Osteoblasts secrete Cxcl9 to regulate angiogenesis in bone

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1 Reeived 2 De 215 Aepted 9 Nov 216 Pulished 14 De 216 DOI: 1.138/nomms13885 OPEN Osteolsts serete to regulte ngiogenesis in one Bin Hung 1,, Wenho Wng 1,, Qinghu Li 1,, Zhenyu Wng 1,BoYn 1, Zhongmin Zhng 1, Ling Wng 1, Minjun Hung 1, Chunhong Ji 2, Jinsen Lu 1, Sihi Liu 2, Hongdong Chen 2, Mngmng Li 2, Dozhng Ci 1, Yu Jing 3, Ddi Jin 1 & Xiohun Bi 1,2 Communition etween osteolsts nd endothelil ells (ECs) is essentil for one turnover, ut the moleulr mehnisms of suh ommunition re not well defined. Here we identify s n ngiostti ftor sereted y osteolsts in the one mrrow miroenvironment. We show tht produed y osteolsts interts with vsulr endothelil growth ftor nd prevents its inding to ECs nd osteolsts, thus rogting ngiogenesis nd osteogenesis oth in mouse one nd in vitro. The mehnisti trget of rpmyin omplex 1 tivtes expression y trnsriptionl upregultion of STAT1 nd inreses inding of STAT1 to the promoter in osteolsts. These findings revel the essentil role of osteolst-produed in ngiogenesis nd osteogenesis in one, nd n e trgeted to elevte one ngiogenesis nd prevent one loss-relted diseses. 1 Ademy of Orthopedis, Gungdong Provine, Deprtment of Orthopedis, The Third Affilited Hospitl, Southern Medil University, Gungzhou 5163, Chin. 2 Stte Key Lortory of Orgn Filure Reserh, Deprtment of Cell Biology, Shool of Bsi Medil Siene, Southern Medil University, Gungzhou 51515, Chin. 3 Deprtment of Phrmology nd Chemil Biology, University of Pittsurgh Shool of Mediine, Pittsurgh, Pennsylvni 15213, USA. These uthors ontriuted eqully to this work. Correspondene nd requests for mterils should e ddressed to X.B. (emil: ix15@smu.edu.n) or to D.J. (emil: nysyddijin@163.om). NATURE COMMUNICATIONS 7:13885 DOI: 1.138/nomms

2 NATURE COMMUNICATIONS DOI: 1.138/nomms13885 During development of the mmmlin skeleton, the formtion of endohondrl one oinides with pillry invsion 1,2, suggesting lose onnetion etween osteogenesis nd ngiogenesis. Blood vessels supply osteolst preursors either y their flow 3 or omponents in their wlls 4,5, nd guide the migrtion of osteolst preursors from the periosteum to the one mrrow 4. Impirment of ngiogenesis deresed treulr one formtion s well s expnsion of the hypertrophi zone into the growth plte 6. Postntl one remodelling lso depends on vsulr formtion. Oxygen, nutrients nd ornuopi of hormones nd growth ftors, whih re importnt for one nd one mrrow development nd homeostsis, re trnsported to one y lood vessels 7,8. Reent studies hve suggested diret role of deresed ngiogenesis in senile nd postmenopusl osteoporosis 9, highlighting the importne of the regultion of ngiogenesis in one. Bone mrrow is highly heterogeneous nd vsulrized tissue. The diverse ell types populting the one mrrow ommunite extensively with eh other, nd the ell-to-ell ross-tlk is vitl for orret one development nd homeostsis 1. The rosstlk etween one-forming osteolsts nd vessel-forming endothelil ells (ECs) is progressively gining strong support in the sientifi ommunity 11. In prtiulr, osteolsts serete ngiogeni ftors, suh s vsulr endothelil growth ftor () 12 nd erythropoietin 13, to medite the ross-tlk etween osteolsts nd ECs. However, moleules tht ouple osteolsts nd ECs to modulte one remodelling nd ngiogenesis hve not een fully defined, nd the signlling pthwys tht ontrol the prodution of these moleules in osteolsts re unler. The mehnisti trget of rpmyin omplex 1 (mtorc1) integrtes diverse intrellulr nd extrellulr signls 14 nd plys entrl role in the regultion of ell growth, prolifertion nd metolism 15. Ativtion of mtorc1 enhnes synthesis to promote ngiogenesis in tumours 16. Although reent studies hve defined mtorc1 signlling s ritil regultor of osteolstogenesis nd one formtion 17,18, the role of mtorc1 in one vessel formtion is unknown. In this study, we found tht mie with onstitutive mtorc1 tivtion in osteolsts demonstrted enhned seretion, ut unexpetedly deresed phosphoryltion of its reeptor (R2) nd downstrem signlling in ECs, nd mrkedly redued vsulture formtion in one. We further identified CXC-hemokine, hemokine (C-X-C motif) lignd 9 () s diret ounter-regultory moleule of signlling onstitutively produed y osteolsts to suppress ngiogenesis nd osteogenesis in one. Mehnistilly, the mtorc1 tivted expression y trnsriptionl upregultion of STAT1 nd inresed inding of STAT1 to the promoter in osteolsts. Thus, our study identified s n ngiostti ftor sereted y osteolsts, supporting s novel trget for stimulting ngiogenesis nd osteogenesis in one. Results Osteolsti mtorc1 regultes one ngiogenesis. Riddle et l. nd our group reported tht tivtion of mtorc1 in osteolst linege ells prevented osteolst mturtion nd one formtion 17,18. As osteogenesis nd ngiogenesis re tightly oupled in one, we determined whether one ngiogenesis ws ffeted in mie with onstitutive mtorc1 tivtion in osteolsts. Osx-re 19 hs previously een reported to trget other ell types esides osteolst linege ells 2. To hieve speifi tivtion of mtorc1 in osteolsts, we rossed floxed Ts1 (mtorc1 negtive regultor) mie 21 with mie expressing the Cre reominse d ps6 ps6/on CD31 + vessels density (mm 2 ) 2,5 2, 1,5 1, 5 2,5 2, 1,5 1, 5 CD31 + vessels density (mm 2 ) CD31 EMCN CD31/EMCN CD31 EMCN CD31/EMCN Type H vessels density (mm 2 ) Type H vessels density (mm 2 ) Figure 1 Altertion of mtorc1 tivity in osteolsts ffets ngiogenesis in mouse one. () Representtive imges of immunostining of ps6 (Ser235/236) nd osteolin (On) in 12-week-old mle mie one. Sle r, 5 mm. () Photogrph of hindlims of 6-week-old mle DTs1 (DT) nd DRptor (DR) mie nd their littermte ontrols (). Sle r, 1 m. () Representtive imges of CD31 þ EMCN þ mirovessels nd quntittive nlysis of type H mirovessel density in femur setions of 12-week-old mle mie. Sle r, 1 mm. n ¼ 9 per group. (d) Consistent numers of CD31 þ vessels in surrounding musle of mouse one. Sle r, 1 mm. Dt re shown s men±s.d. n ¼ 9 per group. Dt re shown s men±s.d. Po.5, Po.1 (Student s t-test). For ll pnels in this figure, dt re representtive for three independent experiments. 2 NATURE COMMUNICATIONS 7:13885 DOI: 1.138/nomms

