Activity Of Mature Osteoclasts Research Articles

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 8 980 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.

Inhibition of receptor activator of nuclear factor kappa-B ligand-mediated osteoclast differentiation and bone resorption by Gryllus bimaculatus extract: An in vitro study.

Excessive bone-resorbing osteoclast activity during bone remodeling is a major feature of bone diseases, such as osteoporosis. Therefore, the inhibition of osteoclast formation and bone resorption can be an effective therapeutic target for various bone diseases. Gryllus biomaculatus (GB) has recently been approved as an alternative food source because of its high nutritional value and environmental sustainability. Traditionally, GB has been known to have various pharmacological properties, including antipyretic and blood pressure-lowering activity, and it has recently been reported to have various biological activities, including protective effects against inflammation, oxidative stress, insulin resistance, and alcohol-induced liver injury. However, the effect of GB on osteoclast differentiation and bone metabolism has not yet been demonstrated. In this study, we confirmed the inhibitory effect of GB extract (GBE) on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. To determine the effect of GBE on RANKL-induced osteoclast differentiation and function, we performed TRAP and F-actin staining, as well as a bone-resorbing assay. The intracellular mechanisms of GBE responsible for the regulation of osteoclastogenesis were revealed by Western blot analysis and quantitative real-time polymerase chain reaction. We investigated the relationship between GBE and expression of osteoclast-specific molecules to further elucidate the underlying mechanisms. It was found that GBE significantly suppressed osteoclastogenesis by decreasing the phosphorylation of Akt, p38, JNK, and ERK, as well as Btk-PLCγ2 signaling, in pathways involved in early osteoclastogenesis as well as through the subsequent suppression of c-Fos, NFATc1, and osteoclastogenesis-specific marker genes. Additionally, GBE inhibited the formation of F-actin ring-positive osteoclasts and bone resorption activity of mature osteoclasts. Our findings suggest that GBE is a potential functional food and therapeutic candidate for bone diseases involving osteoclasts.

Relaxin treatment stimulates the differentiation of mesenchymal stem cells into osteoblasts

Background/purposeSeveral studies have determined that relaxin stimulates differentiation and regulates the activity of mature osteoclasts, but little is known about its effect on the differentiation of mesenchymal cells towards the osteogenic lineage. Therefore, this study aimed to determine the effect of relaxin on the proliferation and differentiation of the osteoblastic lineage of mesenchymal cells derived from human dental pulp (hDPSC). Materials and methodsIn this in vitro study, hDPSC were characterized and treated with relaxin at different doses (10–80 ng/ml) and times (1–21 days). Morphology was assessed by microscopy, and proliferation was assessed using a resazurin assay. Osteoblastic differentiation was evaluated by Alizarin Red staining, alkaline phosphatase (ALP) labeling, and changes in the expression of the osteoblastic differentiation genes RUNX2 and BMP2. ResultsRelaxin treatment did not induce changes in the proliferation or viability of hDPSCs; however, larger cells and increased cytoplasmic prolongation were observed. Relaxin treatment (20 and 80 ng/ml) significantly increased calcified nodule formation on days 14 and 21. The cytochemical signals for ALP, RUNX2, and BMP2 gene expression were significantly (P < 0.05) increased by the relaxin treatment. ConclusionRelaxin treatment does not induce changes in hDPSC proliferation but induces morphological changes, increases ALP detection, calcified nodule formation, and increases expression of RUNX2 and BMP2, suggesting the induction of osteoblastic differentiation of hDPSC.

DNA damage-inducible transcript 3 deficiency promotes bone resorption in murine periodontitis models.

