.Tescin eethen 37 were at chamber sections humidified The a 3% in temperature. in mixture room reaction quenched at TUNEL was the minutes peroxidase with 5 incubated Endogenous for temperature. peroxide room hydrogen at minutes 15 20 o h T 3(,-iehlhao--l-,-ihnlerzlu Bromide) (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium MTT the For emnldoyuloiy rnfrs UPnc n aeig(TUNEL) labeling end nick dUTP transferase deoxynucleotidyl Terminal m ˚ /lo Islto nPSa 4 at PBS in solution PI of g/ml vrih,rsseddi 20 in resuspended overnight, C ora fCl cec 2 (1) 126 Science Cell of Journal 2 O 2 o 0mnts fe B isn,tescin were sections the rinsing, PBS After minutes. 10 for ˚ o 0mnts ahsml a hnpassed then was sample Each minutes. 60 for C m /lo Ns n nuae t37 at incubated and RNase of g/ml 6 nie nakn ouin(Vector Solution Unmasking Antigen Jab1 2 flox/flox n netdwt adenovirus with infected and nSitu In ˚ ,floe yincubation by followed C, iewr eddi 12- in seeded were mice ppoi Detection Apoptosis + ,PI m Jab1 /l for g/ml) 2 cells. ) ˚ ˚ for C for C cKO m M esaitclysignificant. statistically be K uatmc n h idtp itrae ( littermates wild-type the and mice mutant cKO aeilTbeS.Lvl fgn xrsinwr eemndwt the with determined were expression gene of supplementary ( Levels in threshold listed cycle S1. primers as comparative gene-specific RT-PCR Array Table real-time with for 2012) PCR material used al., was et Biosystems) Osteogenesis (Liang (Applied described not Mix an were Master that PCR using genes Green the array, the system, For in USA). included CA, detection Valencia, USA) SABiosciences, PCR CA, (#PAMM-026, per (Carlsbad, Biosystems real-time Applied cDNA (Invitrogen) USA). the CA, first-strand 7500 with Hercules, Kit performed Laboratories, to was (Bio-Rad Mini PCR Kit transcribed Real-time Synthesis RNA reverse cDNA iScript was an PureLink using RNA and instruction. (Invitrogen) manufacturer’s was RNA Reagent 500. analysis. TRIzol of RT-PCR infection real-time of for multiplicity later a days at Transfer 5 Iowa) (Gene extracted of (Ade-GFP) protein University fluorescent Core, green Vector or (Ade-Cre) Cre expressing a efre yStudent’s by performed was p .) 5 MNC,1 P4,1m T,ad1m PMSF, mM 1 and DTT, 0.2 mM onto 1 (Thermo transferred Cocktail and 10 NP-40, SDS-PAGE, Inhibitor USA). Tris–HCl MA, Phosphatase 4–15% 1% Waltham, and Inc., Tris–HCl Protease Scientific mM NaCl, Fisher 50 Halt containing with mM buffer 150 from supplemented lysis extracted the was in 8.0), protein (pH chondrocytes cellular rib total primary mouse (immunoblotting), blotting western For blotting Western upeetr aeilaalbeoln at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.113795/-/DC1 online available Clinic material Supplementary Cleveland The months. [grant 12 from grant after Research release t Institutional grant CCC 119999-IRG-91-022-18-IRG Case number pilot and G.Z.]; to a [NI AR-050953 Center L.B.]; Core Musculoskeletal [grant to (NIH) R03- Health of AR07505 G.Z., Institutes to National R03-DE019190 the by numbers supported is work This Health. of Funding Institutes for National the Schmedlen from by supported CA43703 was Valerie P30 Core Center number Cancer Microscope and grant Comprehensive Imaging Case the and manuscript of Cytometry Facility