3 NATURE COMMUNICATIONS DOI: 1.138/nomms13885 ARTICLE driven y n osteolin (OC) promoter(oc-cre) 22 to produe mie with Ts1 deletion in mture osteolsts (herefter referred to s DTs1 mie). Six-week-old DTs1 mie lerly showed enhned phosphoryltion of S6 (Ser235/236) in osteolsts (positively stined y osteolin) (Fig. 1), inditing tht mtorc1 ws tivted y this geneti mnipultion. Miro-omputed tomogrphy (miro-ct) nlysis reveled the sme high volume of immture woven one in DTs1 mie s reported previously 17, tht is, the high one mss in DTs1 mie ws the result of inresed res of hypominerliztion (Supplementry Fig. 1). At neropsy, we noted ple long ones in DTs1 mie (Fig. 1), inditing redued lood perfusion in the one of these mie. We oserved deresed numer of CD31 þ Endomuin þ vessels, whih hs een reported to ouple ngiogenesis nd osteogenesis in one, in tii setions in DTs1 mie when ompred with their littermte ontrols (Fig. 1). However, numer of vessels in surrounding musle ws not ffeted in DTs1 mie (Fig. 1d), suggesting tht osteolsts with hypertive mtorc1 speifilly suppressed vsulture formtion in one. To test these oservtions in vitro, we ultured immortlized humn umilil vein ECs (HUVECs) with onditionl medium (CM) olleted from primry lvril osteolsts. HUVECs ultured in CM from DTs1 osteolsts exhiited lower prolifertion nd migrtion rte thn those in ontrol CM (Fig. 2,). HUVECs seeded on Mtrigel in the presene of ontrol medium formed rnhing, nstomosing tues, resulting meshwork of pillry-like strutures (Fig. 2). In ontrst, HUVECs remined spheril nd isolted when mintined in CM from DTs1 osteolsts, with smll ellulr nests nd short tues rrely oserved (Fig. 2). These findings suggested tht osteolsts with hypertive mtorc1 inhiited ngiogenesis in mouse one nd in vitro. To definitively estlish the role of osteolsti mtorc1 in regulting ngiogenesis in one, we reted mie with mtorc1 intivtion in osteolsts y rossing the OC-Cre mouse with mouse rrying floxed Rptor (mtorc1-speifi omponent) llele 23 (ontrol). Immunohistohemil stining of S6 phosphoryltion (Ser235/236) onfirmed intivtion of mtorc1 in osteolsts of DRptor mie (Fig. 1). Miro-CT nlysis reveled the sme lower one volume in DRptor mie s tht previously reported 24 (Supplementry Fig. 2). In ontrst to DTs1 mie, the long ones from the Rptor mutnts were more rihly perfused with lood ompred with ontrol ones (Fig. 1). Immunohistohemil stining (Fig. 1) onfirmed inresed CD31 þ Endomuin þ vessel numers in the one of DRptor mie. However, vessel numer in surrounding musle remined unhnged (Fig. 1d). Consistent with the in vivo results, CM from primry DRptor osteolsts indued prolifertion (Fig. 2), migrtion (Fig. 2) nd network formtion (Fig. 2) of HUVECs in vitro. On the sis of these results, we onlude tht osteolsts with impired mtorc1 promote ngiogenesis in one nd in vitro. Osteolsts produe ngiostti ftors. The results desried ove suggested tht mtorc1 regulted the expression of ngiogeni nd (or) ngiostti ftors in osteolsts to modulte the formtion of vsulture in one. Consistent with this notion, enhned expression (Fig. 3) nd seretion (Supplementry Fig. 3) y osteolsts ws oserved in DTs1 mie, inditing tht mtorc1 stimulted expression in osteolsts. Nonetheless, the enhned expression in osteolsts, whih would e expeted to produe more vessels in one, ould not theoretilly e responsile for the impired ngiogenesis of one seen in DTs1 mie. R2 (Kinse insert domin reeptor (KDR)), mjor reeptor trnsduing BrdU-positive ells per totl numer (%) Brdu/DAPI h BrdU-positive ells per totl numer (%) signlling in ECs 25, exhiited onsistent expression, ut deresed phosphoryltion in ECs when exposed to the overll elevted seretion of y DTs1 osteolsts (Fig. 3). In ddition, primry DTs1 osteolsts exhiited elevted expression (Fig. 3) nd seretion (Supplementry Fig. 3) of in vitro, nd CM olleted from these ells repressed trnsdution of the signlling sde in HUVECs, s reveled y the deresed phosphoryltion of KDR nd its downstrem meditors PLCg1 nd ERK1/2 (Fig. 3d). The disjoint etween seretion y osteolsts nd signlling sde trnsdution in ECs ws opied in DRptor mie. Inonsistent with the overll derese in expression (Fig. 3) nd seretion (Supplementry Fig. 3) y DRptor osteolsts, phosphoryltion of KDR ws enhned in ECs in the one mrrow of DRptor mie (Fig. 3). Moreover, while DRptor osteolsts expressed (Fig. 3) nd sereted (Supplementry Fig. 3) redued in vitro, HUVECs showed enhned signl trnsdution when ultured in CM from DRptor osteolsts (Fig. 3d). Together, these dt suggested tht mtorc1 positively regultes expression Tue re (mm 2 ) 18 h Tue re (mm 2 ) Wound losure (%) Wound losure (%) Figure 2 Altertion of mtorc1 tivity in osteolsts ffets ngiogenesis in vitro. () Representtive onfol imges of immunostining of BrdU (green) in HUVECs nd quntittive nlysis of BrdU þ ells over totl ells. Sle r, 5 mm. n ¼ 9 per group. () Representtive photomirogrphs of wounds in HUVECs t h nd fter 18 h; dotted lines highlight the liner srth/wound for eh group of ells. The r grph shows the men perentge of wound losure. Sle r, 2 mm. n ¼ 9 per group. () Representtive photomirogrphs of tue formtion of HUVECs inuted with Mtrigel nd quntittive nlysis of tue re. Sle r, 2 mm. n ¼ 9 per group. Dt re shown s men±s.d. Po.5, Po.1 (Student s t-test). For ll pnels in this figure, dt re representtive for three independent experiments., ontrol. NATURE COMMUNICATIONS 7:13885 DOI: 1.138/nomms

