CD97 inhibits osteoclast differentiation via Rap1a/ERK pathway under compression

Acceleration of tooth movement during orthodontic treatment is challenging, with osteoclast-mediated bone resorption on the compressive side being the rate-limiting step. Recent studies have demonstrated that mechanoreceptors on the surface of monocytes/macrophages, especially adhesion G protein-coupled receptors (aGPCRs), play important roles in force sensing.However, its role in the regulation of osteoclast differentiation remains unclear. Herein, through single-cell analysis, we revealed that CD97, a novel mechanosensitive aGPCR, was expressed in macrophages. Compression upregulated CD97 expression and inhibited osteoclast differentiation;while knockdown of CD97 partially rescued osteoclast differentiation. It suggests that CD97 may be an important mechanosensitive receptor during osteoclast differentiation. RNA sequencing analysis showed that the Rap1a/ERK signalling pathway mediates the effects of CD97 on osteoclast differentiation under compression. Consistently, we clarified that administration of the Rap1a inhibitor GGTI298 increased osteoclast activity, thereby accelerating tooth movement. In conclusion,our results indicate that CD97 suppresses osteoclast differentiation through the Rap1a/ERK signalling pathway under orthodontic compressive force.

Transcriptional reprogramming during human osteoclast differentiation identifies regulators of osteoclast activity

Enhanced osteoclastogenesis and osteoclast activity contribute to the development of osteoporosis,which is characterized by increased bone resorption and inadequate bone formation.As novel antiosteoporotic therapeutics are needed,understanding the genetic regulation of human osteoclastogenesis could help identify potential treatment targets.This study aimed to provide an overview of transcriptional reprogramming during human osteoclast differentiation.Osteoclasts were differentiated from CD14+monocytes from eight female donors.RNA sequencing during differentiation revealed 8980 differentially expressed genes grouped into eight temporal patterns conserved across donors.These patterns revealed distinct molecular functions associated with postmenopausal osteoporosis susceptibility genes based on RNA from iliac crest biopsies and bone mineral density SNPs.Network analyses revealed mutual dependencies between temporal expression patterns and provided insight into subtype-specific transcriptional networks.The donor-specific expression patterns revealed genes at the monocyte stage,such as filamin B(FLNB)and oxidized low-density lipoprotein receptor 1(OLR1,encoding LOX-1),that are predictive of the resorptive activity of mature osteoclasts.The expression of differentially expressed G-protein coupled receptors was strong during osteoclast differentiation,and these receptors are associated with bone mineral density SNPs,suggesting that they play a pivotal role in osteoclast differentiation and activity.The regulatory effects of three differentially expressed G-protein coupled receptors were exemplified by in vitro pharmacological modulation of complement 5 A receptor 1(C5AR1),somatostatin receptor 2(SSTR2),and free fatty acid receptor 4(FFAR4/GPR120).Activating C5AR1 enhanced osteoclast formation,while activating SSTR2 decreased the resorptive activity of mature osteoclasts,and activating FFAR4 decreased both the number and resorptive activity of mature osteoclasts.In conclusion,we report the occurrence of transcriptional reprogramming during human osteoclast differentiation and identified SSTR2 and FFAR4 as antiresorptive G-protein coupled receptors and FLNB and LOX-1 as potential molecular markers of osteoclast activity.These data can help future investigations identify molecular regulators of osteoclast differentiation and activity and provide the basis for novel antiosteoporotic targets.

Involvement of Siglec-15 in regulating RAP1/RAC signaling in cytoskeletal remodeling in osteoclasts mediated by macrophage colony-stimulating factor

DNAX-associated protein 12 kD size(DAP12)is a dominant immunoreceptor tyrosine-based activation motif(ITAM)-signaling adaptor that activates costimulatory signals essential for osteoclastogenesis.Although several DAP12-associated receptors(DARs)have been identified in osteoclasts,including triggering receptor expressed on myeloid cells 2(TREM-2),C-type lectin member 5 A(CLEC5A),and sialic acid-binding Ig-like lectin(Siglec)-15,their precise role in the development of osteoclasts and bone remodeling remain poorly understood.In this study,mice deficient in Trem-2,Clec5a,Siglec-15 were generated.

