Osteoclast Fusion Research Articles

Diabetes Medication Metformin Inhibits Osteoclast Formation and Activity in In Vitro Models for Periodontitis.

Diabetes and periodontitis are comorbidities and may share common pathways. Several reports indicate that diabetes medication metformin may be beneficial for the periodontal status of periodontitis patients. Further research using appropriate cell systems of the periodontium, the tissue that surrounds teeth may reveal the possible mechanism. Periodontal ligament fibroblasts anchor teeth in bone and play a role in the onset of both alveolar bone formation and degradation, the latter by inducing osteoclast formation from adherent precursor cells. Therefore, a cell model including this type of cells is ideal to study the influence of metformin on both processes. We hypothesize that metformin will enhance bone formation, as described for osteoblasts, whereas the effects of metformin on osteoclast formation is yet undetermined. Periodontal ligament fibroblasts were cultured in the presence of osteogenic medium and 0.2 or 1 mM metformin. The influence of metformin on osteoclast formation was first studied in PDLF cultures supplemented with peripheral blood leukocytes, containing osteoclast precursors. Finally, the effect of metformin on osteoclast precursors was studied in cultures of CD14+ monocytes that were stimulated with M-CSF and receptor activator of Nf-κB ligand (RANKL). No effects of metformin were observed on osteogenesis: not on alkaline phosphatase activity, Alizarin red deposition, nor on the expression of osteogenic markers RUNX-2, Collagen I and Osteonectin. Metformin inhibited osteoclast formation and accordingly downregulated the genes involved in osteoclastogenesis: RANKL, macrophage colony stimulating factor (M-CSF) and osteoclast fusion gene DC-STAMP. Osteoclast formation on both plastic and bone as well as bone resorption was inhibited by metformin in M-CSF and RANKL stimulated monocyte cultures, probably by reduction of RANK expression. The present study unraveling the positive effect of metformin in periodontitis patients at the cellular level, indicates that metformin inhibits osteoclast formation and activity, both when orchestrated by periodontal ligament fibroblasts and in cytokine driven osteoclast formation assays. The results indicate that metformin could have a systemic beneficiary effect on bone by inhibiting osteoclast formation and activity.

Osteoclast Stimulatory Transmembrane Protein (OC‐STAMP): A Key Regulator in Osteoclast Fusion

The integrity of bone mass depends on the balance between osteoblastic bone formation and osteoclastic bone resorption. Osteoclasts derived from hematopoietic stem cells in bone marrow via a mechanism mediated mainly by two cytokines: the receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Osteoclast stimulatory transmembrane protein (OC‐STAMP) is a newly identified master fusogen that functions as a regulator of osteoclastogenesis. It is considered a key factor during macrophage fusion and differentiation. Osteoclast formation is mediated by a variety of factors and mechanisms other than OC-STAMP. Furthermore, numerous preclinical studies have investigated drugs that could target osteoclast differentiation, to allow better management of pathological bone metabolism. To improve understanding of osteoclast fusion and differentiation processes, this review will discuss recent findings on OC-STAMP and related signalling pathways.

In vitro and in vivo detection of tunneling nanotubes in normal and pathological osteoclastogenesis involving osteoclast fusion

AbstractOsteoclasts are multinucleated cells formed through specific recognition and fusion of mononuclear osteoclast precursors derived from hematopoietic stem cells. Detailed cellular events concerning cell fusion in osteoclast differentiation remain ambiguous. Tunneling nanotubes (TNTs), actin-based membrane structures, play an important role in intercellular communication between cells. We have previously reported the presence of TNTs in the fusion process of osteoclastogenesis. Here we analyzed morphological details of TNTs using scanning electron microscopy. The osteoclast precursor cell line RAW-D was stimulated to form osteoclast-like cells, and morphological details in the appearance of TNTs were extensively analyzed. Osteoclast-like cells could be classified into three types; early osteoclast precursors, late osteoclast precursors, and multinucleated osteoclast-like cells based on the morphological characteristics. TNTs were frequently observed among these three types of cells. TNTs could be classified into thin, medium, and thick TNTs based on the diameter and length. The shapes of TNTs were dynamically changed from thin to thick. Among them, medium TNTs were often observed between two remote cells, in which side branches attached to the culture substrates and beaded bulge-like structures were often observed. Cell-cell interaction through TNTs contributed to cell migration and rapid transport of information between cells. TNTs were shown to be involved in cell-cell fusion between osteoclast precursors and multinucleated osteoclast-like cells, in which movement of membrane vesicles and nuclei was observed. Formation of TNTs was also confirmed in primary cultures of osteoclasts. Furthermore, we have successfully detected TNTs formed between osteoclasts observed in the bone destruction sites of arthritic rats. Thus, formation of TNTs may be important for the differentiation of osteoclasts both in vitro and in vivo. TNTs could be one target cellular structure for the regulation of osteoclast differentiation and function in bone diseases.

