Journal of Cell Science ugse oefrteC3R–XC1ai na al tg fosteoblas of stage early an in axis CX3CR1–CX3CL1 the Cultured for role a calci suggested reduced and expression the (TRAP5B in 5b decreased phosphatase acid tartrate-resistant (RANKL)/TNFSF11, o:10.1242/jcs.113910 doi: 1032–1045 126, Science Cell of Journal 2012 November 24 Accepted oy eia n etlUiest,Tko1381,Japan 113-8519, Tokyo University, Dental and Medical Tokyo yohrC hmkns ru fpoen unrelated proteins of group A chemokines. CC possessed and cysteines other CCL17 C-terminus of ligand that additional to functional by the identical a is lacks is that but motif and CC CCL22, is residues, a CKLF1 99 has 2006). It of al., CCR4. total et for Wang a 2003; with al., cytokine et a Han characterized 2001; been al., these has et to (CKLF) (Han related factors addition another chemokine-like In of families, CX3C. family chemokine and their categorized C in CC, systematically located CXC, residues to ends: cysteine according N-terminal conserved group subfamilies of G four a arrangement into the comprise transmembrane categorized that are These molecules Chemokines 2006). seven surface Ransohoff, cell and of to (Charo bind inflammation ligands leukocytes of circulating of sites homing to the structurally- direct of that group cytokines a related as identified originally were Chemokines Introduction Osteopenia Osteolysis, fusion, Cell Multinucleation, words: Key NF- of activator receptor as such markers, osteoclastic various for transcripts of number the reduced differences, bone marginal thickness, in showed chemokines cortical parameters of and morphometric roles trabecular physiological the in However, increases destruction. significant bone pathological in involved be metabolism to reported been recently have Chemokines Summary Japan 466-8501, Nagoya Medicine, of School Graduate University Nagoya ` Pathology, Tumor of § Department address: *Present 6 5 4 3 2 1 Hoshino Akiyoshi osteoclasts of and regulation osteoblasts the both through maintaining homeostasis in bone CX3CR1 murine receptor chemokine of Roles 1032 ouaino yli-ieg rcros h ramn fbone-marrow- of treatment The precursors. myeloid-lineage of population nesniltasrpinlfco o sebat,weestegn Osteocalcin gene the whereas osteoblasts, for factor transcriptional essential an bevtosucvrdnvlrlso h XC1C3L xsi axis CX3CR1–CX3CL1 the of fa roles axis novel CX3CR1–CX3CL1 uncovered observations the that suggested points oj Matsushima Kouji uhr o orsodne( correspondence for Authors eateto i-mgn,PoesineCne,EieUiest,To 9-25 Japan 791-0295, Toon University, Ehime Center, Proteoscience Bio-Imaging, of Department lblCne fEclec GO)Porm nentoa eerhCne o oeua cec nTohadBn Diseases, Bone and Tooth in Science Molecular for Center Research Japan International 852-8501, Program, Japan Nagasaki (GCOE) 113-0033, Medicine, Excellence Tokyo of of Tokyo, School Center Japan of University Japan Global 113-8519, University Nagasaki 162-8655, Japan Tokyo The Radiology, Tokyo 650-0047, University, Medicine, of Medicine, Kobe Dental of Department and Inc., and School Health Institute, Medical Graduate Global Research Tokyo Medicine, for Kan Sciences, Preventive Center Dental Molecular National and of Institute, Medical Department Research of Lab, School Director’s Graduate Vice Pathology, Oral of Section 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. 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Results cell bone as of well differentiation as maintaining functional osteoclasts, in and the axis osteoblasts regulating bone CX3CR1–CX3CL1 by the in homeostasis of bone receptors roles chemokine critical the bone the of present roles in Both the the metabolism. in assess that participate further Therefore, of features to suggesting 2010). bone the chemokines investigated al., thus we study, et of ligands, (Hoshino metabolism chemokine networks several hierarchal of levels C1rltdceoielgnsa hrpui agt.Bone targets. using of from therapeutic possibility derived as the cells discuss ligands osteoblasts to of chemokine us function CCR1-related led impaired which the osteoclasts, to and due osteopenia develop XL6(uwgadMnli,20;Sekn n Sirsjo and called Sheikine 2008; (also bone Mentlein, like pathological and CX3CL1 chemokine of in (Ludwig form involved CXCL16 chemokine, membrane-bound unique chemokine a crucial fractalkine), Another al., indicating a et thus 2006), be destruction. 