RUFY4 deletion prevents pathological bone loss by blocking endo-lysosomal trafficking of osteoclasts

Mature osteoclasts degrade bone matrix by exocytosis of active proteases from secretory lysosomes through a ruffled border.However,the molecular mechanisms underlying lysosomal trafficking and secretion in osteoclasts remain largely unknown.Here,we show with GeneChip analysis that RUN and FYVE domain-containing protein 4(RUFY4)is strongly upregulated during osteoclastogenesis.Mice lacking Rufy4 exhibited a high trabecular bone mass phenotype with abnormalities in osteoclast function in vivo.Furthermore,deleting Rufy4 did not affect osteoclast differentiation,but inhibited bone-resorbing activity due to disruption in the acidic maturation of secondary lysosomes,their trafficking to the membrane,and their secretion of cathepsin K into the extracellular space.Mechanistically,RUFY4 promotes late endosome-lysosome fusion by acting as an adaptor protein between Rab7 on late endosomes and LAMP2 on primary lysosomes.Consequently,Rufy4-deficient mice were highly protected from lipopolysaccharide-and ovariectomy-induced bone loss.Thus,RUFY4 plays as a new regulator in osteoclast activity by mediating endo-lysosomal trafficking and have a potential to be specific target for therapies against bone-loss diseases such as osteoporosis.

DEC1 deficiency protects against bone loss induced by ovariectomy by inhibiting inflammation

Studies have shown that differentiated embryo-chondrocyte expressed gene 1(DEC1)promotes osteoblast osteogenesis.To investigate the role of DEC1 in postmenopausal osteoporosis,we used the two genotypes of mice(Dec1+/+and Dec1-/-)to establish an ovariectomy model and found that the bone loss was significantly lower in Dec1-/-ovariectomy mice than in Dec1+/+ovariectomy mice.The expression levels of RUNX2 and OSX were significantly increased in Dec1-/-ovariectomy mice,compared with Dec1+/+ovariectomy mice;however,the expression levels of NFATc1,c-Fos,CTSK,and RANKL/OPG ratio were significantly decreased in Dec1-/-ovariectomy mice,compared with those in Dec1+/+ovariectomy mice.Likewise,DEC1 deficiency also suppressed the expression levels of IL-6 and IL-1β.Further results showed that the mRNA expression levels of Runx2,Osx,and Alp were significantly increased in bone marrow mesenchymal stem cells of Dec1-/-ovariectomy mice,compared with those of Dec1+/+ovariectomy mice.Moreover,the mRNA levels of Il1b,Il6,Tnfa,and Ifng were significantly increased in bone marrow-derived macrophages(BMMs)of Dec1+/+ovariectomy mice,compared with those of Dec1+/+sham mice,but not in Dec1-/-ovariectomy BMMs,when compared with those in Dec1-/-sham BMMs.Additionally,the expression levels of p-IκBαand p-P65 were significantly increased in Dec1+/+ovariectomy BMMs,compared with those in Dec1+/+sham BMMs,but did not increase in Dec1-/-ovariectomy BMMs,compared with those in Dec1-/-sham BMMs.Taken together,DEC1 deficiency inhibited the NF-κB pathway induced by ovariectomy,thereby decreasing cytokines and subsequently inhibiting the decrease of osteo-genesis and the increase of osteoclastogenesis caused by ovariectomy.The findings may provide a novel under-standing of postmenopausal osteoporosis development,and offer potential avenues for the disease intervention.

New insights into inflammatory osteoclast precursors as therapeutic targets for rheumatoid arthritis and periodontitis