the The assistance. of for editorial Murakami Shunichi reading Dr work, critical histology expert for for Behringer Pizzuto R. Teresa Richard Dr thank We Acknowledgements mean as presented were Results analysis Statistical omlzdt h ee of level the to normalized ebae(i-a) h ebaewste lte ih5 o-a r milk dry non-fat 5% with blotted then was membrane The (Bio-Rad). membrane h rmr nioisfr2husa omtmeaueo vrih t4 at overnight or in temperature incubation room by at followed hours 2 Tween-20, for 0.1% antibodies containing primary 7.6) the (pH saline buffered muoitceityadwsenbotn nti td r itdin for listed are antibodies study Primary this analysis. in LDN-193189 blotting in blotting inhibitor S2. cultured western LDN-193189 Table western material of BMP were supplementary before amounts and indicated chondrocytes of hours and immunohistochemistry 24 rib FBS effect 10% primary for containing the TX), medium DMEM For Houston, anti-phospho-Smad2/3 an (Selleckchem, and respectively. TGF- antibody and anti-phospho-Smad1/5 USA) antibody an NJ, Hill, with then Rocky analysis hours, TGF- Peprotech, 12 and ng/ml, for BMP (100 b serum-starved of BMP7 were with analysis re-blotted chondrocytes stimulated Restore the being ECL primary with For before cultured by antibodies antibody. Scientific) response, (Thermo of detected primary Buffer stripped another Piscataway, was Stripping was with Sciences, signal Blot membrane Life Western The the Healthcare Plus Afterwards, temperature. (GE USA). room reagents NJ, detection at blotting hour secondary 1 western peroxidase-conjugated for horseradish with antibody incubated then was membrane xrsinin expression ecinwspromdi rpiaeadrpae na es he independent three data. the least of at reproducibility on the repeated ensure Each to and 1. genotype triplicate as per designated in samples arbitrarily performed values was control reaction with presented was chondrocytes ( gm,Ppoeh o niae ie epciey olwdb etr blot western by followed respectively, times indicated for Peprotech) ng/ml, 1(2 o eltm uniaieR-C nlss oa N a xrce using extracted was RNA total analysis, RT-PCR quantitative real-time For Jab1 Gdf10 K uatcodoye esscnrllittermate control versus chondrocytes mutant cKO Gapdh , Id1 t DD ts ocmaetedfeecsbtenthe between differences the compare to -test , C 6 Ihh t ehd h RAlvlo ahgn was gene each of level mRNA The method. ) RA n h eaiefl hneo gene of change fold relative the and mRNA, tnaddvain S) ttsia analysis Statistical (SD). deviations standard , M oeCne rn ubrP30 number grant Center Core AMS Jab1 , ...Dpstdi M for PMC in Deposited G.Z.]. o Smad7 E110111t .. T32 G.Z., to DE019190-1A1S1 Col2a1-Cre n m 5 fpoen eesprtdby separated were proteins of g and , 3–5). m Snail2 P VFnitrocellulose PVDF m , .5i osdrdto considered is 0.05 rngncmice, transgenic h oe SYBR Power the , b signaling ˚ .The C. Jab1 Journal of Cell Science i,I . to . ae,B n udo,S. Mundlos, and B. Zabel, F., Otto, S., I. Kim, ao .Y n oeaKt,N. Yoneda-Kato, and Y. J. Kato, nd,M,Ysi . oua . iae . euh,K,Hmn,M,St,M., Sato, M., Himeno, K., Deguchi, S., Miyake, S., Nomura, T., Yasui, M., Inada, M. Wan, and X. Cao, X., Liu, C., Lu, H., Yuan, J., Huang, ag .adAge,T. Aigner, and J. Haag, srd,K . etn,K . hn .M n yn,K M. K. Lyons, and