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(BV/TV), 1A). cortical volume (Fig. tissue (Tb.Th.), mice the per of wild-type volume parameters in elevated bone slightly observed showed analyses that Quantitative to the of compared in presence trabeculae mice the cancellous indicated increased images of slightly microCT structure The bone microCT. we using metabolism, the bone investigated in CX3CR1 first of functions the understand bone To as well as formation, resorption bone in roles possible in phenotypes bone The the understand better to chemokine order chemokines. in of in phenotypes roles physiological bone mice the of investigate receptor-deficient it in to understood. Therefore, roles resorption. important fully bone involved is the pathogenic not targeting prevent treatments still the to be the osteoclasts, optimizing chemokines are for crucial regulates is homeostasis of to knowledge Such CX3CR1 bone function in reported that 2007). CX3CR1 al., and suggest also et results Saitoh differentiation was 2009; these al., Although et 1997), (Koizumi exerts osteoclastogenesis (Imai al., that molecule adhesion 2007) and al., et chemoattractant et a Shimaoka as functions 2004; dual al., et Shimaoka 2008; ob ossetwt rvosrpr nwihanti-CX3CL1 which in report previous a appears with which consistent 1D), (Fig. be (N.Oc./B.Pm) (Oc.S/BS) to numbers areas osteoclast surface as associated osteoclast and such parameters resorption, in histomorphometric bone decreases bone with significant the showed importantly, the analysis More between 1C). differences mineralized (Fig. significant the mice no and showed (MAR) (MS/BS) rate surface surface apposition bone per mineral surface (Ob.S/BS), osteoblast (O.Th.), thickness osteoid the ercnl eosrtdthat demonstrated recently We Cx3cr1 dfcetmc oprdt idtp ie although mice, wild-type to compared mice -deficient nvivo in Ccr1 2 and / 2 Cx3cr1 ieso iiihdexpression diminished show mice nvitro in Ccr1 dfcetmc suggest mice -deficient bevtosdemonstrated observations dfcet( -deficient Cx3cr1 Cx3cr1 Cx3cr1 dfcetmice -deficient dfcetmice -deficient Ccr1 2 -deficient / 2 mice ) ¨, XC1C3L xsi oemtbls 1033 metabolism bone in axis CX3CR1–CX3CL1 01 n oemti rtis[olgna (Col1a1), [Collagen1a1 proteins (BGLAP)]. matrix al., Osteocalcin et and and Liu bone (SPARC) 1997; (Runx2 al., Osteonectin and et osteoblast- factors Komori of 1997; 2001) al., transcription transcripts et of (Ducy including Osterix/SP7) levels markers expression related the in changes femurs the the and in in markers wild-type of osteoclast-related osteoclasts and osteoblast- and of osteoblasts levels of of status bones the investigate To osteoclastic in and markers osteoblastic of expression Impaired ta. 09.Atog hs aaeeso QTadfo the differences and (Koizumi from marginal and wild-type mice showed pQCT between of analyses IgG-injected parameters histomorphometric these control bone Although the 2009). al., to resorption et bone compared the for scores index lower exhibited mice Abs-injected mn h ee noigbn arxproteins, matrix bone encoding genes wild-type the the of Among femurs the the whereas in observed mice, that to compared femurs (by upregulated significantly ugs that suggest the between differences significant Cx3cr1 any show not did Collagen1a1 in downregulated, significantly the was osteoblasts, mature of marker and upregulated, significantly was resorption. for roles possible XC1 pcfcaduiu iado XC1 CCR1, CX3CR1, of of bones CCL5 the ligand ligands, in bone-specific CCL9, and unique its and CX3CR1 and receptor, chemokine of another specific lack a CX3CL1, a axis. that RANK–RANKL implying the through 2D), osteoclastogenesis (Fig. impairs mice deficient Interestingly, function. and that both differentiation interactions osteoblast–osteoclast osteoclast essential the an regulates in axis, RANK–RANKL pathway the signaling of the activation the in assessed resorption bone osteoclastic 2C). 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Fig. Cx3cr1 dfcetmc (magnification mice -deficient n 5 Cx3cr1 ;rgtgah.( graph). right 4; dfcetmc.(etpnl)Mliula secat eevsaie ihTA hooei ti notolsi el eie rmwild-type from derived cells osteoblastic on stain chromogenic TRAP with visualized were osteoclasts Multinuclear panels) (Left mice. -deficient n P 5 , dfcetclswssaitclysgiiat[oneuain( [downregulation significant statistically was cells -deficient ) secatcluewt -S n AK ihu sebat eecridota oiiecontrols. positive as out carried were osteoblasts without RANKL and M-CSF with culture Osteoclast 3). .5.Teewr infcn ifrne ewe idtp and wild-type between differences significant were There 0.05). w Rankl epciey( respectively , n sepoeei yteotolsi el eie rmwl-ye(pncrls and circles) (open wild-type from derived cells osteoblastic the by Osteoprotegerin and B h eaieepeso eeso rncit fotolsi rncito atr ( factors transcription osteoblastic of transcripts of levels expression relative The ) Cx3cr1 6 6 Cx3cr1 s.e.m., s.e.m., P dfcetotolsi el eemaue sn LSs(means ELISAs using measured were cells osteoblastic -deficient 6 , Cx3cr1 0) Rgtpnl h ubro TRAP of number The panel) (Right 100). .5.( 0.05). n dfcetmc fle ice eemaue sn TPR(means RT-PCR using measured were circle) (filled mice -deficient 5 n ) n h acu otn ntendlswsmaue n omlzdt h N otn ntelsts(means lysates the in content DNA the to normalized and measured was nodules the in content calcium the and 4), 5 ) D secat eeidcdfo idtp secatpeusr yc-utr ihotolsso wild-type of osteoblasts with co-culture by precursors osteoclast wild-type from induced were Osteoclasts (D) 8). dfcetmc fle ice)a esrdb TPR(means RT-PCR by measured as circles) (filled mice -deficient C P , D , oclueo sebat n secat rmwl-yeand wild-type from osteoclasts and osteoblasts of Co-culture ) .5 ( 0.05. CX3CR1 E h eaieepeso eesof levels expression relative The ) dfcetcells. -deficient w ( ,ND o eetd.