h uattsu n ramn ihdcrn hc lcsTGF- blocks which decorin, with treatment and tissue in sustained mutant are Smad3, the and Smad2 phosphorylated of levels as well inln,ipoe h itptooyo Sharp-1 of histopathology the improves signaling, Hsane l,19) h rgn fatvtdstliecells satellite are activated that of progeny process cytokines The this of in 1996). and implicated differentiation al., been et factors have and (Husmann cells growth proliferation satellite for Many the mitogenic formation cells. on tissue by dependent the satellite followed in is is role This key that tissue. a necrotic play by of macrophages infiltrated include removal activated is which and tissue cells, muscle phases, immune damaged distinct the the During phase, into remodeling. inflammatory tissue divided and formation be tissue inflammation, can satellite on and dependent is cells, muscle of Regeneration 2004). Rudnicki, xrclua arxgnsi yfbolss hs results These TGF- of modulation myofibroblasts. by capacity, part, regenerative in least muscle in at regulates Sharp-1 genes that demonstrate matrix extracellular TGF- inhibits overexpression hsooy ainlUiest fSnaoe 157Singapore. 117597 Singapore, of University National Physiology, ertcfbr,climdpst n irss TGF- fibrosis. and deposits calcium fibers, necrotic eiie n utv .Lv lc,NwYr,N 02,USA. 10029, NY York, New Place, Levy L. Gustave One Medicine, uaaAcharjee Sujata regeneration muscle TGF- regulates Sharp-1 ARTICLE RESEARCH ß eevd1 ue21;Acpe oebr2013 November 6 Accepted 2013; June 11 Received 1 Charge 2002; al., injury et or Huard exercise 2001; following Garry, myofibers and occurs damaged and (Hawke muscle skeletal replace postnatal of to feature a is Regeneration INTRODUCTION Myofibroblast Degeneration, TGF- WORDS: KEY in normally muscle proceed regeneration skeletal injury-induced Sharp-1 of embryonic however, phases Sharp-1; the of initial that Although compromised. absence is the capacity regenerative show in the post-natally, impaired not We is Sharp-1- development mice. in regeneration deficient freeze-injury-induced its understand and muscle to skeletal assessed development order we myogenesis, In post-natal MyoD. and pre- of in role activity the modulating previous of precursor by muscle regulator Our of cells negative differentiation processes. blocks a it cellular is and myogenesis of Sharp-1 skeletal that number demonstrated a have in studies repressor involved transcriptional is (bHLH) helix-loop-helix that basic a is Sharp-1 ABSTRACT Atosfrcrepnec [email protected];[email protected]) ([email protected]; correspondence for *Authors 780336 Nussbaumstr, Psychiatry Mu of Department Neurobiology, Behavioral muscles. eieVercherat Cecile eateto eeomna n eeeaieBooy on ia colof School Sinai Mount Biology, Regenerative and Developmental of Department ¨ 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,5968doi:10.1242/jcs.136648 599–608 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. ce,Germany. nchen, 2 / 2 nvitro In ie uiglt tgs h uatmsl exhibits muscle mutant the stages, late during mice, hr- soitswt md,adits and Smad3, with associates Sharp-1 , b 1, irss eeeain kltlmuscle, Skeletal Regeneration, Fibrosis, , 1 ,Tn-a Chung Teng-Kai *, eia uaGulbagci Tuba Neriman , b n md-eitdepeso of expression Smad3-mediated and - b signaling. 1,2 uaGopinadhan Suma , b b xrsin as expression, 1 3 oizRossner Moritz , 2 inln n skeletal and signaling oeua and Molecular 2 eatetof Department / 2 injured ´ and b irbatclst rlfrt n rdc C htrslsi a in results TGF- that tissues. ECM in produce response and fibrotic proliferate to cells TGF- fibroblast contrast, By cells. al., epithelial differentiation, et of apoptosis Moses induces 1990; TGF- al., general, healing, et In (Barnard 1990). arrest cycle wound cell and apoptosis homeostasis, including processes physiological development, diverse regulate that cytokines nldn hs noigEMpoen.EcsieTGF- Excessive proteins. ECM encoding 1997), al., those et (Massaous 1997). including genes target al., sequence of in et DNA participate regulation and transcriptional CBP/p300 Massaous specific as such bind 1992; coactivators recruit and complexes al., motifs protein et Smad translocate (Wrana Nuclear and nucleus (Smad4) the Smad phosphorylated common into are the Smad3, to and bind (pSmad2/3), Smad2 1992; proteins al., TGF- et Smad of presence (Wrana activated the receptors In 1997). II al., et type Heldin and I type transmembrane fibrosis DMD. of of pathology regulation the that in indicating step key 2007), a al., presents et Cohn 1995; nue eoiino C ttest fijr a edto lead can injury of Although 1998). site al., et the Serini 1996; at Gabbiani, and ECM (Serini fibrosis of deposition induced Se and widely (Lefaucheur are Interestingly, DMD that for 2007). model mutants mouse al., mdx a 1999; in et as regeneration studied al., Zhu improves et 1999; also TGF- Bernasconi of al., 1995; inhibition al., et dystrophic in et Murakami fibrosis cause (Bernasconi (fibrotic) to muscular shown scar muscles been dysfunctional have Duchenne and by compromised, dystrophies TGF- as replaced of is Levels is such process tissue. tissue myopathies regeneration muscle the and In (DMD), 1998). dystrophy al., et (Desmoulie isermdln.Oc h isei eard h contractile the repaired, is tissue the Once of to contributing remodeling. Grinnell, Differentiation further 1994; ECM, tissue produce collagens. Noble, also and Myofibroblasts III Border 1994). 1992; type Ruoslahti, activity and contractile and (Border their augments I myofibroblasts matrix into extracellular type fibroblasts initial fibroblasts an of the produce remodeling, injury in (ECM) of tissue results site the During fusion at present and myofibers. the and growth of by Charge cycle Further 2002; formation characterized al., 2004). et cell (Huard are Rudnicki, nuclei and the located that multiple centrally a myotubes from undergo of presence form they withdraw to and proliferation, differentiate cells of precursor rounds muscle called are hc sasg ficmlt eeeain n soften is and regeneration, incomplete of TGF- sign (fibrosis), elevated tissue by a scar accompanied of fibroblast is formation the excessive which in result and can by persistence proliferation removed are Myofibroblast cells and apoptosis. terminated is myofibroblasts of activity 2 hlaRn Shankar Rani Shilpa , ebr fteTGF- the of Members 3 n ehaTaneja Reshma and ` ee l,19;Srn n abai 96 Serini 1996; Gabbiani, and Serini 1993; al., et re b inln o nypeet irss but fibrosis, prevents only not signaling b b n TGF- and 1 niispoieaino otclsand cells most of proliferation inhibits b 2 1,2, auWang Yaju , uefml r multifunctional are superfamily b * eesadclae deposition collagen and levels b b inl hog heteromeric through signals r lvtdi muscular in elevated are 2 b 2 ,LiLi iad h receptor- the ligand, 1 , b stimulates ´ bille, 599 b ´ -

