INTRODUCTION

Postnatal skeletal growth and bone remodeling are highly coordinated processesprimarilymediated bybone-forming osteoblasts and bone-resorbing osteoclasts.1-4Receptor activator of nuclear factor κ B Ligand(RANKL)signaling is vital for osteoclasts proliferation and differentiation.5-7RANK knockout in mice leads to severe osteopetrosis due to the absence of mature osteoclasts.7-8Up to now,RANKL signaling has been mostly investigated in osteoclastogenesisand itsinhibitor denosumab has been introduced clinically in the treatment of osteoclasts-related diseases like osteoporosis.9-13However,its roles in osteoblastic differentiation and postnatal bone formation have not been clari fi ed.A recent study indicates that RANKL reverse signaling promotes osteoblastic differentiation,14which shows that RANKL signaling could play an important role in bone formation.

RANK is over-expressed on osteosarcoma,15-16a mesenchymal tumor with the osteoblastic origin and regulates osteoblast migration,16essential to bone modeling and remodeling.The current studies imply that RANKL signaling could play a key role in osteoblastic differentiation and bone formation,which has long been underestimated.

In this study,we found that RANK was expressed at the early stage of osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)and was downregulated after the osteogenic differentiation began.RANK inhibited osteoblastic differentiation of BMSCs while had no effects on adipogenesis.BMSCs conditional knockout of RANK with Prx1-Cre mice led to a significantly increased trabecular bone mass,accelerated bone formation rate and showed resistance to ovariectomy-(OVX)induced bone loss.Our results show that RANKL signaling regulates both osteoclasts and osteoblasts by inhibiting BMSCs osteogenic differentiation and promoting osteoclastogenesis.

RESULTS

RANK is expressed in BMSCs

Although RANK is expressed in several osteosarcoma cell lines,its expression in BMSCs has not been determined.We hypothesized that RANK was expressed in BMSCs.We collected bone marrow from patients undergoing fracture surgeries and isolated human BMSCs identified by CD45-/Stro-1+/CD146+.We collected BMSCs from 6-week-old male mice identified by CD34-/CD45-/CD73+/CD90+/CD105+(Figure S1).After passage,BMSCs of the third generation were used.We found that both human and mouse BMSCs highly expressed RANK in the cytoplasm,as assessed by immuno fluorescence(Fig.1a).Flow cytometric analysis showed that RANK and CD90 were co-expressed in 88.3%and 92.8%of human and mice BMSCS,respectively(Fig.1b).Western blot and RT-PCR confirmed the RANK expressions in human and mouse BMSCs(Fig.1c,d).RANK expressions were confirmed by flow cytometry,western blotting and qPCR of positive control with osteoclasts and a negative control with RANK silenced BMSCs(Figure S2a-c).As BMSCs are enriched in PαS cells and calvarial cells are used as osteoblasts,we isolated these two cells and examined(Figure S3a).RANK was expressed in both cell types and decreased after osteogenic differentiation(Figure S3b).In vivo expression was determined by in situ immunostaining with CD90 and RANK in the secondary spongiosa of the distal femur(Fig.1e).The results indicate that RANK is highly expressed in BMSCs.

RANKL signaling inhibits osteogenic differentiation of BMSCs

RANKL has been reported to be expressed in BMSCs.17To determine the changes of RANK during osteogenesis and adipogenesis in vitro,we induced osteogenic differentiation of BMSCs and detected the expressions of RANK with triple immuno fluorescence staining of CD90,RANK,and dentin matrix protein 1(DMP-1),an osteoblast marker at different time points from 0 to 21 d.Results showed that after the osteoblastic differentiation began,the expressions of CD90 and RANK were rapidly downregulated,while the DMP-1 expression was gradually increased(Fig.2a,b).

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Fig.1 BMSCs express RANK.a Both human(h)and mouse(m)BMSCs express RANK assessed by immuno fluorescence staining.Scale bar=20μm.b Flow cytometry confirmed human and mouse BMSCs express RANK.c and d Western blot,RT-PCR analysis.e Immuno fluorescence shows that RANK is expressed in BMSCs in vivo.Scale bar=50μm.