M. A. Chin, N., K. Retting, D., K. Estrada, uy . truk . ree,M,Se,J,Pnr,G,Gofo,V,Aln,M. Amling, V., Geoffroy, G., Pinero, J., Shen, M., Priemel, M., Starbuck, G. P., Karsenty, Ducy, and L. A. Ridall, V., Geoffroy, R., Zhang, P., Ducy, G. Karsenty, and P. Ducy, eg . ad,R,Cede . in,X,Zag . hh .V,Gize W., Grizzle, V., S. Shah, S., Zhang, X., Xiang, W., Cheadle, R., Pardi, Z., Deng, hn . ho .adMny .R. G. Mundy, and M. Zhao, D., E. Chen, Bianchi, A. and D. R. Pardi, Chamovitz, D., Talarico, E., Carminati, S., Carrabino, to . hrel .P,Copo,T,Dne,A,Glor .C,Rswl,I R., I. Rosewell, C., K. Gilmour, A., Denzel, T., Crompton, P., A. Thornell, F., Otto, G. Zhou, and J. C. Hernandez, D., Chen, M., M. Cotter, B., Liang, onhn,M . enkv,I . od,S . hn . oead .G., N. Copeland, H., Chen, G., S. Dodds, N., I. Melnikova, A., M. Bounpheng, la .H,Ry . eBhn . uaa . ari . asn . Quadroni, B., Larsen, S., Maerki, I., Sumara, T., Bihan, Le M., Roy, H., M. Olma, S., Albright, S., A. Aylsworth, B., J. Mulliken, C., A. Mundlos, Vortkamp, F., and Otto, S., M. Mundlos, D. Ornitz, C., M. Naski, C., Kreschel, E., Minina, Metsa G., Zhou, S., J., Garofalo, Hecht, V., A., Baldini, Lefebvre, L., Pastore, L., Zhou, K., Thirunavukkarasu, B., Lee, M. H. Kronenberg, ehOshr . egr .adDbe,W. Dubiel, and M. Seeger, D., Bech-Otschir, ooi . ai . oua . aauh,A,Ssk,K,Dgci . Shimizu, M., K., Y. Deguchi, K., Goh, Sasaki, H., A., Y. Yamaguchi, S., Li, Nomura, S., H., Y. Yagi, T., Lee, Komori, W., J. Jang, S., Cinghu, K., J. Choi, H., J. Felici, Kim, G., J. McNally, S., T. Karpova, R., S. Lee, H., S. Park, J., H. Lee, C., B. Kim, siCawl,M,Fec,C . e . aca . io,K,Prr,A G., A. Perera, K., Bigot, K., Garcha, M., Ye, R., C. French, M., Asai-Coakwell, References ifrnito yCbfa1. by differentiation 14 aaia . iua . au,N tal. et mice. N. Cbfa1-deficient Yasui, in T., chondrocytes Kimura, H., Yamagiwa, complex. checkpoint Rad9-Rad1-Hus1 the of degradation n niisbn opoeei rti signaling. protein morphogenetic bone inhibits and seta olmtBPsgaigdrn atlg development. cartilage during signaling 26 BMP limit to essential omto eodebyncdevelopment. embryonic beyond G. formation Karsenty, and differentiation. osteoblast of activator transcriptional a gene. osteocalcin mouse a of expression control htmrhgnss9(O9 inlsm uui S5rgltsint immune innate G. regulates macrophages. CSN5 H. in subunit Zhang, responses signalosome and (COP9) 9 J. photomorphogenesis Mountz, D., Miller, Factors addt eefrcedcaildslsasnrm,i seta o osteoblast for essential is syndrome, dysplasia al. development. et bone cleidocranial R. and B. for Olsen, differentiation S., gene Mundlos, S., R. candidate Beddington, W., G. Stamp, regulator. JAB1/CSN5 the NEDD8. with of colocalization pattern A. Expression B. Christy, protein. and and A. cDNA N. JAB1 Jenkins, mouse J., D. Gilbert, amla O9sgaooeietfe d1a oesbnto utpeCul4- multiple of subunit core a as ligases. L. Dda1 E3 Pintard, based identifies and signalosome M. COP9 al. Tyers, mammalian et M., Peter, H. M., J. Knoll, W., dysplasia. cleidocranial Henn, cause 779. CBFA1 G., factor transcription W. the involving Cole, D., Lindhout, properties. mechanical bone alters osteoblasts Int. in SOX9 of expression mice. transgenic in collagen/beta-galactosidase II OSF2/CBFA1 type from factor