Periodontitis is a multifactorial inflammatory disease that leads to the destruction of supporting structures of the teeth. DNA damage-inducible transcript 3 (DDIT3) plays crucial roles in cell survival and differentiation. DDIT3 regulates bone mass and osteoclastogenesis in femur. However, the role of DDIT3 in periodontitis has not been elucidated. This research aimed to explore the role and mechanisms of DDIT3 in periodontitis. DDIT3 gene knockout (KO) mice were generated using a CRISPR/Cas9 system. Experimental periodontitis models were established to explore the role of DDIT3 in periodontitis. The expression of DDIT3 in periodontal tissues was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). The alveolar bone phenotypes were observed by micro-CT and stereomicroscopy. The inflammation levels and osteoclast activity were examined by histological staining, immunostaining, and qRT-PCR. Bone marrow-derived macrophages (BMMs) were isolated to confirm the effects of DDIT3 on osteoclast formation and function in vitro. The increased expression of DDIT3 in murine inflamed periodontal tissues was detected. DDIT3 knockout aggravated alveolar bone loss and enhanced expression levels of inflammatory cytokines in murine periodontitis models. Increased osteoclast formation and higher expression levels of osteoclast-specific markers were observed in the inflamed periodontal tissues of KO mice. In vitro, DDIT3 deficiency promoted the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts and the bone resorption activity of mature osteoclasts. Our results demonstrate that DDIT3 deletion aggravated alveolar bone loss in experimental periodontitis through enhanced inflammatory reactions and osteoclastogenesis. The anti-inflammation and the inhibition of bone loss by DDIT3 in murine periodontitis provides a potential novel therapeutic strategy for periodontitis.

The Mechanism of Osteoprotegerin-Induced Osteoclast Pyroptosis In Vitro.

Osteoprotegerin (OPG) is a new member of the tumor necrosis factor (TNF) receptor superfamily, which can inhibit the differentiation and activity of osteoclasts by binding to nuclear factor kappa B receptor activator (RANK) competitively with nuclear factor kappa B receptor activator ligand (RANKL). The previous experiments found that OPG can induce apoptosis of mature osteoclasts in vitro, which can inhibit the activity of mature osteoclasts, thereby exerting its role in protecting bone tissue. In addition, pyroptosis is a new type of cell death that is different from apoptosis. It is unclear whether OPG can induce mature osteoclast pyroptosis and thereby play its role in protecting bone tissue. In this study, the results showed that compared with the control group, the survival rate of osteoclasts in the OPG group was significantly reduced, and the contents of IL-1β, IL-18, and LDH in the supernatant both increased. Many osteoclast plasma membranes were observed to rupture in bright fields, and OPG induced loss of their morphology. Flow cytometry was used to analyze the pyroptosis rate; OPG significantly increased the osteoclast pyroptosis rate. To further reveal the mechanism of OPG-induced osteoclast pyroptosis, we examined the expression level of pyroptosis-related genes and proteins, and the results found that OPG increased the expression of NLRP3, ASC, caspase-1, and GSDMD-N compared with the control group. In summary, OPG can induce osteoclast pyroptosis, and its mechanism is related to the expression levels of ASC, NLRP3, caspase 1 and GSDMD, which were included in the classical pathway of pyroptosis.

Aromatic Bis[aminomethylidenebis(phosphonic)] Acids Prevent Ovariectomy-Induced Bone Loss and Suppress Osteoclastogenesis in Mice.

Osteoporosis is a skeletal disease associated with excessive bone turnover. Among the compounds with antiresorptive activity, nitrogen-containing bisphosphonates play the most important role in antiosteoporotic treatment. In previous studies, we obtained two aminomethylidenebisphosphonates—benzene-1,4-bis[aminomethylidene(bisphosphonic)] (WG12399C) acid and naphthalene-1,5-bis[aminomethylidene(bisphosphonic)] (WG12592A) acid—which showed a significant antiproliferative activity toward J774E macrophages, a model of osteoclast precursors. The aim of these studies was to evaluate the antiresorptive activity of these aminobisphosphonates in ovariectomized (OVX) Balb/c mice. The influence of WG12399C and WG12592A administration on bone microstructure and bone strength was studied. Intravenous injections of WG12399C and WG12592A bisphosphonates remarkably prevented OVX-induced bone loss; for example, they sustained bone mineral density at control levels and restored other bone parameters such as trabecular separation. This was accompanied by a remarkable reduction in the number of TRAP-positive cells in bone tissue. However, a significant improvement in the quality of bone structure did not correlate with a parallel increase in bone strength. In ex vivo studies, WG12399C and WG12592A remarkably bisphosphonates reduced osteoclastogenesis and partially inhibited the resorptive activity of mature osteoclasts. Our results show interesting biological activity of two aminobisphosphonates, which may be of interest in the context of antiresorptive therapy.