Journal of Cell Science Sa23admoirbat oiiefrsot uceactin muscle smooth for positive (SMA myofibroblasts and pSmad2/3 yfbols el n hsipcsseea uceregeneration. muscle skeletal impacts this and cells myofibroblast icda htm Ymd n iaoo 05 u tal., of et overexpression that Sun shown 2005; have studies Miyamoto, previous and Our 2007a). (Yamada and rhythms progression tumor circadian arrest, cellular cycle in cell roles apoptosis, complex differentiation, plays that factor transcription loop-helix governing understood. mechanisms fully not molecular are repair the and steps regeneration regeneration, distinct the muscle of regulation in in implicated are pathways several ARTICLE RESEARCH 600 in as well in as expressed development, is embryonic Sharp-1 during that muscles demonstrated skeletal previously have Sharp-1 We in development muscle Skeletal RESULTS of stages late muscle at skeletal defect a we TGF- in exhibit Here, Sustained role mutants 2013). regeneration. null al., TGF- a Sharp-1 et regulating injury, plays Wang by 2012; Sharp-1 Gulbagci al., regeneration et that 2004; to Ling al., ability demonstrate 2009; et their al., (Azmi blocks et differentiation preadipocytes terminal or undergo myoblasts in Sharp-1 rbbyb neatn ihSa3adihbtn its inhibiting TGF- and limit to Smad3 that essential indicate studies with is our Sharp-1 (TIMP1) together, interacting Taken 1 activity. metalloproteinase transcriptional by of inhibitor probably tissue and collagens TGF- inln mloae uceptooyaddgnrto in degeneration and pathology muscle Sharp-1 ameliorates signaling hr- as nw sDc n HH4)i ai helix- basic a is BHLHE41) and Dec2 as known (also Sharp-1 b + nSharp-1 in ) inln n naoie md-eedn xrsinof expression Smad3-dependent antagonizes and signaling 2 / 2 ie edmntaeta hr- ietyregulates directly Sharp-1 that demonstrate We mice. 2 / 2 eeeaigtsu.Ihbto fTGF- of Inhibition tissue. regenerating b xrsinicesslvl of levels increases expression b b inln.I epneto response In signaling. 2 / n md inln in signaling Smad3 and 2 mice b kltlmslsocr omlyi h bec fSharp-1. of Pax7 absence of the number the in in of changes normally development no the area Moreover, occurs that cross-section muscles indicating mean panel), skeletal the right in 1C, or (Fig. (CSA) 1B) (Fig. architecture muscle ygnssi idtp W)adltemt Sharp-1-null littermate and (WT) postnatal and wild-type embryonic (Sharp-1 role in analyzed the examine we further myogenesis To Sharp-1, 2013). al., endogenous 2004; et al., of Wang et 2012; Azmi al., 2003; al., et et Ling through (Azmi myogenesis activity MyoD inhibits of overexpression regulation its and tissues, adult uli(i.2) e n ite as(1 n D16 and In (D10 myotubes. formed days newly evident nucleated was sixteen centrally mice of WT and presence in regeneration by Ten injury, central post containing 2A). respectively) myotubes (Fig. formed newly nuclei of presence the with iea 2 ihn akdhsooia ifrne paetat apparent differences histological marked no with D2, at mice Sharp-1 and Sharp-1-null 2- WT from of muscles 3-month-old Quadriceps potential injury. to freeze regenerative to response the in mutants examined mutants Sharp-1-null then in impaired We is regeneration muscle Skeletal hssae(aantson.Fv asatrijr (D5), injury after days Sharp-1 and Five WT both shown). in not evident was (data regeneration stage this oe ewe TadSharp-1 and WT between noted eeldetniemoie aaei ohW n Sharp-1 histologically. and WT sections both analyzed muscle in injured damage was myofiber of extensive staining regeneration revealed (HE) and eosin and 2007b) Hematoxylin al., et ifrnei ygnnepeso a paeti Tand WT in apparent was expression Sharp-1 myogenin in difference itlgclaayi fqarcp uce rmautW and WT adult from muscles Sharp-1 quadriceps of analysis Histological 2 2 2 / / ora fCl cec 21)17 9–0 doi:10.1242/jcs.136648 599–608 127, (2014) Science Cell of Journal 2 2 / 2 uatmc Rsnre l,20) oovert No 2008). al., et (Rossner mice mutant ) ieas i o eelayovosdfcsin defects obvious any reveal not did also mice mro tebync()dy1. Fg 1A). (Fig. 16.5 day (E) embryonic at embryos ifrn ewe TadSharp-1 and WT significantly between not different was size fiber mean ( muscle Sharp-1 skeletal in of Development 1. Fig. -ot-l T(/)adSharp-1 and (+/+) WT from 2-month-old muscles Quadriceps (B) microscopy. fluorescence by followed antibody anti-myogenin with immunostaining needn TadSharp-1 and four WT from independent cells satellite the of analyze percentage to used were sections muscle Pax7-stained (E) with hematoxylin. counterstained were satellite Sections detect cells. to antibody anti-Pax7 with yfbr nbt eoye ( genotypes both in of myofibers area cross-section the to determine used were of muscles sections quadriceps H&E-stained (C) staining. H&E Sharp-1 mean are Data cl as 100 bars: Scale ygnssi T(/)adSharp-1 and (+/+) WT in myogenesis ie( mice 2 / 2 mro tE65wsaaye by analyzed was E16.5 at embryos ) n 2 5 2 / 2 )wr nlzdhsooial by histologically analyzed were 4) 2 / 2 2 / uat Fg 1D,E). (Fig. mutants 2 uce eeimmunostained were muscles / 2 iewr nue (Sun injured were mice mutants. 6 m m. ..()W and WT (D) s.e. + aelt el was cells satellite A Embryonic (A) 2 2 n / 5 2 / 2 ) The 4). mice. 2 2 / muscles 2 / 2 2 mice. ( 2 / 2 / 2 2 / ) 2

Journal of Cell Science peuae uigrgnrto nbt TadSharp-1 and WT both were in levels MyoD regeneration myoblasts. proliferating during of upregulated marker a is which D0adD6atrijr) ucedgnrto saprn nSharp-1 in apparent is degeneration muscle injury), after D16 and (D10 acfcto,wihi alako ytohctissues mutant the dystrophic in Sharp-1 in 2C). of detectable indicating (Fig. staining was hallmark Trichrome Red, Masson’s fibrosis by a addition, muscles Alizarin regenerating In is with 2B). (Fig. which positively quadriceps calcification, mutant stained Indeed, of 2A). signs (Fig. muscles overt revealed degeneration tissue and mutant necrosis the muscles, WT to contrast ARTICLE RESEARCH i.2 lee eeeaiersos pnfez nuyi Sharp-1 in injury freeze upon response ( regenerative Altered 2. Fig. 3A). (Fig. analysis blot Moreover, muscle. MyoD WT (SMA western with many smooth-muscle-actin-positive compared revealed tissue several mutant by tissue the injured in cells seen D10 of Immunostaining as mutants 1 fe nuyrvae h rsneo acu eoisadclae eoiin(lesann)rsetvl nSharp-1 in respectively staining) (blue deposition collagen and deposits calcium of presence the revealed injury after D16 2 / 2 oeaietemcaim neligdfcieregeneration defective underlying mechanisms the examine To iea 5 1 n 1 olwn nuy tD,nwyfre ytbswt eta uliaeeieti ohW n uatmsls tltrstages later At muscles. mutant and WT both in evident are nuclei central with myotubes formed newly D5, At injury. following D16 and D10 D5, at mice ) 2 / 2 uce efrtaaye h xrsino MyoD, of expression the analyzed first we muscle, + ,clswr also were cells ), 2 2 2 / / / 2 2 2 + mice. uat.(,)Aiai e tiig() n asnsTihoesann C,at (C), staining Trichrome Masson’s and (B), staining Red Alizarin (B,C) mutants. hshrlto fSa2adSa3 hc oma form which Smad3, TGF- and ligand, Smad2 by of activation phosphorylation Upon TGF- Increased remodeling. for tissue role of additional steps an late reflect the might in due Sharp-1 but not defect, is differentiation higher (D10/D16) a stages a to late with at regeneration that correlated compromised suggest results the which these Together, 3C,D). tissue, (Fig. and mutant index regeneration WT the in injured myotubes of Immunostaining embryonic Sharp-1 an antibody. using (eMHC) differentiation examined MHC then We tissue mutant 3B). the (Fig. in myofibroblasts of presence the indicating cells, paeti Sharp-1 in apparent A itlgclaayi fmsl eeeaini T(/)adSharp-1 and (+/+) WT in regeneration muscle of analysis Histological (A) 2 / ora fCl cec 21)17 9–0 doi:10.1242/jcs.136648 599–608 127, (2014) Science Cell of Journal 2 ucea 5rvae ihrnme feMHC of number higher a revealed D5 at muscle b inln nSharp-1 in signaling 2 / 2 uceta i o ooaiewt MyoD with colocalize not did that muscle 2 2 / / 2 2 uce.Saebr:100 bars: Scale muscles. nue muscles injured b eetr induce receptors m 601 m. 2 / 2 + +