Fig.2 RANK inhibits osteoblastic differentiation of BMSCs in vitro.a Immuno fluorescence confocal analysis of CD90,RANK,and DMP-1 after osteogenic induction of human BMSCs from 0 to 21 d.Scale bar=50μm.b Western blot analysis of RANK(66kD)expressions after osteogenic induction with RANK overexpression and knockdown in human BMSCs at 0 d/14 d/21 d.c Western blot analysis of RUNX2(56kD)after osteogenic induction with RANK overexpression and knockdown in human BMSCs at 0 d/14 d/21 d.d Western blot analysis of ALP(39kD)after osteogenic induction with RANK overexpression and knockdown in human BMSCs at 0 d/14 d/21 d.β-actin was used as an endogenous reference for normalization.e and f ALP and alizarin red staining after 21 days of osteogenic induction of human BMSCs.

To test its function,we used RANKL to activate RANK in vitro and found that RANKL(50 ng·mL-1)significantly inhibited calcium nodules formation by alizarin red staining(Figure S4).Then we over-expressed and knocked-down RANK in BMSCs(Figure S5)and induced osteogenic and adipogenic differentiation.Western blotanalysis showed thatosteoblastic differentiation was significantly enhanced after rank was silenced demonstrated by significantly increased alkaline phosphatase(ALP)and runt-related transcription factor 2(RUNX2)expressions,while RANK overexpression significantly inhibited osteoblastic differentiation(Fig.2c,d).ALP and alizarin red staining results showed the osteoblastic differentiation of BMSCs was consistent with western blotting(Fig.2e,f).

Fig.3 Increased bone formation during bone remodeling in rank-/-mice.a Representative micro-CT images of distal femurs from 8-weekfemale adult rank+/+and rank-/-mice.b-e Quantitative micro-CT analysis of the secondary spongiosa of distal femora.b Bone surface density(BS/TV),c Trabecular number(Tb.N),d Volumetric BMD,e Bone volume fraction.f H&E staining of the distal femora.Scale bar=200 μm.g Trabecular area analysis.h Oil Red O staining of the distal femora.Scale bar=200 μm.i Lipid droplet analysis.j Calcein double labeling of the metaphyseal trabecular bone(scale bar=200 μm).k Trabecular mineral apposition rate analysis.l Trabecular bone(TB)formation rate.All data are mean±s.e.m.n=8.*P＜0.05,**P＜0.01 by one-way analysis of variance(ANOVA)followed by t-test.

Interestingly,RANK did not affect adipogenic differentiation of BMSCs.After the adipogenic differentiation was induced,CD90 rapidly decreased while RANK expression did not change significantly(Figure S6a).Western blot showed that overexpression or knockdown did not affect adipogenic differentiation by lipoprotein lipase(LPL)and peroxisome proliferator-activated receptor γ2(PPARγ2),two adipogenesis markers(Figure S6c-d)and oil red O staining(Figure S6e).The results suggest a crucial role of RANK in regulating osteoblastic differentiation of BMSCs at an early stage.

Knockout of rank in BMSCs increases bone formation in mice

As whole-body loss-of-function approach will inevitably affect osteoclastogenesis,we used a cell-specific gene knockout approach.We crossed floxed rank mice with Prx1-Cre mice to generate MSCs conditional rank knockout mice(named rank-/-)(Figure S7a-b).Rank fl ox/ fl oxcontrol littermates were referred to as rank+/+.We compared sex-matched littermates using paired statistical tests to evaluate difference significance of independent experiments.Both male and female mice were used in the study and all the conclusions were based on analysis of both male and female mice.But only female data were presented.The body weight and length of rank-/-and rank+/+mice were similar at birth,but they increased faster in rank-/-mice for both male and female(Figure S7c).No difference was detected for bone analysis between rank+/+and WT mice.

BMD was greater in 8-week-old rank-/-mice than in their rank+/+littermates of the same age and gender.Rank-/-mice had a significantly higher trabecular bone volume,trabecular number,and bone surface in distal femur but not in the spine relative to their rank+/+littermates(P＜0.05)(Fig.3a-e,Figure S8a,c).However,the cortical bone mineral density,porosity,and thickness were not statistically different in two groups(Figure S8b).Similar results were found in H&E-stained sections and in histomorphometric analyses(Fig.3f,g).Oil red O staining showed that the number of fat vacuoles in rank-/-mice was not significantly different from that in their rank+/+littermates(Fig.3h,i).Calcein double labeling results showed a significantly higher bone formation rate(BFR)and mineral apposition rate(MAR)of trabecular bone in rank-/-mice than in rank+/+mice(Fig.3j,l).The above results indicate RANK ablation promoted trabecular bone formation in adult mice.

