Osteoclasts are not a source of SLIT3

时间：2025-01-07

Na Li,Kazuki Inoue,Jun Sun,Yingzhen Niu,Sarfaraz Lalani,Alisha Yallowitz,Xu Yang,Chao Zhang,Rong Shen,Baohong Zhao,Ren Xu and Matthew B.Greenblatt

INTRODUCTION

Drugsfor the treatment of skeletal disorders,such asosteoporosis,typically fall into one of two categories.These drugs function to either block bone resorption by osteoclasts or augment bone formation by osteoblasts.However,current treatment options still have strong limitations,due in part to both the few anabolic agents available and restrictions on the use of these agents.1–2To continue to advance therapy for disorders of low bone mass,the identi fication of new strategies to promote bone formation is critical.

Axon guidance cues have emerged as agentsof great interest in this regard,as they control bone formation by a variety of mechanisms,including shaping the skeletal microenvironment to support osteogenesis.3–7Among them,SLIT3 was recently identi fied as an osteoanabolic agent that ameliorated the bone loss in mouse models in two recent reports.8–9

SLIT3 was first discovered as a repulsive axon guidance cue during neuronal migration.10SLIT3 is now increasingly recognized to play additional physiological roles outside of the nervous system,asit isinvolved in immunoregulation,stem cell regulation,and cancer development.11–13More recently,SLIT3 was characterized as having angiogenic functions in nonbone tissues,and SLIT3 knockout mice displayed severe developmental vascular defects in the diaphragm.14–16In vitro,recombinant SLIT3 stimulation could enhance angiogenesis by increasing the proliferation,migration and tube formation of endothelial cells.15In contrast,SLIT3 signaling inhibition signi ficantly decreased functional blood vessel formation in human engineered tissue.17

Our prior study showed that SLIT3 is highly expressed in osteoblasts,where it acts as a proangiogenic factor in bone to increase the levels of skeletal vascular endothelium and thereby increase bone formation.Through these angiogenic effects,recombinant SLIT3 was found to have therapeutic activity in mouse models of fracture healing and postmenopausal osteoporosis.8,15Meanwhile,a separate study reported that SLIT3 is critical for skeletal physiology and also found osteopenia and a reduction in the skeletal vascular endothelium inSlit3-/-mice.9However,in this report,osteoclasts,as opposed to osteoblasts,were nominated as a key source of SLIT3 to control coupling between osteoblasts and osteoclasts.Given these con flicting data and the fundamental importance of SLIT3 as a promising osteoanabolic agent and physiologic signal linking bone metabolism to skeletal angiogenesis,further studies to clarify the cellular sources and targets of SLIT3 are needed.

RESULTS

The expression of SLIT3 in osteoclastogenesis

To clarify the cellular sources of SLIT3,we first examinedSlit3expression in parallel with other osteoclast makers using real-time PCRduring in vitro osteoclastogenesis.Osteoclast formation was further monitored by tartrate-resistant acid phosphatase(TRAP)staining(Fig.1a–c).Unlike the robustSlit3expression observed in the brain and primary osteoblasts,we were unable to detectSlit3mRNA expression during bone marrow macrophage(BMM)-derived osteoclastogenesis at the mRNA level(Fig.1b).To further con firm this observation,we analyzed RNA-sequencing(RNA-seq)transcriptional pro filing data from macrophages,osteoclasts,and osteoblasts derived from wild-type mice.This approach also showed thatSlit3expression in osteoclasts was negligible relative to its expression in osteoblasts.Western blotting using a polyclonal SLIT3 antibody validated for speci ficity(Fig.1e)also failed to detect SLIT3 expression during osteoclastogenesis(Fig.1f).These observations are consistent with those of prior analyses of axon guidance cue expression during osteoclast and osteoblast differentiation published by other groups.5,18Moreover,SLIT3 expression was also not detected in prior studies of human osteoclast differentiation.8,19Thus,we were unable to find evidence that SLIT3 is expressed in osteoclast lineage cells,which contrasts with the robust expression of SLIT3 observed in osteoblasts.8

