Background:Skeletal tuberculosis(TB)remains a persistent clinical and research chal-lenge due to its chronic course,osteolytic destruction,and the limitations of existing animal models,which often require high-level b...Background:Skeletal tuberculosis(TB)remains a persistent clinical and research chal-lenge due to its chronic course,osteolytic destruction,and the limitations of existing animal models,which often require high-level biosafety containment or fail to repli-cate human skeletal pathology.Methods:This study developed a biosafe,accessible,and versatile murine model of skeletal TB using Mycobacterium smegmatis,a fast-growing,nonpathogenic myco-bacterial species with high genomic homology to Mycobacterium tuberculosis.Three infection routes-subperiosteal calvarial injection,intratibial injection,and intra-cardiac inoculation-were systematically evaluated for their ability to induce lo-calized versus disseminated bone infection under standard biosafety level(BSL)-1 conditions.Results:Subperiosteal calvarial and intratibial injection of M.smegmatis induced local-ized bone lesions characterized by osteolysis,sequestrum formation,granulomatous inflammation,and increased osteoclast activity.Intratibial infection additionally trig-gered compartment-specific immune responses,including neutrophil and macrophage expansion,transient B-cell depletion,and activation of interferon-γ^(+)(IFN-γ^(+))T cells,reflecting active immune remodeling at the infection site.Systemic dissemination via intracardiac injection reproducibly generated progressive vertebral and tibial bone destruction with organized granuloma formation and immune cell infiltration but without prominent sequestrum formation.Compared to intratibial infection,intracar-diac delivery exhibited lower intragroup variability and more closely recapitulated the diffuse progression of extrapulmonary skeletal tuberculosis.Conclusions:This M.smegmatis-based murine model provides a straightforward,reliable,and immunopathologically relevant platform for exploring host-pathogen dynamics,immune-driven bone destruction,and early-stage therapeutic testing in skeletal TB,all within standard BSL-1 laboratories.This model fills a critical gap by enabling BSL-1 research into skeletal TB mechanisms and drug development.展开更多
Osteoporosis(OP)is a common age-related disease characterized by a deterioration of bone mass and structure that predisposes patients to fragility fractures.Pharmaceutical therapies that promote anabolic bone formatio...Osteoporosis(OP)is a common age-related disease characterized by a deterioration of bone mass and structure that predisposes patients to fragility fractures.Pharmaceutical therapies that promote anabolic bone formation in OP patients and OP-induced fracture are needed.We investigated whether a neutralizing antibody against Siglec-15 can simultaneously inhibit bone resorption and stimulate bone formation.We found that the multinucleation of osteoclasts was inhibited in SIGLEC-15 conditional knockout mice and mice undergoing Siglec-15 neutralizing antibody treatment.The secretion of platelet-derived growth factor-BB(PDGF-BB),the number of tartrate-resistant acid phosphatase-positive(TRAP+)mononuclear cells,and bone formation were significantly increased in the SIGLEC-15 conditional knockout mice and antibody-treated mice.The anabolic effect of the Siglec-15 neutralizing antibody on bone formation was blunted in mice with Pdgfb deleted in TRAP-1"cells.These findings showed that the anabolic effect of the Siglec-15 neutralizing antibody was mediated by elevating PDGF-BB production of TRAP4 mononuclear cells.To test the therapeutic potential of the Siglec-15 neutralizing antibody,we injected the antibody in an ovariectomy-induced osteoporotic mouse model,which mimics postmenopausal osteoporosis in women,and in two fracture healing models because fracture is the most serious health consequence of osteoporosis.The Siglec-15 neutralizing antibody effectively reduced bone resorption and stimulated bone formation in estrogen deficiency-induced osteoporosis.Of note,the Siglec-15 neutralizing antibody promoted intramembranous and endochondral ossification at the damaged area of cortical bone in fracture healing mouse models.Thus,the Siglec-15 neutralizing antibody shows significant translational potential as a novel therapy for OP and bone fracture.展开更多