Journal of Cell Science erssi Sharp-1 in in necrosis TGF- 2007). al., enhanced fibrosis et the Li whether against 1996; assess al., used To et (Isaka successfully decorin proteoglycan been the is has mice one that and 2007) blocker al., et such (Cohn mice mdx in pathology muscle improve in SMA of role number TGF- increased crucial an of exhibited levels a high sustained play with that (Desmoulie myofibroblasts fibrosis of differentiation eeas ihri Sharp-1 in higher also were nuy(i.4)wihwsi otatt Ttsu,weefew where tissue, WT to contrast in was SMA which 4C) (Fig. injury EERHARTICLE RESEARCH otaaea ucewsijce ihPS Decorin-injected The PBS. 602 later. with days injected 3 was analysis muscle histological contralateral performed and muscle TGF- blocking that demonstrated have studies Previous TGF- of Inhibition TGF- 4B). (Fig. muscles WT with compared D10 Sharp-1 and WT in analysis blot TGF- western examined TGF- we by fibrosis, of exhibit measure thus mutants levels a the pSmad2/3 of as to Estimation translocate serves 1992). al., then et and (Wrana Smad4 nucleus with complex heterotrimeric Sa23lvl yimnsann Fg B.Interestingly, TGF- 4B). increased the (Fig. with whether immunostaining correlation examine by in To levels mutants. pSmad2/3 Sharp-1-null D10. TGF- and in increased D5 at D10 decline to and started TGF- levels TGF- contrast, the mice, By and levels WT D2, elevated In with at injury, 4A). apparent upon (Fig. induced strongly injury was after expression points time various + yfbolsswr paeta n stage. any at apparent were myofibroblasts b b b inln mloae ucenecrosis muscle ameliorates signaling eutdi nrae inln,w analyzed we signaling, increased in resulted ` 2 ee l,19;L ta. 04.Consistent 2004). al., et Li 1993; al., et re xrsinwsssanda ihlvl tD5 at levels high at sustained was expression / 2 ie eijce eoi nSharp-1 in decorin injected we mice, 2 b / 2 inln.BcueSharp-1 Because signaling. b nue uce tD and D5 at muscles injured b b inln,temtn tissue mutant the signaling, + inln nele muscle underlies signaling ees Sa23levels pSmad2/3 levels, el tD n 1 after D10 and D5 at cells b xrsinlevels expression 2 / 2 uce at muscles b induces b 2 2 can / / 2 2 b nue uce rmSharp-1 from muscles injured Fg C.A xetd Sa23sann a ihrin decorin. with higher TGF- injected was of that inhibition staining with with consistent pSmad2/3 compared Moreover, expected, tissue antibody pSmad2/3 uninjected As with immunostained 5C). (Fig. were decorin that muscles TGF- confirm reduced injured muscles To to 5B). decorin-injected led (Fig. indeed in muscle uninjected pathology to relative reduced damaged the indicated in of increase Quantification area significant 5A). (Fig. a myofibers and regenerating myofibers degenerating in decrease 7–6 fSap1(i.6) oevr hr- colocalized Sharp-1 Moreover, 6B). (Fig. Smad3. Sharp-1-bHLH acids Sharp-1 amino between the of with resides region 173–265 Smad3 the with with that interacted interaction indicating seen for domain, essential bHLH domain, the was lacks which orange association mutant, mutants no the deletion However, the lacks and which Sharp-1, assays full-length Immunoprecipitation Sharp-1 TGF- Smad3. that with of with were indicated 2012) or associate presence al., that alone et Sharp-1 the (Ling transfected, mutants in deletion in domains Sharp-1 the various apparent Smad3, define To was 6A). Smad2/3 (Fig. of presence total and The absence and the in pathway. harvested TGF- TGF- were the the lysates of and mediator cells, of key a Smad3, components b with Sharp-1 with of interaction interacts deregulated for Sharp-1 account might that TGF- mechanisms the examine To TGF- regulates Sharp-1 5D). (Fig. muscle decorin-treated in reduced also were cells epnewsaaye.Mctge hr- a rnfce in transfected was Sharp-1 Myc-tagged analyzed. was response b b nitrcino hr- ihedgnu pSmad2/3 endogenous with Sharp-1 of interaction An . D xrsinadsgaig efrtivsiae whether investigated first we signaling, and expression ,lcigteCtria ein n Sharp-1 and region, C-terminal the lacking C, ora fCl cec 21)17 9–0 doi:10.1242/jcs.136648 599–608 127, (2014) Science Cell of Journal b signaling ecnaeo eMHC the of determining percentage by calculated was index Regeneration (D) antibody. anti-eMHC with Sharp- 1 and WT from muscles of quadriceps Sections (C) antibodies. anti- SMA and anti-MyoD with immunostained udiesmslsfo TadSharp- 1 and WT of from Sections muscles (B) quadriceps control. internal an as to EF1 injury) expression. after MyoD D10 detect and D5 (D2, tissues lee nSharp-1 not in is altered differentiation Myogenic 3. Fig. rti yae rmW ++ n Sharp- and (+/+) 1 WT from with lysates performed protein was blotting Western (A) 6 Sharp-1 b 2 2 2 s.e. / / / 2 2 2 2 inln,uijce n injected and uninjected signaling, iea 5atrijr eestained were injury after D5 at mice were injury after D10 at mice ( / 2 2 / 2 2 nnue D)adinjured and (D0) uninjured ) iesoe considerable a showed mice / 2 ie( mice n 2 5 / + 2 ) aaaemeans are Data 4). ytbsi Tand WT in myotubes b muscles. ciiy SMA activity, a a used was D O, b +

Journal of Cell Science fijrdmslsfo TadSharp-1 and WT from muscles injured of S rti ln eeicbtdwith incubated were or alone GST–Sharp-1 performed of protein we amounts Smad3, GST Equivalent with assays. pull-down interacts GST directly Sharp-1 whether EERHARTICLE RESEARCH ihSa3i h rsneo TGF- of presence the in Smad3 with TGF- Increased 4. Fig. 1 2 / 2 eeeaigmsl ( muscle regenerating b n Sa23lvl nSharp-1 in levels pSmad2/3 and n 5 )atr2 n 0dy fijr n nlzduiganti-TGF- using analyzed and injury of days 10 and 5 2, after 4) 2 / 2 iewr muotie ihatbde gis Sa23()adSA()a 5adD0atrinjury. after D10 and D5 at (C) SMA and (B) pSmad2/3 against antibodies with immunostained were mice b Fg C.T determine To 6C). (Fig. 35 -aee Smad3. S-labeled 2 / 2 eeeaigtissue. regenerating n md-eedn rncitoa epne sn h 3TP-Lux the 6D). using responses transcriptional (Fig. Smad3-dependent protein and GST with apparent no was expected, Smad3 as of whereas, interaction Smad3, with interacted directly Sharp-1 A etr ltigwspromdwt rti xrcsfo Tn Sharp- WTand from extracts protein with performed was blotting Western (A) enx netgtdteefc fSap1epeso nTGF- on expression Sharp-1 of effect the investigated next We ora fCl cec 21)17 9–0 doi:10.1242/jcs.136648 599–608 127, (2014) Science Cell of Journal b nioy EF1 antibody. a a sda nitra oto.(,)Sections (B,C) control. internal an as used was 603 b