( detected]. not N.D. ), A + sebatcclsioae rmwl-ye(pnbr)and bars) (open wild-type from isolated cells Osteoblastic ) XC1C3L xsi oemtbls 1037 metabolism bone in axis CX3CR1–CX3CL1 Cx3cr1 utncerotolsswscutd(means counted was osteoclasts multinuclear dfcetcls ihsgiiaturglto eoe by denoted upregulation significant with cells, -deficient Cx3cr1 , Cx3cl1 F 6 hmkns(C5adCL)i h culture the in CCL9) and (CCL5 Chemokines ) ... duplicated, s.e.m., and 6 6 Runx2 s.e.m., s.e.m. Ccr1 Cx3cr1 Cx3cr1 and n yteimtr n aueosteoblastic mature and immature the by n 5 5 dfcetmc.()Terelative The (C) mice. -deficient P ) h ttsia infcnewas significance statistical The 8). # dfcetmc fle ice)were circles) (filled mice -deficient ) h ifrnebtentewild- the between difference The 6). 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(Fig. downregulated that and RT-PCR osteoclast by points, of markers noticed time levels lineage distinct myeloid expression and two time-dependent at the and analyzed rmCX3CL1 M-CFS with by stimulated RANKL cultures osteoclast differentiation marrow-derived axis CX3CR1–CX3CL1 osteoclast the of in roles temporal the dissociate expression To marker osteoclastic after, delays not but stimulation to, RANKL prior CX3CL1 recombinant with Treatment oto el,adwsdlydi h rae el.The cells. treated the in time- delayed a was Consistently, of and expression 8A). (Fig. cells, the control control of the downregulation in dependent those than atrso hs akr n hmkn eetr Fg 8B). (Fig. receptors expression chemokine temporal and their markers of these the terms of RANKL in the However, patterns cells between after control differences control. 4 obvious and any treated day to in lead on not upregulated did initiated stimulation rmCX3CL1 was with it treatment while level, Ccr1 S1P Rank 1 ntetetdclswsssanda low a at sustained was cells treated the in , XC1poen nosteoblasts and in CX3CR1 proteins of CX3CL1 expression The 4. Fig. rmr rbcle(B.Tearw indicate arrows The (TB). and trabeculae cartilage primary (GP) plate growth the between border 20 bars: observation. Scale morphological for section obtained each were of images Differential (DIC) blue). contrast (in interference nuclei with detect counterstained to were DAPI Sections red). (in Fluor 568 Alexa with conjugated by detected antibodies were secondary CX3CL1 and 4-week- CX3CR1 from mice. tibiae old of sections on out CX3CL1 carried were and CX3CR1 against antibodies specific vitro hw ntergtpnl.Saebr:50 bars: Scale panels. right the are images in Merged shown blue). (in nuclei detect to green), with DAPI (in counterstained 488 were and Sections red) respectively. (in 568 with Fluor conjugated Alexa antibodies secondary were by CX3CL1 detected and on CX3CR1 out cells. carried MC3T3-E1 was CX3CL1 and CX3CR1 CB, ( marrow; bone. bone cortical BM, cells. lining endosteum and Nfat-c1 . Irf8 ( A muoloecnesuisusing studies Immunofluorescence ) a utie thge levels higher at sustained was , nvitro in , Trap m B .Tedte ie eact the demarcate lines dotted The m. obeimnsann for immunostaining Double ) Cx3cr1 etetdtebone- the treated we , n ahpi was K Cathepsin and Cx3cr1 ee bevdin observed level a readily was nvivo in m and m. in Journal of Cell Science S et n niaeasgiiaturglto n oneuain epciey( respectively downregulation, and upregulation significant a indicate # and (means * vehicle control test. with HSD stimulated osteoblasts wild-type the with compared fotolsi akr and markers osteoblastic of al tg fotols ifrnito.Frhroe the Furthermore, an in differentiation. role osteoblast a of plays axis stage CX3CR1–CX3CL1 3E), early (Fig. the differentiation that together osteoblast suggest findings, sub- of cultured These patterns their during stages. expression observed in temporal later downregulated the in with be and state; to state pre-confluent confluent appeared osteoblastic their they in cultured processes however, the In cellular in and detectable osteoblast 2012). were cytoplasm of CX3CL1 and al., stage CX3CR1 beta-catenin cells, et early MC3T3-E1 an (Watanabe active cells and function formation cuboidal exhibited osteoblast the active bone that and signaling demonstrated trabecular diaphysis. the flat work of covering the bone cortical previous in the of detected Our surface were the covering on signals cells cells faint lining cuboidal while proteins the bone, two in these trabecular predominantly that the expressed revealed osteoblast were specimens investigated bone in and on further axis CX3CR1 CX3CL1 for CX3CR1–CX3CL1 we staining the Immunofluorescence have differentiation. documented, of differentiation role fully functional osteoblast been in not axis indicated CX3CR1–CX3CL1 function study and differentiation histomorphometric osteoblast in bone impaired