Bone remodeling is precisely coordinated by bone resorption and formation.Apoptotic osteoclasts generate large amounts of apoptotic bodies(ABs)marking the end of the bone resorption phase,whereas the functions of oste...Bone remodeling is precisely coordinated by bone resorption and formation.Apoptotic osteoclasts generate large amounts of apoptotic bodies(ABs)marking the end of the bone resorption phase,whereas the functions of osteoclast-derived ABs remain largely unknown.Here,we identified the molecular profile of ABs derived from osteoclasts at distinct differentiation stages and investigated their corresponding functions.ABs were isolated from apoptotic bone marrow macrophages,preosteoclasts,and mature osteoclasts induced by staurosporine.Proteomic signature analysis with liquid chromatography-tandem mass spectrometry suggested marked protein cargo differences among the different ABs.Further bioinformatic analysis showed that the proteomic signatures of the ABs were highly similar to those of their parental cells.Functionally,pOC-ABs induced endothelial progenitor cell differentiation and increased CD31hiEmcnhi endothelial cell formation in a murine bone defect model via their PDGF-BB cargo.mOC-ABs induced osteogenic differentiation of mesenchymal stem cells and facilitated osteogenesis via RANKL reverse signaling.In summary,we mapped the detailed proteomic landscapes of ABs derived from osteoclasts and showed that their potential biological roles are important in coupling bone formation with resorption during bone remodeling.展开更多
The molecular control of osteoclast formation is still not clearly elucidated. Here, we show that a process of cell recognition mediated by Siglec15-TLR2 binding is indispensable and occurs prior to cell fusion in RAN...The molecular control of osteoclast formation is still not clearly elucidated. Here, we show that a process of cell recognition mediated by Siglec15-TLR2 binding is indispensable and occurs prior to cell fusion in RANKL-mediated osteoclastogenesis. Siglec15 has been shown to regulate osteoclastic bone resorption. However, the receptor for Siglec15 has not been identified, and the signaling mechanism involving Siglec15 in osteoclast function remains unclear. We found that Siglec15 bound sialylated TLR2 as its receptor and that the binding of sialylated TLR2 to Siglec15 in macrophages committed to the osteoclast-lineage initiated cell fusion for osteoclast formation, in which sialic acid was transferred by the sialyltransferase ST3 Gal1. Interestingly, the expression of Siglec15 in macrophages was activated by M-CSF, whereas ST3 Gal1 expression was induced by RANKL. Both Siglec15-specific deletion in macrophages and intrafemoral injection of sialidase abrogated cell recognition and reduced subsequent cell fusion for the formation of osteoclasts, resulting in increased bone formation in mice. Thus, our results reveal that cell recognition mediated by the binding of sialylated TLR2 to Siglec15 initiates cell fusion for osteoclast formation.展开更多
Antiresorptive drugs are widely used for treatment of osteoporosis and cancer bone metastasis,which function mainly through an overall inhibition of osteoclast.However,not all osteoclasts are“bone eaters”;preosteocl...Antiresorptive drugs are widely used for treatment of osteoporosis and cancer bone metastasis,which function mainly through an overall inhibition of osteoclast.However,not all osteoclasts are“bone eaters”;preosteoclasts(pOCs)play anabolic roles in bone formation and angiogenesis through coupling with osteoblasts and secreting platelet derived growth factor-BB(PDGF-BB).In this study,a bone-targeted pH-responsive nanomaterial was designed for selectively eliminating mature osteoclasts(mOCs)without affecting pOCs.Biocompatible cerium nano-system(CNS)was guided to the acidic extracellular microenvironment created by mOCs and gained oxidative enzymatic activity.Oxidative CNS decreased the viability of mOCs through accumulating intracellular reactive oxygen species and enhancing calcium oscillation.Non-acid secreting anabolic pOCs were thus preserved and kept producing PDGF-BB,which lead to mesenchymal stem cell osteogenesis and endothelial progenitor cell angiogenesis via PI3K-Akt activated focal adhesion kinase.In treating osteoporotic ovariectomized mice,CNS showed better protective effects compare with the current first line antiresorptive drug due to the better anabolic effects marked by higher level of bone formation and vascularization.We provided a novel anabolic therapeutic strategy in treating bone disorders with excessive bone resorption.展开更多