Journal of Cell Science EERHARTICLE RESEARCH rnfce ihFA–md n y–hr- eeaaye sn niFA n niSap1atbde.Nce eesandwt AI D Equival (D) 604 DAPI. with stained were with Nuclei incubated antibodies. were anti-Sharp-1 alone and GST anti-FLAG or using GST–Sharp-1 analyzed of were amounts lysates. Myc–Sharp-1 in and Sharp-1 FLAG–Smad3 and with M Smad3 transfected bHLH, Myc–Sharp-1 of Myc–Sharp-1, expression and FLAG–Smad3 shows expressing Input plasmids antibody. with transfected were Cells (B) lysates. Sharp-1 the in Sharp-1 and Smad3 of expression Smad3. b with interacts Sharp-1 6. Fig. was Smad3, with interact to Sharp-1- failed the which contrast, By mutant, activity. bHLH reporter repression Smad3-induced in resulted of Sharp-1 full-length of Co-expression 1992). TGF- been monitor has to used and widely promoter TGF- (PAI-1) inhibitor-1 a activator plasminogen contains which reporter, o or yae eeimnpeiiae ihat-y grs ed n muolte ihat-Sa23 md n y niois nu sho Input antibodies. Myc and Smad3 anti-pSmad2/3, with immunoblotted and beads agarose anti-Myc with immunoprecipitated were Lysates hour. 1 for 1 D rMyc–Sharp-1 or O D .4 or fe rnfcin yae eeimnpeiiae ihFA–grs ed olwdb etr ltwt anti-Myc with blot western by followed beads FLAG–agarose with immunoprecipitated were lysates transfection, after hours 48 C. b n mdsgaig(rn tal., et (Wrana signaling Smad and A 21 el eetasetdwt xrsinvcosfrMcSap1 el eelf nrae rtetdwt TGF- with treated or untreated left were Cells Myc–Sharp-1. for vectors expression with transfected were cells C2C12 (A) b epneeeetfo the from element response 35 S-labeled b atnwsue sa nenlcnrl N,annseii g ad C 21 el co- cells C2C12 (C) band. IgG non-specific a *NS, control. internal an as used was -actin nvitro in rnltdSa3 0 fiptwsrno h e sacontrol. a as gel the on run was input of 10% Smad3. translated Fg B.T xmn h ehnssudryn h inhibitory the activity underlying mechanisms Smad3 the examine regulates To Sharp-1 7B). (Fig. (siSharp-1) endogenous siRNA that (siRNA) Sharp-1 siRNA confirming scrambled with with transfected cells control transfected to Smad3-dependentrelative cells of in was activity repression increased reporter Conversely, 7A). in (Fig. activity transcriptional effective less significantly ora fCl cec 21)17 9–0 doi:10.1242/jcs.136648 599–608 127, (2014) Science Cell of Journal ucencoi nSharp-1 reduces in signaling necrosis pSmad muscle of Inhibition 5. Fig. orst fmc.Dt r means are Data mice. of the from sets of representative four area are injured shown total Results the muscle. to of area ratio a necrotic as the determined was and decorin absence of the presence in pathology of percentage muscles injured ( contralateral the pathology with muscle compared of reduction striking show a muscles D10 Decorin-injected at staining. analyzed H&E were by sections cross and injury A eoi,o h oto eil PS was (PBS) Sharp-1 vehicle into control injected the or Decorin, (A) netdmsls( muscles injected decorin- in which signaling pSmad2/3 antibodies reduced (D) revealed anti-SMA and (C) 3 anti-pSmad2/ with immunostained were sections * n P 5 , )ta eeijce ihPS B The (B) PBS. with injected were that 4) .5 CD oto n decorin-injected and Control (C,D) 0.05. n 5 2 ) cl a:100 bar: Scale 3). / 2 uce asafter days 7 muscles 2 / 2 mutants. 6 s.d.; m m. ent yc– ws

Journal of Cell Science EERHARTICLE RESEARCH Fg C.Tgte,teedt eosrt htSap1de not antibody does Sharp-1 anti-Myc that demonstrate with data not these Together, but 7C). was (Fig. anti-FLAG, which SBE, were the with on evident supershifted complex was A FLAG–Smad4 Smad4 probe. Nuclear and SBE Smad3 Myc–Sharp-1. radioactive containing a of and with presence incubated were or binding extracts absence FLAG–Smad3 Smad the a whether in to transfected bind investigated (SBE). to we Smad3 activity, of element ability Smad3 the impacts on Sharp-1 Sharp-1 of effect ee uha olgnA (COL1 collagen1A1 myofibroblasts as to such SMA. cells genes of other of TGF- expression and synthesis requires and fibroblasts by of proteins characterized Differentiation (ECM) are matrix They extracellular remodelling. tissue in proteins Smad of binding alter SBE. it the does to nor SBE, the to bind directly yfbolssaehtrgnu noii n lyakyrole key a play and origin in heterogenous are Myofibroblasts ora fCl cec 21)17 9–0 doi:10.1242/jcs.136648 599–608 127, (2014) Science Cell of Journal b inln hc eut nepeso fECM of expression in results which signaling LG(ae7 n anti-Myc- and 7) anti- (lane with FLAG out carried were assays Supershift 5,6). (lanes and Myc–Sharp-1 Smad3/4 of amount and increasing 4), 3, (lanes of FLAG–Smad3/4 amount increasing 1,2), (lanes cells non-transfected from extracts prepared nuclear with incubated was h xrsino COL1 of expression The TGF- with treated (DOX) and doxycycline with uninduced induced left or were cells NIH3T3 8). (D) and 7 (lanes antibodies (C) means are Data was assayed. activity luciferase further hours, a 24 after Then, as figure. ng) in (100 indicted Smad3 and ng) (200 p3TP-Lux cells with Sharp-1 transfected later, were hours 24 of nM. concentration 100 final a at siRNA scrambled control with and transfected siSharp-1 were Cells (B) means s.d. are Data activity. luciferase for assayed after and hr transfection, 48 harvested indicated. were as Cells ng) (25 and bHLH ng) Sharp-1 (25 and Sharp-1 absence of the presence in ng) (100 with Smad3 ng) (200 p3TP-Lux with reporter transfected were Cells (A) myofibroblasts. in TGF- signaling inhibits Sharp-1 7. Fig. *** 6 TGF- of and presence absence the SMA in and promoters PAI-1 the at and Smad2/3 Sharp-1 of to occupancy performed determine were assays ChIP Q-PCR. (E) by analyzed was TIMP-1 and P .. * s.d.; a 32 , ) olgnA (COL1 collagen1A2 1), -aee B oligonucleotide SBE P-labeled 0.001. P , .5 ** 0.05; b aaaemeans are Data . P , b 0.01; o 4hours. 24 for a ,COL3 1, 6 s.d. b a 605 a 2), 6 1

Journal of Cell Science uattsu,a enb h nrae ubro eMHC of however, number Sharp- increased the Intriguingly, in by stages seen as regeneration. early tissue, at mutant 1 during augmented is mutants differentiation myogenic the in TGF- is factor regulatory negative such One 2004). ciiyta ol one h niioyefcso TGF- Its Sharp-1 MyoD of 2013). in effects increased inhibitory al., stages in the et counter result Wang could that presumably 2012; activity al., would of et 2004; therefore inhibitor Ling al., absence 2006; et potent al., (Azmi a et differentiation is Morosetti myogenic Sharp-1 and activity because MyoD occurs probably This inln n ubro SMA tissue of as number and TGF- such increased signaling the inhibitors Given (TIMP-1). metalloprotease metalloprotease of inhibitor matrix of expression TGF- addition, (COL3 collagen3A1 and ARTICLE RESEARCH 606 to ability in its Sharp-1 through for TGF- role regeneration limit novel muscle a and demonstrate remodeling study tissue this of results The DISCUSSION usqett ygncdfeetain rbbyrslsi more in results probably SMA differentiation, myogenic to subsequent epnet nuyi nacd neetnl oee,despite however, Interestingly enhanced. Sharp-1 differentiation, is productive injury to response dt o hw) h aa xrsino COL1 of expression basal TGF- The shown). TGF- with not treated (data were inhibited cells when seen were results significantly COL1 of expression doxycycline with treated with that were TGF- 2010) by cells induced al., fibroblast et (Liu TGF- NIH3T3 Sharp-1 express myofibroblasts. inducibly in expression sought TGF- we regulates activity, Sharp-1 whether Smad3 determine antagonize to to Sharp-1 of ability the and ise rvossuishv eosrtdta TGF- that demonstrated have studies Previous tissue. nijrdseea uce(ie l,20) h resulting The of hindering tissue. 