The effect of recombinant SLIT3 on osteoclastogenesis in vitro To assess the effects of exogenous SLIT3 on osteoclastogenesis,we treated wild-type BMMs undergoing osteoclast differentiation with recombinant SLIT3 at a level that showed bioactivity in multiple other cellular assays(1 μg·mL-1),including tube formation assays in endothelial cells.8,11,15Although SLIT3 treatment modestly inhibited the expression of osteoclast marker genes,we were not able to observe a signi ficant change in the number of TRAP-positive osteoclasts compared with controls(Fig.2a–c).Similar negative results were obtained during a dose titration of SLIT3 during osteoclast differentiation (Fig.2d).Moreover,recombinant SLIT3 treatment did not affect the osteoclast activity during an in vitro mineral resorption assay(Fig.2e).These findings are consistent with our prior report that osteoclast numbers and serum levels of crosslinked C-telopeptide of type Icollagen(CTX),a marker of osteoclast activity,were not substantially altered inSlit3-/-mice.8

Deletion ofSlit3did not affect osteoclast progenitor cells in vivo To assess the role of SLIT3 in osteoclastogenesis in vivo,we first analyzed CD117+CD11bdimCD115+osteoclast precursors(OCPs)in maleSlit3-/-mice using flow cytometry and found no apparent differences in frequency(Fig.3a,b),20despite the severe osteopenic phenotype observed in these mice.Next,as osteoblast-derived SLIT3 is crucial for bone mass accrual,we asked whether osteoblast-derived SLIT3 would speci fically affect osteoclast precursors.To this end,we bredSlit3f/fmice to the Osxcre deleter strain in which osteoblasts were targeted(Slit3osx).Similar to observations made following the global deletion of SLIT3,the number of osteoclast precursors in maleSlit3osxmice was unaltered despite the severe osteopenia observed in these mice8(Fig.3c).Finally,to con firm the role of SLIT3 in osteoclasts,we bredSlit3f/fmice to a cre deleter strain in which mature osteoclasts were targeted,CTSK-cre(Slit3ctsk)mice.No apparent difference in the overall abundance of osteoclast precursors in maleSlit3ctskmice was observed,indicating that the deletion of SLIT3 in CTSK-positive cells did not disrupt osteoclast precursors(Fig.3d).Furthermore,fl uorescence-activated cell sorting(FACS)-isolated osteoclast precursors derived fromSlit3ctskmice displayed intact osteoclast differentiation capacity in vitro(Fig.3e).Taken together,these results indicate that SLIT3 is dispensable for the generation and homeostasis of osteoclast progenitors.

Bone mass and skeletal vasculature are normal inSlit3ctskmice In contrast with the growth retardation and perinatal lethality observed inSlit3-/-andSlit3osxmice,Slit3ctskmice developed normally without any gross abnormality or decrease in body weight(Fig.4a,b).Micro-CTshowed that neither male nor femaleSlit3ctskmice displayed detectable alterations in bone mass in either the trabecular or cortical compartments of long bones at 5 weeks of age relative to their littermate controls(Fig.4c,d).We further examined 4-month-old maleSlit3ctskmice versus littermate controls and also observed no detectable skeletal phenotype(Fig.4e).Consistent with this finding,histomorphometric analysis of the vertebral bone showed that the mineralization rate,bone formation rate,and numbers of osteoblasts and osteoclasts were also not affected inSlit3ctskmice.(Fig.4f).In addition,in contrast to the vascular phenotype observed inSlit3-/-andSlit3osxmice,neither the visualization of endothelial cells with CD31 or EMCN immuno fluorescence nor endothelial cell flow cytometry detected vascular alterations inSlit3ctskmice(Supplementary Fig.1a–c).8–9Taken together,these data indicate that the deletion of SLIT3 in CTSK-positive osteoclasts was unable to alter bone remodeling and skeletal vascular endothelium,in contrast to the strong effects on both of these parameters in osteoblast lineage cells observed with the deletion of SLIT3.Finally,to further evaluate the role of SLIT3 in osteoclast lineage cells,we bredSlit3f/fmice to the cre deleter strain LysM-cre in which osteoclasts were targeted in addition to macrophages and neutrophils(Slit3lysM).21Similar to observations inSlit3ctskmice,femaleSlit3lysMmice at 8 weeks of age did not show a discernible change in bone mass accrual(Fig.5a,b).