4 NATURE COMMUNICATIONS DOI: 1.138/nomms13885 /On /On + OBs per totl OBs (%) ps6k(t389) S6K ps6(s235/236) KDR KDR/CD31 pkdr pkdr/cd31 + OBs per totl OBs (%) S6 A KDR + ECs per totl ECs (%) d pkdr(y1175) KDR pplcγ1(s1248) pkdr + ECs per totl ECs (%) PLCγ1 perk1/2 ERK1/ Figure 3 Regultion of in osteolsts y mtorc1 does not explin the vsulr phenotypes of mutnt mouse one. () Representtive imges of immunostining of nd On in 12-week-old mle mie one nd quntittive nlysis of þ osteolsts ompred with totl osteolsts. Sle r, 5 mm. n ¼ 9 per group. () Representtive imges of immunostining of CD31, KDR nd pkdr (Y1175) in 12-week-old mle mie one, nd quntittive nlysis of KDR þ nd pkdr þ ECs ompred with totl ECs in one mrrow. Sle r, 5 mm. () Western lot of in primry osteolsts. (d) Western lot of phosphoryltion of KDR, PLCg1 nd ERK1/2 in HUVECs treted with CM from primry osteolsts for 1 min. Dt re shown s men±s.d. Po.5, Po.1 (Student s t-test)., ontrol. nd seretion in osteolsts, whih did not ount for the orresponding ngiogenesis ltertions in mutnt mouse one nd in vitro, suggesting tht osteolsts my produe ngiostti ftor(s) to lok signlling in one. is onstitutively produed y osteolsts. To sreen for the ngiostti ftor(s), we developed glol mrna expression profile in DTs1 or ontrol lvril osteolsts using mirorry. Among the upregulted mrnas, hs een reported s diret ounter-regultory moleule of signlling within the liver 26. Importntly, ws found to e expressed onstitutively in osteolsts in ells residing in the one (Fig. 4), nd its reeptor, CXCR3, ws reveled to e expressed y ECs in one mrrow nd y ultured HUVECs (Fig. 4,). CXCR3 presented onsistent expression in the two trnsgeni mouse models nd their respetive ontrols (Fig. 4), ut ws expressed nd sereted into one mrrow nd serum t signifintly greter levels y DTs1 osteolsts (Fig. 4,d). Furthermore, mrna expression showed sevenfold inrese in primry DTs1 osteolsts versus ontrol ells (Fig. 4e). A similr inresed pttern ws further onfirmed t the protein expression (Fig. 4f) nd seretion level (Fig. 4g). In ontrst to DTs1 mie, DRptor osteolsts hd signifintly lower levels of expression (Fig. 4) nd seretion (Fig. 4d). mrna (Fig. 4e), protein expression (Fig. 4f) nd seretion (Fig. 4g) were ll deresed in DRptor lvril osteolsts ultured in vitro. On the sis of these oservtions, we onlude tht mtorc1 positively regultes expression nd seretion in osteolsts, inditing possile explntion for the ngiogenesis phenotypes of mutnt mie desried ove. inhiits ngiogenesis in one. We next sought to determine whether is responsile for the vsulture ltertions seen in the two mouse models. DTs1 mie were treted with nti- ntiody to neutrlize endogenous. Interestingly, ntiody ginst signifintly inresed the numer of one CD31 þ Endomuin þ vessels in DTs1 mie to more thn tht in ontrol mie (Fig. 5). As depited ove, HUVECs mintined in DTs1 CM exhiited impired prolifertion nd migrtion. When the ells were grown in the sme medium supplemented with nti-, prolifertion (Supplementry Fig. 4) nd migrtion rtes (Supplementry Fig. 4) of the ells were signifintly elevted to levels higher thn ells treted with the ontrol medium. Moreover, n endothelil network ws formed when HUVECs were grown in DTs1 CM with supplementry nti-, ompred with those ultured in CM from DTs1 osteolsts nd ontrol osteolsts (Fig. 5). Importntly, redued phosphoryltion of KDR nd its downstrem meditor in HUVECs were oth inresed to ove sl level (Fig. 5). In ddition, ws downregulted in DTs1 osteolsts y sirna, CM from whih lso promoted the prolifertion, migrtion nd tue formtion of HUVECs (Supplementry Fig. 5). These dt suggested tht elevted in osteolsts is responsile for the redued vsulture in one of DTs1 mie. As the ngiostti effet of ws eliminted, trnsdution of signlling reovered nd the undnt expressed in DTs1 osteolsts exerted their potentil effet on promoting ngiogenesis in one nd in vitro. To test whether redued in DRptor osteolsts ontriuted to inresed vsulture in one, we treted DRptor mie with. DRptor mie reeiving hd signifintly fewer vessels thn those treted with phosphte-uffered sline (PBS) nd ontrol mie (Fig. 5). lso signifintly reversed 4 NATURE COMMUNICATIONS 7:13885 DOI: 1.138/nomms

5 NATURE COMMUNICATIONS DOI: 1.138/nomms13885 ARTICLE /Runx2 -positive OBs per totl OBs (%) -positive OBs per totl OBs (%) CXCR3/CD31 CXCR3 CXCR3-positive ECs per totl ECs (%) CXCR3-positive ECs per totl ECs (%) CXCR3/DAPI HUVECs d (ng ml 1 ) BM Serum (ng ml 1 ) BM Serum e mrna level y qpcr (/GAPDH 1 4 ) f 14 g (pg ml 1 ) Figure 4 mtorc1 regultes in osteolsts. () Representtive imges of in situ hyridiztion of mrna in onjuntion with immunostining of Runx2 in femur setions of 12-week-old mle mie one. Boxed re is enlrged in the ottom right orner. þ osteolsts out of totl osteolsts were lso quntified. Sle r, 5 mm. n ¼ 9 per group. () Representtive photomirogrphs of immunostining of CXCR3 in CD31 þ ECs in one mrrow nd quntittive nlysis of CXCR3 þ ECs out of totl ECs in 12-week-old mle mie one. Sle r, 5 mm. n ¼ 9 per group. () Representtive photomirogrphs of immunostining of CXCR3 in ultured HUVECs. Sle r, 1 mm. (d) onentrtions ssessed y ELISA in one mrrow (BM) nd serum. n ¼ 5 per group. (e) Quntittive PCR nlysis of mrna in primry osteolsts. (f) Western lot of in primry osteolsts. (g) Conentrtions of in CM of primry osteolsts ssessed y ELISA. n ¼ 5 per group. Dt re shown s men±s.d. Po.5, Po.1 (Student s t-test)., ontrol. the promotion of prolifertion nd migrtion y DRptor CM in HUVECs nd further redued prolifertion (Supplementry Fig. 6) nd migrtion (Supplementry Fig. 6) to rtes lower thn those in ells mintined in ontrol medium. In the Mtrigel ssy, the network in HUVECs mintined in DRptor medium supplemented with ws less formed thn tht grown in ontrol medium (Fig. 5). Immunolotting nlysis of lystes of the ell sets reveled the underlying mehnism. As trnsdution NATURE COMMUNICATIONS 7:13885 DOI: 1.138/nomms