2004). and muscle deposition fibrosis of al., in regeneration underlies resulting complete et proteins, myofibroblasts matrix (Li extracellular myofibroblasts muscle of into TGF- skeletal overproliferation fibroblasts Moreover, injured 2001). of al., in differentiation et induces Liu myogenic 1987; (Massague also Boxhorn, and MyoD and proliferation of Allen myoblast inhibition by inhibit differentiation to Charge reported 1996; been al., et regulators (Husmann negative or regeneration positive as of act which cytokines, and factors fibrosis. and proliferating calcification myonecrosis, cells, by non-myogenic characterized and myogenic stages late at defect ygnssde o pert eoetyprubdi Sharp- in perturbed overtly be to appear 1 not does myogenesis niiino t rncitoa ciiyi yfbols cells. myofibroblast in activity transcriptional its of inhibition antagonizes Sharp-1 that evidence TGF- provide TGF- results these a significant together, in a fibrosis, plays in which role the PAI-1, and SMA inhibitor of promoters activator the plasminogen to bind Smad3 and Sharp-1 exogenous both that of showed assays absence immunoprecipitation chromatin the Moreover, activity. in even Sharp-1 TGF- by suppressed were etaiigatbde n lcescnb sda potential of as Blockade 2007). used al., et Li be 1996; al., can et (Isaka blockers agents antifibrotic and antibodies neutralizing 2 xrsinadsgaigo TGF- of signaling and Expression growth many of interplay the involves myogenesis Postnatal / 2 + b b b mro,dfeetaino ygncpeusrclsin cells precursor myogenic of differentiation embryos, sepce,teepeso fclae n IP1was TIMP-1 and collagen of expression the expected, As . niaiga mato hr- nedgnu Smad3 endogenous on Sharp-1 of impact an indicating , yfbolssada nraei irssi h mutant the in fibrosis in increase an and myofibroblasts inlig rbbyb soito ihSa3and Smad3 with association by probably signalling, b inln nmoirbat.Atog embryonic Although myofibroblasts. in signaling b b 2 / 2 Fg D.Itrsigy nuto fSharp-1 of induction Interestingly, 7D). (Fig. niisEMdgaainb increasing by degradation ECM inhibits a ,COL3 1, uat.SsandTGF- Sustained mutants. a )i md-eedn anr In manner. Smad3-dependent a in 1) b rsosv anr(i.7) Taken 7E). (Fig. manner -responsive a + n IP1(i.7) Similar 7D). (Fig. TIMP-1 and 1 el nSap1ijrdmuscle, injured Sharp-1 in cells 2 b / 2 r utie thg levels high at sustained are ieso regeneration a show mice b idcdEMgene ECM -induced b ´ a eesa step a at levels n COL3 and 1 ´ n Rudnicki, and b b ta. 1986; al., et TGF- . o 8hours 48 for b -mediated b tearly at + b cells. has a b b b 1 - iess u nsvrlohrtsusa el lhuhterl of role the Although well. as tissues other cell-type- several in a but al., diseases, in et Wang Sirt1 activity 2012; transcriptional 2001), al., Smad3 et al., manner. (Ling specific block et G9a to or co- of (Garriga-Canut 2007) recruits 2013) binding al., Sharp-1 HDAC1 impact et that as it (Fujimoto plausible does is such It nor not DNA. factors sites, to does binding proteins Sharp-1 Smad Smad because the data and binding to to due DNA Smad3, bind Our not for is with competition antagonism 2004). This interacts direct activity. (Flanders, transcriptional directly its Sharp-1 inhibits inhibitors that MMP genes demonstrate ECM increasing of and of MMPs expression of expression expression enhancing decreasing collagens, by as fibrosis such in role central tde rvd oe nihsit h oeo hr- in Sharp-1 of role to the therapies. anti-fibrotic lead into of development TGF- that insights mechanisms novel controlling the provide to studies regard TGF- with inappropriate understood be TGF- yncoi nSharp-1 in myonecrosis tiigwtotpiayatbd evda eaiecnrl For control. negative with a incubated as were sections served paraffin-embedded antibody immunofluorescence, primary CA). without Burlingame, Laboratories, Staining (Vector using kit visualized ABC and was Elite 2007b) VECTASTAIN al., the Immunohistochemistry et (Sun previously immunohistochemistry. used described as were and performed area damaged histology comparable with for area. genotypes damaged both largest with the from stained of Sections was identification slides for ten (HE) every eosin and of hematoxylin out one and tissues, muscle embedded (8 HRP- cross-sections Serial anti-rabbit immunohistochemistry and Histology and from anti-mouse antibodies secondary Sigma. OR; from antibodies Fluor Eugene, secondary conjugated Alexa Probes, anti-rabbit Molecular anti MA; and Sigma; Billerica, Millipore, from anti-mouse from from anti-eMHC anti-SMA antibody and (Ser465/467) Bank; phospho-Smad2/3 anti-Pax7 Hybridoma NY; Studies Placid, Developmental Lake Inc., anti-TGF- anti Biotechnology TX; IL; Dallas, Grove, Cruz, Buffalo Santa from immunohistochemistrywere for used antibodies anti-pSmad2/3 and Anti-MyoD Antibodies Sharp-1 muscle skeletal of Regeneration METHODS AND MATERIALS aoe l,21) oee,wehrSap1drcl inhibits TGF- reported. directly been 2012; not Sharp-1 have al., whether effect et However, TGF- (Montagner 2012). and TGF- cancer al., prostate expression et and Sato Sharp-1 breast in between activity correlation inverse xrsinadsgaigacut,a es npr,frdefective for part, in Sharp-1 least in at regeneration accounts, signaling and expression 0ad1 asatrijr) l nmlpooosfloe institutional followed protocols 5, animal (2, All point time TGF- injury). per For after guidelines. analyzed days were described 16 mice four and as least 10 At performed 2007b). al., was et (Sun regeneration injury-induced Freeze-crush TGF- sdsrbdaoe ee asatrijr,20 injury, after days Seven above. described as 04.PS(n01 S)wsijce sacnrli h contralateral and al., the injection et decorin in after (Li control days histologically. a described 3 analyzed as collected as were injected sites Muscles was muscle. six BSA) injured at 0.1% injected (in PBS was 2004). MO) Louis, St (Sigma, irsspssamjrosal oteay o nyi muscle in only not therapy, to obstacle major a poses Fibrosis ossetwt u idns eetsuishv hw an shown have studies recent findings, our with Consistent b b b 2 inligi irssi eletbihd uhrmisto remains much established, well is fibrosis in signalling ihdcrnide eutdi ery3%dces in decrease 35% nearly a in resulted indeed decorin with inln,adtemcaim neligisinhibitory its underlying mechanisms the and signaling, / 2 ora fCl cec 21)17 9–0 doi:10.1242/jcs.136648 599–608 127, (2014) Science Cell of Journal iehv endsrbdpeiul Rsnre l,2008). al., et (Rossner previously described been have mice b b b blockade m inln n eeomn ffboi.Our fibrosis. of development and signaling hc)wr olce ln h nielnt of length entire the along collected were thick) m inln nmoirbat n the and myofibroblasts in signaling b 2 atnfo im;anti-EF1 Sigma; from -actin 2 / 2 / 2 muscle. nvivo in ie hrfr,ehne TGF- enhanced Therefore, mice. udiesmslswr injured were muscles quadriceps , b inln n md lya play Smad3 and signaling b a rmNovocastra, from was m (50 l a m )o decorin of g) rmUpstate from b b