Lack of Adiponectin Drives Hyperosteoclastogenesis in Lipoatrophic Mice.

Long bones from mammals host blood cell formation and contain multiple cell types, including adipocytes. Physiological functions of bone marrow adipocytes are poorly documented. Herein, we used adipocyte-deficient PPARγ-whole body null mice to investigate the consequence of total adipocyte deficiency on bone homeostasis in mice. We first highlighted the dual bone phenotype of PPARγ null mice: one the one hand, the increased bone formation and subsequent trabecularization extending in the long bone diaphysis, due to the well-known impact of PPARγ deficiency on osteoblasts formation and activity; on the other hand, an increased osteoclastogenesis in the cortical bone. We then further explored the cause of this unexpected increased osteoclastogenesis using two independent models of lipoatrophy, which recapitulated this phenotype. This demonstrates that hyperosteoclastogenesis is not intrinsically linked to PPARγ deficiency, but is a consequence of the total lipodystrophy. We further showed that adiponectin, a cytokine produced by adipocytes and mesenchymal stromal cells is a potent inhibitor of osteoclastogenesis in vitro and in vivo. Moreover, pharmacological activation of adiponectin receptors by the synthetic agonist AdipoRon inhibited mature osteoclast activity both in mouse and human cells by blocking podosome formation through AMPK activation. Finally, we demonstrated that AdipoRon treatment blocks bone erosion in vivo in a murine model of inflammatory bone loss, providing potential new approaches to treat osteoporosis.

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.

Grape Seed Proanthocyanidin Extract Prevents Bone Loss via Regulation of Osteoclast Differentiation, Apoptosis, and Proliferation.

Dietary procyanidin has been shown to be an important bioactive component that regulates various pharmacological activities to maintain metabolic homeostasis. In particular, grape seed proanthocyanidin extract (GSPE) is a commercially available medicine for the treatment of venous and lymphatic dysfunction. This study aimed to investigate whether GSPE protects against lipopolysaccharide (LPS)-induced bone loss in vivo and the related mechanism of action in vitro. The administration of GSPE restored the inflammatory bone loss phenotype stimulated by acute systemic injection of LPS in vivo. GSPE strongly suppressed receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation and bone resorption activity of mature osteoclasts by decreasing the RANKL-induced nuclear factor-κB transcription activity. GSPE mediates this effect through decreased phosphorylation and degradation of NF-κB inhibitor (IκB) by IκB kinaseβ, subsequently inhibiting proto-oncogene cellular Fos and nuclear factor of activated T cells. Additionally, GSPE promotes osteoclast proliferation by increasing the phosphorylation of components of the Akt and mitogen-activated protein kinase signaling pathways and it also inhibits apoptosis by decreasing the activity of caspase-8, caspase-9, and caspase-3, as corroborated by a decrease in the Terminal deoxynucleotidyl transferase dUTP nick end labeling -positive cells. Our study suggests a direct effect of GSPE on the proliferation, differentiation, and apoptosis of osteoclasts and reveals the mechanism responsible for the therapeutic potential of GSPE in osteoclast-associated bone metabolism disease.

The human sialic acid-binding immunoglobulin-like lectin Siglec-9 and its murine homolog Siglec-E control osteoclast activity and bone resorption.