was our there that of results The osteoblast in axis CX3CR1–CX3CL1 differentiation the of roles The Discussion of Enhancement 5. Fig. Cx3cr1 dfcetmc Fg C.Sneterlso the of roles the Since 1C). (Fig. mice -deficient Osterix Runx2 Cx3cl1 xrsin hc r soitdwith associated are which expression, and nwl-yeotolsstetdwt mXC1(0 0ad10n/l eedtrie yR-C.Tedt were data The RT-PCR. by determined were ng/ml) 100 and 50 (10, rmCX3CL1 with treated osteoblasts wild-type in Osterix xrsinb ramn ihrcmiatC3L nads-eedn manner. dose-dependent a in CX3CL1 recombinant with treatment by expression Cx3cr1 and Cx3cl1 6 s.e.m., XC1C3L xsi oemtbls 1039 metabolism bone in axis CX3CR1–CX3CL1 motn oe aoiga al tg fosteoblast its of of stage regulation the early to contribute maturation. an functional thus plays proper favoring together, axis and Taken CX3CR1–CX3CL1 differentiation, roles the 5). (Fig. that important proteins suggest matrix findings these bone of expression expression the stimulated mouse-derived of rmCX3CL1 wild-type with of that cultures Treatment osteoblastic may 1C). of (Fig. which deposition bones mice the deposition, in deficient volume osteoid mineral matrix increased the impaired to contribute temporal in bone results inverse eventually disordered the that spatiotemporally as such possible factors transcription Runx2 is of al., upregulation et and it (Yoshida downregulation manner Therefore, independent reciprocally 2012). a and in stage of Recently, late expression 2002). commitment sustained al., et that Komori Runx2 Nakashima reported 2006; 2001; al. osteoblast al., al., et et et Yoshida Liu calcium (Kanatani 1997; stage for al., early et decreased an at factors and differentiation markers, transcription 3A,B). osteoblastic (Fig. deposition of expression temporal the patterns and in changes suggests complicated CX3CL1 showed also mice proteins between interactions two trans osteoblasts. these in and CX3CR1 of cis possible localization subcellular h utrdotolsi el sltdfrom isolated cells osteoblastic cultured The Runx2 n P 5 , and niie sebatdfeetainfo rceigt a to proceeding from differentiation osteoblast inhibited ) h ttsia infcnewsdtrie yteTukey–Kramer the by determined was significance statistical The 3). .5 ewe oto n mXC1stimulation. rmCX3CL1 and control between 0.05) Osterix, and Osterix swsobserved was as u a niioyefcso the on effects inhibitory had but , Runx2 and nvitro in Osterix h eaieepeso levels expression relative The Fg B,mycause may 3B), (Fig. noeessential encode Cx3cr1 Osterix -deficient Cx3cr1 and - Journal of Cell Science sn olgnbsdzmgah.BM ellstsfo oemro-eie arpae (10 macrophages bone-marrow-derived from lysates cell BMM, zymography. collagen-based using uprpnl)o 7dy lwrpnl)adclue eesandwt nat-ahpiKplcoa nioy(e) n o -ci gen n nuc and myeloi (green) and F-actin beta3; for and alphaV and integrins (red), osteoclast-specific antibody protease; CathepsinK polyclonal S1P and anti-CathepsinK markers TRAP5B an enzymes, with bone-specific NFATC1; stained factor were transcription cultures and 50 panels) bar: (lower Scale days 17 or panels) (upper secatlsts( n 10 and (1 lysates osteoclast and wild-type of eiin ieuigMCFadRNLtreatment. RANKL and M-CSF using mice deficient hogottecluepro Fg E.A epreviously we As 3E). (Fig. from period in culture isolated observed the cells throughout osteoblastic Sgiiatyupregulated, *Significantly i.6 mareto secatfntosb XC1deficiency. CX3CR1 by functions osteoclast of Impairment 6. Fig. 1040 ta. 00.Ide,w bevddfeecsi h temporal the in in differences observed of involved we patterns (Hoshino Indeed, or networks expression discussed 2010). previously al., sustains a signaling we et in as chemokine that differentiation, CCR1 osteoblast of the loop upstream amplifies acts amplification CX3CR1 chemokine-dependent that significantly were suggest CCL9, findings and CCL5 in as reduced such CCR1, for ligands el,wihmyla otetmoal iodrdepeso of expression in disordered temporally changes the to chemokine cause osteoblastic lead to CX3CR1 may the signaling which of chemokine cells, that of loss inputs complex dysfunction is the spatiotemporal the the osteoblastic by deficiency impaired and for CX3CR1 networks formation reason with bone possible associated of one cultures cell the phenotypes Therefore, osteoblastic wild-type and in 3E). stage stage (Fig. later a early in an augmented at upregulated eas bevdasgiiatsprsinof suppression significant a observed also We ora fCl cec 2 (4) 126 Science Cell of Journal 1 m Cx3cr1 n R-]i idtp oe as and bars) (open wild-type in IRF-8] and Ccr1 .( m. Cx3cr1 B 2 h eaieepeso eeso rncit fotolsi ifrnito akr ndy4atrclueRN n t downstream its and culture[RANK after 4 day on markers differentiation osteoclastic of transcripts of levels expression relative The ) / dfcetotolsi el Fg F.These 3F). (Fig. cells osteoblastic -deficient 2 dfcetotolss cl a:2 bar: Scale osteoclasts. -deficient sebatccls h eeso chemokine of levels the cells, osteoblastic # Cx3cr1 infcnl oneuae oprdwt idtp controls, wild-type with compared downregulated significantly m rti/ae,respectively. protein/lane), g and Ccr1 Cx3cr1 wherein , Ccr1 nvitro In Cx3cr1 dfcetmice -deficient xrsinwas expression m .