The Novel Role of Checkpoint Inhibitor PD-1H/VISTA in Osteoclast Activation and Multiple Myeloma Bone Disease

IntroductionMultiple myeloma (MM)-induced bone disease remains one of its most devastating complications, caused by increased bone resorption by overactivated osteoclasts coupled with impaired bone formation. MM cells produce osteoclast-activating factors that induce osteoclast activation and extensive bone resorption. Our previous work demonstrated that matrix metalloproteinase 13 (MMP-13) is a critical osteoclastogenic factor that is highly secreted by MM cells (Fu J etc. JCI. 2016). We also identified that the checkpoint inhibitor, programmed death-1 homolog (PD-1H/VISTA), serves as the MMP-13 receptor in osteoclasts and mediates MMP-13-dependent osteoclastogenic function which is largely blocked in Pd-1h -/-osteoclasts (Fu J etc. ASH 2019, 2020). While the inhibitory role of PD-1H/VISTA in T-cells has recently been described (ElTanbouly MA etc. Science 2020), its cellular binding proteins remain unclear, and its role in osteoclast activation andMM bone disease have not been addressed.Methods and ResultsTo identify its interacting proteins, PD-1H-His 6 recombinant protein was expressed in mouse bone marrow mononuclear cells, the associated proteins pulled down by Ni-NTA agarose beads from cell lysates and identified by mass spectrometry. Functional annotation charting of the 75 proteins enriched in PD-1H pull-down samples (with signal ratio of PD-1H-His 6 pull-down vs control >2) indicated that almost 30% of the interacting targets were either cytoskeletal or cytoskeleton-associated proteins.Given that the F-actin cytoskeleton undergoes dynamic reorganization during osteoclast differentiation and plays critical roles in bone resorption, we further addressed the role of PD-1H in F-actin cytoskeleton regulation. Initially, osteoclasts form F-actin-rich adhesive structures, termed podosomes. At later stages, podosomes collectively rearrange into clusters and rings and finally into sealing belts to mediate osteoclast spreading, migration and bone resorption (Teitelbaum SL. Ann N Y Acad Sci. 2011). By confocal immunofluorescence microscopy, we found that PD-1H co-localized with F-actin podosome clusters, rings and sealing belts during osteoclast differentiation (Figure 1A). The functional role of PD-1H in F-actin cytoskeleton reorganization was addressed using Pd-1h -/- osteoclast wherein Pd-1h knockout lead to the disruption of podosome clusters at early stages relative to WT controls, while at later stages, Pd-1h -/-osteoclasts exhibited significantly fewer F-actin rings and belts (Figure 1B). Further, binding of MMP-13 to PD-1H increased the number of osteoclasts forming F-actin rings and belts, as well as the size of F-actin belts, which was blocked in Pd-1h -/- osteoclasts.To determine the role of PD-1H in the development of myeloma-induced lytic bone lesions, 5TGM1 myeloma cells were bilaterally intratibially injected into Pd-1h wtRag2 -/- or Pd-1h -/-Rag2 -/- mice (n=10) to induce lytic bone lesions. Three weeks following intratibial 5TGM1 injection, tibiae were harvested for micro-computed tomography. Subsequent quantitative histomorphology analyses of the trabecular and cortical bones confirmed that the knockout of Pd-1h reduced MM-induced bone destruction with significantly less decrease in trabecular bone volume (Tb. [BV/TV]), trabecular bone number (Tb. N.), trabecular bone thickness (Tb. Th.), as well as less increase in trabecular bone spacing (Tb. Sp.) and bone specific surface (Tb. [BS/BV]) compared to Pd-1h wtRag2 -/- mice. Similar effects were observed in cortical bone with less decrease in cortical bone thickness (CT. Th.), cortical bone area fraction (CT. [BA/TA]), and cortical tissue mineral density (CT. TMD) in 5TGM1 bearing Pd-1h -/-Rag2 -/- mice vs Pd-1h wtRag2 -/- mice (Table 1).ConclusionsTaken together, our study, for the first time, reveals the novel role of checkpoint inhibitor, PD-1H/VISTA, in osteoclasts and myeloma bone disease. PD-1H associates with cytoskeleton proteins and regulates the F-actin cytoskeleton reorganization which is critical for osteoclast bone resorption activity. Further, PD-1H mediates MMP-13-induced osteoclast fusion, F-actin belts formation, and osteoclast activation. Pd-1h -/-in recipient mice significantly impairs MM-induced bone loss, demonstrating that PD-1H/VISTA plays a critical role in MM bone disease. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

OAB-020: The role of checkpoint inhibitor PD-1H/VISTA in Multiple Myeloma bone disease