Estrogen deficiency after menopause accelerates bone loss by stimulating osteoclast formation and activity,but the molecular pathways that link estrogen signaling to osteoclast regulation remain incompletely defined.H...Estrogen deficiency after menopause accelerates bone loss by stimulating osteoclast formation and activity,but the molecular pathways that link estrogen signaling to osteoclast regulation remain incompletely defined.Here,we identify the sialyltransferase ST3GAL-I as a key mediator of RANKL-induced osteoclastogenesis.RANKL activates c-FOS to drive ST3GAL1 transcription,whereas estrogen-bound ERαcompetes with TRAF6 and suppresses this c-FOS–dependent induction.In a clinical cohort of pre-menopausal and post-menopausal women with or without osteoporosis,serum total andα-2,3-linked sialic acid levels increased with age and were highest in post-menopausal osteoporotic patients.Single-cell RNA sequencing of human bone revealed that osteoclasts form a prominent cluster only after menopause,where FOS,CTSK,and ST3GAL1 are strongly co-expressed,and the estrogen-responsive gene PGR is down-regulated.Additionally,in vivo experiments showed that sialidase treatment in estrogen-deficient models effectively reduced osteoclast-mediated bone loss,mimicking the effects of estradiol.These findings define a direct molecular link between loss of estrogen and activation of a FOS–ST3GAL1 sialylation pathway in osteoclasts,providing mechanistic insight into the enhanced bone resorption characteristic of post-menopausal osteoporosis.展开更多
Endochondral bone formation is an important route for bone repair.Although emerging evidence has revealed the functions of long non-coding RNAs(lncRNAs)in bone and cartilage development,the effect of lncRNAs in endoch...Endochondral bone formation is an important route for bone repair.Although emerging evidence has revealed the functions of long non-coding RNAs(lncRNAs)in bone and cartilage development,the effect of lncRNAs in endochondral bone repair is still largely unknown.Here,we identified a lncRNA,named Hypertrophic Chondrocyte Angiogenesis-related lncRNA(HCAR),and proved it to promote the endochondral bone repair by upregulating the expression of matrix metallopeptidase 13(Mmp13)and vascular endothelial growth factorα(Vegfa)in hypertrophic chondrocytes.Lnc-HCAR knockdown in hypertrophic chondrocytes restrained the cartilage matrix remodeling and decrease the CD31hiEmcnhi vessels number in a bone repair model.Mechanistically,we proved that lnc-HCAR was mainly enriched in the cytoplasm using fluorescence in situ hybridization(FISH)assay,and it acted as a molecular sponge for miR-15b-5p.Further,in hypertrophic chondrocytes,lnc-HCAR competitively bound to miR-15b-5p to increase Vegfa and Mmp13 expression.Our results proved that lncRNA is deeply involved in endochondral bone repair,which will provide a new theoretical basis for future strategies for promoting fracture healing.展开更多
The skeletal system is a dynamically balanced system, which undergoes continuous bone resorption and formation to maintain bone matrix homeostasis. As an important ADP-ribosylase and NAD+-dependent deacylase, SIRT6 (S...The skeletal system is a dynamically balanced system, which undergoes continuous bone resorption and formation to maintain bone matrix homeostasis. As an important ADP-ribosylase and NAD+-dependent deacylase, SIRT6 (SIR2-like protein 6) is widely expressed on various kinds of bone cells, such as chondrocytes, osteoblasts, osteoclasts. The aberration of SIRT6 impairs gene expression (e.g., NF-κB and Wnt target genes) and cellular functions (e.g., DNA repair, glucose and lipid metabolism, telomeric maintenance), which disturbs the dynamic balance and ultimately leads to several bone-related diseases. In this review, we summarize the critical roles of SIRT6 in the onset and progression of bone-related diseases including osteoporosis, osteoarthritis, rheumatoid arthritis, and intervertebral disc degeneration, as well as the relevant signaling pathways. In addition, we discuss the advances in the development of SIRT6 activators and elucidate their pharmacological profiles, which may provide novel treatment strategies for these skeletal diseases.展开更多