( m. secatclue rmwl-yeand wild-type from cultures osteoclast Cx3cr1 dfcet(ildbr)pesecat eemaue yR-C (means RT-PCR by measured were preosteoclasts bars) (filled -deficient D h olgndgsinatvt fwl-yeand wild-type of activity digestion collagen The ) Ccr1 ( A was ,Otolsi el eeidcdfo h oemro fwl-yeand wild-type of marrow bone the from induced were cells Osteoclastic ), in h oetsu of tissue bone both the of the downregulation along Rankl The signaling diminished axis. deficiency with RANK–RANKL CX3CR1 associated largely from be resulting could dysfunction osteoclast The maintenance the precursors in osteoclast axis of CX3CR1–CX3CL1 the of roles The segnctasrpinfcosadbn arxpoen,thus proteins, deposition. matrix mineral impaired bone in and resulting factors transcription osteogenic xiie eakberdcin nthe in reductions remarkable exhibited secat,telvl of levels the in osteoclasts, activity of osteoclast-supporting downregulation the with in Cx3cr1 of reduction pre-osteoclasts presence functional the confirming a wild-type 3C), (Fig. osteoclasts of TRAP-positive from cells Co-cultures osteoblastic 3C). (Fig. eedcesd hl h eeso yli-ieg markers myeloid-lineage of levels the while decreased, were utrdotolsi el sltdfrom isolated cells osteoblastic Cultured n h peuaino sepoeei,wr bevdin observed were Osteoprotegerin, of upregulation the and , P dfcetotolsi el,ms ieydet the to due likely most cells, osteoblastic -deficient , .5 ( 0.05. m rti/ae;W n O idtp and wild-type KO, and WT protein/lane); g C Cx3cr1 cnigeeto irgah fteptfrainassay formation pit the of micrographs electron Scanning ) Cx3cr1 Rankl dfcetpeusr eecridotfr1 days 10 for out carried were precursors -deficient Cx3cr1 dfcetmc Fg 2D). (Fig. mice -deficient Rank utemr,in Furthermore, . Cx3cr1 dfcetmc aldt induce to failed mice -deficient n te secatcmarkers osteoclastic other and dfcetotolsswsmeasured was osteoclasts -deficient Rankl Cx3cr1 6 s.e.m., Cx3cr1 xrsinlevel expression dfcetmice -deficient Cx3cr1 -ieg cell d-lineage n Rank 5 -deficient Cx3cr1 e (blue). lei 5). -deficient and - Journal of Cell Science ouainaayi fRN nCD45 in RANK of analysis population oe as and bars) (open upplto fCD11b of subpopulation hmkn eetrCR n oerltdceoie CL n C5 xrce rmotolsi el fwl-ye(pnbr)and bars) (open wild-type of cells (means osteoclastic RT-PCR from a extracted using CCL5) measured and were (CCL9 bars) chemokines (filled bone-related and and wild-type CCR1 of receptor marrow chemokine bone the from induced of receptors chemokine of oto g sae itga) lt n itgasaesona h ersnaiedt ftreidpneteprmns n h ouainrati population the and experiments, independent three of data representative the as shown percentage are mean histograms and the Plots histogram). (shaded IgG control asi utr a nlzdb lwctmty itgaswr ae nteCD11b the on gated were Histograms cytometry. flow by analyzed was culture in days lgtices ntenme fmeodpeusr,bta but cells) precursors, gated myeloid in (R2 expression of precursors RANK lower osteoclastic number with in a the decrease was in significant there 2009). after that increase al., cells demonstrated marrow slight et stimulation bone RANKL Koizumi of and analysis 1997; M-CSF cytometric al., flow our et and/or Indeed, cells, (Bazan osteoblastic and CX3CL1 cells to, endothelial express as such to prior cells, where of interaction environment type marrow other occurs bone RANK–RANKL the in initial between osteoclastogenesis possibly induce the interaction CX3CR1 with, physiological concomitantly and the RANKL after CX3CL1 level 5A), basal (Fig. the to stimulation downregulated the that readily and was 2009) cells al., et of (Koizumi cells expression lineage of the osteoclast level to the detectable prior in functional no cells to these that due of Given be potential culture. differentiation could the cells in wild-type changes in in those of to functions expression compared the and in markers differences These various 6B). (Fig. increased were deficiency. CX3CR1 by caused population precursor osteoclasic the and axis CCR1-mediated the in Changes 7. Fig. Gapdh n niaesgiiaturglto n oneuain epciey oprdwt h eeso a ( 0 day on levels the with compared respectively, downregulation, and upregulation significant indicate # and * . Cx3cr1 Cx3cr1 6 dfcetotolsi fle as el eemaue sn LSs(means ELISAs using measured were cells bars) (filled osteoclastic -deficient Ccr1 ...( s.e.m. + CD115 nwl-yemuedrvdosteoclastic mouse-derived wild-type in and n + 5 Cx3cr1 secatcpeusr.Tesraeepeso eeso AK(odln)aeoeli nclssandwt subclass-matched with stained cells on overlaid are line) (bold RANK of levels expression surface The precursors. osteoclastic 3). Cx3cr1 + CD11b ywl-yepeusr eemaue sn TPR(means RT-PCR using measured were precursors wild-type by Cx3cr1 + dfcetcls(i.7D; (Fig. cells -deficient CD115 Cx3cr1 Cx3cl1 dfcetosteoclasts -deficient 6 s.e.m., + dfcetmc ytetetwt -S n AK.