B. transcription Crombrugghe, osteoblast-specific the dysplasia. G. of cleidocranial Karsenty, binding and P. DNA Ducy, V., Geoffroy, 332-336. nefc ewe inltasuto n bqii-eedn proteolysis. ubiquitin-dependent 115 and transduction signal between interface neato fFF h/tl,adBPsgaigitgae hnrct proliferation chondrocyte differentiation. integrates signaling hypertrophic BMP and and Ihh/Pthlh, FGF, of Interaction of arrest maturational to owing al. formation et bone M. of lack Inada, complete H., osteoblasts. a Y. in Gao, results T., Cbfa1 R. Bronson, Y., al. et H. RUNX3. Wee, of S., degradation to and K. localization binding Lee, Z., by X. Chi, signaling beta degradation. J. factor its S. promoting growth and Kim, transforming Smad7 and regulates K. signalosome, D. Lee, A., nopeepntac n hntpcvraiiycaatrz Gdf6-attributable characterize al. variability et A. phenotypic Mushegian, phenotypes. and B., oculo-skeletal Chanda, penetrance C., S. Incomplete Mema, K., Staehling-Hampton, 1209-1225. , 2498-2510. , 467-473. , 90 76-89. , 22 MORep. EMBO 233-241. , Cell 89 .Cl Sci. Cell J. 20) eeomna euaino h rwhplate. growth the of regulation Developmental (2003). 20) eiiigteCP inlsm satranscriptional a as signalosome COP9 the Revisiting (2009). 755-764. , 19) hrceiaino rmr utrso chondrocytes of cultures primary of Characterization (1994). 19) ba-eedn eei aha otosbone controls pathway genetic Cbfa1-dependent A (1999). 20) u ciaindmi-idn rti id Smad5 binds 1 protein domain-binding activation Jun (2006). 10 a.Genet. Nat. 19) w itntotols-pcfccsatn elements cis-acting osteoblast-specific distinct Two (1995). eh Dev. Mech. 352-358. , u.Ml Genet. Mol. Hum. Blood eeEp.Patterns Expr. Gene 122 20) amla O9signalosome. COP9 Mammalian (2009). e.Cell Dev. Gene 1035-1044. , 117 20) a1CN,acmoeto h COP9 the of component a Jab1/CSN5, (2004). 16 4796-4804. , 80 20) oemrhgntcproteins. morphogenetic Bone (2004). 307-310. , Cell e.Dyn. Dev. o.Cl.Biol. Cell. Mol. 242 20) a1CN nue h cytoplasmic the induces Jab1/CSN5 (2009). 159-170. , .Cl.Biochem. Cell. J. 41-50. , 3 89 ee Dev. Genes 439-449. , eedrn uieembryogenesis: murine during gene 19) isnemttosabolishing mutations Missense (1997). 20) h O9sgaooe tthe at signalosome: COP9 The (2002). 20) nitrcinntoko the of network interaction An (2009). 18 765-771. , 21) ln ooou constitutive homologue Plant (2011). rna . uro .adDe and E. Vuorio, M., ¨ranta, 19) auainldsubneof disturbance Maturational (1999). 1110-1121. , o.Cl.Biol. Cell. Mol. 19) euaino chondrocyte of Regulation (1999). 214 4 423-431. , rhii Rheum. Arthritis 20) hrceiaino the of Characterization (2000). 19) agtddsuto of disruption Targeted (1997). 279-290. , Cell 24 .Ml Biol. Mol. J. 13 20) a1mdae protein mediates Jab1 (2007). arxBiol. Matrix 2251-2262. , 1025-1036. , 107 89 747-754. , 557-565. , .Bn ie.Res. Miner. Bone J. 19) Osf2/Cbfa1: (1997). 15 19) Mutations (1997). 21) md is Smad6 (2011). 1858-1869. , 19) ba,a Cbfa1, (1997). 21) Ectopic (2012). 371 54 acf Tissue Calcif. 14 Cell 3878-3884. , 514-527. , ee Cells Genes Nature 329-335. , .Cl Sci. Cell J. 89 Growth (2004). (2002). (2009). 