Rheumatoid arthritis(RA)and periodontitis are chronic inflammatory diseases leading to increased bone resorption.Preventing this inflammatory bone resorption is a major health challenge.Both diseases share immunopathogenic similarities and a common inflammatory environment.The autoimmune response or periodontal infection stimulates certain immune actors,leading in both cases to chronic inflammation that perpetuates bone resorption.Moreover,RA and periodontitis have a strong epidemiological association that could be explained by periodontal microbial dysbiosis.This dysbiosis is believed to be involved in the initiation of RA via three mechanisms.(i)The dissemination of periodontal pathogens triggers systemic inflammation.(ii)Periodontal pathogens can induce the generation of citrullinated neoepitopes,leading to the generation of anti-citrullinated peptide autoantibodies.(iii)Intracellular danger-associated molecular patterns accelerate local and systemic inflammation.Therefore,periodontal dysbiosis could promote or sustain bone resorption in distant inflamed joints.Interestingly,in inflammatory conditions,the existence of osteoclasts distinct from“classical osteoclasts”has recently been reported.They have proinflammatory origins and functions.Several populations of osteoclast precursors have been described in RA,such as classical monocytes,a dendritic cell subtype,and arthritis-associated osteoclastogenic macrophages.The aim of this review is to synthesize knowledge on osteoclasts and their precursors in inflammatory conditions,especially in RA and periodontitis.Special attention will be given to recent data related to RA that could be of potential value in periodontitis due to the immunopathogenic similarities between the two diseases.Improving our understanding of these pathogenic mechanisms should lead to the identification of new therapeutic targets involved in the pathological inflammatory bone resorption associated with these diseases.

Role of Iron Accumulation in Osteoporosis and the Underlying Mechanisms

Osteoporosis is prevalent in postmenopausal women.The underlying reason is mainly estrogen deficiency,but recent studies have indicated that osteoporosis is also associated with iron accumulation after menopause.It has been confirmed that some methods of decreasing iron accumulation can improve the abnormal bone metabolism associated with postmenopausal osteoporosis.However,the mechanism of iron accumulation-induced osteoporosis is still unclear.Iron accumulation may inhibit the canonical Wnt/β-catenin pathway via oxidative stress,leading to osteoporosis by decreasing bone formation and increasing bone resorption via the osteoprotegerin(OPG)/receptor activator of nuclear factor kappa-B ligand(RANKL)/receptor activator of nuclear factor kappa-B(RANK)system.In addition to oxidative stress,iron accumulation also has been reported to inhibit either osteoblastogenesis or osteoblastic function as well as to stimulate either osteoclastogenesis or osteoclastic function directly.Furthermore,serum ferritin has been widely used for the prediction of bone status,and nontraumatic measurement of iron content by magnetic resonance imaging may be a promising early indicator of postmenopausal osteoporosis.

IgSF11-mediated phosphorylation of pyruvate kinase M2 regulates osteoclast differentiation and prevents pathological bone loss

Osteoclasts are primary bone-resorbing cells,and receptor-activated NF-k B ligand(RANKL)stimulation is the key driver of osteoclast differentiation.During late-stage differentiation,osteoclasts become multinucleated and enlarged(so-called“maturation”),suggesting their need to adapt to changing metabolic demands and a substantial increase in size.Here,we demonstrate that immunoglobulin superfamily 11(Ig SF11),which is required for osteoclast differentiation through an association with the postsynaptic scaffolding protein PSD-95,regulates osteoclast differentiation by controlling the activity of pyruvate kinase M isoform2(PKM2).By using a system that directly induces the activation of Ig SF11 in a controlled manner,we identified PKM2 as a major Ig SF11-induced tyrosine-phosphorylated protein.Ig SF11 activates multiple Src family tyrosine kinases(SFKs),including c-Src,Fyn,and Hc K,which phosphorylate PKM2 and thereby inhibit PKM2 activity.Consistently,Ig SF11-deficient cells show higher PKM2activity and defective osteoclast differentiation.Furthermore,inhibiting PKM2 activities with the specific inhibitor Shikonin rescues the impaired osteoclast differentiation in Ig SF11-deficient cells,and activating PKM2 with the specific activator TEPP46 suppresses osteoclast differentiation in wild-type cells.Moreover,PKM2 activation further suppresses osteoclastic bone loss without affecting bone formation in vivo.Taken together,these results show that Ig SF11 controls osteoclast differentiation through PKM2 activity,which is a metabolic switch necessary for optimal osteoclast maturation.