( RANKL. and M-CSF with treatment by mice -deficient secatcpeusr sltdfo h oemro fwl-yeand wild-type of marrow bone the from isolated precursors osteoclastic a expressed was n 5 ) .. o eetd ( detected. not n.d., 8). XC1C3L xsi oemtbls 1041 metabolism bone in axis CX3CR1–CX3CL1 deino secatpeusr yotolssi h bone subsequent the the in have and osteoblasts the by attraction may in precursors initial which role osteoclast the of relevant precursors, in adhesion a roles osteoclastic favorable plays of axis osteoclastic maintenance of CX3CR1–CX3CL1 that sustaining differentiation suggest findings the the current in to our Collectively, contribution osteoclasts. axis smaller mature a findings CX3CR1–CX3CL1 with These role precursors, a 8B). performed the suggest (Fig. treatment was CX3CL1 myeloid-lineage 4 for of treatment timing day distinct or on the this regarding stimulation osteoclastic when RANKL observed either after 8A (Fig. were in myeloid- markers upregulated the changes were while obvious and markers, markers significant osteoclastic to lineage precursor cells led of stimulation osteoclastic RANKL osteoclast the downregulation to of prior the rmCX3CL1 treatment with stages, in osteoclast immature axis CX3CR1–CX3CL1 for role populations. critical precursor osteoclast a of suggesting regulation strongly 1), Table ossetwt hs da eadn h oe fthe of roles the regarding ideas these with Consistent hi C RANK noeosCL n C9i h utr uentn fwild-type of supernatant culture the in CCL9 and CCL5 Endogenous ) + R-ae)sbouainadCD11b and subpopulation (R1-gated) 6 6 ... xeiet efre nduplicate, in performed experiments s.e.m., s.d., n 5 ) h eesaenraie oteexpression the to normalized are levels The 5). B h xrsinlvl ftasrpso the of transcripts of levels expression The ) ( P A , h eprlepeso profiles expression temporal The ) .5.Otolsi el were cells Osteoclastic 0.05). Cx3cr1 lo RANK dfcetmc fe 4 after mice -deficient Cx3cr1 ) oee,no However, . Cx3cl1 dull dfcetmice -deficient ssonas shown is o n (R2-gated) 5 ) ( 3). nthe in D )A Journal of Cell Science XC1O91.93 CX3CR1KO ( C1O91.75 87.73 CCR1KO Wild-type enpercentage mean controls, utrdwt -S n AK o 0dy,siuae ihrC3L 5 gm)frdy –0(rage) n o as41 fle ice) hnwere then circles), (filled 4–10 days for and (triangles), 1–10 days of for and expression ng/ml) mRNA, the (50 upregulated to rmCX3CL1 *Significantly normalized with circles). were stimulated (open levels days, osteoclasts The 10 unstimulated analysis. stimulation. for with RT-PCR RANKL RANKL compared a to and prior using CX3CL1 M-CSF measured recombinant with were with cultured markers treatment differentiation after osteoclastic expression marker of osteoclastic transcripts in Delay 8. Fig. CD45 in RANK of analysis Population 1. Table 1042 q ouainrtoo CD11b of ratio Population P , .5 n erae ( decreased and 0.05) P , ora fCl cec 2 (4) 126 Science Cell of Journal .5(means 0.05 6 ...( s.e.m. 6 n w hi s.e.m., 5 P R-ae)sbouainadCD11b and subpopulation (R1-gated) ) Saitclysgiiat( significant *Statistically 3). , .5,respectively. 0.05), CD45 n 5 ) h ifrne ewe ntmltdotolssadrC3L-rae secat eesgiiatyincreased significantly were osteoclasts rmCX3CL1-treated and osteoclasts unstimulated between differences The 3). 6 6 6 + .314.35 1.53 .39.67 13.40 2.33 4.28 % CD11b (%) + P CD11b , .5,bsdon based 0.05), lo + R-ae)sbouaino CD11b of subpopulation (R2-gated) CD115 eiin mice deficient + CD115 6 6 6 + .3 8.26 0.43* post-hoc .013.00 1.90 .510.99 0.95 secatcpeusr fwild-type, of precursors osteoclastic + % CD11b (%) eto n-atrfcoilANOVA. factorial one-factor of test # infcnl oneuae RAcmae otedy1 day the to compared mRNA downregulated significantly + CD115 + 6 secatcpeusr i i.5 ssonas shown is 5) Fig. (in precursors osteoclastic 6 6 .0 0.63 0.30* hi .81.01 1.98 .41.89 0.94 % CD11b (%) Ccr1 h eaieepeso eesof levels expression relative The dfcetand -deficient Gapdh 6 6 6 0.02* 0.09* lo 0.55 el were Cells . (%) Cx3cr1 - Journal of Cell Science oe n a infcnl eue in reduced significantly was the fact, In and we 2010). al., which bones et deficiency, (Hoshino study CCR1 Ccr1 previous with a associated in reported those to similar al. et Koizumi 2009). by al., reported et previously (Koizumi as environment, marrow aoaoisadbccosdfregtt e eeain nteC57BL/6 the on generations ten to eight for backcrossed Japan. and CLEA from laboratories obtained were male) Cx3cr1 (6–9-week-old, mice C57BL/6 Standard Mice Methods and Materials of upregulation Ccl9 significant while 2010), al., et (Hoshino suppressed that ligands chemokine major Ccr1 a are they to G that 2010), transduce al., likely formation et is (Hoshino osteoclast treatment it PTX therefore, inhibited by that CCL9 to level comparable and antibodies neutralizing CCL5 with blockade against the Simultaneous in 2010). 