无人驾驶时代，交通肇事罪是否存在问题，目前有两种截然相反的观点。一种观点认为，“由于控制无人驾驶汽车的是自动系统，而非人类，因此从道路交通肇事的犯罪构成上说，在无人驾驶的状态因为不符合交通肇事罪的主体和主观要件，因此不可能构成交通肇事罪。”[11]一种观点认为，交通肇事罪不会成为“水中月”，“交通肇事罪在构造的本身上并不存在问题，立法仍然能够涵盖无人驾驶这一新兴的领域。”[12]

“玉笛谁家听落梅”乃是肉菜，一条小羊坐臀、一条小猪耳朵、一条小牛腰子、一条獐腿肉加兔肉，拼作一盘，难免有油腻过头的嫌疑。但因为江南式精巧的刀工“肉条形如笛子”，和繁复的口味组合“有二十五变，合五五梅花之数”而深得老叫花之心。

To determine whether the increased BMD in the rank-/-mice was the result of decreased osteoclasts,we measured the number of mature osteoclasts by tartrate-resistant acid phosphatase(TRAP)staining and serum TRAcp5b,CTX-1 levels.The number of TRAP+mature osteoclasts in rank-/-mice was not significantly different from that in rank+/+littermates(Fig.4a).Formation of TRAP-positive cells from bone marrow mononuclear cells from rank-/-and rank+/+mice and osteoclasts quantification showed no significant difference(Fig.4b,c).The serum level of TRAcp5b in rank-/-mice was slightly lower than that in rank+/+mice,but the difference was not statistically significant(Fig.4d).Similar results were found for CTX-1(data not shown).Moreover,the tooth eruption of rank-/-mice was normal(data not shown).The results demonstrate that conditional knockout of RANK in BMSCs has little effects on osteoclastogenesis.

Then we measured the number of mature osteoblasts by immunostaining femur sections.The number of osteocalcin(OCN)positive mature osteoblasts on the bone surfaces and the serum OCN levels of rank-/-mice were significantly higher than that in rank+/+littermates(Fig.4e,f).We then measured the numbers of osteoblasts at different stages of differentiation by immunostaining femur sections.The numbers of runx2+(Fig.4g,h)and osterix+osteoprogenitors(Fig.4i,j)on the trabecula of rank-/-mice were significantly higher than those in their rank+/+littermates.

Fig.4 Increased osteoblast maturation in rank-/-mice.a Light micrographs of TRAP staining performed on trabecular bone sections from distal femora.Scale bar=100 μm.b Formation of TRAP-positive cells from bone mononuclear cells isolated from rank-/-and rank+/+mice.Scale bar=100 μm.c Quantification of mature osteoclasts.d Serum TRAcp5b levels of 8-week-female adult rank+/+and rank-/-mice.e Osteocalcin immunohistological staining of the metaphyseal trabecular bone at distal femora.Scale bar=100 μm.f Serum OCN levels of 8-week-female adult rank+/+and rank-/-mice.g-h Immunohistochemical analysis of runx2 in the metaphyseal trabecular bone at distal femora.Scale bar=20μm.i-j Immunohistochemical analysis of osterix in the metaphyseal trabecular bone at distal femora.Scale bar=20μm.All data are mean±s.e.m.n=8.*P＜0.05,**P＜0.01 by one-way ANOVA followed by t-test.

For molecular mechanisms,after RANKL activates RANK,several pathways,including NF-κB,MAPK and Akt are activated.NF-κB was reported to inhibit osteoblastic bone formation.18We hypothesized that NF-κB pathway was vital.RANK activation or overexpression led to increased phosphorylation and translocation of p65 as well as decreased β-catenin expressions.β-catenin is a crucial component of the Wnt pathway,which is vital for osteoblastic differentiation.22β-catenin and p65 could form a dynamic complex which could undergo temporal dissociation to allow for p65 activation and the decreased β-catenin is associated with increased p65 activity.23The upstream kinase of p65,IKKβ is also a β-catenin kinase that phosphorylates the conserved degron motif to prime the β-catenin for β-transducing repeat-containing protein(βTrCP)-mediated ubiquitination and degradation in MSCs.24Thereafter,adipogenesis is increased while the osteogenesis is inhibited.Therefore,how RANKL signaling regulates BMSCs osteoblastic differentiation is generally clear.Once the RANK is activated by RANKL,TRAF6 is recruited and IKKβ is activated and phosphorylates IκB and β-catenin.Then the complex dissociates with p65 released,phosphorylated and translocated into the nucleus and β-catenin phosphorylated and degraded through inducing βTrCP expression.The βTrCP targets both β-catenin and IκB to induce ubiquitination and subsequent degradation,25forming a positive feedback for the NF-κB pathway sustained activation and Wnt/β-catenin inhibition.