Regulation of osteoclast differentiation and function is a central element in bone homeostasis. While the role of soluble factors, such as cytokines, hormones and growth factors, in controlling osteoclast differentiation has been intensively characterized, the function of surface receptors is less well understood. Sialic acid-binding immunoglobulin-like lectin (Siglec)-9 and its murine homolog Siglec-E are sialic acid-recognizing inhibitory receptors from the CD33-related Siglec-family and mainly expressed on myeloid cells. We found Siglec-9 and Siglec-E to be expressed at all stages of human and murine osteoclastogenesis, respectively. Siglec-E knockout mice displayed lower bone mass despite unchanged osteoclast numbers and an increased bone formation rate. Ex vivo osteoclast assays using Siglec-E knockout cells or a blocking antibody against human Siglec-9 confirmed the suppressive effect of Siglec-9/Siglec-E on osteoclast function. Although osteoclast numbers were unchanged or even slightly decreased, the blockade/absence of Siglec-9/Siglec-E resulted in an augmented resorption activity of mature osteoclasts. This increased resorption activity was associated with enlarged actin rings. Together, our results suggest Siglec-9/Siglec-E to inhibit osteoclast activation independently from osteoclast differentiation and thereby propose a new mechanism for the control of local bone resorption.

The role of small leucine zipper protein in osteoclastogenesis and its involvement in bone remodeling

Bone remodeling is critical to maintain the quality of bone tissues and to heal bone tissue injury. Osteoclasts and osteoblasts are special types of cells involved in this event. In particular, the resorption activity of mature osteoclasts is required for the formation of new bones. Human small leucine zipper protein (sLZIP) is known to induce the osteoblast differentiation of mesenchymal stem cells. However, the roles of sLZIP in osteoclast differentiation and bone remodeling have not been explored. In this study, we investigated the roles of sLZIP in regulating osteoclast formation and in the bone remodeling process using sLZIP transgenic (TG) mice. Tibiae from sLZIP TG mice contained more osteoclasts than those from wild type (WT) mice. Bone marrow-derived macrophages (BMM) from sLZIP TG mice showed increased differentiation into osteoclasts compared with BMM from WT mice. sLZIP bound to the promotor and induced the expression of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and its target osteoclastogenic genes. To understand the role of sLZIP in bone remodeling, a bone-defect model was generated. Results of micro-CT scanning and histologic analysis demonstrated that sLZIP TG mice have faster bone formation during healing compared with WT mice. Notably, the soft callus around the defect area was replaced faster by hard callus in sLZIP TG mice than in WT mice. These findings suggest that sLZIP promotes osteoclast differentiation and plays an important role in bone remodeling.

Mitofusin 2, a mitochondria-ER tethering protein, facilitates osteoclastogenesis by regulating the calcium-calcineurin-NFATc1 axis

Mitofusin 2 (Mfn2) is a mitochondrial outer membrane protein that participates in tethering mitochondria to the ER. Mitochondria-ER tethering has been demonstrated to play important roles in many cellular activities by regulating homeostasis of metabolites and calcium. Intracellular calcium signaling is crucial for the differentiation of osteoclasts, the bone-resorbing cells. In this study, we investigated whether Mfn2 plays a role in osteoclastogenesis by receptor activator of nuclear factor kappa B (RANKL) in primary cells. We found that RANKL increased Mfn2 expression during osteoclast formation from mouse bone marrow-derived macrophages (BMMs). When Mfn2 expression was suppressed in BMMs by using a siRNA-mediated gene knock-down system, osteoclast differentiation and activity of mature osteoclasts were reduced. Mfn2 knock-down also decreased the RANKL-mediated induction of NFATc1, the key transcription factor for osteoclast gene expression, without affecting c-Fos level. This effect on NFATc1 was associated with decreased calcium oscillation and calcineurin activity in Mfn2-deficient osteoclasts. Taken together, our results indicate that Mfn2 positively contributes to RANKL-induced osteoclast differentiation by regulating the calcium-calcieurin-NFATc1 axis, raising the importance of a previously under-recognized role of mitochondria in osteoclastogenesis.

Neferine suppresses osteoclast differentiation through suppressing NF-κB signal pathway but not MAPKs and promote osteogenesis.