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Kobe, CX3CL1 Institute, and were anti-CX3CL1 Research CX3CR1 IgG (Kan MA). Hamster murine hamster (Cambridge, Imai Systems. control for CX3CL1/fractalkine Toshio USA), and antibodies Dr mouse R&D NJ, neuAb) by Hill, of from (anti-CX3CL1 provided (Rocky form antibodies purchased Inc. neutralizing soluble was PeproTech Systems recombinant and (rmCX3CL1) R&D The from USA) purchased of MN, respectively. were (Minneapolis, Institute RANKL and Inc. Research M-CSF murine the Recombinant Tokyo, University. Dental Reagents of and Medical Tokyo University and Japan the local of the Center of approval of Medical the the International with for and committees Research Guidelines in Institutional Animals ethics the Laboratory of to Use according and Care performed were experiments experiments All and care animal Laboratory tiigwssqetal odce sn nioisaantC3R and CX3CR1 against antibodies using fixed then conducted and CA) sequentially immunofluorescence were Jose, Double Cells culture. San was after 2012). Falcon, days and 4 al., (BD staining and slides et 2 paraformaldehyde culture (Watanabe NY), 4% with 2-well described on previously Island, seeded as were cultured Grand and maintained Japan). Technologies, Kumamoto, Laboratories, (DOJINDO Life antibodies DAPI 568 with Fluor counterstained Alexa Probes, Paraffin- by 2012). followed MA), al., (Molecular Cambridge, et (Abcam, (Watanabe CX3CL1 and described 5 C57BL/6 previously of 4-week-old sections as embedded from conducted dissected was tibiae mice the of staining Immunohistochemical staining Immunohistochemical in culture 200 rmCX3CL1 by days. of induced absence three or every was presence replaced the were with The in media stimulated 10) the culture were days. day The indicates cells to ng/ml). marrow 1 RT-PCR gene 4 (50 bone (day Osteoprotegerin RANKL rmCX3CL1, an for and of differentiation the M-CSF the presence by For of RANKL the 2010). al., in level monitored et ng/ml analysis (Hoshino expression 20 cells was stromal/osteoblastic low of and presence cells a G-10 as M-CSF stromal/osteoblastic then Sephadex analysis, were ng/ml with of cells 10 filled the contamination and column pre-osteoclasts, with NJ), a generate Piscataway, cultured mesenchymal To through Biosciences, cells. bone passed (Amersham osteoblastic microspheres adherent were for cells and collected bone-marrow- non-adherent cells, for were collected osteoclastic cells were stromal and cells al., et non-adherent macrophages (Hoshino the described derived cultures, previously these as in In cultures 2010). cultured cell osteoblastic cells and osteoclastic cells marrow osteoblastic bone and osteoclasts Murine bone-marrow-derived of culture Cell those 1987). are Bone al., for study et Society present (Parfitt American the Research the in of Mineral Committee and used al., Nomenclature units the et and by (Hoshino recommended symbols described nomenclature, previously The as 2010). performerd was histomorphometry Bone histomorphometry Bone efre yiouaigbn-arwdrvdpeusr (1 precursors bone-marrow-derived inoculating by (Wako performed WAKO content DNA from the using E-test normalized lysates. Calcium were The the results the PA). of was the and using deposition Warrington, Japan), mineral Inc., Osaka, quantified Chemicals, The (Polysciences, was days. method content 3 Kossa’s calcium every von once using replaced evaluated was media culture The n iea eerhsadrs(idbade l,19) h microstructure The al., 1999). et (Ito al., previously described et Bone as 2005). for calculated (Hildebrand Society three-dimensionally American standards were the parameters to Research according defined Mineral (Hoshino were previously and scores The described 2010). as al., Germany), et Pforzheim, GmbH, Medizintechnik h oersrto ciiyo h ocluesuiswr locnutdusing 2010). conducted al., also et were 2010). (Hoshino studies al., previously et co-culture described the as (Hoshino slices of described osteoblast- bone activity previously with resorption as days 21 bone plates for The 24-well cultured been in had media that cells inducing osteoblastic of layer a onto h sebatccl ie CT-1(IE OC skb,Jpn,was Japan), Tsukuba, BOCC, (RIKEN MC3T3-E1 line, cell osteoblastic The cells stromal mesenchymal marrow bone adherent in differentiation Osteoblastic oclueeprmnswt secatpeusr n sebat were osteoblasts and precursors osteoclast with experiments Co-culture m sobcai,1 mM 10 acid, ascorbic M m eesandwt pcfcatbde gis CX3CR1 against antibodies specific with stained were m a MMcnann segncfcos(0 FBS, (10% factors osteogenic containing -MEM b gyeohsht n 0n dexamethasone). nM 10 and -glycerophosphate a MMwr sda h ore of sources the as used were -MEM + 6 ytm(Stratec system 10 5 cells/well) Journal of Cell Science niaeasgiiaturglto n oneuain epciey( respectively # and downregulation, and * and 3B–D significant. USA). Fig. not upregulation PA, NS: significant Reading, analysis, comparisons, Software, a (Synergy indicate (multiple (non-parametric Windows for Student’s test program Mann–Whitney software and HSD 8A,B) Wilcoxon Fig. Tukey–Kramer 1), 2C), Table Fig. 7A–C; Fig. a with determined idr .B,Neerie,B,Hfmn,O,Sag,R,Pp . tli,T M., T. Stulnig, T., Pap, R., Stange, O., Hoffmann, B., Niederreiter, B., N. Binder, aa,J . ao,K . admn . ag . o,K,Rsi . Greaves, D., Rossi, K., Soo, W., Wang, G., Hardiman, B., K. Bacon, T., F., J. Miyata, Bazan, K., Brasel, T., Sudo, T., Inada, O., Ohneda, T., Miyamoto, F., Arai, References H19- number [grant Iimura; number Welfare T. and K.Y.]