<h3>Background</h3> Multiple myeloma (MM) bone disease remains one of the most devastating complications of this incurable cancer, causing bone fractures, pain, mobility issues, and neurological deficits. MM cells secret pro-osteoclastogenic factors which lead to osteoclast (OCL) activation. Our previous work showed that matrix metalloproteinase 13 (MMP-13) is a critical osteoclastogenic factor highly secreted by MM cells and induces OCL fusion and bone resorption independently of its proteolytic activity (JCI 2016). We recently reported that MMP-13 binds to checkpoint inhibitor programmed death-1 homolog (PD-1H/VISTA), a surface receptor that is expressed on OCLs and mediates MMP-13 induced OCL fusion and bone resorption activity (ASH 2019). Bone resorption activity is significantly impaired in Pd-1h-/- OCLs in vitro. However, the function of PD-1H in MM bone disease has not been defined. <h3>Methods</h3> The role of PD-1H in MM bone disease was investigated using the intratibial 5TGM1 Rag2-/- MM bone disease mice model. Pd-1h-/-Rag2-/- mice were generated by crossbreeding Pd-1h-/- with Rag2-/- mice. Firefly luciferase-expressing 5TGM1 cells were intratibially injected into age and sex-paired Rag2-/- or Pd-1h-/-Rag2-/- mice (N=10). 3 weeks later, tibiae were harvested for quantitative micro-CT followed by histological analysis. <h3>Results</h3> Morphological analyses of trabecular and cortical bones confirmed that Pd-1h-/- recipient mice exhibited significantly less 5TGM1-induced bone loss (P<0.05). In trabecular bone, 5TGM1 induced 40.0% decrease of bone volume fraction in Rag2-/- mice vs 12.5% in Pd-1h-/-Rag2-/- mice, 7.3% decrease of trabecular bone numbers in Rag2-/- mice vs 0.6% in Pd-1h-/-Rag2-/- mice, 12.1% decrease of trabecular bone thickness in Rag2-/- mice vs 4.2% in Pd-1h-/-Rag2-/- mice, 8.1% increase of trabecular bone spacing in Rag2-/- mice vs 0.6% in Pd-1h-/-Rag2-/- mice, and 22.2% increase of specific bone surface in Rag2-/- mice vs 5.5% in Pd-1h-/-Rag2-/-mice. Similar effects were observed in cortical bone. 5TGM1 induced 18.9% decrease of cortical bone thickness in Rag2-/- mice vs 1.6% in Pd-1h-/-Rag2-/- mice, 7.28% decrease of cortical bone area fraction in Rag2-/- mice vs 2.7% in Pd-1h-/-Rag2-/- mice, and 8.19% decrease of cortical tissue mineral density in Rag2-/- mice vs 1.7% in Pd-1h-/-Rag2-/- mice. <h3>Conclusions</h3> Taken together, our study, for the first time, reveals that checkpoint inhibitor PD-1H/VISTA is the critical receptor for MMP-13 in osteoclasts, thereby mediating MMP-13-induced osteoclast fusion, activation, and bone resorption. MM-induced trabecular bone loss was significantly lower in Pd-1h-/- mice, demonstrating that PD-1H/VISTA plays a critical role in MMP-13-induced MM bone disease. Given the checkpoint role of PD-1H/VISTA in cancer immunosuppression, we further posit that targeting the interaction of MMP-13 and PD-1H may represent a novel therapeutic strategy to treat MM bone disease and modulate the MM immune environment.

Diterbutyl phthalate attenuates osteoarthritis in ACLT mice via suppressing ERK/c-fos/NFATc1 pathway, and subsequently inhibiting subchondral osteoclast fusion.

Osteoarthritis (OA) is the most common arthritis with a rapidly increasing prevalence. Disease progression is irreversible, and there is no curative therapy available. During OA onset, abnormal mechanical loading leads to excessive osteoclastogenesis and bone resorption in subchondral bone, causing a rapid subchondral bone turnover, cyst formation, sclerosis, and finally, articular cartilage degeneration. Moreover, osteoclast-mediated angiogenesis and sensory innervation in subchondral bone result in abnormal vascularization and OA pain. The traditional Chinese medicine Panax notoginseng (PN; Sanqi) has long been used in treatment of bone diseases including osteoporosis, bone fracture, and OA. In this study we established two-dimensional/bone marrow mononuclear cell/cell membrane chromatography/time of flight mass spectrometry (2D/BMMC/CMC/TOFMS) technique and discovered that diterbutyl phthalate (DP) was the active constituent in PN inhibiting osteoclastogenesis. Then we explored the therapeutic effect of DP in an OA mouse model with anterior cruciate ligament transaction (ACLT). After ACLT was conducted, the mice received DP (5 mg·kg-1·d-1, ip) for 8 weeks. Whole knee joint tissues of the right limb were harvested at weeks 2, 4, and 8 for analysis. We showed that DP administration impeded overactivated osteoclastogenesis in subchondral bone and ameliorated articular cartilage deterioration. DP administration blunted aberrant H-type vessel formation in subchondral bone marrow and alleviated OA pain assessed in Von Frey test and thermal plantar test. In RANKL-induced RAW264.7 cells in vitro, DP (20 μM) retarded osteoclastogenesis by suppressing osteoclast fusion through inhibition of the ERK/c-fos/NFATc1 pathway. DP treatment also downregulated the expression of dendritic cell-specific transmembrane protein (DC-STAMP)and d2 isoform of the vacuolar (H+) ATPase V0 domain (Atp6v0d2) in the cells. In conclusion, we demonstrate that DP prevents OA progression by inhibiting abnormal osteoclastogenesis and associated angiogenesis and neurogenesis in subchondral bone.

Can we talk about myoblast fusion?

Can we talk about myoblast fusion?