基金Southwest Hospital Boqing Innovation Fund,Grant/Award Number:2024BQCXJJ-9Fundings for Young Investigators of PLA,Grant/Award Number:2022-JCJQ-QT-004+3 种基金NSFC Key Projects of the Regional Innovation and Development Joint Fund,Grant/Award Number:U23A20413China Postdoctoral Science Foundation,Grant/Award Number:2023M744280National Natural Science Foundation of China,Grant/Award Number:82103778,82172449 and 82172489Southwest Hospital Postdoctoral Starting Fund,Grant/Award Number:5175ZA36BP。
文摘Background:Skeletal tuberculosis(TB)remains a persistent clinical and research chal-lenge due to its chronic course,osteolytic destruction,and the limitations of existing animal models,which often require high-level biosafety containment or fail to repli-cate human skeletal pathology.Methods:This study developed a biosafe,accessible,and versatile murine model of skeletal TB using Mycobacterium smegmatis,a fast-growing,nonpathogenic myco-bacterial species with high genomic homology to Mycobacterium tuberculosis.Three infection routes-subperiosteal calvarial injection,intratibial injection,and intra-cardiac inoculation-were systematically evaluated for their ability to induce lo-calized versus disseminated bone infection under standard biosafety level(BSL)-1 conditions.Results:Subperiosteal calvarial and intratibial injection of M.smegmatis induced local-ized bone lesions characterized by osteolysis,sequestrum formation,granulomatous inflammation,and increased osteoclast activity.Intratibial infection additionally trig-gered compartment-specific immune responses,including neutrophil and macrophage expansion,transient B-cell depletion,and activation of interferon-γ^(+)(IFN-γ^(+))T cells,reflecting active immune remodeling at the infection site.Systemic dissemination via intracardiac injection reproducibly generated progressive vertebral and tibial bone destruction with organized granuloma formation and immune cell infiltration but without prominent sequestrum formation.Compared to intratibial infection,intracar-diac delivery exhibited lower intragroup variability and more closely recapitulated the diffuse progression of extrapulmonary skeletal tuberculosis.Conclusions:This M.smegmatis-based murine model provides a straightforward,reliable,and immunopathologically relevant platform for exploring host-pathogen dynamics,immune-driven bone destruction,and early-stage therapeutic testing in skeletal TB,all within standard BSL-1 laboratories.This model fills a critical gap by enabling BSL-1 research into skeletal TB mechanisms and drug development.
基金This research was partially supported by a grant from NextCure,Inc.and the NIH National Institute on Aging under Award Number P01AG066603.
文摘Osteoporosis(OP)is a common age-related disease characterized by a deterioration of bone mass and structure that predisposes patients to fragility fractures.Pharmaceutical therapies that promote anabolic bone formation in OP patients and OP-induced fracture are needed.We investigated whether a neutralizing antibody against Siglec-15 can simultaneously inhibit bone resorption and stimulate bone formation.We found that the multinucleation of osteoclasts was inhibited in SIGLEC-15 conditional knockout mice and mice undergoing Siglec-15 neutralizing antibody treatment.The secretion of platelet-derived growth factor-BB(PDGF-BB),the number of tartrate-resistant acid phosphatase-positive(TRAP+)mononuclear cells,and bone formation were significantly increased in the SIGLEC-15 conditional knockout mice and antibody-treated mice.The anabolic effect of the Siglec-15 neutralizing antibody on bone formation was blunted in mice with Pdgfb deleted in TRAP-1"cells.These findings showed that the anabolic effect of the Siglec-15 neutralizing antibody was mediated by elevating PDGF-BB production of TRAP4 mononuclear cells.To test the therapeutic potential of the Siglec-15 neutralizing antibody,we injected the antibody in an ovariectomy-induced osteoporotic mouse model,which mimics postmenopausal osteoporosis in women,and in two fracture healing models because fracture is the most serious health consequence of osteoporosis.The Siglec-15 neutralizing antibody effectively reduced bone resorption and stimulated bone formation in estrogen deficiency-induced osteoporosis.Of note,the Siglec-15 neutralizing antibody promoted intramembranous and endochondral ossification at the damaged area of cortical bone in fracture healing mouse models.Thus,the Siglec-15 neutralizing antibody shows significant translational potential as a novel therapy for OP and bone fracture.