6 NATURE COMMUNICATIONS DOI: 1.138/nomms A CD31/EMCN + Type H vessel density (mm 2 ) Type H vessel density (mm 2 ) A + +A +A + + Tue re (mm 2 ) Tue re (mm 2 ) A + pkdr(y1175) 25 KDR 152 pplcγ1(s1248) 15 PLCγ1 15 perk1/2 ERK1/ Figure 5 is responsile for vsulr phenotypes in mutnt mouse one. () Representtive onfol imges of mirovessels immunostined y CD31 nd EMCN, nd quntittive nlysis of mirovessel density in tii setions of DTs1 mie dministered with ntiody (A) nd DRptor mie injeted with suutneously. Sle r, 1 mm. n ¼ 9 per group. () Representtive Mtrigel tue formtion ssy imges nd quntittive nlysis of tue re with ultures of HUVECs using CM with or without ddition of or -neutrlizing ntiody s indited. Sle r, 1 mm. n ¼ 9 per group. () Western lot of phosphoryltion of KDR, PLCg1 nd ERK1/2 in HUVECs treted with CM from primry osteolsts with or without ddition of or -neutrlizing ntiody s indited for 1 min. Dt re shown s men±s.d. Po.5, Po.1 (Student s t-test)., ontrol. of signlling ws improved in HUVECs mintined in DRptor CM, signl trnsdution in ells ws loked y supplementtion of in the sme medium (Fig. 5). These oservtions indited tht deresed expression in osteolsts ws responsile for the inresed vsulture in one of DRptor mie. As ws downregulted in DRptor osteolsts, loking signl trnsdution y ws llevited in ECs, nd relesed y DRptor osteolsts ws le to exert its pro-ngiogeni role more effetively nd led to the formtion of more vessels in the one. Together, these findings indite tht ntgonizes signlling to prevent ngiogenesis in one. We next determined 6 NATURE COMMUNICATIONS 7:13885 DOI: 1.138/nomms

7 NATURE COMMUNICATIONS DOI: 1.138/nomms13885 ARTICLE the mehnisms y whih ntgonizes signlling trnsdution in ECs. We first exmined whether CXCR3 ( reeptor) medited the ntingiogeni effets of in our model. As CXCR3 ws rogted y its ntgonist supplemented in DTs1 CM, HUVECs ultured in the CM retined low rte of prolifertion (Fig. 6) nd migrtion (Fig. 6), nd poor pity to form tue (Fig. 6). Furthermore, loked CXCR3 tivity ut filed to llevite the inhiition of signlling trnsdution in ECs y in DTs1 CM (Fig. 6d), inditing tht CXCR3 is not required for to suppress ngiogenesis in this model. On the other hnd, supplementtion with mrkedly reversed the inhiition of ngiogenesis y DTs1 CM (Fig. 6 d). In light of these oservtions, we hypothesized tht ould intert with nd ttenute its inding to ECs. We further mixed reominnt mouse nd 164 in vitro nd immunopreipitted using n nti- ntiody. As shown in Fig. 6e, the nti- ntiody suessfully preipitted 164 from the mixture of 164 nd, ut filed to preipitte 164 from the solution ontining 164 lone. These results suggest tht interts with 164 in vitro. To investigte the effet of on the inding of to h 18 h BrdU-positive ells per totl numer (%) Wound losure (%) d pakt(s473) psr(y416) 6 6 Tue re (mm 2 ) e + pkdr(y1175) KDR 152 pplcγ1(s1248) 15 f PLCγ1 perk1/2 ERK1/ Speifi 125 I 164 inding (.p.m. per well) 6, 4, 2, (μg ml 1 ) Figure 6 ntgonizes signlling trnsdution in ECs y interting with nd preventing its inding to ECs. () Representtive onfol imges of immunostining of BrdU (green) in HUVECs nd quntittive nlysis of BrdU þ ells over totl ells. Sle r, 1 mm. n ¼ 9 per group. () Representtive photomirogrphs of wounds in HUVECs t h nd fter 18 h; dotted lines highlight the liner srth/wound for eh group of ells. The r grph shows the men perentge of wound losure. Sle r, 2 mm. n ¼ 9 per group. () Representtive photomirogrphs of tue formtion of HUVECs inuted with Mtrigel nd quntittive nlysis of tue re. Sle r, 2 mm. n ¼ 9 per group. (d) Western lot of phosphoryltion of Akt (S473), Sr (Y416), KDR, PLCg1 nd ERK1/2 in HUVECs treted with DTs1 CM with or without ddition of (CXCR3 ntgonist) or s indited for 1 min. (e) Reominnt mouse nd 164 were mixed nd immunopreipitted with nti- ntiody nd exmined y immunolotting with n nti- ntiody. (f) Binding of 125 I 164 to ECs in the presene of inresing onentrtions of. Shown is the speifi inding, whih ws lulted y sutrting the nonspeifi inding from the totl inding. Dt re shown s men±s.d. Po.1 (Student s t-test). NATURE COMMUNICATIONS 7:13885 DOI: 1.138/nomms

8 NATURE COMMUNICATIONS DOI: 1.138/nomms13885 ECs, HUVECs were inuted with 1 ng ml I 164 nd inresing onentrtions of. As expeted, ws le to inhiit the speifi inding of 125 I 164 to ECs (Fig. 6f). Together, these findings reveled tht interts with nd prevents from inding to ECs, nd thus ntgonizes signl trnsdution in ECs. suppresses osteogenesis. Given tht ngiogenesis nd osteogenesis re oupled in one, we next investigted whether hs ny diret role in one formtion. After tretment with the ntiody depited ove, DTs1 mie exhiited prtil reovery of osteolsti differentition, minerliztion of one mtrix nd normliztion of one struture (Supplementry Fig. 1). In ontrst, DRptor mie reeiving showed impirment of osteolsti differentition nd more severe loss of one mss (Supplementry Fig. 2). As CXCR3 expression in osteolsts is onsistent etween the two knokout mie nd their ontrols (Supplementry Fig. 7), we suspeted tht my exert inhiitory effet on the funtion of osteolsts. To etter hrterize the role of in osteolsts, we dded reominnt to ultured MC3T3-E1 ells, hd CXCR3 expression (Supplementry Fig. 7). demonstrted mrked inhiitory effet on prolifertion (Fig. 7), differentition (Fig. 7) nd minerliztion of MC3T3-E1 ells (Fig. 7). Sine inhiition of CXCR3 y filed to reverse the rogtion of osteogenesis y (Fig. 7 ), we suspeted tht my inhiit osteogenesis through CXCR3-independent mehnism. Interestingly, supplementtion with signifintly reversed the inhiitory effet of on the osteogenesis of MC3T3-E1 ells (Fig. 7 ), inditing tht my prevent osteogenesis y interting with. Indeed, ws le to rogte the inding of to MC3T3-E1 ells in vitro (Fig. 7d). Furthermore, inhiited prolifertion, differentition nd minerliztion of rt one mrrow stem ells (BMSCs) nd prevented inding of to these ells s well (Supplementry Fig. 8). Tken together, these oservtions indite tht my suppress osteogenesis in one nd in vitro y interting with nd rogting inding of to osteolsts. mtorc1 regultes in osteolsts vi STAT1. We next investigted the mehnism y whih mtorc1 regultes in osteolsts. The trnsription ftor, signl trnsduer nd tivtor of trnsription 1 (STAT1), is known to e regulted y mtor 27,28 nd is involved in the regultion of gene trnsription 29 in mny ell types. We thus exmined the potentil regultion of STAT1 y mtorc1 in osteolsts nd found tht RNA trnsripts for STAT1 were upregulted in DTs1 lvril osteolsts nd were signifintly redued in DRptor ells (Fig. 8). Aordingly, STAT1 protein prodution nd phosphoryltion ws reveled to e inresed in DTs1 nd deresed in DRptor osteolsts (Fig. 8). The funtion of mny trnsription ftors is ssoited with hnges in their intrellulr loliztion etween the ytoplsm nd the nuleus. We then found tht STAT1 ws more onentrted in the nuleus of DTs1 osteolsts (Fig. 8). In ontrst, DRptor osteolsts showed STAT1 umultion in the ytoplsm (Fig. 8). These findings suggested tht mtorc1 promoted STAT1 expression nd trnslotion into the nuleus in osteolsts. We next investigted the mehnisms y whih mtorc1 drives STAT1 expression nd tivtion. To ssess the involvement of S6K1 in the regultion of STAT1 expression y mtorc1, we downregulted S6K1 in primry lvril osteolsts using sirna. S6K1 redution led to deresed STAT1 in ontrol osteolsts nd reversed the upregultion of STAT1 expression in DTs1 osteolsts (Fig. 8d), suggesting tht S6K1 medited the positive regultion of STAT1 expression y mtorc1 in osteolsts. STAT1 phosphorylted on serine 727 enhnes its trnsriptionl tivity 3. As shown in Fig. 8e, mtor immunopreipittes phosphorylte STAT1 t Ser727 in vitro nd onstitutive tivted mtorc1 from DTs1 lvril ells enhned phosphoryltion, suggesting tht mtor diretly phosphoryltes STAT1 t Ser727 nd promotes its trnsriptionl tivity. We lso performed eletrophoreti moility shift ssy (EMSA) to delinete STAT1 inding to the gene promoter. Nuler protein of osteolsts ound speifilly to n oligonuleotide proe ontining onsensus STAT1-speifi inding sequene + + BrdU-positive ells per totl numer (%) Runx2 On d Speifi 125 I 164 inding (.p.m. per well) + 5, 4, 3, 2, 1, CxCl9 (μg ml 1 ) 5 38 Figure 7 suppresses osteogenesis y interting with nd rogting inding of to osteolsts. () BrdU stining of MC3T3-E1 ells nd quntittive nlysis of BrdU þ ells out of totl ells. Sle r, 1 mm. () Western lot nlysis of osteolsti mrker On nd Runx2 expression in MC3T3-E1 ells on the seventh dy of osteogeni indution. () Alizrin red stining of differentited MC3T3-E1 ells on the fourteenth dy. Sle r, 1 m. (d) Binding of 125 I 164 to MC3T3-E1 ells in the presene of inresing onentrtions of. Shown is the speifi inding, whih ws lulted y sutrting the nonspeifi inding from the totl inding. Dt re shown s men±s.d. Po.5, Po.1 (Student s t-test). 8 NATURE COMMUNICATIONS 7:13885 DOI: 1.138/nomms

9 NATURE COMMUNICATIONS DOI: 1.138/nomms13885 ARTICLE mrna level y qpcr (STAT1/GAPDH 1 3 ) pstat1(y71) pstat1(s727) STAT S6K1 ps6k1 STAT1 NC si-s6k d e Input IgG kinse IP:mTOR kinse STAT1/DAPI STAT GST-pSTAT1(S727) pstat1(s727) GST-STAT1 STAT1 mtor f STAT1 supershift STAT1 omplex Free proe Nuler extrt Unlled proe Anti-STAT1 C T C T C T C R C R C R g STAT1 NC si-stat Figure 8 mtorc1 regultes CXCL9 in osteolsts vi STAT1. () Quntittive PCR (qpcr) nlysis of STAT1 mrna in primry osteolsts. n ¼ 3per group. Dt re shown s men±s.d. Po.5 (Student s t-test). () Western lot of totl STAT1 protein nd phosphoryltion (Y71 nd S727) of STAT1 in primry osteolsts. () Representtive onfol imges show suellulr lotion of STAT1 (red) in primry osteolsts. (d) Controlnd DTs1 (DT) primry osteolsts were treted with S6K1 sirna nd negtive ontrol (NC) for 48 h nd then immunolotting ws rried out to detet STAT1 expression. (e) Cultured primry lvril ells were immunopreipitted with nti-mtor ntiody nd the preipitted mtor ws ssyed for kinse tivity ginst reominnt glutthione S-trnsferse (GST)-tgged full-length STAT1. (f) Nuler extrts from primry DTs1 (T), DRptor (R) nd ontrol (C) osteolsts were nlysed for inding of STAT1 to promoter using EMSA. Binding of STAT1 to iotin-lelled DNA proes is shown s STAT1 omplex. To ompete with the inding, n unlelled STAT1-inding-site DNA proe ws dded to the retion in 2 times molr exess. Adding nti-stat1 ntiody to the retions used redution of STAT1-DNA inding nd nds of supershift. (g) ControlndDTs1 primry osteolsts were treted with STAT1 sirna nd NC for 48 h nd then immunolotting ws rried out to detet expression., ontrol. IFN-γ Bone formtion Bone regenertion Angiogenesis GAS STAT1 STAT1 P Osteolst P gene expression R R STAT1 mtorc1 R R Endothelil ell Figure 9 Model of sereted y osteolsts in regulting ngiogenesis nd osteogenesis in one. expression is positively regulted y mtorc1 nd downstrem STAT1 in osteolsts. inds with, prevents from inding to its reeptors, loks signlling trnsdution in ECs nd thus inhiits ngiogenesis in one. In ddition, suppresses osteogenesis y interting with nd rogting its inding to osteolsts. IFN-g, interferon-gmm. NATURE COMMUNICATIONS 7:13885 DOI: 1.138/nomms

10 NATURE COMMUNICATIONS DOI: 1.138/nomms13885 in the promoter region of (Fig. 8f). The ddition of ntiody to STAT1 in the nuler extrt displed the inding nd (Fig. 8f), inditing tht this inding omplex ontined STAT1. Importntly, nuler protein from DTs1 osteolsts showed elevted inding of STAT1 to the proe, while DRptor nuler protein showed deresed inding (Fig. 8f). These oservtions indited tht STAT1 ws ound to the gene promoter in osteolsts, nd mtorc1 promoted this inding. The role of STAT1 in mediting mtorc1-regulted expression of ws finlly determined y sirna knokdown of STAT1 mrna in osteolsts. In ontrol ells, the sl level of ws deresed signifintly y STAT1 sirna (Fig. 8g). Moreover, si-stat1 mrkedly reversed the upregultion of y mtorc1 tivtion in DTs1 ells (Fig. 8g). These results onfirmed tht STAT1 is involved in the regultion of gene trnsription y mtorc1 in osteolsts. Disussion Using mouse models with mtorc1 tivtion or inhiition in osteolsts, we identified s n essentil ftor sereted y osteolsts, whih modulte ngiogenesis nd osteogenesis in one. We found tht ws expressed onstituently in osteolsts in ells residing in the one, nd its expression ws positively regulted y mtorc1 upstrem of STAT1. inhiited lood vessel formtion y interting with nd preventing its inding to ECs, whih ws suffiient to reverse the ngiogeni effet of. suppressed osteogenesis in one nd in vitro y ntgonizing s well. Thus, is novel ngiostti ftor tht medites the ommunition etween osteolsts nd ECs during one regenertion (Fig. 9). Bone regenertion ours in lose sptil nd temporl ssoition with ngiogenesis, thus the rte of one formtion nd lood flow in the niml