Low Intensity Vibration Treatment Reduces Myeloma Cell-Induced Osteoclast Formation By Inhibiting IL-6 and GFI-1 Production By MM Cells

Multiple myeloma (MM) bone disease is characterized by increased osteoclast-mediated resorption of bone adjacent to tumor growth. Fusion and activation of osteoclasts (OC), indicated by elevated tartrate-resistant acid phosphatase (TRAP) production, are induced by a myriad of inflammatory factors; e.g. TGF-β, growth factor independent 1 transcription factor (GFI-1), RANKL, and IL-6 that are produced by myeloma cells and cells in the marrow microenvironment. Current therapies reduce tumor burden and block osteoclast formation and activity, thereby decreasing bone destruction. However, MM bones rarely heal. While the anabolic effects of exercise on healthy bone are well-documented, patients with compromised skeletal tissue, such as in MM, may not be able to participate in regimented exercise for fear of developing a fracture. Low intensity vibrations (LIV) that deliver subtle mechanical signals, on the order of those induced by fast-twitch muscle fibers on bone, can enhance mesenchymal stem cell differentiation towards osteoblasts. LIV also significantly preserves trabecular bone while reducing tumor burden in femora of mice harboring U266 MM cells. We hypothesize that LIV is both anti-resorptive and anabolic, through differential effects on MM cells and bone cells. To test this hypothesis, we used a vertically oscillating, vibratory platform that delivered mechanical signals (<1 x Earth's gravitational acceleration; where 1g=9.8m/s2) to culture plates.5TGM1 murine MM cells (5x105 cells/well) were cultured in RPMI with 10% FCS and subjected to LIV or Sham-LIV. Each 6-well plate received either LIV (frequency=90Hertz, acceleration=0.3g) or Sham-LIV (SH; unpowered, static platform) twice per day for 20min per treatment for 48hrs. MM cell lysates were then collected using RIPA buffer. IL-6, GFI-1, and RANKL levels were determined via Western Blot using specific monoclonal antibodies. GAPDH was used as the loading control. Conditioned media (CM) were isolated from SH- and LIV-treated MM cultures. Purified primary murine OC precursors (non-adherent marrow mononuclear cells) were expanded with 10ng/mL of MCSF for 3 days and then these cells were isolated by trypsinization and replated in 12-well plates at a density of 5x105 cells/well in α-MEM, 10% FCS, and 30% v/v CM. All cultures were then incubated for 36hrs. OC number and morphology were confirmed via TRAP staining (counterstained with hematoxylin) of the cultures. TRAP+ OC's containing at least three nuclei were quantified in the cultures by microscopy.Expression levels of IL-6 and GFI-1 were 53% (p<0.01) and 50% (p<0.02) lower, respectively, in lysates from LIV-exposed 5TGM1 cells as compared to those that were SH-treated. Expression of RANKL was not significantly different between these groups. TRAP+ OC's were 61% (p<0.005) lower in LIV-treated cultures compared with SH cultures. The morphology of OC's exposed to LIV-conditioned media from MM cultures was distinct from control cultures, with fewer filopodia than OC's in cultures treated with SH-conditioned media. Based on these data we then examined the direct effect of LIV-treatment on committed OC-precursors treated with RANKL (50ng/mL) to induce formation of mature OC's. After 48hrs of LIV-treatment, committed OC-precursors had 36% (p<0.0001) fewer nuclei per OC than in SH-treated cultures. The cultures of committed OC-precursors directly treated with LIV had fewer filopodia in comparison to SH-treated cultures upon examination of OC morphology.These findings suggest that mechanical signals in the form of LIV may influence the secretion of IL-6 and GFI-1 by myeloma cells, which, in turn, can decrease the capacity of MM cells to induce OC formation. The morphology of OC's and expression of TRAP by OC's exposed to LIV further suggest a distinct mechanosensitive effect on osteoclast formation and fusion that may impact their bone resorbing capacity. Thus, LIV has a significant effect on the capacity of MM cells to secrete inflammatory cytokines as well as directly affect OC formation. These results further suggest that low intensity vibrations may be a useful, non-invasive approach for treating myeloma bone disease. DisclosuresRoodman:Amgen Denosumab Trial: Membership on an entity's Board of Directors or advisory committees.

Neoplastic Monocyte-Derived Osteoclasts Are Highly Proliferative, but Sub-Functional in MPN-Associated Myelofibrosis