Osteoporosis is an ageing disease characterized by elevated osteoclastic bone resorption resulting in bone loss, decrease bone strength, and elevated incidence of fractures. Neferine, a natural compound isolated from the traditional Chinese medicine Nelumbo nucifera (Lotus), has been reported exhibit anti-inflammatory, antioxidant, and anticancer properties. However, its effect on bone remains to be determined. Here we showed that Neferine inhibits RANKL-induced osteoclast formation in a dose- and time-dependent manner. Furthermore, Neferine also demonstrated antiresorptive properties by effectively ameliorating the bone resorptive activity of mature osteoclasts. Mechanistically, Neferine suppressed RANKL-induced activation of NF-κB signaling pathway. This in turn hindered the induction and activation of NFATc1 resulting in downregulation of osteoclast marker genes closely related to differentiation, fusion as well as bone resorption. Interestingly, we found Neferine enhanced the differentiation and bone mineralization activity of MC3T3-E1 preosteoblast cells. Finally, mice treated with Neferine was protected against ovariectomy (OVX)-induced bone loss. The Neferine treatment improved bone volume following ovariectomy and also exhibited less TRAP-positive osteoclasts on bone surface. Collectively our data provide promising evidence that Neferine could be a potential therapeutic application for against osteolytic bone conditions such as osteoporosis.

Daidzin inhibits RANKL-induced osteoclastogenesis in vitro and prevents LPS-induced bone loss in vivo.

Osteoclasts are multinuclear giant cells responsible for bone resorption in bone loss diseases, including rheumatoid arthritis, periodontitis, and the aseptic loosening of orthopedic implants. Because of injurious side effects with currently available drugs, it is necessary to continue research novel bone-protective therapies. Daidzin, a naturally occurring isoflavone found in leguminous plants, has numerous beneficial pharmacologic effects, including anti-cancer, anti-cholesterol, and anti-angiocardiopathy, promoting osteoblasts differentiation, and even anti-osteoporosis. However, the effect of daidzin on the regulation of osteoclast activity has not yet been investigated. In this study, our study showed that daidzin significantly inhibited receptor activator of nuclear factor-kB ligand (RANKL)-induced osteoclast differentiation of bone marrow macrophages and the hydroxyapatite-resorbing activity of mature osteoclasts by inhibiting RANKL-induced NF-kB signaling pathway. In addition, daidzin could inhibit the expression of osteoclast marker genes, including nuclear factor of activated T cells cytoplasmic 1 (NFATc1), cellular oncogene fos (c-Fos), tartrate-resistant acid phosphatase (TRAP), and cathepsin K (CTSK). Consistent with in vitro results, daidzin inhibited lipopolysaccharide-induced bone loss by suppressing the osteoclast differentiation. Together our data demonstrated that daidzin inhibits RANKL-induced osteoclastogenesis through suppressing NF-ĸB signaling pathway and that daidzin is a promising agent in the treatment of osteolytic diseases.

Interleukin 29 inhibits RANKL-induced osteoclastogenesis via activation of JNK and STAT, and inhibition of NF-κB and NFATc1

Interleukin (IL)-29 is known to modulate immune functions of monocytes or macrophages. In this study, we investigated the effect and its underlying mechanism of IL-29 on receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis using murine macrophage cell line RAW264.7 cells and bone-marrow-derived monocyte/macrophage precursor cells (BMMs), and human peripheral blood mononuclear cells (PBMCs). In response to human recombinant IL-29, cell viability and apoptosis were assessed by Cell Counting Kit-8 and flow cytometry; the osteoclast formation and activity by tartrate-resistant acid phosphatase (TRAP) staining and pit formation assay, respectively; the expression and activation of molecules that associated with osteoclastogenesis by real time-PCR, immunoblotting or immunofluorescent analysis. IL-28 receptor α (IL-28Rα), a specific receptor of IL-29 was expressed on RAW264.7 cells. Although IL-29 did not affect the viability and apoptosis of RAW264.7 cells, it inhibited multinucleated cells in the differentiation of osteoclastogenesis, the bone-resorbing activity of mature osteoclasts and osteoclastic specific genes expression including TRAP, cathepsin K (CTSK), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), C-Fos and matrix metallopeptidase 9 (MMP-9). This inhibitory effect of IL-29 was confirmed on BMMs and PBMCs and mediated via IL-28Rα through the activation of Stat1 and 3 and the suppression of nuclear factor kappa B (NF-κB) and NFATc1 nuclear translocation in RAW264.7 cells. In conclusion, IL-29 inhibited osteoclastogenesis via activation of STAT signaling pathway, prevention of NF-κB activation and NFATc1 translocation, and suppression of downstream osteoclastogenic genes expression.