基金This work was funded by a grant from the National Natural Science Foundation of China(81802166)a grant from the State Key Program of National Natural Science of China(81930067)a first-class General Financial Grant from the China Postdoctoral Science Foundation(2017M613315).
文摘Bone remodeling is precisely coordinated by bone resorption and formation.Apoptotic osteoclasts generate large amounts of apoptotic bodies(ABs)marking the end of the bone resorption phase,whereas the functions of osteoclast-derived ABs remain largely unknown.Here,we identified the molecular profile of ABs derived from osteoclasts at distinct differentiation stages and investigated their corresponding functions.ABs were isolated from apoptotic bone marrow macrophages,preosteoclasts,and mature osteoclasts induced by staurosporine.Proteomic signature analysis with liquid chromatography-tandem mass spectrometry suggested marked protein cargo differences among the different ABs.Further bioinformatic analysis showed that the proteomic signatures of the ABs were highly similar to those of their parental cells.Functionally,pOC-ABs induced endothelial progenitor cell differentiation and increased CD31hiEmcnhi endothelial cell formation in a murine bone defect model via their PDGF-BB cargo.mOC-ABs induced osteogenic differentiation of mesenchymal stem cells and facilitated osteogenesis via RANKL reverse signaling.In summary,we mapped the detailed proteomic landscapes of ABs derived from osteoclasts and showed that their potential biological roles are important in coupling bone formation with resorption during bone remodeling.
基金supported by US National Institutes of Health grants AR 071432 (to X.C.)
文摘The molecular control of osteoclast formation is still not clearly elucidated. Here, we show that a process of cell recognition mediated by Siglec15-TLR2 binding is indispensable and occurs prior to cell fusion in RANKL-mediated osteoclastogenesis. Siglec15 has been shown to regulate osteoclastic bone resorption. However, the receptor for Siglec15 has not been identified, and the signaling mechanism involving Siglec15 in osteoclast function remains unclear. We found that Siglec15 bound sialylated TLR2 as its receptor and that the binding of sialylated TLR2 to Siglec15 in macrophages committed to the osteoclast-lineage initiated cell fusion for osteoclast formation, in which sialic acid was transferred by the sialyltransferase ST3 Gal1. Interestingly, the expression of Siglec15 in macrophages was activated by M-CSF, whereas ST3 Gal1 expression was induced by RANKL. Both Siglec15-specific deletion in macrophages and intrafemoral injection of sialidase abrogated cell recognition and reduced subsequent cell fusion for the formation of osteoclasts, resulting in increased bone formation in mice. Thus, our results reveal that cell recognition mediated by the binding of sialylated TLR2 to Siglec15 initiates cell fusion for osteoclast formation.
基金This work was funded by the State Key Program of National Natural Science of China(No.81930067)the Nature Science Foundation of China(81802166)AMU Southwest Hospital funding for young investigators(XZ-2019-505-005).
文摘Antiresorptive drugs are widely used for treatment of osteoporosis and cancer bone metastasis,which function mainly through an overall inhibition of osteoclast.However,not all osteoclasts are“bone eaters”;preosteoclasts(pOCs)play anabolic roles in bone formation and angiogenesis through coupling with osteoblasts and secreting platelet derived growth factor-BB(PDGF-BB).In this study,a bone-targeted pH-responsive nanomaterial was designed for selectively eliminating mature osteoclasts(mOCs)without affecting pOCs.Biocompatible cerium nano-system(CNS)was guided to the acidic extracellular microenvironment created by mOCs and gained oxidative enzymatic activity.Oxidative CNS decreased the viability of mOCs through accumulating intracellular reactive oxygen species and enhancing calcium oscillation.Non-acid secreting anabolic pOCs were thus preserved and kept producing PDGF-BB,which lead to mesenchymal stem cell osteogenesis and endothelial progenitor cell angiogenesis via PI3K-Akt activated focal adhesion kinase.In treating osteoporotic ovariectomized mice,CNS showed better protective effects compare with the current first line antiresorptive drug due to the better anabolic effects marked by higher level of bone formation and vascularization.We provided a novel anabolic therapeutic strategy in treating bone disorders with excessive bone resorption.