Background: Myeloproliferative neoplasms (MPN) are clonal disorders characterized by the proliferation of myeloid progenitors. A defining hallmark of MPN-associated myelofibrosis (MF) is the deposition of fibrous tissue inside the bone marrow (BM) stroma, arising either as a primary process (PMF), or secondary to the essential thromocythemia (post-ET MF) or polycythemia vera. In at least 1/3 of cases, BM fibrosis is accompanied by the osteosclerotic thickening of the bone trabeculae. It was generally held that these changes are a reaction of mesenchymal cells to the stimuli from the malignant clone. However, the recent discovery of neoplastic monocyte-derived fibrocytes capable of directly inducing BM fibrosis in situ and in vivo has led us to hypothesize that other tissue remodeling cells of the myeloid lineage may play similar roles in the pathogenesis of MPN-associated MF. Osteoclasts (OC) are giant multinuclear cells formed by the fusion of multiple monocytic progenitors. Apart from being exclusively bone-resorbing cells, OCs have a critical role in regulating bone formation by the mesenchymal cell-derived osteoblasts (OB) and maintaining the hematopoietic BM microenvironment. The aim of this study was to examine the status of these cells in MF patients and explore the effects that the neoplastic OC progenitors exert on the development of MPN-associated osteosclerosis.Methods: In order to quantitate bone remodeling cells, trephine biopsies were obtained from patients with PMF (n = 32) and post-ET MF (n = 27), as well as healthy controls (n = 3). A tyramide signal amplification-based fluorescence IHC approach was employed using TRAP and cathepsin K as OC-specific and CD56and osterix as OB-specific markers. In addition, CD14+ low-density peripheral blood cells from the representative MF patients and healthy controls were cultured in the presence of M-CSF and RANKL. After 3 weeks mature OCs were visualized using fluorescently labeled F-actin and TRAP with DAPI as the nuclear counterstain, and the JAK2V617F allele frequency was assessed via RT-PCR. For the OC functional assay, cells were plated on a mineralized surface, which was subsequently stained using a modified von Kossa method. To assess the OC fusion capacity, two populations of progenitors labeled with different lipophilic probes were mixed in a 1:1 ratio and the colocalized signal was quantified for each mature cell. All imaging was conducted using an automated multispectral or a dual spinning disk confocal system.Results: Based on the multiplex BM imaging analysis, we observed a significant increase in the number of OCs in MF patients compared to healthy controls (median: 21.8 vs. 7.0 per 100 mm of bone perimeter; p < 0.0001) that was followed by a substantial increase in the total number of OBs and the density of trabecular bone. After inducing differentiation of JAK2+ monocytes from MF patients into mature TRAP+ OCs in vitro, these cells consistently showed a high mutant allele frequency. Compared to OCs cultured from healthy monocytes, MF patients' OCs exhibited a 40.5% decrease in the median number of nuclei per cell (p= 0.024) and a tendency to incompletely develop F-actin rings. Simultaneously, there was no change in the mean TRAP positivity between the two groups. Finally, MF OCs were significantly less apt at resorbing mineral-coated areas in contrast to healthy cells (median: 21.5% vs. 7.3%; p= 0.016), and there was a 3-fold decrease in the proportion of fused mature OCs from MF monocytes compared to healthy ones (p= 0.008).Conclusions: The development of OCs is highly skewed towards proliferation in both primary and secondary MPN-associated MF. We were able to demonstrate that neoplastic monocytic progenitors retain their aberrant genetic constitution even after differentiating into mature OCs. However, fusion of such progenitors seems to be profoundly impaired, and MF OCs are unable to fully acquire the phenotypical features associated with efficient bone resorption, particularly multinucleation and the development of actin-rich structures. At least to a certain extent, this process happens independently of the non-malignant stroma, although it seems to be impacting the OBs as well. Taken together, we show evidence that even though MF OCs are hyperproliferative, their function is intrinsically supressed due to the inherited neoplastic burden, which in turn contributes to the osteosclerotic dysplasia of the MPN-affected BM. DisclosuresVerstovsek:Blueprint Medicines Corp: Research Funding; Seattle Genetics: Research Funding; Genentech: Research Funding; Incyte: Research Funding; Galena BioPharma: Research Funding; Bristol Myers Squibb: Research Funding; Bristol Myers Squibb: Research Funding; Celgene: Research Funding; Roche: Research Funding; Pfizer: Research Funding; Lilly Oncology: Research Funding; Astrazeneca: Research Funding; Incyte: Research Funding; Promedior: Research Funding; Genentech: Research Funding; Galena BioPharma: Research Funding; Pfizer: Research Funding; Gilead: Research Funding; NS Pharma: Research Funding; Seattle Genetics: Research Funding; Astrazeneca: Research Funding; Lilly Oncology: Research Funding; Roche: Research Funding; CTI BioPharma Corp: Research Funding; NS Pharma: Research Funding; Blueprint Medicines Corp: Research Funding; Gilead: Research Funding; CTI BioPharma Corp: Research Funding; Celgene: Research Funding; Promedior: Research Funding.

Vitamin D receptor activated by vitamin D administration alleviates Mycobacterium tuberculosis-induced bone destruction by inhibiting NFκB-mediated aberrant osteoclastogenesis.

Clinically, bone destruction caused by Mycobacterium tuberculosis was serious especially in patients with vitamin D (VD) deficiency. However, the role of VD in M. tuberculosis-induced bone destruction remains clear. In this context, we investigate the role of VD and vitamin D receptor (VDR) in the M. tuberculosis-induced bone destruction. First, we infected RAW264.7 and bone marrow-derived macrophages (BMMs) with Mycobacterium bovis Bacillus Calmette-Guérin (M. bovis BCG) in vitro. Then, we activated VDR through VD administration. TRAP and FAK staining, bone resorption assays, immunofluorescence staining, qPCR, and western blot were carried out. In vivo, the M. tuberculosis-induced osteolytic model on the murine skull was established and the μCT and histological analyses were performed. We found that VDR and TRAP were upregulated in bone tuberculosis tissue and proved that M. tuberculosis infection promoted osteoclastogenesis in RAW264.7 and BMMs. VD could inhibit osteoclasts differentiation, fusion, and bone resorption dose-dependently. However, when VDR was knocked down, the inhibitory effect of VD on osteoclasts disappeared. In mechanism, activation of VDR inhibits the phosphorylation of IκB α, thereby inhibiting NFκB signaling pathway and alleviating osteoclastogenesis. Furthermore, in the skull osteolysis model, VD administration reduced osteolysis, but not in VDR-/- mice. Our study, for the first time, demonstrates that activation of VDR by VD administration inhibits M. tuberculosis-induced bone destruction. Our results reveal that VD and VDR are potential therapeutic targets for M. tuberculosis-induced bone destruction, and are of great clinical significance for the development of new therapeutic strategies.