Ablation of SLIT3 in early osteoclast lineage cells leads to normal bone mass accrual

As CTSK-cre mediates gene deletion relatively late in osteoclast differentiation,22we created bone marrow chimeras fromSlit3-/-donors to assess the importance of SLIT3 production by early osteoclast lineage cells.23Bone marrow was collected from 6-week-old maleSlit3+/+mice andSlit3-/-mice and then injected into lethally irradiated 6-week-old male WTmice.ACD45.1/CD45.2 congenic system was used to validate the ef ficiency of hematopoietic reconstitution 8 weeks after bone marrow transplantation.Flow cytometric analysis showed that B220+cells,Gr1/CD11b+myeloid cells and osteoclast precursors all showed nearly 100%CD45.1+donor origin.CD3+cells were approximately 80%CD45.1+at this time point(Supplementary Fig.2 a–c).Thus,high levels of donor chimerism were observed,particularly within the osteoclast precursor compartment most relevant to this study.The resulting chimeras were maintained for 12 weeks before the analysis outlined in Fig.6a.Micro-CT analysis showed that the mice reconstituted with bone marrow cells isolated fromSlit3-/-mice had normal bone mass,unlike those reconstituted with WT bone marrow cells,as illustrated by analysis of the trabecular bone volume/total volume and cortical bone thickness(Fig.6a–c).Similarly,no alterations could be detected in the density of the bone vasculature in the recipients ofSlit3-/-bone marrow(Fig.6d).Consistent with this,the shRNA-mediated suppression of SLIT3 expression in BMMs had no effect on osteoclastogenesis in vitro(Supplementary Fig.3a–c).Collectively,the results of both the conditional deletion ofSlit3in osteoclast lineage cells and development of bone marrow chimeras lacking SLIT3 in their hematopoietic cells,including all of the osteoclast lineage cells,indicate that osteoclasts are not a physiologically relevant source of SLIT3.

DISCUSSION

Here,we address the discrepancies between our recent finding that osteoblasts are the major source of SLIT3 in the skeleton and another recent report nominating osteoclasts as the major source of SLIT3.In summary,multiple approaches were unable to provide evidence supporting osteoclast lineage cells as a source of SLIT3.Two separate cre lines mediating deletion in osteoclast lineage cells and bone marrow chimeras lacking SLIT3 expression in all osteoclast lineage cells displayed no detectable alteration in bone mass.These findings are in contrast to the robust phenotypes observed with either the osx-cre-or dmp1-cre-mediated deletion of SLIT3 in osteoblast lineage cells.8Consistent with the absence of an in vivo function of SLIT3 in osteoclasts,we were also unable to observe evidence of SLIT3 expression in osteoclasts,a finding corroborated by other studies.Taken together,our resultsindicate that osteoclasts are not a physiologically relevant source of SLIT3 under the conditions examined,leaving osteoblasts as the major source of SLIT3 in bone.

We previously reported that SLIT3 mediates its osteoanabolic effects by promoting skeletal angiogenesis.As osteoclast lineage cells have independent mechanisms to regulate the skeletal vasculature through the secretion of PDGF-BB,the finding that the angiogenic functions of SLIT3 are restricted to osteoblasts is signi ficant as it suggests that osteoblasts and osteoclasts each have orthogonal mechanisms to participate in the regulation of angiogenesis.This further opens the possibility of fundamental differences in osteoblast-regulated versus osteoclast-regulated vascular responses through the differential actions of SLIT3 versus PDGF-BB.23Moreover,if SLIT3 is primarily derived from osteoblasts,it is an attractive candidate as a key mediator of the angiogenic responses induced by osteoblast-targeting anabolic agents.

In a study by Kim et al,the deletion of SLIT3 using CTSK-cre resulted in modest osteopenia at 16 weeks of age.Here,however,we did not observe a similar bone phenotype in mice of the same age and sex as those in the Kim et al.report and also did not observe phenotypes with the other approaches used here to delete SLIT3 in osteoclasts.Notably,we previously observed robust phenotypeswith the cre lines used in this study.24–25Some of these discrepancies may be due to differences in theSlit3 floxed allele mice,as the two reports utilized different mouse lines,though the deletion strategies used in each report were similar.However,the finding that bone marrow chimeras made fromSlit3-/-donors failed to display skeletal phenotypes suggests that these discrepancies are not attributable solely to any idiosyncrasies of theSlit3 floxed allele utilized in the present report.