RANKL signaling increases degradation and inhibits synthesis of β-catenin

Since BMSCs express RANKL,the ligand for RANK,we explored the roles of RANKL in BMSCs osteogenic differentiation.Similar to RANK,RANKL overexpression significantly inhibited BMSCs osteogenic differentiation while RANKL silencing promoted BMSCs osteogenic differentiation demonstrated by ALP and alizarin red stainings(Figure S11a-b).RANKL overexpression significantly activated phosphorylation ofp65 and promoted nucleus translocation of p65 while silencing inhibited p65 activation(Figure S11c-d).RANKL overexpression significantly decreased βcatenin while silencing increased β-catenin(Figure S11e).Interestingly,RANKL expression is consistent with RANK(Figure S11f).

To investigate the possible underlying mechanisms,we explored the effects of RANKL signaling on β-catenin,a key transcriptor for BMSCs osteoblastic differentiation.Overexpression of RANK significantly increased phosphorylation which was decreased by RANK knockdown(Fig.5a).Exogenous RANKL(10ng·mL-1)added increased p65 nucleus translocation(Fig.5b,c).RANK and β-catenin expressions were reversely correlated.Overexpression of RANK significantly reduced β-catenin content(Fig.5d).CHX stands for cycloheximide and is used to inhibit protein synthesis.G132 isMG132 (Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal),a proteasome inhibitor used to inhibit protein ubiquitylation and degradation.RANK overexpression increased β-catenin degradation and inhibited its synthesis(Figs.5e,f and 7a).

To further illustrate the relationship between p65 and β-catenin,we evaluated the effects of p65 and β-catenin silence on the osteogenic differentiation of BMSCs.Western blotting results showed that p65 knockdown significantly promoted osteogenic differentiation demonstrated by higher runx2 and ALP expressions compared with induction group,which was consistent with the previous report,18while knockdown of β-catenin significantly abolished the effects.Silencing both p65 and β-catenin signi ficantly inhibited osteogenesis.The results implied that p65 inhibited BMSCs osteogenic differentiation dependenton β-catenin (Figure S10).RANKL signaling inhibits osteoblastic differentiation of BMSCs through inhibiting β-catenin synthesis and promoting its degradation.

每一条道路上都有出发的人，每个人头顶上都有一方天空，改变为道，道路坦平。鸡东工匠王世君，兢兢业业，变通革新，扎根公路、以路为家，因工作业绩突出，多次被评为鸡西市交通系统“先进个人”。

RANK knockout BMSCs ameliorates ovariectomy-induced bone loss

To explore the effects of RANK knockout in BMSCs on pathological bone loss,we used the OVX-induced bone loss model.After 6 weeks of ovariectomy,rank+/+mice showed a significant bone loss by HE staining and micro-CT analysis of the distal femora(Fig.6a).Rank-/-mice showed significant resistance to OVX-induced bone loss(Fig.6a).BMD,Tb.N,BV/TV and BS/TV were greater in rank-/-ovariectomized mice than in their ovariectomized rank+/+littermates and similar to their sham rank-/-mice(P＞0.05)(Fig.6b).But rank-/-mice had a significant decrease in BMD,Tb.N,BV/TV and BS/TV in the vertebrae (P＜0.05)(Figure S12).Similar results were found in HE-stained sections and in histomorphometric analyses(Fig.6c).The number of fat vacuoles in rank-/-ovariectomized mice was not statistically different from that of the rank+/+ovariectomized mice(Fig.6e).The ameliorated bone loss induced by OVX is attributed to the increased bone formation.