CD13 is a critical regulator of cell\u2013cell fusion in osteoclastogenesis

The transmembrane aminopeptidase CD13 is highly expressed in cells of the myeloid lineage, regulates dynamin-dependent receptor endocytosis and recycling and is a necessary component of actin cytoskeletal organization. Here, we show that CD13-deficient mice present a low bone density phenotype with increased numbers of osteoclasts per bone surface, but display a normal distribution of osteoclast progenitor populations in the bone marrow and periphery. In addition, the bone formation and mineral apposition rates are similar between genotypes, indicating a defect in osteoclast-specific function in vivo. Lack of CD13 led to exaggerated in vitro osteoclastogenesis as indicated by significantly enhanced fusion of bone marrow-derived multinucleated osteoclasts in the presence of M-CSF and RANKL, resulting in abnormally large cells containing remarkably high numbers of nuclei. Mechanistically, while expression levels of the fusion-regulatory proteins dynamin and DC-STAMP1 must be downregulated for fusion to proceed, these are aberrantly sustained at high levels even in CD13-deficient mature multi-nucleated osteoclasts. Further, the stability of fusion-promoting proteins is maintained in the absence of CD13, implicating CD13 in protein turnover mechanisms. Together, we conclude that CD13 may regulate cell–cell fusion by controlling the expression and localization of key fusion regulatory proteins that are critical for osteoclast fusion.

Extracellular pH is a critical regulator of osteoclast fusion, size and activation

Extracellular pH is a critical regulator of osteoclast fusion, size and activation

Apremilast inhibits inflammatory osteoclastogenesis.

Psoriatic arthritis (PsA) is associated with bone erosion and inflammation-induced bone loss, which are mediated by osteoclasts (OC) and modulated by inflammatory cytokines. Apremilast (APR) (a selective phosphodiesterase 4 inhibitor) is efficacious in PsA and acts by inhibiting cytokine production. However, there are no direct data informing whether and how APR affects osteoclast formation in humans. Osteoclastogenic cytokine production by activated human peripheral blood mononuclear cells (PBMCs) was measured in the presence and absence of APR. Effects of APR on osteoclast differentiation were tested (i) in co-cultures of activated PBMCs and human CD14+ blood monocytes as well as (ii) in CD14+ blood monocytes stimulated with activated-PBMCs supernatant, TNF or IL-17A. Bone resorption was measured on OsteoAssay plates. Effects of APR on ex vivo osteoclast differentiation were compared in PsA, pre-PsA and psoriasis patients, as well as in healthy controls. APR significantly impaired the expression of key osteoclastogenic cytokines in activated PBMCs. Furthermore, APR dose-dependently and significantly inhibited activated PBMC-driven osteoclast differentiation and ex vivo osteoclast differentiation of PBMCs derived from PsA and pre-PsA patients, but not from psoriasis patients or healthy controls. TNF and IL-17A-enhanced osteoclastogenesis and osteolytic activity of CD14+ blood monocytes from PsA patients was also significantly inhibited by APR. Finally, APR inhibited expression of the key osteoclast fusion protein dendritic cell-specific transmembrane protein. Phosphodiesterase 4 targeting by APR not only inhibits osteoclastogenic cytokine production, but also directly suppresses inflammation-driven osteoclastogenesis. These data provide initial evidence that APR has the potential to provide a direct bone protective effect in PsA.

Protocatechualdehyde inhibits receptor activator of nuclear factor kappa-B ligand-induced osteoclastogenesis and attenuates lipopolysaccharide-induced inflammatory osteolysis.

Inflammatory osteolysis as a consequence of chronic bacterial infection underlies several lytic bone conditions, such as otitis media, osteomyelitis, septic arthritis, periodontitis, periprosthetic infection, and aseptic loosening of orthopedic implants. In consideration of the lack of effective preventive or treatments options against infectious osteolysis, the exploitation of novel pharmacological compounds/agents is critically required. The present study assessed the effect of protocatechualdehyde (PCA), a natural occurring polyphenolic compound with diverse biological activities including but not limited to antibacterial and antiinflammatory properties, on nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis in vitro and lipopolysaccharide (LPS)-induced bone loss in vivo. In the present study, it was found that PCA potently inhibited RANKL-induced osteoclast formation, fusion, and activation toward bone resorption in a dose-dependent manner via the suppression of the ERK/c-Fos/nuclear factor of activated T-cells, cytoplasmic 1 signaling axis. It was further demonstrated that the in vivo administration of PCA could effectively protect mice against the deleterious effects of LPS-induced calvarial bone destruction by attenuating osteoclast formation and activity in a dose-dependent manner. Collectively, these findings provided evidence for the potential therapeutic application of PCA in the prevention and treatment of infectious osteolytic conditions, and potentially other osteoclast-mediated bone diseases.

Inhibitory Effects of Combined Bone Morphogenetic Protein 2, Vascular Endothelial Growth Factor, and Basic Fibroblast Growth Factor on Osteoclast Differentiation and Activity.