We are unable to fully exclude the possibility that osteoclasts are an additional target of SLIT3 activity based on the observation of modest effects on osteoclast gene expression after SLIT3 treatment.These effects may be mediated by the low-level expression of ROBO3 in osteoclasts.8However,we were unable to detect an overall effect on the endpoint of osteoclast differentiation based on the formation of TRAP+multinucleated osteoclasts in vitro or the number of either osteoclast progenitors or mature TRAP+osteoclasts in vivo.Similarly,we were previously unable to detect alterations in markers of osteoclast resorptive activity inSlit3-/-orSlit3osxmice displaying severe osteopenia.In the context of the robust angiogenic responses evoked by SLIT3 in vivo and in vitro,this finding reinforces our model that osteoblast-derived SLIT3 acts on endothelial cells to evoke osteoanabolic responses.The differences regarding the in vitro effects of SLIT3 between the report by Kim et al.and the present report may re flect differences in the utilized source of SLIT3 or other factors.Notably,in addition to the failure of exogenous SLIT3 to exert a robust effect,in vitro genetic approaches had no effect.By serving as an endothelium-targeted osteoanabolic agent,SLIT3 may have utility when combined with traditional osteoblast-or osteoclast-targeted agents.Given that increasing evidence indicates that the optimal therapy for disorders of low bone mass may require sequential or combination therapy with multiple agents,the development of osteoanabolic agents with orthogonal action at the cellular level would be highly desired.26–27

MATERIALSAND METHODS

Animals

OSX-Cre,LysM-Cre,CTSK-Cre,Slit3-/-,andSlit3f/fmice were obtained or generated as described in previous studies.8,21,25All mice were maintained under a standard 12 h dark–light cycle with chow ad libitum.All animal experiments were conducted according to the guidelinesapproved by the Weill Cornell Medical College subcommittee on animal care.

Osteoblast and osteoclast culture

Murine osteoclast differentiation assay was set up as previously described.28Brie fly,the whole bone marrow cells were flushed from the femora and tibiae dissected from the mutant mice and their littermates and plated on 10 cm dish for 3 days culture inα-MEM supplemented with 10% FBS in the presence of CMG14–12 supernatant as a source of M-CSF.Subsequently,we scraped the attached BMMs and seeded at a density of 4.5×104/cm2for osteoclast formation with the stimulation of RANKL(40 ng·mL-1).We evaluate osteoclastogenesis using TRAPstaining.Murine primary osteoblast differentiation assay was prepared as previously described.29

Osteoclast activity assay

Mineral resorption pit assay was used to evaluate the osteoclast activity as previously described.28Brie fly,BMMs were initially seeded on 96-well Corning Osteo Assay Surface Plates(Corning,Tewksbury,MA,USA)at a seeding density of 2×104per well.Subsequently, osteoclast differentiation was driven by CMG14–12 supernatant and 40 ng·mL-1RANKL.Once we observed mature osteoclasts,the cells on the plate were removed by adding 10%sodium hypochlorite solution.After twice PBS washing,von Kossa staining was performed to visualize resorptive pits.Light microscopic images were captured for each well and pit areas were quanti fied by Image J(National Institutes of Health,Bethesda,MD,USA).