Propionate and butyrate attenuate macrophage pyroptosis and osteoclastogenesis induced by CoCrMo alloy particles

Background:Wear particles-induced osteolysis is a major long-term complication after total joint arthroplasty.Up to now,there is no effective treatment for wear particles-induced osteolysis except for the revision surgery,which is a heavy psychological and economic burden to patients.A metabolite of gut microbiota,short chain fatty acids(SCFAs),has been reported to be beneficial for many chronic inflammatory diseases.This study aimed to investigate the therapeutic effect of SCFAs on osteolysis.Methods:A model of inflammatory osteolysis was established by applying CoCrMo alloy particles to mouse calvarium.After two weeks of intervention,the anti-inflammatory effects of SCFAs on wear particle-induced osteolysis were evaluated by micro-CT analysis and immunohistochemistry staining.In vitro study,lipopolysaccharide(LPS)primed bone marrow-derived macrophages(BMDMs)and Tohoku hospital pediatrics-1(THP-1)macrophages were stimulated with CoCrMo particles to activate inflammasome in the presence of acetate(C2),propionate(C3),and butyrate(C4).Western blotting,enzyme-linked immunosorbent assay,and immunofluorescence were used to detect the activation of NLRP3 inflammasome.The effects of SCFAs on osteoclasts were evaluate by qRT-PCR,Western blotting,immunofluorescence,and tartrate-resistant acid phosphatase(TRAP)staining.Additionally,histone deacetylase(HDAC)inhibitors,agonists of GPR41,GPR43,and GPR109A were applied to confirm the underlying mechanism of SCFAs on the inflammasome activation of macrophages and osteoclastogenesis.Results:C3 and C4 but not C2 could alleviate wear particles-induced osteolysis with fewer bone erosion pits(P<0.001),higher level of bone volume to tissue volume(BV/TV,P<0.001),bone mineral density(BMD,P<0.001),and a lower total porosity(P<0.001).C3 and C4 prevented CoCrMo alloy particles-induced ASC speck formation and nucleationinduced oligomerization,suppressing the cleavage of caspase-1(P<0.05)and IL-1β(P<0.05)stimulated by CoCrMo alloy particles.C3 and C4 also inhibited the generation of gasdermin D-N-terminal fragment(GSDMD-NT)to regulate pyroptosis.Besides,C3 and C4 have a negative impact on osteoclast differentiation(P<0.05)and its function(P<0.05),affecting the podosome arrangement and morphologically normal podosome belts formation.Conclusions:Our work showed that C3 and C4 are qualified candidates for the treatment of wear particle-induced osteolysis.

Glycobiology in osteoclast differentiation and function

Glycans,either alone or in complex with glycan-binding proteins,are essential structures that can regulate cell biology by mediating protein stability or receptor dimerization under physiological and pathological conditions.Certain glycans are ligands for lectins,which are carbohydrate-specific receptors.Bone is a complex tissue that provides mechanical support for muscles and joints,and the regulation of bone mass in mammals is governed by complex interplay between bone-forming cells,called osteoblasts,and bone-resorbing cells,called osteoclasts.Bone erosion occurs when bone resorption notably exceeds bone formation.Osteoclasts may be activated during cancer,leading to a range of symptoms,including bone pain,fracture,and spinal cord compression.Our understanding of the role of protein glycosylation in cells and tissues involved in osteoclastogenesis suggests that glycosylation-based treatments can be used in the management of diseases.The aims of this review are to clarify the process of bone resorption and investigate the signaling pathways mediated by glycosylation and their roles in osteoclast biology.Moreover,we aim to outline how the lessons learned about these approaches are paving the way for future glycobiology-focused therapeutics.

Mesenchymal stem/stromal cells-derived exosomes for osteoporosis treatment

Osteoporosis is a systemic bone disease,which leads to decreased bone mass and an increased risk of fragility fractures.Currently,there are many anti-resorption drugs and osteosynthesis drugs,which are effective in the treatment of osteoporosis,but their usage is limited due to their contraindications and side effects.In regenerative medicine,the unique repair ability of mesenchymal stem cells(MSCs)has been favored by researchers.The exosomes secreted by MSCs have signal transduction and molecular delivery mechanisms,which may have therapeutic effects.In this review,we describe the regulatory effects of MSCs-derived exosomes on osteoclasts,osteoblasts,and bone immunity.We aim to summarize the preclinical studies of exosome therapy in osteoporosis.Furth-ermore,we speculate that exosome therapy can be a future direction to improve bone health.