Bone morphogenetic protein 2 (BMP-2), vascular endothelial growth factor (VEGF), and basic fibroblast growth factors (bFGF) are important regulators of bone development and bone remodeling involving the coordination of osteoblast-mediated bone formation and osteoclast-mediated bone resorption. The synergistic promotions of these growth factors on osteogenesis in the appropriate combination have been confirmed by a lot of studies, but the effect of this combined application on osteoclastogenesis still remains ambiguous. On the basis of comparing the osteoclastic potentials under stimulation of BMP-2, VEGF, or bFGF alone, this study focused on their combined effects on the differentiation and activity of osteoclasts. Our results showed that osteoclastogenesis was enhanced to some extent under the stimulation of BMP-2, VEGF, or bFGF alone, and the potential of these three growth factors to stimulate osteoclastogenesis was VEGF > BMP-2 > bFGF. However, the treatment with the combination of BMP-2 (50 ng/mL), VEGF (1 ng/mL), and bFGF (10 ng/mL), the most suitable dose combination for osteogenesis optimized in our previous study, weakened osteoclast differentiation confirmed by smaller tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, lower TRAP activity, and lower expression of dendritic cell-specific transmembrane protein, an important molecule regulating osteoclast fusion. Moreover, BMP-2, VEGF, and bFGF in combination also moderately inhibited the bone-resorbing activity of mature osteoclasts by suppressing the expression of osteoclast-specific genes cathepsin K, and matrix metalloproteinase-9. The underlying molecular mechanisms involved the suppression of the receptor activator of nuclear factor-κB ligand-induced c-Fos levels and the activation of nuclear factor of activated T cells c1, two major transcription factors in osteoclast differentiation. Taken together, our study showed that the combination of BMP-2 (50 ng/mL), VEGF (1 ng/mL), and bFGF (10 ng/mL) promoted osteoblastogenesis but inhibited osteoclastogenesis. Thus, the simultaneous use of BMP-2 (50 ng/mL), VEGF (1 ng/mL), and bFGF (10 ng/mL) in an appropriate combination might improve efficacious bone regeneration in a clinical setting. Impact statement Few studies have addressed the combined effects of multiple growth factors on osteoclasts. This study demonstrated that the simultaneous use of bone morphogenetic protein 2 (BMP-2; 50 ng/mL), vascular endothelial growth factor (VEGF; 1 ng/mL), and basic fibroblast growth factors (bFGF; 10 ng/mL), the most suitable dose combination for osteogenesis optimized in our previous study, showed inhibitory effects on the differentiation and activity of osteoclasts. Our results suggest that the growth factor signaling pathways in osteoclasts may interact with each other. Furthermore, this study could provide new insights into the optimal application of BMP-2, VEGF, and bFGF for bone repair and regeneration.

Osteoclasts Differentiation from Murine RAW 264.7 Cells Stimulated by RANKL: Timing and Behavior.

Simple SummaryThe formation of multinucleated cells is critical for mature osteoclast generation. For this to happen, mononuclear pre-osteoclasts migrate in proximity to other pre-osteoclasts to fuse together. This finely regulated process is dependent on the “feeling” and “recognition” between neighboring cells. In this study, we focused on pre-osteoclast fusion timing and behavior, and highlighted changes in morphological and cytoskeletal organization during 4 days of osteoclast differentiation. For the first time, interesting cellular extensions have been described, with the presumed function to serve as migration mediators. The sub-cellular localization of key proteins correlated with the osteoclast maturation timing. In particular, for the first time, a relationship of mutual exclusion from the nuclei has been shown between two mitogen activated protein kinases and a master transcription factor. The different trends in the expression of some genes involved in the osteoclast fusion process were related to osteoclast differentiation timing. Although further investigation is needed, we are confident that this study will contribute to the understanding of the mechanisms regulating the initial processes of osteoclastogenesis, including migration and fusion, which in turn are of fundamental importance for the management of many bone-related diseases, such as osteoporosis, osteopetrosis and rheumatoid arthritis.The development of multi-nucleated cells is critical for osteoclasts (OCs) maturation and function. Our objective was to extend knowledge on osteoclastogenesis, focusing on pre-OC fusion timing and behavior. RAW 264.7 cells, which is a murine monocyte-macrophage cell line, provide a valuable and widely used tool for in vitro studies on osteoclastogenesis mechanisms. Cells were treated with the receptor activator of nuclear factor κ-B ligand (RANKL) for 1–4 days and effects on cell morphology, cytoskeletal organization, protein distribution, and OC-specific gene expression examined by TEM, immunofluorescence, and qPCR. Multinucleated cells began to appear at two days of Receptor Activator of Nuclear factor κ-B Ligand (RANKL) stimulation, increasing in number and size in the following days, associated with morphological and cytoskeletal organization changes. Interesting cellular extensions were observed in three days within cells labeled with wheat germ agglutinin (WGA)-Fluorescein isothiocyanate (FITC). The membrane, cytoplasmic, or nuclear distribution of RANK, TRAF6, p-p38, pERK1/2, and NFATc1, respectively, was related to OCs maturation timing. The gene expression for transcription factors regulating osteoclastogenesis (NFATc1, c-fos, RelA, MITF), molecules involved in RANKL-signaling transduction (TRAF6), cytoskeleton regulation (RhoA), fusion (DC-STAMP), migration (MMP9), and OC-specific enzymes (TRAP, CtsK), showed different trends related to OC differentiation timing. Our findings provide an integrated view on the morphological and molecular changes occurring during RANKL stimulation of RAW 264.7 cells, which are important to better understand the OCs’ maturation processes.