Journal of Cell Science tlat50nce eecutdfrec nml n h aapresented data the animals. and four animal, of each average for an counted calculated. was are were cells nuclei satellite 500 of least percentage At the and animal, each for fields ctn,aeoexln 11,xln n eete one.Calcium analysis mounted. Morphological then in were washes staining. by orange-red and followed as viewed solution xylene were Red deposits (1:1), Biosystem, Alizarin 2% acetone:xylene (Diagnostics in and acetone, kit seconds deparaffinized were 30 a sections for staining, stained using Red Alizarin Masson’s performed For microscope. CA). fluorescence Pleasanton, was a staining under analyzed Trichrome antibody and secondary Fluor with Alexa detection from by followed antibody primary appropriate ARTICLE RESEARCH efre ntilct n eetda es wc.Vle r eotdas reported are Values twice. least were at DNA. the means transfections Empty repeated All and using total control. triplicate WI). performed of in Fitchburg, internal performed were (Promega, amount assays system an the luciferase luciferase as normalize dual transfection, after luciferase to hours added Renilla 48 was of vector ng the expression 5 with transfected figures with the were in indicated along cells as constructs plasmid assays, various and reporter 3TP-Lux reporter For (Invitrogen, NY). 2000 Lipofectamine Island, were or transfections Grand Transient Plus 2010). Lipofectamine been al., using et have (Liu performed Sharp-1 induce cells to 2 hours NIH3T3 with 48 treated Sharp-1-inducible were (BS). and described serum DMEM containing bovine in DMEM in maintained cultured 10% (FBS), were cells were serum NIH3T3 serum. cells horse bovine 2% C2C12 with fetal differentiation, 10% Eagle’s 2012). For and modified respectively. Dulbecco’s al., 20% in containing et maintained (DMEM) (Ling were medium cells previously 293T described and been C2C12 have GST–Sharp-1 Myc–Sharp-1 Canada); and Toronto, Hospital, Sinai Francisco, (Lunenfeld-Tanenbaum Mount Wrana San Institute, Jeff Research Francisco, by provided San kindly California was 3TP-Lux of CA); (University provided Derynck kindly Rik were by FLAG–Smad4 and knockdown Sharp-1 FLAG–Smad3 and FLAG–Smad2, assays luciferase culture, cell Plasmids, a containing under DAPI viewed and microscope. in Laboratories) fluorescence fluorescence- (Vector mounted Vectastain by were from followed medium Cells mounting after antibodies antibodies. FLAG 0.5% secondary and hours slides PBS), conjugated 24 with Myc in with BSA treated (10% together. incubated blocking paraformaldehyde, After were PBS. 4% with or washed in and X-100 fixed Triton alone were cells FLAG–Smad3 transfection, AX10 Myc–Sharp-1, Zeiss at slides a with chamber cells using in C2C12 Smad3, seeded and imaged Sharp-1 were of and localization the determine CA) H-1200, To microscope. (Vectashield Burlingame, DAPI Sections Laboratories, containing antibodies. medium and Vector secondary mounting MyoD in dye-conjugated and mounted SMA Fluor were against Alexa antibodies hour, with 1 primary for detection with buffer staining blocking in by incubated followed were (SUPPLIER?). sections Histoclear retrieval, in antigen After deparaffinised were sections embedded Paraffin analysis Immunofluorescence hematoxylin. were with Pax7 mice counterstained and 2-month-old and myonuclei from antibody Both muscle anti-Pax7 with quadriceps stained of sections Paraffin number cell Satellite eMHC of number the counting by assessed was Regeneration index Regeneration (CSAs, areas section cross and areas Damaged ilsa ecie rvosy(u ta. 2007b). selected al., randomly ten et to (Sun five previously in described nuclei as of fields number total the to normalized nue ucet h oa rao h nue ucesection. muscle injured the the of of area or area total necrotic with the the pathology of least to ratio of at muscle a extent as injured and determined The analyzed was measured. were treatment decorin genotype were without each muscle quadriceps of per CSA, old) myofibers the months 500 measuring (3 For mice four NIH). from 1.36b, (version software ImageJ 6 ..T nc onSap1 21 el eetasetdwith transfected were cells C2C12 Sharp-1, down knock To s.d. + uliwr one rmsvrlrandom several from counted were nuclei , 0 ofune el eetransfected were Cells confluence. 30% m /ldxccie(O)fr2 and 24 for (DOX) doxycycline g/ml m m 2 eemaue using measured were ) + fibers ncdw el eetasetdwt h eotradplasmid and reporter For above. the siRNA described as transfection, RNAiMAX with TGF- measured experiments, was all after transfected activity Lipofectamine luciferase hours and were 24 using constructs cells NY). siRNA or Island, knockdown CA), Valencia, Grand scrambled (Qiagen, (Invitrogen, Sharp-1 control mouse for with specific siRNA nM 100 osdrdsgiiat(* significant considered G) idn ecin otie 000cmpoe 1 probe, cpm 40,000 contained reactions Binding GGA). Poea icbr,WI). Fitchburg, (Promega, S-hr- rGTi idn ufr ape eerno D esand gels SDS on run were Samples buffer. binding in GST or GST-Sharp-1 eetdb uoaigah SnadTnj,2000). Taneja, and (Sun autoradiography by detected eeinmtnsSap1bL,Sharp-1 bHLH, and Myc–Sharp-1 for Sharp-1 full-length mutants with probed between transfected deletion domains were interaction and cells the Smad3, and map (Sigma) Sharp-1 To Smad3. beads and pSmad2/3 Myc–agarose endogenous ng/ml using 5 with hour immunoprecipitated 1 for with treated were transfected cells TGF- were later, cells hours assays, 24 Co-IP Then, described for Myc–Sharp-1. as Briefly, 2000). done Taneja, were and assays (Sun down GST-pull and assays Co-immunoprecipitation down GST-pull and Co-immunoprecipitation Student’s analysis Statistical 2011). al., et Science, COL3 following Applied the was using (Roche TGGACCGGCGATTTCAGAG-3 amplified Q-PCR LC480 5 was Forward in and Sharp-1 primers: green Mouse CA) Sybr Germany). Hercules, Mannheim, reverse Roche was (Bio-Rad, RNA using Briefly, iSCRIPT performed 2012). al., using et (Ling transcribed described as done was Q-PCR PCR real-time Quantitative TGF- ng/ml 5 of presence medium or absence differentiation the in in cultured days were 2 confluency for 80% at cells C2C12 assay immunoprecipitation Chromatin with incubated were samples TGF- transfected with from 32 stimulated prepared and were extracts Nuclear transfected cells later, were hours Myc–Sharp-1 48 and FLAG–Smad4 (EMSA) FLAG–Smad3, assay shift mobility Electrophoretic o uesit nioiswr de n nuae o 0minutes 10 for incubated and probe. added radiolabeled with were gels. incubation polyacrylamide following antibodies 5% on supershift, fractionated and For minutes 20 for temperature 1.5 (+ extract nuclear C,1 MHPS H79 0m C,01m DA mM 5 EDTA, mM 0.1 KCl, mM 80 7.9, pH HEPES, MgCl mM 10 dC), ih[ (1:1000, translated antibody was with Smad3 anti-Myc assays, using down GST-pull blotting For western mM Sigma). by 1 beads analyzed FLAG-agarose NaCl, and with (Roche were incubated (Sigma) mM were protein inhibitors Lysates 50 total blotting. protease of western 8.0, for amounts and pH loaded Equal PMSF Germany). Tris-HCl, mM Mannheim, 0.5 Science, mM Applied 50 X-100, twice washed Triton in were 0.1% lysed cells EDTA, PBS, later, hours cold 48 with FLAG-Smad3. with together 2012) ih1 omleyeadCI saswr efre sn Millipore using 2 performed Massachusetts). were (Billerica, assays kit ChIP ChIP and formaldehyde 1% with Ebr ta. 2008). al., et (Elberg 5 are: PAI-1 TGTGC-3 mouse for sequences CACAAAGAGCGAGCCCTCAG-3 Primer primers regions. with ( SMAD-responsive promoter Germany) quantitative PAI-1 Mannheim, mouse by to amplified Science, specific was Applied DNA (Roche used. RT-PCR were Cruz) (Santa antibodies -aee B rb (5 probe SBE P-labeled b 2 a 35 . Mdtitrio,1%gyeo n nraigaonsof amounts increasing and glycerol 10% dithiothreitol, mM 0.5 , eoelsn.Cnrlclswr o rae ihTGF- with treated not were cells Control lysing. before aebe ecie rvosy(ege l,21;adUezumi and 2010; al., et (Meng previously described been have 1 ]ehoieuigteTTculdrtclct yaesystem lysate reticulocyte TNT-coupled the using S]methionine t 9 ora fCl cec 21)17 9–0 doi:10.1242/jcs.136648 599–608 127, (2014) Science Cell of Journal ts a sdt efr ttsia nlssand analysis statistical perform to used was -test rmr o M rmtrhv endsrbdpreviously described been have promoter SMA for Primers . 9 -AACACTGGGGCATTTGGAGA-3 b m ;+ 3 ++ g; a sda ng/ml. 5 at used was P , 35 9 -CTCTATCAATTGGTCTAGACTTAACC- -aee md a nuae ihpurified with incubated was Smad3 S-labeled .5 ** 0.05; m 9 ) h ecinwsicbtdi room in incubated was reaction The g). rmr o IP1 COL1 TIMP-1, for Primers . 2 9 P 0 to 600 m , n 5 and fat-hr- n anti-Smad3 and anti-Sharp-1 of g .1 *** 0.01; D OorSharp-1 9 2 -CCAGAGGGCATGAAA- 0 p otiigTGF containing bp) 800 b P , 5n/l o hour. 1 for ng/ml) (5 0.001). b nvitro in 9 el eefixed were Cells . D n ees 5 Reverse and m Ln tal., et (Ling C b fpoly(dI- of g hr- was Sharp-1 . P , n labeled and .5was 0.05 a 1and 607 b 9 - 9 /

Journal of Cell Science sk,Y,Bes .K,Ieaa . aea . mi . ol,N .and A. N. Se Noble, and E., P. Imai, J. Y., Lefaucheur, Kaneda, K., Ikegaya, K., D. Brees, Y., Isaka, H. F. Fu, and Y. Li, J., Huard, umn,I,Sue,L,Gurn J. Gautron, L., Soulet, I., Husmann, P. Dijke, ten and K. Miyazono, H., J. C. D. Heldin, Garry, and J. T. Hawke, odr .A n ol,N A. N. Noble, and A. W. Confalonieri, Border, S., Galbiati, L., Morandi, M., Mora, C., Blasi, Di P., Bernasconi, L. Mora, R., H. Barresi, R., Moses, Brugnoni, P., and Confalonieri, E., Torchiana, M. P., Bernasconi, R. Lyons, A., J. Barnard, R. Taneja, and A. Ozog, S., Azmi, R. Taneja, and A. Ozog, H., Sun, S., Azmi, ubgi .T,L,L,Ln,B,Gpnda,S,Wlh . ose,M,Nave, M., Rossner, M., Walsh, S., Gopinadhan, B., Ling, L., Li, T., N. Gulbagci, F. Grinnell, J. N. Buckley, and A. Roopra, M., Garriga-Canut, h manuscript. the ...... adNTG efre experiments; performed N.T.G. and Sharp-1 C.V. provided L.L., M.R. Y.W., S.R.S., S.G., T.K.C., S.A., contributions Author interests. financial competing no declare authors The interests Competing vectors. expression Massague J. thank We Acknowledgements ARTICLE RESEARCH 608 Lisi, C., E. Desmoulie Klein, A., A. Soleimani, P., J. Habashi, C., Erp, van D., R. Cohn, Charge E. Ruoslahti, and A. W. Border, K. L. Boxhorn, and E. R. Allen, References [grant Fund Research Academic MOE the R.T.]. by to part T13-0802-P28 in number supported was work This Funding lnes .C. K. Flanders, A. M. Turman, and J. C. Logan, D., M. Chan, D., Elberg, L., Chen, G., Elberg, uioo . aauh,H,Hsia . aaua . aaoo . Sato, T., Kawamoto, T., Nakamura, T., Hashiba, H., Hamaguchi, K., Fujimoto, a emdfe nvv yimn etaiaino ai irbatgrowth fibroblast basic of neutralization immune by vivo in modified be can kidney. rat in disease fibrotic prevents A. W. Border, atr nseea uceregeneration. muscle skeletal in factors research. proteins. SMAD through nucleus to membrane biology. molecular fibrosis. dystrophies. muscular congenital in R. fibrosis and Mantegazza, and F. Cornelio, P., with correlates R. cytokine. muscles fibrogenic Mantegazza, patient a of dystrophic and role in Pathogenetic fibrosis. L. 1 factor-beta Morandi, growth F., transforming Cornelio, M., beta. factor growth factors. transforming transcription myogenic of repression Chem. through differentiation activity box E of repressor expression. transcriptional Stra13 a and as functions protein helix-loop-helix yrgltn h ciiyo C/EBP. of activity the regulating R. by Taneja, and A. K. deacetylase- histone a by mechanism. transcription deacetylase-independent 276 represses histone and sharp-1, dependent protein, helix .T,Gmat . pRy,C . om .M,Les .L tal. et L. B. of states. Loeys, failure myopathic M., TGF-beta-induced multiple T. attenuates in Holm, blockade regeneration M., receptor muscle 1 C. type Rhys, II ap Angiotensin M., Gamradt, T., M. regeneration. muscle repair. tissue of side dark the factor-beta. growth transforming by differentiation Biol. ua ea pteilcells. alpha- epithelial renal TGF-beta1-induced human mediates MKL1 fibroblasts. cultured growing and quiescent Biol. in and granulation myofibroblasts in expression tissue actin muscle alpha-smooth induces 1 factor-beta growth rncitoa ersinb h ai ei-ophlxpoenDc:multiple Dec2: protein elements. helix-loop-helix E-box basic through the mechanisms by repression Transcriptional F.,Noshiro,M.,Bhawal,U.K.,Suardita,K.andKato,Y.Pathol. 14821-14828. , ,S .adRdik,M A. M. Rudnicki, and B. S. ´, 122 124 279 85 r,A,Gio,A,Gbin,F n abai G. Gabbiani, and F. Gabbiani, A., Geinoz, A., `re, .Eg.J Med. J. Engl. N. 103-111. , 401-404. , .Bn on ug Am. Surg. Joint Bone J. 47-64. , 19) irbat,moirbat,adwudcontraction. wound and myofibroblasts, Fibroblasts, (1994). 52643-52652. , 20) md samdao ftefboi response. fibrotic the of mediator a as Smad3 (2004). 19) eeteayb kltlmsl xrsino decorin of expression muscle skeletal by therapy Gene (1996). .Ap.Physiol. Appl. J. n .Drnkfrrpre osrcsadFLAG–Smad and constructs reporter for Derynck R. and ´ 2 hso.Rev. Physiol. 20) HR1DC niisaioei differentiation adipogenic inhibits SHARP1/DEC2 (2009). / 2 ble A. ´bille, .Bo.Chem. Biol. J. ie ..adRT eindeprmnsadwrote and experiments designed R.T. and S.A. mice; 331 m .Pyil ea Physiol. Renal Physiol. J. Am. 20) uceijre n ear urn rnsin trends current repair: and injuries Muscle (2002). 1286-1292. , .Ci.Invest. Clin. J. 18) niiino kltlmsl aelt cell satellite muscle skeletal of Inhibition (1987). 19) rnfriggot factor- growth Transforming (1992). 20) ygncstliecls hsooyto physiology cells: satellite Myogenic (2001). 19) rnfriggot factor growth Transforming (1994). ici.Bohs Acta Biophys. Biochim. 20) hr-/E2ihbt kltlmuscle skeletal inhibits Sharp-1/DEC2 (2004). atly .adBrial,D. Barritault, and I. Martelly, , 19) ucergnrto olwn injury following regeneration Muscle (1995). 20) ellradmlclrrglto of regulation molecular and Cellular (2004). 84-A MORep. EMBO 91 84 yoieGot atrRev. Factor Growth Cytokine 19) rnfriggot factor-beta1 growth Transforming (1999). 534-551. , 209-238. , a.Med. Nat. n.J o.Med. Mol. J. Int. 278 822-832. , mohmsl ci xrsinin expression actin muscle smooth 20098-20109. , 19) TGF- (1997). 