. to Labor [grant to Health, Foundation nano-012 of Science Imaging’ Ministry Takeda the on Iimura]; Live and T. Research ‘Fluorescence to Scientific Sports, 22113002 Culture, for Areas Education, Grant-in-Aid of Innovative Ministry Technology the and and Iimura]; Diseases’ Science Science T. Bone and [grant and ‘International Tooth Sports, A.Y. in Program, Science to Science Culture, Excellence Molecular to for of of Education, Center Foundation Research Center Naito Promotion of the Global the Ministry Iimura]; Technology, T. the for and A.Y. A.H.; Society Research A.H., Scientific to Japan for KAKENHI their Grant-in-Aid the a by for from supported was Ltd work Satoshi This Instech., Tamai, Nikon Funding Mami at processing. to image Ohba and thanks microscopy Norio on expertise sincere and express Takiguchi authors The Acknowledgements means the as presented are data The analyses Anti-mouse CA). Statistical Diego, control CA). (San Pharmingen Diego, BD subclass-matched from (San purchased and were eBioscience (1D3) Fc from (R12-31) to purchased antibodies CD265/RANK were (104), CD45.2 antibodies to (AFS98), PerCP-Cy5.5-, antibodies APC-, anti-mouse previously PE-, CD115 biotin-conjugated FITC-, as or Blue-, performed Pacific APC-Cy7 were 2010). PE-Cy7-, al., precursors et osteoclast (Hoshino of described analysis Ltd, cytometric Co Flow Biotech respectively. cytometry were (Cusabio Japan), Flow system, Tokyo, kit supernatants Inc., (Immunodiagnostic (SRL, ELISA culture kit kit ELISA BALP and NTx assay murine the serum and EIA the DE) the Wilmington, TRAP AZ), in Hills, murine NTx Fountain the and using BALP measured phosphatase MAB463 acid (TRAP5b), CCL5, tartrate-resistant for Murine 5b ELISA systems). (R&D using antibodies determined BAF463 were and levels NTx chemokine and murine BALP The TRAP, 2010). chemokines, al., of et Measurement (Hoshino previously described as system CA) detector City, PCR sequence Taqman Foster 7700 quantitative Biosystems, ABI real-time the (Qiagen, the A using the CA). kit and performed using Carlsbad, was RNeasy cDNA the (Invitrogen, analysis into the at kit (RT-PCR) reverse-transcribed marrow using then RT was bone III isolated RNA of Superscript total was removal and The the flushing) CA). cells after Valencia, osteoblastic by tibia cells, regions proximal osteoclastic (the metaphysial of sample cultures tissue the bone from as RNA cellular out analysis Total (RT-PCR) PCR carried quantitative Real-time were osteoclasts 2010). al., Japan). cultured et Tokyo, (Hoshino (NIKON, was previously of program described processing software staining Image Elements Japan). NISE Immunohistochemical ECLIPSE Tokyo, the an and using (NIKON, using images conducted captured microscope Fluorescence were fluorescence DAPI. images Ni-E (DIC) with contrast counterstained interference were differential slides and CX3CL1, 1044 ak . re,R . mln .S n elc,K. Redlich, and S. J. Smolen, G., R. Erben, M., Mack, .R,Zonk .adShl,T J. T. Schall, motif. CX3C and a A. with chemokine Zlotnik, R., D. nuclear of activator receptor receptors. and (RANK) c-Fms kappaB of expression factor sequential the by cells T. precursor Suda, and M. D. Anderson, ora fCl cec 2 (4) 126 Science Cell of Journal c 242,L6/G(B-C) D1 M/0 n CD19 and (M1/70) CD11b (RB6-8C5), Ly6C/6G (2.4G2), R H post-hoc eeEpeso nlsssfwr rga (Applied program software Analysis Expression Gene t eto n-atrfcoilAOA(i.3;Fg 5; Fig. 3C; (Fig. ANOVA factorial one-factor or test ts ohrfgrs sn h KaleidaGraph the using figures) (other -test 19) omtetaddfeetaino osteoclast of differentiation and Commitment (1999). Nature .Ep Med. Exp. J. 385 19) e ls fmembrane-bound of class new A (1997). 6 640-644. , ...Saitclsgiiac was significance Statistical s.e.m. 190 1741-1754. , 20) Estrogen-dependent (2009). P , H 0.05). 4.0 si,M,Ee,J . luce,F,MirShlesem . ak,Y,Vce,J., Vacher, Y., Saeki, M., Meier-Schellersheim, F., Klauschen, G., J. Egen, M., Ishii, mi . isia . akl,C,Bb,M,Ngr,M,Nsiua . Kakizaki, M., Nishimura, M., Nagira, M., Baba, C., Haskell, K., Hieshima, T., Imai, ohn,A,Imr,T,Uh,S,Hnd,S,Mroa . aaaa . Suzuki, M., Mayahara, Y., Maruoka, S., Hanada, S., Ueha, T., Iimura, A., Hoshino, P. P. Tak, and P. Reinders-Blankert, J., T. Smeets, J., J. Haringman, ol,H,Brhgn .adWbr C. Weber, and Vyas, J. A., Bernhagen, B. McCormick, H., S., Noels, Jung, L., Landsman, X., Gu, S., Brand, H., J. Niess, idbad . ab . Mu A., Laib, T., Hildebrand, Y., Chen, Y., Zheng, Y., Liu, S., Shi, M., Rui, L., Wang, M., Xu, P., Ding, W., Han, b,Y,Le .W,Erih .A,Cug .Y,Jlnk .F,Cladr .S., N. Callander, F., D. Jelinek, Y., H. Chung, A., L. 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