Osteoclast fusion and bone loss are restricted by interferon inducible guanylate binding proteins

Chronic inflammation during many diseases is associated with bone loss. While interferons (IFNs) are often inhibitory to osteoclast formation, the complex role that IFN and interferon-stimulated genes (ISGs) play in osteoimmunology during inflammatory diseases is still poorly understood. We show that mice deficient in IFN signaling components including IFN alpha and beta receptor 1 (IFNAR1), interferon regulatory factor 1 (IRF1), IRF9, and STAT1 each have reduced bone density and increased osteoclastogenesis compared to wild type mice. The IFN-inducible guanylate-binding proteins (GBPs) on mouse chromosome 3 (GBP1, GBP2, GBP3, GBP5, GBP7) are required to negatively regulate age-associated bone loss and osteoclastogenesis. Mechanistically, GBP2 and GBP5 both negatively regulate in vitro osteoclast differentiation, and loss of GBP5, but not GBP2, results in greater age-associated bone loss in mice. Moreover, mice deficient in GBP5 or chromosome 3 GBPs have greater LPS-mediated inflammatory bone loss compared to wild type mice. Overall, we find that GBP5 contributes to restricting age-associated and inflammation-induced bone loss by negatively regulating osteoclastogenesis.

Aqueous extract of Chrysanthemum morifolium Ramat. inhibits RANKL-induced osteoclast differentiation by suppressing the c-fos/NFATc1 pathway

ObjectiveThe flower of chrysanthemum, used worldwide as a medicinal and edible product, has shown various bioactivities, such as anti-inflammatory, antioxidant, anti-tumorigenic, and hepatoprotective activities, as well as cardiovascular protection. However, the effect of Chrysanthemum morifolium Ramat. on the regulation of osteoclast differentiation has not yet been reported. In this study, we aimed to investigate the inhibitory effect of Chrysanthemum morifolium Ramat. water extract (CME) on RANKL-induced osteoclast differentiation in mouse bone marrow-derived macrophages (BMMs). Study designBone marrow-derived macrophages (BMMs) isolated from the C57BL/6 J mice. The viability of BMMs was detected with MTT assays. Inhibitory effects of CME on osteoclast differentiation and bone resorption was measured by TRAP staining and Pit assay. Osteoclast differentiation-associated gene expression were assessed by Real-time quantitative polymerase chain reaction. Intracellular signaling molecules was assessed by western blot. ResultsCME significantly inhibited osteoclast differentiation in BMMs without cytotoxicity, besides inhibiting MAPK/c-fos and PLCγ2/CREB activation. The inhibitory effects of CME on differentiation-related signaling molecules resulted in significant repression of NFATc1 expression, which is a key transcription factor in osteoclast differentiation, fusion, and activation. ConclusionOur results confirmed the inhibition of RANKL-induced PLCγ2/CREB/c-fos/NFATc1 activation by CME during osteoclast differentiation. The findings collectively suggested CME as a traditional therapeutic agent for osteoporosis, RA, and periodontitis.

RNF219/α-Catenin/LGALS3 Axis Promotes Hepatocellular Carcinoma Bone Metastasis and Associated Skeletal Complications.

The incidence of bone metastases in hepatocellular carcinoma (HCC) has increased prominently over the past decade owing to the prolonged overall survival of HCC patients. However, the mechanisms underlying HCC bone‐metastasis remain largely unknown. In the current study, HCC‐secreted lectin galactoside‐binding soluble 3 (LGALS3) is found to be significantly upregulated and correlates with shorter bone‐metastasis‐free survival of HCC patients. Overexpression of LGALS3 enhances the metastatic capability of HCC cells to bone and induces skeletal‐related events by forming a bone pre‐metastatic niche via promoting osteoclast fusion and podosome formation. Mechanically, ubiquitin ligaseRNF219‐meidated α‐catenin degradation prompts YAP1/β‐catenin complex‐dependent epigenetic modifications of LGALS3 promoter, resulting in LGALS3 upregulation and metastatic bone diseases. Importantly, treatment with verteporfin, a clinical drug for macular degeneration, decreases LGALS3 expression and effectively inhibits skeletal complications of HCC. These findings unveil a plausible role for HCC‐secreted LGALS3 in pre‐metastatic niche and can suggest a promising strategy for clinical intervention in HCC bone‐metastasis.

Effect of nitrogen-containing bisphosphonates on osteoclasts and osteoclastogenesis: an ultrastructural study.

We have previously indicated that a single injection of alendronate, one of the nitrogen-containing bisphosphonates (NBPs), affects murine hematopoietic processes, such as the shift of erythropoiesis from bone marrow (BM) to spleen, disappearance of BM-resident macrophages, the increase of granulopoiesis in BM and an increase in the number of osteoclasts. NBPs induce apoptosis and the formation of giant osteoclasts in vitro and/or in patients undergoing long-term NBP treatment. Therefore, the time-kinetic effect of NBPs on osteoclasts needs to be clarified. In this study, we examined the effect of alendronate on mouse osteoclasts and osteoclastogenesis. One day after the treatment, osteoclasts lost the clear zone and ruffled borders, and the cell size decreased. After 2 days, the cytoplasm of osteoclasts became electron dense and the nuclei became pyknotic. Some of the cells had fragmented nuclei. After 4 days, osteoclasts had euchromatic nuclei attached to the bone surface. Osteoclasts had no clear zones or ruffled borders. After 7 days, osteoclasts formed giant osteoclasts via the fusion of multinuclear and mononuclear osteoclasts. These results indicate that NBPs affect osteoclasts and osteoclastogenesis via two different mechanisms.