20) Sap1DC,abasic a mSharp-1/DEC2, (2003). 90 Nature ermsu.Disord. Neuromuscul. 1-7. , 10 .Cl.Physiol. Cell. J. 2 .Ci.Invest. Clin. J. 20) h ai helix-loop- basic The (2001). a.Med. Nat. 418-423. , 19) h elbooyof biology cell The (1990). 79-86. , 390 19 19) xrsinof Expression (1995). 294 1032 b 19) Transforming (1993). 925-932. , 465-471. , F1116-F1128. , inligfo cell from signalling 13 79-87. , 204-210. , .Bo.Chem. Biol. J. 96 19) Growth (1996). 133 b 7 1137-1144. , 249-258. , n.J Exp. J. Int. ndisease: in b 567-572. , 9 ntissue in 28-33. , .Biol. J. (2007). (2007). (2008). .Cell J. .Cell J. Massague i,J . hn,T . i .adTnj,R. Taneja, and J. Li, K., T. R. Chung, J., Derynck, J. Liu, and L. B. Black, D., Liu, eii .adGbin,G. Gabbiani, and G. Serini, T., Kawamoto, K., U. Bhawal, D., Jin, H., Sato, Y., Wu, H., Kawamura, F., Sato, and Reinecke, C., M. C. Wehr, L., Baker, Reinecke, P., K., S. Wichert, Koishi, H., Oster, J., I., M. Rossner, Nonaka, S., I. McLennan, N., A. Murakami, J. Pietenpol, and Y. E. Yang, L., H. Moses, ig .M,Gpnda,S,Kk .K,Sakr .R,Gpl . Bharathy, P., Gopal, R., S. Shankar, and K., X. W. Xiao, Kok, W., S., Foster, Gopinadhan, Y., M., Tang, B. M., Ling, Branca, B., Sun, J., Zhu, J., Li, Y., Li, and J. Cummins, Y., Tang, V., Prisk, Y., Chan, M., B. Deasy, W., Foster, Y., Li, ooet,R,Mrbla . luiz,C,Bocln,A,D nei,L., Angelis, De A., Broccolini, C., Gliubizzi, M., Mirabella, R., Morosetti, Rampazzo, A., Parenti, F., Zanconato, M., Forcato, E., Enzo, M., Montagner, u,H n aea R. Taneja, and H. Sun, L. Zardi, L., Borsi, A., Geinoz, P., Ropraz, L., M. Bochaton-Piallat, G., Serini, eg .M,Hag .R,Cug .C,Qn . ho . grsi . u W., Ju, P., Igarashi, X., Shao, W., Qin, C., A. Chung, R., X. Huang, M., X. Meng, A. Hata, and J. Massaous, u,H,Gafr,S n aea R. Taneja, and S. Ghaffari, H., Sun, h,J,L,Y,Se,W,Qa,C,Aboi,F,Lvsn,M,Nzk,M., Nozaki, M., Lavasani, F., Ambrosio, C., Qiao, W., Shen, Y., Li, J., Zhu, K. Miyamoto, and K. Yamada, u,H,L,L,Vrhrt . ubgi .T,Ahre,S,L,J,Cug .K., T. Chung, J., Li, S., Acharjee, T., N. Gulbagci, C., Vercherat, L., Li, H., Sun, ag . hna,S . hr . ig .M n aea R. Ca Taneja, L., and Attisano, L., M. J. B. Wrana, Ling, D., Kher, R., M., S. Segawa, Shankar, T., Y., Yoneda, Wang, N., Shimizu, D., Morikawa, T., Ito, A., Uezumi, ellrrsos oDAdamage. DNA to response cellular factors transcription myogenic of Smad3. repression by functional through differentiation acetccne xC3cells. BxPC-3 TGF- cancer al. inhibits pancreatic et DEC2 H. factor Seino, transcription M., Noshiro, K., Fujimoto, A. K. mice. mutant Nave, double and and single Sharp-2 e2762. R. and Sharp-1 Taneja, in G., resetting Eichele, J., disorders. muscle skeletal G. Hammond-Tooke, insights. mechanistic new proliferation: cell of inhibition . ag .adTnj,R. Taneja, differentiation. muscle and skeletal of Y. inhibition Wang, N., regeneration. muscle J. Huard, muscle in event I. key a muscle: factor skeletal injured in growth fibrogenesis. cells fibrotic into insulin-like cells myogenic or J. 1 Huard, beta factor Neuroimmunol. growth J. transforming factor, eonibat rmicuinbd ystsmuscle. human al. myositis of USA et differentiation inclusion-body E. myogenic from Roncaglia, defective M., mesoangioblasts restores Papacci, expression T., MyoD Gidaro, (2006). al. M., Sampaolesi, hypoxia- et E., of Tagliafico, S. degradation Bicciato, promoting A., by Rosato, metastasis factors. inducible G., cancer Leo, breast suppresses G., Basso, E., yfbolsi hntp nuto ytasomn rwhfactor-beta1. growth transforming by Biol. Cell induction J. phenotype G. myofibroblastic Gabbiani, and factor- study. growth vitro transforming in by and fibroblasts in expression otne,E .adLn .Y. H. fibrosis. Lan, renal and Smad3-mediated P. E. Bottinger, Biol. Cell Trends n erse rncito hog itn ectls (HDAC)-dependent deacetylase 4063. mechanisms. histone HDAC-independent through and transcription represses and rwhfco-ea,mottn n eoi:ipiain o kltlmuscle skeletal for implications decorin: J. and multiple fibrosis. Huard, myostatin, by and factor-beta1, F. growth regulated M. are Branca, expressions gene their stimuli. BHLHB3; extracellular and BHLHB2 complex. receptor kinase protein rnfriggot atri nihbtro ygncdifferentiation. myogenic of USA inhibitor Sci. an Acad. is factor growth transforming hn .H n aea R. Taneja, and H. T. Thin, cancer. and development 120. in factors ag .F n Massague and F. X. Wang, myogenesis. in function and regulates G9a Chem. sharp-1 Biol. methyltransferase J. factor histone the transcription of helix-loop-helix recruitment basic the of Sumoylation in al. progenitor et mesenchymal Y. common muscle. Miyagoe-Suzuki, a skeletal from M., originate M. adipogenesis Matev, and Fibrosis R., Ogawa, M., Yamaguchi, signaling. Notch antagonizing 103 ,J,Ceft,S,Ed,T n aa-iad B. Nadal-Ginard, and T. Endo, S., Cheifetz, J., ´, .Bo.Chem. Biol. J. 16995-17000. , ora fCl cec 21)17 9–0 doi:10.1242/jcs.136648 599–608 127, (2014) Science Cell of Journal 20) rnfriggot atrbt1idcstedfeetainof differentiation the induces factor-beta1 growth Transforming (2004). 20) eoi eetase rmtsmsl eldfeetainand differentiation cell muscle promotes transfer gene Decorin (2007). ee Dev. Genes 142 m .Pathol. J. Am. 288 873-881. , 83 .Cl Sci. Cell J. 7 Nature 57 on earRegen. Repair Wound 187-192. , 17654-17662. , 8206-8210. , rn.Biosci. Front. 85-91. , 20) ta3epeso sascae ihgot arrest growth with associated is expression Stra13 (2000). 19) h irnci oanE- scuilfor crucial is ED-A domain fibronectin The (1998). o.Ther. Mol. 19) rnfriggot atrbt2i lvtdin elevated is factor-beta2 growth Transforming (1999). 282 15 487 19) TGF- (1997). ,J. ´, uceNerve Muscle 2950-2966. , 25852-25863. , 20b.Sr1 euae aelt elatvto by activation cell satellite regulates Stra13 (2007b). 124 164 380-384. , rao . etla . od,J,Lio M., Laiho, J., Doody, A., Zentella, J., ´rcamo, 20) ai ei-ophlxtasrpinfactors, transcription helix-loop-helix Basic (2005). .Cl Biol. Cell J. 19) ouainof Modulation (1996). 19) G easgastruhaheteromeric a through signals beta TGF (1992). .A.Sc Nephrol. Soc. Am. J. 3654-3664. , 1007-1019. , 15 n.J o.Med. Mol. J. Int. 20) eainhp ewe transforming between Relationships (2007). Cell 21) 9 eitsSharp-1-dependent mediates G9a (2012). 10 21) md rtcsaantTGF-beta/ against protects Smad2 (2010). 1616-1622. , ESLett. FEBS 3151-3171. , 71 b b rc al cd c.USA Sci. Acad. Natl. Proc. rnltoa Oncogenomics Translational 1003-1014. , idcdtmrporsini human in progression tumor -induced 20a.bL-rnetranscription bHLH-Orange (2007a). inligtruhteSa pathway. Smad the through signalling 22 4 177 278-287. , o.Bo.Cell Biol. Mol. 20) G-eaihbt muscle inhibits TGF-beta (2001). 889-898. , 21) h ai helix-loop-helix basic The (2012). 19) G-easiuainand stimulation TGF-beta (1990). 647-657. , 584 21) hr- ouae the modulates Sharp-1 (2010). 20) itre clockwork Disturbed (2008). 30 619-624. , 495-501. , 21 a Cell sot uceactin muscle -smooth 1477-1487. , rc al cd Sci. Acad. Natl. Proc. b 23 sfrs ni vivo in an isoforms: 63 4778-4785. , 18) yebeta Type (1986). 21) SHARP1 (2012). 245-247. , LSONE PLoS rc Natl. Proc. 97 2 4058- , (2013). (2011). 107- , 3 ,

Journal of Cell Science