773- , 423 , a,M,Co . u . a,S,W,L,Si . ag .adCo X. Cao, and N. Wang, X., Shi, L., Wu, S., Bai, Y., Wu, X., Cao, M., Wan, a,M n a,X. Cao, and M. Wan, ea . wmt,M,Kntn,N,Ysia . i,Y,EoooIaoo M., Enomoto-Iwamoto, Y., Liu, C., Yoshida, N., Kanatani, M., Iwamoto, C., Ueta, Y. J. Kato, and S. Yamanaka, A., Fukumoto, N., Yoneda-Kato, K., Tomoda, in . eg . aat .M,Lvnai . rks . hn,Q,Parker- Q., Zhang, E., Drakos, V., Leventaki, M., J. G. Parant, Karsenty, G., Peng, and L., P. Tian, Ducy, J., M. Owen, P., J. Bonnamy, S., Takeda, og . srd,K .adLos .M. K. Lyons, and D. K. Estrada, B., Song, hu . hn,Q,Egn . uie,E,Ce,Y,Sbl,E,Kao,D and D. Krakow, E., Sebald, Y., Chen, E., Munivez, F., Engin, Q., Zheng, G., Zhou, aatn,M,Snio . as,V,Dgin,C,Csrt,G,Mnii E., Montini, G., Casorati, C., Doglioni, V., Basso, F., Sanvito, M., Panattoni, R. R. Behringer, and G. Ogunrinu, M., J. Deng, A., D. Ovchinnikov, h,B,Zag . i,R,Wn,M,Jn . o,W,Tn,D,Hri,S E., S. Gla Harris, S., D., Sitte, Tang, W., Hou, H., Jin, M., Wang, R., Xie, M., Zhang, B., Shu, H., I. Choi, J., T. Cho, A., D. Fenstermacher, J., G. Feldman, M., Xu, M., E. Shore, X. F. Claret, and J. T. Shackleford, C. Chang, and R. Serra, hu . hn . hu . hrnvkaau . eh,J,Ciaa,D,Gelb, D., Chitayat, J., Hecht, K., Thirunavukkarasu, L., Zhou, B. Y., Lee, Chen, and G., Zhou, X. Garcia-Rojas, Y., Chen, R., Morello, G., Zhou, Hong, Q., L., Zheng, M. Bouxsein, D., G. Cuny, C., C. Hong, S., C. Lai, Y., D. Deng, B., P. Yu, cree,T . esn .D n etnof .J. J. Westendorf, and D. E. Jensen, M., T. Schroeder, S. C. Hill, and S. Ross, M. K. Lyons, and S. B. Yoon, B., Song, N., K. Retting, ohd,C . aaoo . uia . uuci . t,K,Ioe . Yamana, K., Inoue, K., Ito, T., Furuichi, T., Fujita, H., Yamamoto, A., C. Yoshida, R. Derynck, and W. K. X. Wharton, Deng, and G. Serino, N., Wei, W. X. Deng, and N. Wei, on .S n yn,K M. K. Lyons, and S. B. Yoon, French, T., Footz, P., Sundaresan, M., Asai-Coakwell, M., K. Berry-Wynne, M., Ye, a1atgnzsTFbt inln yidcn md degradation. Smad4 inducing 171-176. by signaling TGF-beta antagonizes Jab1 Commun. Res. Biophys. hoi . nmt,H,Nkt,K,Tkd,K tal. et in form K. negative dominant Takada, its or K., Cbfa1 Nakata, of chondrocytes. overexpression H., by caused Enomoto, malformations T., Ohmori, 6125-6137. utpefntoso a1aerqie o al mroi eeomn n growth and development embryonic early mice. for in required are potential Jab1 of functions repair. Multiple DNA and damage al. DNA et spontaneous Y. survival, S. cell Lin, in H., Dai, Jab1 J., of T. Shackleford, J., Thornburg, eeomn n regeneration. and development rc al cd c.USA Sci. Acad. Natl. Proc. B. Lee, Cbfa1- rescues partially and differentiation chondrocyte mice. ability hypertrophic deficient its uncovers induce chondrocytes nonhypertrophic to in Cbfa1 of expression Continuous inlsm mar utpesae fTcl development. cell T of 477. stages multiple R. impairs Pardi, and signalosome A. Mondino, R., J. Bender, n nesl euae a iadadBl expression. Bcl6 and ligand Fas regulates inversely and A. development. bone sporadic endochondral and during Sci. al. Cell maturation inherited et J. and J. causes proliferation R. ACVR1 chondrocyte O’Keefe, receptor Y., Mishina, I al. type et M. BMP LeMerrer, progressiva. ossificans L., the fibrodysplasia D. Glaser, in P., mutation Delai, M., J. Connor, disorders. nlssadfntoa orlto ihpeoyi aiblt ncleidocranial in variability phenotypic al. with et R. correlation C. Greenberg, functional