re usully oupled 31. However, our trnsgeni mouse models showed the ontrry. The DTs1 mie exhiited inresed one volume ut low vsulriztion, while the DRptor mie hd low one mss ut inresed vsulriztion. These inonsistenies might revel protetive mehnism in these mie to mintin pproprite one mss. Hypertive mtorc1 in osteolsts resulted in exess one mss in DTs1 mie (Supplementry Fig. 1). In this extreme sitution, redution of vsulriztion ws enefiil for deesing one mss. In ontrst, s Rptor defiieny in osteolsts indued low one volume (Supplementry Fig. 2), regining one mss requires undnt lood flow in DRptor mie. Despite no oupling etween ngiogenesis nd osteogenesis ws oserved, ross-tlk etween one-forming osteolsts nd ECs medited y ws witnessed in these geneti models. Moreover, s exerts dul inhiitory role in ngiogenesis nd osteogenesis, oth of whih re essentil for mintining one mss, inhiition is promising therpeuti strtegy for one loss diseses. Ativtion of expression hs een reported in severl stress-relted situtions, inluding mlignnies 32, hemotherpy 33, grft-versus-host diseses 34 nd infetions 35,36. For the first time, we demonstrted tht is expressed onstitutively in osteolsts. In ontrst, Lisignoli et l. 37 found tht ws undetetle in ultured humn osteolsts 37. We speulte tht these different results my hve risen due to different ell types nd sensiility of the detetion methods for. We filed to detet protein in osteolsts in one when performing n ntiody-dependent immunohistohemil ssy. However, in situ hyridiztion reveled mrked mrna expression in osteolsts. As seretory protein, the mjority of is sereted in extrellulr fluid fter synthesis from mrna, leving smll quntity in the ell elow detetion. High-level seretion provides preonditions for to exert its role in the miroenvironment of one mrrow. generlly exerts its effet y inding with its reeptor CXCR3 (refs 38,39); however, our dt showed tht rogte ngiogenesis nd osteogenesis independent of CXCR3. Insted, interts with nd prevents its inding to ECs nd osteolsts. Anlogously, PF4, nother CXC-hemokine s well s CXCR3 lignd, hs lso een reported to e le to ind nd inhiit its reeptor-inding ility 4. On the other hnd, s immunohistohemil stining of CXCR3 reveled its expression in other ells esides ECs nd osteolsts residing in one mrrow, we ould not rule out tht n indiret effet of on these ells would ontriute to the vsulr nd one phenotypes oserved in our mouse model. However, ECs nd osteolsts ultured in vitro were sujeted to the sme effets s those in one mrrow when exposed diretly to, whih onsolidted our onlusion tht is responsile for the vsulr nd one phenotypes in mouse one. Moreover, my exert its other iologil effets in ddition to inhiiting ngiogenesis nd osteogenesis on other ell types. Thus, other potentil ltertions in one in our mouse model my hve ourred, whih re outside the sope of this study. Our dditionl dt showed tht the numer of osteolsts per one perimeter ws inresed in DTs1mie ut deresed in DRptor mie (Supplementry Fig. 9), whih my hve prtilly ontriuted to the respetive ltertion in seretion in one mrrow nd ser in these two mouse models. However, s mrna expression, lirted y GAPDH, ws reveled to e elevted in the primry DTs1 osteolsts nd deresed in DRptor osteolsts, expression in single ells should e inresed in DTs1 osteolsts nd redued in DRptor osteolsts. This evidene shows tht mtorc1 positively regultes expression in osteolsts. Mehnistilly, we show tht mtorc1 regultes in osteolsts y modulting STAT1 expression nd tivity. expression is determined lrgely y the ontrol of STAT1 nuler ontent nd inding of STAT1 to gene promoters 29. A onsiderle quntity of STAT1 ws shown to e present in the nuleus of ontrol osteolsts (Fig. 8), explining the onstituent expression of in osteolsts. Moreover, STAT1 mrna nd protein were inresed in osteolsts with tivted mtorc1 nd were deresed in those with impired mtorc1, whih demonstrted positive trnsriptionl regultion of STAT1 y mtorc1 in osteolsts. In support of these oservtions, EI-Hshemite et l. 27 demonstrted mrked inrese in STAT1 expression in tumours nd mouse emryo firolst ell lines tht lked either Ts1 or Ts2 (ref. 27). In summry, our study identified s n ngiostti ftor sereted y osteolsts to regulte ngiogenesis nd osteogenesis in one nd reveled mtorc1 signlling nd STAT1 s ritil upstrem meditors. Phrmeutil oordintion of the pthwys nd gents my e enefiil in one formtion. Methods Mie. We purhsed the Ts1 flox/flox, Rptor flox/flox nd OC-Cre mouse strins from Jkson Lortory. The kground of Ts1 flox/flox mie is 129S4/SvJe, nd these mie were krossed to mie with C57BL/6 kground for eight genertions efore use. We performed genotyping using genomi DNA isolted from til iopsies, nd the primers used were s follows: loxp Ts1 llele forwrd, 5 -GTCACGACCGTAGGAGAAGC-3 nd reverse, 5 -GAATCAACCCCAC AGAGCAT-3 ; loxp Rptor llele forwrd, 5 -CTCAGTAGTGGTATGTGCTCA G-3 nd reverse, 5 -GGGTACAGTATGTCAGCACAG-3 ; OC-Cre forwrd, 5 -CAAATAGCCCTGGCAGATTC-3 nd reverse, 5 -TGATACAAGGGACA TCTTCC-3. The mie (mle, 4-week-old, three mie per eh group) were suutneously dministered mouse CXCL9 ntiody (R&D System, #AF-492-NA, 1 mg per 5 ml) 1 NATURE COMMUNICATIONS 7:13885 DOI: 1.138/nomms