Osteoblasts increase in rank-/-mice

产业创新速度对创新效益的作用机制如图1所示，包括微观叠加机制和宏观作用机制。所谓微观叠加机制，起源于产品创新速度，即若干企业的若干产品创新速度的提升，带来整个产业创新效益的提升;所谓宏观作用机制，主要指产业整体的环境要素对企业创新速度的影响机制与方式。

Fig.5 RANK affects β-catenin synthesis and degradation.a Western blot analysis of p-p65 versus p65(65kD)with RANK over-expressed and silenced in BMSCs from mice.b Western blot analysis of p65(65kDa)content in nucleus versus total of BMSCs.c Immuno fluorescence images of p65 in BMSCs with sRANKL introduction(10ng·mL-1),Scale bar=50 μm.d Western blot analysis of β-catenin(98 kD)with RANK overexpressed and silenced in BMSCs.e Analysis of β-catenin degradation rate with RANK overexpressed and silenced.f Analysis of β-catenin synthesis with RANK overexpressed and silenced.All data are the mean±s.e.m.of triplicate cultures.**P＜0.01 by one-way ANOVA followed by t-test.

DISCUSSION

RANKL signaling is essential for osteoclast development and RANK-/-mice showed profound osteopetrosis due to a lack of mature osteoclasts.7-8RANK-/-mice were characterized by small body size,shortened limbs and doming of the skull.For decades studies of RANK have been focusing on osteoclastogenesis.19-20 And RANKL inhibitor denosumab has been used clinically to inhibit osteoclastogenesis as an anti-resorptive agent.9

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BMSCs are progenitor cells of osteoblasts in bone marrow.RANK expression in BMSCs has not been determined.In 2015,a study showed that RANK was expressed in osteosarcoma tumor cells and associated with osteoblast chemotactic migration.16Based on the above findings,we hypothesized that RANKL signaling could play an important role in bone formation regulation.

Immuno fluorescence and immunohistological assay

As the ligand for RANK receptor,RANKL has been reported to couple bone resorption and formation.14Vesicular RANK secreted from the maturing osteoclasts binds osteoblastic RANKL and promotes bone formation by triggering RANKL reverse signaling.Schena et al.reported that RANKL-deficient BMSCs displayed an osteogenic differentiation defect in vitro and in vivo.21We replicated the in vitro experiments and unexpectedly found that RANKL-deficient BMSCs showed increased osteogenic capacities and RANKL overexpression inhibited BMSCs osteogenic differentiation,which is controversial to reported study.The in vivo protocol employed is ectopic bone formation assay,which could not fully reflect the roles of RANKL-deficient BMSCs.For,an in vivo study by Jinhu Xiong et al.reported that mice with RANKL deletion in committed osteoblast progenitors with Osx1-Cre showed a normal cancellous architecture with no evidence of bone formation defect.17Generally,the roles of RANKL on BMSCs in regulating osteoblastogenesis are under debate,which needs further study.

Fig.6 RANK-/-in BMSCs resists OVX-induced pathological bone loss.a Representative micro-CT images of distal femurs from 14-weekfemale adult rank+/+and rank-/-mice after 6 weeks of ovariectomy.b Analysis of bone mineral density(BMD),bone surface density(BS/TV),trabecular number(Tb.N),bone volume over total volume(BV/TV).c HE staining of the distal femora.Scale bar=200 μm.d Trabecular area analysis.e Analysis of fat vacuole number.All data are mean±s.e.m.n=8.*P＜0.05,**P＜0.01 by one-way analysis of variance(ANOVA)followed by t-test.

As the Prx1-Cre transgene causes deletion in both chondrocytes and osteoblast-lineage cells,to exclude the interference of chondrocytes,we isolated chondrocytes and performed the immunohistological staining of RANK in vitro and in vivo on the distal femur sections from rank+/+and rank-/-mice at 8-week.It showed that the chondrocyte did not express RANK(Figure S9a-c).The results show that RANKL signaling negatively regulates osteoblast formation in mice.

In this study,we expanded the understanding for RANKL signaling and found that it not only promoted osteoclastogenesis but negatively regulated osteoblastic differentiation of BMSCs and postnatal bone formation in bone modeling and remodeling(Fig.7b).