Reverse transcription and real-time PCR

Total RNA(DNA-Free)was extracted from culture cells and tissues using an RNeasy Mini Kit(Qiagen,Germantown,MD,USA)and reverse transcription was carried on by First Strand cDNA Synthesis Kit(Thermo Fisher Scienti fic,Waltham,MA,USA).We performed real-time PCRusing Fast SYBRGreen Master Mix in the QuantStudio5 Real-Time PCRSystem(Applied Biosystems,Foster City, CA) according to the manufacturer’s instructions.Glyceraldehyde-3-phosphate dehydrogenase(Gapdh)or hypoxanthine guanine phosphoribosyl transferase(Hprt)was used as a control for normalization.The following primers were used:Slit3-forward,5′-TCCAGTGTTCCTGAAGGCTCCT-3′,andSlit3-reverse,5′-T GGCAATGCCAGGCTCCTTGTA-3′;Ctsk-forward,5′-AAGATATTGGTG GCTTTGG-3′,andCtsk-reverse,5′-ATCGCTGCGTCCCTCT-3′;Dcstam p-forward,5′-TTTGCCGCTGTGGACTATCTGC-3′,andDcstampreverse,5′-AGACGTGGTTTAGGAATGCAGCTC-3′;Nfatc1-forward,5′-CCCGTCACATTCTGGTCCAT-3′,andNfatc1-reverse,5′-CAAGTAAC CGTGTAGCTCCACAA-3′;Itgb3-forward,5′-CCGGGGGACTTAATGAG ACCACTT-3′,andItgb3-reverse,5′-ACGCCCCAAATCCCACCCATAC A-3′;Calcr-forward,5′-CTGAAGCTTGAGCGCCTGAGTC-3′,andCalcrreverse,5′-TGGGGTTGGGTGATTTAGAAGAAG-3′;Trap-forward,5′-A CCAGCAAGGATTGCGAGGCAT-3′,andTrap-reverse,5′-GGATGAC AGACGGTATCAGTGG-3′;Tnfrsf11b-forward,5′-CGGAAACAGAGAA GCCACGCAA-3′,andTnfrsf11b-reverse,5′-CTGTCCACCAAAACAC TCAGCC-3′;Gapdh-forward,5′-ATCAAGAAGGTGGTGAAGCA-3′,andGapdh-reverse,5′-GTCGCTGTTGAAGTCAGAGGA-3′;andHprt-forward,5′-CTGGTGAAAAGGACCTCTCGAAG-3′,andHprt-reverse,5′-C AAGATATCGTTGAAACGTGGA-3′.

Transcriptional expression pro filing by RNA-seq analysis

We puri fied total RNA using an RNeasy Mini Kit(Qiagen,Germantown,MD,USA)and used true-seq RNA Library preparation kits(Illumina,San Diego,CA,USA)to purify poly-A+transcripts and generate libraries with multiplexed barcode adapters according to the manufacturer’s protocols.After the quality of all samples passed control analysis using a Bioanalyzer 2100(Agilent,Lexington,MA,USA),we constructed RNA-seq libraries per the Illumina TrueSeq RNA sample preparation kit and carried out high-throughput sequencing using the Illumina HiSeq 4000 in the Genomics Resources Core Facility of Weill Cornell Medical College.For analysis,we used STAR(version 2.3.0e)30with the default parameters to align the reads to mm9 mouse transcripts.Then we sorted and indexed the resulting bam files using SAMtools.To obtain gene counts,we applied feature counts(version 1.4.3)31to sorted bam files and excluded the genes without any expression counts.We normalize gene count data using the DESeq2(version 1.4.5)Rpackage.32

Immunoblotting

The extracts from cultured cells and total tissue were prepared using RIPA buffer or a lysis buffer[150 mmol·L-1Tris-HCl(p H 6.8),6%SDS,30%glycerol,and 0.03%bromophenol blue]with 10%2-ME.The lysates were subjected to 7.5%SDS-PAGE and transferred to Immobilon-Pmembranes(Millipore,Billerica,MA,USA).The transferred membranes were blocked with 5%skimmed milk and incubated with speci fic antibodies.The following primary antibodies were used:SLIT3(1:1 000;R&D),Nfatc1(1:1 000;BD Pharmagen),c-Fos(1:1 000;Santa Cruz),p38(1:3 000;Santa Cruz),and HSP90(1:2 000;Sigma).We detected protein bands using Western Lightning plus-ECL(PerkinElmer,Waltham,MA,USA).

Skeletal analysis

The whole-body radiographs of all mice were taken by the Faxitron X-ray system.Three dimensional skeletal images and assessments of trabecular and cortical bone from distal femur were obtained by a Scanco MedicalμCT 35 system,which was calibrated weekly by scanning manufacturer-provided,resinembedded phantoms.All sample scans were performed in 70%ethanol with an isotropic voxel size of 7μm in the condition of an X-ray tube:55 kVp,0.145 mA,600 ms integration time.The threshold of trabecular bone was 211 per mille,which corresponds to~270 mg hydroxyapatite/ccm.The threshold of cortical bone was 350 per mille,which corresponds to~570 mg hydroxyapatite/ccm.We reduced noise in the thresholded images using a Gaussian noise filter applied for murine bone analysis.