11 NATURE COMMUNICATIONS DOI: 1.138/nomms13885 ARTICLE or rmumig/cxcl9 (PrimeGene Bio-Teh, #221-9, 1 ng per 5 ml) every other dy for 2 months. Mie importtion, trnsporttion, housing nd reeding were onduted ording to the reommendtions of The use of non-humn primtes in reserh. The mie were killed y ervil dislotion to prevent suffering. The Southern Medil University Animl Cre nd Use Committee pproved ll proedures involving the mie. Miro-CT nlysis. Femor were disseted from 12-week-old mle mie (five mie per eh group), fixed for 48 h in 4% prformldehyde nd nlysed t 12 mm resolution on miro-ct Snner (Viv CT4; Sno Medil AG, Bssersdorf, Switzerlnd). In rief, we snned the lower growth plte in the femor nd extended proximlly for 3 slies. We strted morphometri nlysis with the first slie in whih the femorl ondyles were fully merged nd extended for 1 slies proximlly. Using ontouring tool, we segmented the treulr one from the ortil shell mnully on key slies, nd morphed the ontours utomtilly to segment the treulr one on ll slies. The three-dimensionl struture nd morphometry were onstruted nd nlysed for treulr one volume frtion (BV/TV), treulr thikness (T. Th), treulr numer (T. N) nd treulr seprtion (T. Sp). We lso performed miro-ct imging in the mid-diphysis of the femur nd performed midshft evlution of 1 slies to quntify the ortil thikness (Ct. Th) nd periostel perimeter (Ps. Pm). Immunostining of slies nd ells nd histomorphometri nlyses. Hindlim tissues from the mie were fixed using 4% prformldehyde in PBS t 4 C for 48 h nd then delified in 15% ethylenediminetetreti id (EDTA; ph 7.4) t 4 C for 14 dys. The tissues were emedded in prffin or optiml utting temperture ompound (Skur Finetek), nd 2 5 mm sgittloriented setions were prepred for histologil nlyses. For immunohistohemistry, we inuted primry ntiodies tht reognized mouse phospho-s6 (Ser235/236) (Cell Signling, #2211, 1:1), osteolin (Am, #93876, 1:5), CD31 (Am, #28364, 1:5), (Proteinteh Group, #193-1-AP, 1:2),phospho-R2 (Y1175) (Cell Signling, #2478, 1:1) nd CXCR3 (Snt Cruz Biotehnology, #s-13951,1:5) overnight t 4 C. Susequently, we used seondry ntiodies onjugted with fluoresene t room temperture for 1 h. We ounted the numers of positively stined ells or vessels (CD31 þ ) in the whole diphysel periosteum or four rndom visul fields of metphysis per femur or tii in three sequentil setions per mouse in eh group. We lulted þ osteolsts/totl osteolsts (%) s the numers of ells doule-stined with nd osteolin ompred with osteolin-positive ells, nd determined mirovessel density s the numer of CD31 (nd endomuin (EMCN))- positive vessels per re of one mrrow. In situ hyridiztion with digoxinlelled proes ws performed using mrna in situ hyridiztion kit (Boster, #MK3237). We used FluoView FV1 onfol mirosopy (Olympus) or n Olympus BX51 mirosope for imging smples. For immunoytohemil stining, we inuted ultured ells with primry ntiody to mouse STAT1 (Proteinteh, # AP, 1:1) overnight t 4 C. Susequently, we used seondry ntiodies onjugted with fluoresene t room temperture for 1 h while voiding light. FluoView FV1 onfol mirosopy (Olympus) ws used for imging. Cells. Primry osteolsti ells were prepred from the lvri of neworn mie. In rief, lvrie were disseted from the mie (24 h fter irth), rinsed with PBS nd digested in freshly mde.1 mg ml 1 ollgense type II (Thermo Fisher Sientifi, #171115) in -miniml essentil Egle s medium t 37 C for 2 min; the digestion ws repeted five times. After digestion, superntnts were omined nd entrifuged to pellet ells 41,. Cells were then mintined in -MEM (Gio) supplemented with 1% fetl ovine serum (Gio), 1 U ml 1 peniillin nd 1 mg ml 1 streptomyin sulfte, t 37 C with 5% CO2. After rehing onfluene in 6 mm ulture dishes, the medium ws repled with -MEM (Gio) supplemented with 1% ovine serum lumin, nd the ells were ultured for 16 h efore olleting the CM. Mouse CXCL9 ntiody (R&D System, #AF-492-NA, 2 mgml 1 ), rmumig/cxcl9 (PrimeGene Bio-Teh, #221-9, 25 ng ml 1 ), CXCR3 ntgonist -7433(R&D System, #4528, 1 mm) or mouse reominnt 164 (R&D System, #493-MV-25, 1 ng ml 1 ) ws dded to the CM s indited. HUVECs were purhsed from the Amerin Type Culture Colletion nd were ultured in EC sl medium 2 (EBM2) ontining low serum (2% fetl lf serum) nd EC growth supplement (Promo Cell). Cells were serum-deprived for 16 h efore CM ws dded. Cell prolifertion ssy. HUVECs were serum-strved in EBM2 medium (.1% fetl ovine serum without growth ftor; Lonz, USA) for 16 h, nd then seeded (2 ml ontining 8, ells per well) into the hole of onfol dish (Bioimger, #135) nd inuted for 4 h. The ells were then treted with BrdU lelling regent (Invitrogen) for 2 h ording to the mnufturer s instrutions nd wshed with PBS. The ells were fixed with 7% ethnol for 25 min t room temperture, nd then stined for immunoytohemil nlysis. Nine res in eh group were ounted y two independent oservers linded to the groups. We sored BrdU-positive ells over totl ells visully nd with Imge Pro Plus softwre. In vitro migrtion ssys. HUVECs were seeded (1 1 5 ells per well) in 1% geltin-oted 24-well pltes (Corning, Shiphol, Netherlnds). Confluent ells were serum-deprived for 16 h, nd liner wound ws reted in monolyers y srthing with sterile pipette tip (2 ml yellow tip). Monolyers were wshed with PBS to remove floting ells nd the CM ws dded. After n dditionl 18 h, ell migrtion into the wound ws ssessed y mirosopy using digitl inverted mirosope. The degree of wound losure ws mesured s the perentge of the re overed y migrting ells in the initil wound in nine wounds per test ondition, using Imge Pro Plus softwre. In vitro tue formtion ssy. HUVECs were serum-strved for 16 h nd then seeded t density of 1, per well on growth ftor-depleted Mtrigel (BD Biosienes, NSW, Austrli) in 24-well pltes. CM ws dded, nd the results were quntified 6 h lter. Mirosopi fields ontining the tue strutures formed in the gel were photogrphed t low mgnifition ( 1). Nine fields per test ondition were exmined. Before they were photogrphed, the ells were fixed with 4% prformldehyde. Tue re ws quntified using Imge Pro Plus softwre. Colletion of one mrrow superntnt. Two-month-old mle mie (five per eh group) were killed nd the one mrrow ws exposed y utting two ends of the tii. Smples were then entrifuged for 15 min t 3, r.p.m. nd 4 C to otin one mrrow superntnts, whih were then stored t 8 C until ELISA nlysis. ELISA nlysis. We used the Mouse ELISA Kit (Elsiene Biotehnology, #E-EL-M5) nd Mouse CXCL9/MIG (Monokine indued y interferongmm) ELISA Kit (Elsiene, # E-EL-M2) to nlyse nd in serum, one mrrow superntnt nd CM, respetively. We performed the ELISA nlysis ording to the mnufturers instrutions. Western lot. We lysed ells with 2% SDS, 2 M ure, 1% glyerol, 1 mm Tris-HCl (ph 6.8), 1 mm dithiothreitol nd 1 mm phenylmethylsulfonyl fluoride. The lystes were entrifuged nd the superntnts were seprted y SDS polyrylmide gel eletrophoresis nd lotted