MATERIALS AND METHODS

BMSCs isolation and induction

Human BMSCs were isolated from three premenopausal female patients receiving fracture surgeries.The procedure was approved by the Ethics committee of Shanghai Changhai hospital and the informed consent was obtained from all subjects.Bone marrow cells were flushed from the cavities of femurs and tibias of 8-weekold rank-/-mice and rank+/+control littermates and were plated in α-MEM with 15%FBS(Fetal bovine serum)and 1%penicillin/streptomycin(Invitrogen)at 37°C in a 5%CO2incubator.After 48h of culture,the medium was changed to remove nonadherent cells.Then cells were cultured for an additional 4 days with a single medium change until BMSCs reached con fl uency.Cells were passaged with 0.25%trypsin-EDTA digestion(Invitrogen)and reseeded at 5×105cells/6-well or 1×105cells/12-well.Third-passage BMSCs were subjected to induction of adipogenic and osteogenic differentiation,and transfection of plasmids.For osteogenic induction,BMSCs were cultured in media(α-MEM containing 15% FBS,1% penicillin/streptomycin,50 μg·mL-1 ascorbic acid and 10 mmol·L-1 β-glycerophosphate)for 21 days.Alizarin Red staining was carried out to determine the mineralization.For adipogenic differentiation assay,BMSCs were cultured in 6-well plates with adipogenesis induction medium(α-MEM,10%FCS,0.5mmol·L-13-isobutyl-1-methylxanthine,5 μg·mL-1insulin,and 1 μmol·L-1dexamethasone)for 14 days.Oil Red O staining was performed to detect mature adipocytes.

Fig.7 Illustrations of BMSCs osteoblastic differentiation regulations.a Molecular mechanisms of RANK in regulating the osteogenic differentiation of BMSCs.b Schematic diagram RANKL signaling regulating the osteogenic differentiation of BMSCs.The RANKL-RANK interaction between BMSCs restrains osteoblastic differentiation.Soluble RANKL released from osteoblasts negatively regulates osteoblastic differentiation of BMSCs.

Animal experiments

Protein quantification was performed.Protein lysate was introduced to SDS-PAGE and subsequently electrotransferred to a polyvinylidene fluoride membrane.Western blotting was carried outaspreviously reported.26The antibodiesused were listed as follows:anti-RANK(Abcam,ab200369,1:800),anti-RUNX2(Cell Signaling Technology,12486,1:1000),anti-ALP(Millipore,06-942,1:1000),anti-pp65(Abcam,ab76307,1:5000),anti-p65(Active Motif,39159,1:1000),anti-β catenin(Cell Signaling Technology,4824,1:200).

Histology and TRAP staining

For histological analysis,decalcified distal femur and vertebrae samples were embedded in paraffin and then sectioned at 4-μm thickness.TRAP,OCN staining was performed following standard protocols.

对于准静水压实验，衍射谱的一维积分图见图1.图中可以看到铬的三个峰(110)、(200)、(211)一直存在，而且铬一直保持bcc结构到实验的最高压力47 GPa.

Bone structure analysis

The bone structure of the distal femur and vertebra was analyzed using micro-computed tomography (Micro CT)(Skyscan1172,Antwerp,Belgium).The analysis parameters were 80 kv,124 μA and resolution was 8 μm.Structural parameters of metaphyseal area and trabecular bone were analyzed with the built-in software.Total bone mineral density(BMD),bone volume/total volume(BV/TV),trabecular number(Tb.N),bone surface area expressed per unit total volume(BS/TV)were analyzed.Two-dimensional and three-dimensional bone structure image slices were reconstructed.

RT-PCR

Total RNA was extracted following the instruction of TRIzol Reagent(Invitrogen).RNA(2µg)was used to synthesize cDNA by reverse transcription Master Mix(Promega).Quantitative real-time PCR was performed with iTaq Universal SYBR Green Supermix(Bio-Rad)on a CFX384 Real-Time PCR system(Bio-Rad).Data were normalized by 18S rRNA transcript and calculation was performed with the ΔΔCt method.

Rank-RT(human)-F:CTGTCTTCAGGAGATTGGCA,rank-RT(human)-R:ACTTGGAAATCTG-GCAGCTC;RANK(mouse)-RT-F:ATCCAGCAGG GAAGCAAA,RANK(mouse)-RT-R:GGGACACGGGCATAGAGT.