基金funded by a grant from the National Natural Science Foundation of China(82572785,82172489,82172449)funding for young investigators of PLA(2022-JCJQ-QT-004)。
文摘Estrogen deficiency after menopause accelerates bone loss by stimulating osteoclast formation and activity,but the molecular pathways that link estrogen signaling to osteoclast regulation remain incompletely defined.Here,we identify the sialyltransferase ST3GAL-I as a key mediator of RANKL-induced osteoclastogenesis.RANKL activates c-FOS to drive ST3GAL1 transcription,whereas estrogen-bound ERαcompetes with TRAF6 and suppresses this c-FOS–dependent induction.In a clinical cohort of pre-menopausal and post-menopausal women with or without osteoporosis,serum total andα-2,3-linked sialic acid levels increased with age and were highest in post-menopausal osteoporotic patients.Single-cell RNA sequencing of human bone revealed that osteoclasts form a prominent cluster only after menopause,where FOS,CTSK,and ST3GAL1 are strongly co-expressed,and the estrogen-responsive gene PGR is down-regulated.Additionally,in vivo experiments showed that sialidase treatment in estrogen-deficient models effectively reduced osteoclast-mediated bone loss,mimicking the effects of estradiol.These findings define a direct molecular link between loss of estrogen and activation of a FOS–ST3GAL1 sialylation pathway in osteoclasts,providing mechanistic insight into the enhanced bone resorption characteristic of post-menopausal osteoporosis.
基金This work was supported by Key Program of Natural Science Foundation of China(No.81930067)General Program of Nature Science Foundation of China(No.31870962)+2 种基金the Key Project of Logistics Research Plan of the PLA(No.AWS17J004-02-06)the Medical Science and Technology Youth Cultivation Project of PLA(No.20QNPY022)Medical innovation capability upgrading Plan of Southwest Hospital(No.SWH2018LJ-03).
文摘Endochondral bone formation is an important route for bone repair.Although emerging evidence has revealed the functions of long non-coding RNAs(lncRNAs)in bone and cartilage development,the effect of lncRNAs in endochondral bone repair is still largely unknown.Here,we identified a lncRNA,named Hypertrophic Chondrocyte Angiogenesis-related lncRNA(HCAR),and proved it to promote the endochondral bone repair by upregulating the expression of matrix metallopeptidase 13(Mmp13)and vascular endothelial growth factorα(Vegfa)in hypertrophic chondrocytes.Lnc-HCAR knockdown in hypertrophic chondrocytes restrained the cartilage matrix remodeling and decrease the CD31hiEmcnhi vessels number in a bone repair model.Mechanistically,we proved that lnc-HCAR was mainly enriched in the cytoplasm using fluorescence in situ hybridization(FISH)assay,and it acted as a molecular sponge for miR-15b-5p.Further,in hypertrophic chondrocytes,lnc-HCAR competitively bound to miR-15b-5p to increase Vegfa and Mmp13 expression.Our results proved that lncRNA is deeply involved in endochondral bone repair,which will provide a new theoretical basis for future strategies for promoting fracture healing.
文摘The skeletal system is a dynamically balanced system, which undergoes continuous bone resorption and formation to maintain bone matrix homeostasis. As an important ADP-ribosylase and NAD+-dependent deacylase, SIRT6 (SIR2-like protein 6) is widely expressed on various kinds of bone cells, such as chondrocytes, osteoblasts, osteoclasts. The aberration of SIRT6 impairs gene expression (e.g., NF-κB and Wnt target genes) and cellular functions (e.g., DNA repair, glucose and lipid metabolism, telomeric maintenance), which disturbs the dynamic balance and ultimately leads to several bone-related diseases. In this review, we summarize the critical roles of SIRT6 in the onset and progression of bone-related diseases including osteoporosis, osteoarthritis, rheumatoid arthritis, and intervertebral disc degeneration, as well as the relevant signaling pathways. In addition, we discuss the advances in the development of SIRT6 activators and elucidate their pharmacological profiles, which may provide novel treatment strategies for these skeletal diseases.