Histology,histomorphometry,and immunohistochemistry

The mutant mice and relative controls were subcutaneously injected with a dose of 20 mg·kg-1on day 5 and 1 before scari fication.Plastic embedding,sectioning,TRAP staining,toluidine blue staining and von Kossa staining were conducted as previously described.4Histomorphometric analyses were carried out using the Osteomeasure Analysis System(OsteoMetrics,Atlanta,USA)as previously described29Frozen sectioning and immuno fluorescence staining were conducted according to a published protocol.33

Cell sorting and flow cytometry analysis

For OCP frequency analysis,bone marrow cells collected fromSlit3+/+andSlit3-/-mice,Slit3f/fandSlit3osxmice,Slit3f/fandSlit3ctskmice were flushed out and removed debris using a 70μm cell strainer.For skeletal endothelial cell analysis,the femurs and tibias fromSlit3f/fandSlit3ctskmice were isolated,and the surrounding muscles and connective tissues were cleaned.We crushed the bones in Hank’s balanced salt solution with 10 mmol·L-1HEPES.For the enzymatically digestion,2.5 mg·mL-1Collagenase A and 1 U·mL-1Dispase IIwere added and mixed thoroughly for 15 min incubation at 37°C with gentle agitation.Next,we added PBScontaining 0.5%FBSand 2mmol·L-1EDTAto stop digestion and the resulting suspensions filtered through a 40μm cell strainer and washed twice with PBS.After washing,antimouse CD16/CD32 antibody(BD)was used to block unspeci fic staining for 15min on ice.For OCPanalysis,the cells were stained with BUV395-conjugated B220 antibody(BD),PerCP-Cy5.5-conjugated CD11b(BioLegend),FITC-conjugated CD115(BioLegend)and PE/Cy7-conjugated CD117(BioLegend)for 30 min on ice.For skeletal endothelial cell analysis,the cells were stained with FITC-conjugated CD45(BioLegend),APC/Cy7-conjugated Ter119(Bio-Legend),PE-conjugated CD31(eBioscience)and APC-conjugated EMCN antibody(eBioscience)for 30 min on ice.DAPI solution(1 μg·mL-1)was used for live/dead exclusion.Cell sorting was performed with FACSAria II(BD,San Jose,CA,USA)and analyzed using FlowJo software(Tree Star,Ashland,OR,USA).

Bone marrow transplantation

We followed a published protocol for bone marrow transplantation.34Brie fly,recipient mice(6-week-old BALB/c mice)were irradiated with a lethal dose of 875 rads 1 day before transplantation.The whole bone marrow cells fromSlit3+/+andSlit3-/-mice were harvested,and 5 million bone marrow cells per donor mouse were transplanted into each irradiated recipient via intravenous tail vein injection.The resulting bone marrow chimeras were euthanized 16 weeks after transplantation.Congenic CD45.1/CD45.2 mice were used to validate the reconstitution ef ficiency of bone marrow transplantation in our protocol described in Supplementary 2.

Statistical methods

All data statistical analysis in our study was performed using GraphPad Prism software(v6.0a;GraphPad,La Jolla,CA,USA).Two-tailed Student’sttest was used to determine signi ficance for only two groups compare.One-way ANOVAwith Tukey’s post-hoc tests was used to determine signi ficance for compare between multiple groups.APvalue＜0.05 indicated statistical signi ficance.Error bars are presented as mean±s.e.m.

ACKNOWLEDGEMENTS

R.X.holds an Award Program for the Minjiang Scholar Professorship in Fujian Province,a start-up grant from Xiamen University and support from the National Natural Science Foundation of China(81972034 to RX).M.B.G.is supported by the NIH grant DP5OD021351 and the grant NF150055 from the Department of Defense,and holds a Career Award for Medical Scientistsfrom the Burroughs Wellcome Fund and a March of Dimes Basil O’Connor Award.B.Z.issupported by NIHgrants(R01AR068970,AR071463).We thank Dr Edward Fung and Dr Douglas Ballon from Citigroup Biomedical Imaging Center for their expert assistance with micro-CTstudies.

AUTHORCONTRIBUTIONS

R.X.,B.Z.,and M.B.G designed the experimental plan.N.L.and K.I.executed the experimental plan.Y.N.,J.S.,S.L.,A.Y.,X.Y.,and R.S.assisted with mouse studies.R.X.,B.Z.,and M.B.G.wrote the manuscript and supervised the project.B.Z.is a co-senior author.