Invasive breast carcinoma with osteoclast-like stromal giant cells:A case report

BACKGROUND Invasive breast carcinoma with osteoclast-like stromal giant cells(OGCs) is an extremely rare morphology of breast carcinomas.To the best of our knowledge,the most recent case report describing this rare pathology was published six years ago.The mechanism controlling the development of this unique histological formation is still unknown.Further,the prognosis of patients with OGC involvement is also controversial.CASE SUMMARY We report the case of a 48-year-old woman,who presented to the outpatient department with a palpable,growing,painless mass in her left breast for about one year.Sonography and mammography revealed a 26.5 mm ×18.8 mm asymmetric,lobular mass with circumscribed margin and the Breast Imaging Reporting and Data System was category 4C.Sono-guided aspiration biopsy revealed invasive ductal carcinoma.The patient underwent breast conserving surgery and was diagnosed with invasive breast carcinoma with OGCs,grade Ⅱ,with intermediate grade of ductal carcinoma in situ(ER:80%,3+,PR:80%,3+,HER-2:negative,Ki 67:30%).Adjuvant chemotherapy and post-operation radiotherapy were initiated thereafter.CONCLUSION As a rare morphology of breast cancer,breast carcinoma with OGC occurs most often in relatively young women,has less lymph node involvement,and its occurrence is not racedependent.

Effects of acupotomy on the activity of osteoclasts and osteoblasts in the subchondral bone of rabbits with early and mid-stage knee osteoarthritis models

Objective:To investigate whether acupotomy could inhibit subchondral bone remodeling in knee osteoarthritis(KOA)rabbits by regulating the activity of osteoblasts and osteoclasts.Methods:KOA rabbits were prepared by immobilization for 6 and 9 weeks by Videman method.Nine groups of rabbits(control,6 weeks and 9 weeks model,6 weeks and 9 weeks acupotomy,6 weeks and 9 weeks electroacupuncture,and 6 weeks and 9 weeks drug groups)received acupotomy,electroacupuncture and risedronate sodium intervention,respectively,for 3 weeks.Results:Acupotomy can inhibit the activity of osteoclasts and osteoblasts in subchondral bone by reducing the proteins expression of cathepsin K(CK)and tartrate-resistant acid phosphatase(TRAP)and decreasing the proteins expression of osteocalcin(OCN)and alkaline phosphatase(ALP),to intercept the abnormal bone resorption and bone formation of subchondral bone in 6-week and 9-week immobilization-induced KOA rabbits.Conclusion:These findings indicated that acupotomy may be more advantageous than risedronate sodium intervention in modulating subchondral bone remodeling in KOA rabbits,especially in 9-week immobilization-induced KOA rabbits.

Research progress of bone sialoprotein in osteoclast differentiation and bone resorption

Bone sialoprotein(BSP)is an important non-collagen extracellular matrix protein(EMC)that promotes bone formation and induces bone resorption.BSP is secreted by odontoblasts,it plays an important role in cementum,alveolar bone formation and mineralization,and periodontal function.Bone resorption is controlled by a complex molecular network,and BSP can promote osteoclast differentiation and bone resorption.It is also associated with the metastasis of a range of malignancies.Osteoclasts(OC)are thought to be the only cells involved in bone resorption and play an important role in bone formation and late developmental remodeling.Osteoporosis and periodontal disease are caused by excessive bone resorption.This article will summarize the osteoclasts differentiation,the biological function of bone resorption,and explore the progress of the prevention and treatment of the related bone resorption diseases such as osteoporosis and periodontal disease through the regulation of osteoclasts.

miR-483-5p regulates osteoclast generation by targeting Timp2

Objective:To investigate whether miR-483-5p regulates osteoclast generation by targeting Timp2.miR-483-5p can promote osteoclast differentiation and bone destruction.Methods:Target genes of miR-483-5p were predicted by miRNAs target gene prediction software TargetScan8.0,and wild type and mutant 3'UTR plasmids were constructed.Dual luciferase reporter genes were used to verify whether target genes had a targeted regulatory relationship with miR-483-5p.Western blotting was used to detect the corresponding changes in the expression level of target protein after adjusting the level of miR-483-5p in cells.Cells were transfected or infected with target gene siRNA or target protein lentivirus,and TRAP staining and q-PCR assays were performed.In addition,for osteoclast induction experiment,RAW264.7 cells were co-transfected with ago-miR-483-5p and target protein-overexpressed lentiviruses q-PCR and TRAP staining were performed respectively.Results:Bioinformatics software was used to predict the target gene of miR-483-5p,and the Timp2 gene was found to regulate osteoclasts,and the dual luciferase reporter detection system found that miR-483-5p could be associated with the 3-UTR of the predicted target gene Timp2 gene.There are complementary loci and targeted regulatory relationship between them.Subsequently,we upregulated miR-483-5p in RAW264.7 cells to reduce the expression of Timp2.Compared with the normal group,the number of osteoclasts and the expression of osteoclast-specific genes increased significantly after the induction of Timp2 in knockdown cells.After co-transfection of target gene and miR-483-5p into cells,the number of osteoclasts and the expression of specific genes decreased significantly compared with the normal group.Conclusion:Timp2 is a downstream target gene of miR-483-5p and is involved in and inhibits osteoclast generation.