Western blotting

To generate conditional rank knockout mouse models for investigating postnatal bone formation and remodeling,we first crossed female rank fl ox/ fl oxmice(B6.Cg-Tnfrsf11atm1.1Irw/J)with male Prx1-Cre mice(B6.Cg-Tg(Prx1-Cre)1Cjt/J)from the Jackson laboratory and obtained Prx1-Cre:rank fl ox/-mice among which male mice were crossed with rank fl ox/ fl oxmice to obtain Prx1-Cre:rank fl ox/ fl oxmice for study.All skeletal analyses were performed on both female and male mice unless otherwise speci fi ed.Data of female mice were provided.For serum TRAcp5B,CTX-1 and OCN assay,serum was collected from 8-week-old mice without fasting following the manufacturer's instructions.

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Silence and overexpression of RANK and RANKL in mouse BMSCs by lentivirus

A siRNA sequence complementarily binding to mouse RANK(NM_017206.1)was chosen.The target sequences of siRNA(5′-GGTTGTCTACTTCACTGCT-3′)are homologous to RANK,respectively.The target sequences of siRNA(5′-GCGTACCTACAGACT ATCT-3′)are homologous to RANKL.The oligonucleotide templates of these shRNAs were chemically synthesized and cloned into the linear pSIH1-H1-copGFP shRNA Vector(System Biosciences)which was obtained through digestion by BamH I and EcoR I(Takara,Dalian,China)and puri fi cation by agarose gel electrophoresis.An invalid siRNA sequence(5′-GTTGTCATGTC TATCTCGC-3′)was used as an NC(negative control).The CDS(Coding sequence)of mouse RANK was ampli fi ed using the primers 5′-GGAATTCGCCACCATGGCCACCAAGGAGAAG-3′and 5′-CGGGATCCTCACATCATGGTCTCCAC-3′,which contain an EcoRI cutting site and Kozak sequence and a BamHI cutting site,respectively.The cDNA was prepared by reverse transcription RNA isolated from rat brain tissue.The PCR product was digested and cloned into a pcDH1-CMV lentiviral expression vector(System Biosciences);the recombinant vector was named pcDH1-RANK.The products of the vectors were confirmed by DNA sequencing,and endotoxin-free DNA was prepared.

Packaging of lentivirus was carried out in 293 virus packaging cellline.One day before transfection,293TN cellswere seeded into 10-cm dishes.Two micrograms of each shRNA and expression vector and 10 μg pPACK Packaging Plasmid Mix(System Biosciences)was co-transfected using Lipofectamine 2000(Invitrogen)in accordance with the manufacturer's protocol.The medium was replaced with DMEM plus 1%FBS.Fortyeight hours later,the supernatant was collected and then cleared by centrifugation at 5 000×g at 4°C for 5min,and passed through a 0.45µm PVDF membrane(Millipore,MI,USA).The titer of the virus was determined by gradient dilution.The packaged lentiviruses were named as Lv-shRNA-RANK,Lv-NC,and Lv-RANK.

P3 mouse BMSCs in logarithmic phase were seeded on 6-well plates at 5×105cells/well.One day later,the viral solution was added at an MOI of 20.The infection efficiency was evaluated by observing and analyzing the fluorescent mark 72 h after infection.And total RNA or total protein were isolated from the cells and subjected to real-time PCR for RANK mRNA or protein level,respectively.

In this study,we first isolated human and mice BMSCs and identified RANK expressions in vitro and in vivo.After the osteogenic differentiation began,the expression was rapidly downregulated.Intriguingly,RANK expression was not affected during adipogenic differentiation.Thus,we speculated that RANK functioned in regulating osteoblastic differentiation.RANKL signaling inhibited osteoblastic differentiation of BMSCs in vitro.To avoid interfering with osteoclastogenesis by general knockout,we used the conditional knockout technique to specifically knockout RANK in BMSCs.We found that RANK elimination in BMSCs significantly increased bone formation with more osteoblasts by BMSCs and adipogenesis by BMSCs was not affected.The osteoclasts seemed not being affected.Thus,the increased bone formation by loss of RANK in BMSCs is independent of osteoclastogenesis.

根据信度检验,在本研究中SRRS量表的4个维度的Cronbach's α如表2。可以看出,按不同样本地即各个学校来看,SRRS量表的4个维度的Cronbach's α系数均在0.709～0.943之间,总量表的Cronbach's α在0.709～0.782之间,均高于0.6的最低标准,组合信度在0.951～0.967之间;按总样本数来看,Cronbach's α系数也均大于0.6,组合信度为0.962,说明问卷具有良好的信度。

hMSCs and mMSCs were fixed with formaldehyde(4%in PBS),permeabilized with Triton X-100(0.4%in PBS),incubated with blocking buffer(10%donkey serum in PBS),and stained with primary antibody overnight at 4°C.Then,cells were treated with secondary antibodies for 1h at room temperature.Hoechst 33342(Invitrogen)was used for nuclear DNA staining.The antibodies used are as follows:anti-RANK (ab12008,1:100),anti-CD90(Abcam,ab21624,1:200),anti-DMP1(Santa Cruz,sc-17320,1:100).