onto nitroellulose memrne (Bio-Rd Lortories). The memrne ws then inuted with speifi ntiodies to phospho-s6k (T389) (Cell Signling Tehnology, #9234, 1:1,), S6K (Snt Cruz Biotehnology, #s-8418, 1:2,), phospho-s6 (S235/236) (Cell Signling Tehnology, #2211, 1:1,), S6 (Snt Cruz Biotehnology, #s-74459, 1:2,), (Proteinteh Group, #193-1-AP, 1:1,), phospho-r2 (Y1,175) (Alonl Tehnology, #AP382, 1:1,), R2 (Alonl Tehnology, #A7695, 1:1,), phospho-plcg1(s1,248) (Cell Signling Tehnology, #8713, 1:1,), PLCg1 (Alonl Tehnology, #A7711, 1:1,), phospho-erk1/2 (Thr22/Tyr24) (Cell Signling Tehnology, #437, 1:1,), ERK1/2 (Alonl Tehnology, #A229, 1:1,), phospho-akt (Ser473) (Cell Signling Tehnology, #46, 1:1,), phospho-sr (Tyr416) (Cell Signling Tehnology, #211, 1:1,), (R&D System, #AF-492-NA, 1:2,), Runx2 (Bioworld Tehnology, #BS8734, 1:1,), osteolin (Snt Cruz Biotehnology, #s-2379, 1:2,), phospho-stat1(y71) (Alonl Tehnology, #AP135, 1:1,), phospho-stat1(s727) (Alonl Tehnology, #AP453, 1:1,), mtor ((Cell Signling Tehnology, #2983, 1:1,) nd STAT1 (Proteinteh Group, # AP, 1:1,). The memrne ws then visulized y enhned hemiluminesene (ECL Kit, Amershm Biosienes). Unropped western lots sns re provided in the Supplementry Fig. 1 k. Rel-time quntittive PCR nd mirorry nlysis. Totl RNA ws isolted from ell pellets with TRIzol Regent (Life Tehnologies, # ) nd reverse trnsried (2.5 mg per smple in 5 ml retion volume) using PrimeSript Reverse Trnsriptse ording to the mnufturer s protool (Tkr, #268B). A volume of 2ml of DNA (orresponding to 1 ng of totl RNA) ws used for rel-time PCR using SYBR Premix Ex Tq (Tkr, #RRA). Primers for, nd STAT1 were s follows: forwrd, 5 -CCACGTCAGAGAGCAA- CATCA-3 nd reverse, 5 -TCATTCTCTCTATGTGCTGGCTTT-3 ; forwrd, 5 -GGAGTTCGAGGAACCCTAGTG-3 nd reverse 5 -GGGATTTG- TAGTGGATCGTGC-3 ; STAT1 forwrd, 5 -TCACAGTGGTTCGAGCTTCAG-3 nd reverse, 5 -GCAAACGAGACATCATAGGCA-3. For mrna rry ssy, smples were sumitted to Shnghi Biotehnology Corportion for hyridiztion on n Agilent Whole Mouse Genome Mirorry 4x44K G4122F (Proe Nme version). Eh mirorry hip ws hyridized to single smple lelled with Cy3. Bkground sutrtion nd normliztion were performed. Finlly, mrnas with expression levels differing y t lest threefold etween ontrol nd DTs1 osteolsts were seleted (Po.5). Mirorry dt hve een deposited in GEO dtse under ession ode GSE NATURE COMMUNICATIONS 7:13885 DOI: 1.138/nomms

12 NATURE COMMUNICATIONS DOI: 1.138/nomms13885 Co-immunopreipittion ssy. Reominnt mouse (PrimeGene Bio-Teh, #221-9, 5 ng ml 1 ) nd 164 (R&D System, #493-MV-25, 3 ng ml 1 ) were mixed in ie-old lysis uffer (4 mm HEPES (ph 7.4), 2 mm EDTA, 1 mm pyrophosphte, 1 mm glyerophosphte,.3% CHAPS nd one tlet of EDTA-free protese inhiitors (Rohe, Bsel, Switzerlnd) per 25 ml). The mixture were then inuted with nti- ntiody (R&D System, #AF-492-NA, 1:5) for 2 h t 4 C, followed y ddition of 3 ml of 5% slurry of protein G Sephrose eds for nother 1 h. Beds were then wshed four times with lysis uffer, trnsferred into 2 SDS smple uffer, oiled for 5 min t 1 C nd sujeted to western lot ssy for. In vitro kinse ssy for mtorc1. Primry lvril ells were lysed in ie-old uffer (4 mm HEPES (ph 7.4), 2 mm EDTA,1 mm pyrophosphte, 1 mm glyerophosphte,.3% CHAPS nd one tlet of EDTA-free protese inhiitors (Rohe, Bsel, Switzerlnd) per 25 ml). Superntnts were inuted with ntimtor ntiody for 2 h t 4 C, followed y ddition of 3 ml of 5% slurry of protein G Sephrose eds for nother 1 h. Beds were then wshed four times with lysis uffer nd one with kinse uffer (25 mm HEPES (ph7.4), 5 mm KCl, 1 mm MgCl 2 nd 25 mm ATP). A unit of.4 mg of reominnt glutthione S-trnsferse-tgged full-length STAT1 peptide ws dded to 3 ml kinse uffer. Kinse ssys were performed for 3 min t 3 C, nd terminted y the ddition of the 2 SDS smple uffer followed y oiling for 5 min. Binding ssy. Iodintion of mouse reominnt 164 ws performed using iodogen (Piere, Rokford, IL) ording to the mnufturer s inditions. The speifi tivities of the 125 I 164 were out 1 5.p.m. ng 1. Confluent HUVECs grown in 24-well dishes were wshed twie with ie-old PBS efore inding nd inuted with the indited onentrtions of 125 I 164 nd in DMEM ontining 2 mmol l 1 HEPES (ph 7.4) nd.15% geltin for 2 h t 4 C. Nonspeifi inding of 125 I 164 ws determined in the presene of 1 mgml At the end of the inding the ells were wshed with nd lysed using.5 M NOH. Smples were ounted in g-ounter (Sint-Quentin- Yvelines, Frne). Speifi inding ws determined y sustrting nonspeifi inding from totl inding. Eletrophoreti moility shift ssy. A totl of 2 mg of nuler protein extrted from primry lvril ells ws inuted with iotin-lelled STAT1-indingsite DNA proe in inding uffer (EMSA kit; Thermo Sientifi) for 3 min t room temperture. The proe used for the retion ontined the STAT1-inding site of the promoter (g-re1 site) with sequene of 5 -CCTTACTA- TAAACTCC-3. After inution, the smples were seprted on 6% polyrylmide gel in trisorte EDTA, trnsferred onto nylon memrne nd fixed on the memrne y ultrviolet rosslinking. The iotin-lelled proe ws deteted with streptvidin-horserdish peroxidse (EMSA kit; Thermo Sientifi). A proe lking nuler extrts ws used s negtive ontrol. The speifiity of the identified STAT3-DNA inding tivity ws onfirmed using 2-fold exess of unlelled proe ontining the sme sequene. For supershift nlysis, 1 mg STAT1 ntiody (Proteinteh) ws inuted with nuler extrts for 3 min efore the ddition of the iotin-lelled DNA proe. sirna knokdown. We trnsiently trnsfeted ells with STAT1 sirna using Lipofetmine RNAi MAX (Invitrogen, Crlsd, CA, USA) in Opti-MEM medium (Invitrogen), ording to the mnufturer s instrutions. The effiieny of trnsfetion ws mesured y western lot. The sequene of STAT1 sirna ws s follows: 5 -AAGGAAAAGCAAGCGTAATCT-3 (GenePhrm, Shnghi, Chin) 43. Sttistis. All results re presented s the men±s.d. Curve nlysis ws determined using Prism (GrphPd). The dt in eh group were nlysed using the unpired, two-tiled Student s t-test. The level of signifine ws set t Po.5. Dt vilility. 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