The Role of T Lymphocytes in Bone Remodeling

Bone remodeling is a tightly regulated resorption and formation of bone matrix for physiological processes or to maintain bone function. Bone remodeling involves the synchronized differentiation and activity of bone-related cell types including bone matrix-depositing osteoblasts, bone matrix-resorbing osteoclasts, collagen/extracellular matrix-producing chondrocytes, and the progenitors of these cell types. T and B cells are adaptive immune cells that can influence bone remodeling by directly regulating the function of bone-related cells under normal and pathophysiological conditions. The specific mechanisms through which T cells control remodeling are not well defined. Here, we review the impact and influence of T cells and their products on the differentiation and function of bone cells during bone remodeling. Synthesizing new connections and highlighting potential mechanisms may promote additional avenues of study to elucidate the full role that immune cells play in regulating bone homeostasis.

鼻窦筛骨成骨细胞及破骨细胞染色方法比较

为了在慢性鼻窦炎的病理研究中更清晰地展示筛骨成骨细胞及破骨细胞的形态结构,对慢性鼻窦炎筛窦骨重新塑形的组织病理学变化进行评估,用三种不同方法进行比较染色.

A Simplified Method for Purifying Osteoclasts from Human Giant Cell Tumor of Bone

Objective: To purify and identify the osteoclasts from the tissue of humangiant cell tumor of bone. Methods: We have developed a new method that allows the purification oflarge numbers of authentic osteoclasts (OCs). The OCs were isolated from tissue of human giant celltumor of bone by 0.25% trypsin and collagenase. We characterized OCs in terms of the expression ofdifferent phenotypic markers of OCs. The phenotypic markers of OC included Tartrate-resistant acidphosphatase staining (TRAP). The expression of calcitonin receptor (CTR), cathepsin K and receptoractivator of necrosis factor κB (RANK) mRNA were examined by RT-PCR. Results: The OC cell purifiedby above method functioned normally in vitro. The purity was about 79.7%. They showed the normalosteoclast phenotypes markers of OC. Conclusion: The method provides a system for performingbiochemical and molecular studies of OCs. The study indicates that the method of purifying theosteoclasts from human GCT cell can be used for research of bone metabolism.

伴破骨细胞样间质巨细胞的乳腺癌3例

伴破骨细胞样间质巨细胞的癌（Carcinoma with osteoclast-like stroma giant cells）是一种罕见的乳腺癌类型,《2012版乳腺肿瘤WHO分类》将其归入非特殊型浸润性癌中[1]。此病例罕见,对破骨细胞样间质巨细胞的来源与作用目前尚有争议,笔者收集3例此病例（其中一例癌为化生性癌）,对其组织病理学、免疫表型和临床特征进行分析讨论,

Osteoporosis in diabetes mellitus: Possible cellular and molecular mechanisms

Osteoporosis,a global age-related health problem in both male and female elderly,insidiously deteriorates the microstructure of bone,particularly at trabecular sites,such as vertebrae,ribs and hips,culminating in fragility fractures,pain and disability.Although osteoporosis is normally associated with senescence and estrogen deficiency,diabetes mellitus(DM),especially type 1 DM,also contributes to and/or aggravates bone loss in osteoporotic patients.This topic highlight article focuses on DM-induced osteoporosis and DM/ osteoporosis comorbidity,covering alterations in bone metabolism as well as factors regulating bone growth under diabetic conditions including,insulin,insulin-like growth factor-1 and angiogenesis.Cellular and molecular mechanisms of DM-related bone loss are also discussed.This information provides a foundation for the better understanding of diabetic complications and for development of early screening and prevention of osteoporosis in diabetic patients.