dysplasia. A., and S. Berry, analysis S., Pirinen, D., B. ossification. [corrected] al. 1369. et heterotopic C. reduces Sachidanandan, inhibition T., Katagiri, receptor M., P. McManus, W., D. ietdepeso fCercmiaei ifrnitn hnrctsi transgenic in chondrocytes differentiating in recombinase mice. Cre of expression directed oto n cancer. and control osteoblasts. Today maturing Embryo and C developing in Res. transcription gene of organizer formation. Biol. bone Cell endochondral for required is Smad5 and Development Smad1 through signaling yeXclae eerglto yRn2cnrbtsdrcl oishypertrophic its to directly contributes Runx2 vivo. by in regulation expression gene chondrocyte-specific collagen X Type of induction through growth limb regulates Runx2 hedgehog. Indian and al. et maturation, Y. Ito, chondrocyte K., Takada, A., Zanma, K., protease. uaino h oemrhgntcpoenGF assoua n skeletal al. and et ocular T. causes Biochem. W. Cell. GDF3 J. Allison, protein N., Corbett, morphogenetic C., bone V. the anomalies. Fleisch, of M., Mutation Abitbol, R., C. disease. and development 19 261-286. , 21) A1i seta o eldvlpetadgria etrformation center germinal and development cell B for essential is JAB1 (2012). Genesis 20) oiac fSX ucinoe UX uigskeletogenesis. during RUNX2 over function SOX9 of Dominance (2006). a1adcodoyedfeetain243 differentiation chondrocyte and Jab1 se,J,Jluoa . esl . aatn,M,Pri .adGessner, and R. Pardi, M., Panattoni, F., Weisel, J., Jellusova, J., ¨sner, rnsBohm Sci. Biochem. Trends it eet e.CEby Today Embryo C Res. Defects Birth 40 u.Ml Genet. Mol. Hum. u.Ml Genet. Mol. Hum. 124 383-408. , 136 .Cl Biol. Cell J. 26 3428-3440. , ee Dev. Genes 145-146. , 1093-1104. , ee Dev. Genes 93 .Bo.Chem. Biol. J. elDiv. Cell 93-103. , 20) o h md euaetranscription. regulate Smads the How (2008). 75 20) M inln nseea development. skeletal in signaling BMP (2005). 20) G-easgaigi ua kltladpatterning and skeletal human in signaling TGF-beta (2003). 328 20) h O9signalosome. COP9 The (2003). 213-225. , Development 153 15 103 651-657. , 20) utpefntoso Msi chondrogenesis. in BMPs of functions Multiple (2004). 8 5 19 18 467-481. , 2311-2316. , 26. , 20) Gbt aiysgaig oe nihsin insights novel signaling: family TGFbeta (2009). 87-100. , yoieGot atrRev. Factor Growth Cytokine 33 19004-19009. , 287-298. , 952-963. , 279 592-600. , 21) A1CN:anwpae ncl cycle cell in player new a JAB1/CSN5: (2010). 43013-43018. , a.Genet. Nat. 21) M2 u o M4 scuilfor crucial is BMP4, not but BMP2, (2011). 20) h O9sgaooe oeta a than more signalosome: COP9 The (2008). 136 .Cl Biol. Cell J. 20) ux n ux r seta for essential are Runx3 and Runx2 (2004). 20) agtdiatvto fteCOP9 the of inactivation Targeted (2008). 3691-3697. , 69 20) mdsgaigi skeletal in signaling Smad (2009). 333-351. , 38 525-527. , 162 20) M aoia Smad canonical BMP (2009). .Immunol. J. nu e.Cl e.Biol. Dev. Cell Rev. Annu. 833-842. , 20) ux:amaster a Runx2: (2005). 19) BA mutation CBFA1 (1999). .Ep Med. Exp. J. 20 21) seta roles Essential (2010). a.Med. Nat. 379-388. , 20) recurrent A (2006). 20) M yeI type BMP (2008). 20) Skeletal (2001). 188 n.J Biochem. J. Int. 20) Col2a1- (2000). MORep. EMBO Oncogene it Defects Birth 2677-2686. , 205 14 Biochem. 1363- , (2002). (2004). (2001). (2003). (2010). 465- , 29 3 , ,