Flow cytometry

目前，我国全面提倡实施素质教育，同时，提倡减轻学生的学业负担。“变题”的方法和技术成为教学中常用的教学方法。通过“变题”练习把规律性的问题联系在一起，不仅对此类题型做了归纳总结，对解题方法有了进一步的认识，避免了学生的“题海”战术，减轻学生的课业负担，提高课堂的教学效率，对知识的掌握，思维和能力培养都起着非常重要的作用。

The procedure was carried out according to the protocals.The antibodies used are as follows:anti-RANK(ab13918,1:50),anti-Runx2(ab192256,1:150),anti-ALP(ab197781,1:50),anti-CD73(AF4488,1:200),anti-CD90(ab3105,1:100),anti-CD105(ab221675,1:100),anti-CD34(ab8158,1:100),anti-CD45(ab10558,1:100),anti-PDGFRα(ab203491),anti-SCA1(ab93537).

Statistical analysis

于是在一个春天的早晨，依旧是十八年前的那些人把我送到门口，这里面少了几个，也多了几个。还是和那次一样，看不见我姐姐的影子，那次是我没有等待她，这次是我找不到她的坟墓。一个叔父和一个堂兄弟到车站送我，十八年前他们也送过我一段路程。

The data were expressed as means±SD.The two independentsample t-test was used for comparisons between two groups.In cases of a comparison involving more than 2 groups,a oneway ANOVA was used.Statistical significance was considered as P＜0.05.

近年来，有学者提出了不同的看法。虽然肥胖可增加肾透明细胞癌(clear-cell renal cell carcinoma, ccRCC)的发病风险[19]，但一项针对2 119例ccRCC病例的研究发现，超重或肥胖患者较体质量正常者疾病进展风险降低了近40%，而且BMI越高，ccRCC相关死亡率越低[20]。基因分析表明，肥胖者脂肪酸代谢相关基因表达更低，其中部分基因已被发现在肿瘤中过表达，并与肿瘤生长相关，提示肥胖患者肿瘤细胞生长较缓慢[20]。

ACKNOWLEDGEMENTS

We thank the Clear-Medtransstudio for language polishing and Shanghai Geekbiotech Company for technical support.We thank Prof.Wang from Fudan University for his careful technical guidance which was very helpful and essential.Special thanks go to Dr.Ying Wang and Dr.Longjuan Qin for their help at the beginning of the study.We would like to thank Shanghai Model Organisms for animal housing and breeding.This work was supported by the National Natural Science Foundation(NNSF)Key Research Program in Aging(91749204);National Natural Science Foundation of China(81871099,31370958,81701364,81771491,81501052);Shanghai Municipal Science and Technology Commission Key Program(15411950600,18431902300);Municipal Human Resources Development Program for Outstanding Leaders in Medical Disciplines in Shanghai(2017BR011).

AUTHOR CONTRIBUTIONS

C.X.designed the study.Z.Z.and W.J.conducted experiments.C.X.and Z.Z.analyzed the data and prepared the manuscript.S.J.C.generally guided and financially supported this study.Data availability:The authors declare that all data supporting the findings of this study are available within the paper and its supplementary information files.

当前，钢贸企业的发展不容乐观，主要表现在钢材交易受到国际国内市场环境的影响出现了大规模的下滑，这就使得很大钢贸企业的经济利润得不到保证，导致企业的经营面临着巨大的挑战。钢贸企业想要在市场中保持强大的活力，就必须要做好成本控制工作。不过，当前钢贸企业财务管理中成本控制现状不容乐观，还存在着诸多的问题，只有针对性地解决这些问题，才能确保成本控制的有效性。

ADDITIONAL INFORMATION

The online version ofthisarticle (https://doi.org/10.1038/s41413-018-0035-6)contains supplementary material,which is available to authorized users.

Competing interests:The authors declare no competing interests.

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