Dynamic Analyses of the Short-Term Effects of Different Bisphosphonates Using Intravital Two-Photon Microscopy.

ABSTRACTBisphosphonates are commonly used for the treatment of bone disorders such as osteoporosis; however, the mechanism by which they affect the dynamics of living mature osteoclasts in vivo remains unknown. Here, we describe the short‐term effects of different bisphosphonates on controlling the bone resorptive activity of mature osteoclasts in living bone tissues of mice using intravital two‐photon microscopy with a pH‐sensing chemical fluorescent probe. Three types of nitrogen‐containing bisphosphonates, risedronate, alendronate, and minodronate, inhibited osteoclastic acidification during osteoporotic conditions just 12 hours after i.v. injection. Among the three types of drugs, risedronate was the most effective at increasing osteoclast motility and changing the localization of proton pumps, which led to an inhibition of bone resorption. Together, these results demonstrate that the intravital imaging system is a useful tool for evaluating the similarities and differences in currently used antibone resorptive drugs. © 2018 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Transglutaminase activity regulates differentiation, migration and fusion of osteoclasts via affecting actin dynamics.

Osteoclasts, bone resorbing cells, derive from monocyte/macrophage cell lineage. Increased osteoclast activity is responsible for bone destruction in diseases such as osteoporosis, periodontitis and rheumatoid arthritis. Transglutaminases (TGs), protein crosslinking enzymes, were recently found involved in osteoclastogenesis in vivo, however their mechanisms of action have remained unknown. In this study, we have investigated the role of TG activity in osteoclastogenesis in vitro using four TG inhibitors, NC9, Z006, T101, and monodansyl cadaverine. Our results showed that all TG inhibitors were capable of blocking the entire osteoclastogenesis process. The most potent of the inhibitors, NC9 when added to cultures at different phases of osteoclastogenesis, inhibited differentiation, migration, and fusion of pre-osteoclasts as well as resorption activity of mature osteoclasts. Further investigation into the mechanisms revealed that NC9 increased RhoA levels and blocked podosome belt formation suggesting that TG activity regulates actin dynamics in pre-osteoclasts. The inhibitory effect of NC9 on osteoclastogenesis as well as podosome belt formation was completely reversed with a Rho-family inhibitor Exoenzyme C3. Microtubule architecture, acetylation, and detyrosination of α-tubulin were not affected. Finally, we demonstrated that macrophages and osteoclasts expressed mRNA of three TGs:TG1, TG2, and Factor XIII-A which were all differentially regulated in these cells during differentiation. Immunofluoresence microscopic analysis showed that all three enzymes co-localized to podosomes in osteoclasts. Taken together, our data suggests that TG activity regulates differentiation, migration and fusion of osteoclasts via affecting actin dynamics and that this may involve contribution from all three TG enzymes.

Ethanolic Extract of Rubus coreanus Fruits Inhibits Bone Marrow-Derived Osteoclast Differentiation and Lipopolysaccharide-Induced Bone Loss

The inhibition of osteoclast differentiation/bone resorption is a well-known therapeutic strategy for controlling pathological and postmenopausal bone loss. Natural products that specifically inhibit osteoclastogenesis could therefore be developed as antiresorptive drugs for the treatment of metabolic bone disorders characterized by excessive osteoclastic bone resorption. We therefore examined the effects of Rubus coreanus extract (eeRc) on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced differentiation of bone marrow macrophages (BMMs) into osteoclasts and pit formation in vitro. Additionally, the in vivo effects of the eeRc were observed in mice with lipopolysaccharide (LPS)-induced bone erosion. In this study, we found that the ethanolic extract of Rubus coreanus fruits considerably suppressed the RANKL-induced differentiation of primary BMMs into osteoclasts and bone-resorbing activity of mature osteoclasts. Oral administration of eeRc attenuated LPS-induced bone loss in vivo, as demonstrated by the reversal of LPS-induced reduction in bone volume per tissue volume, bone mineral density, and trabecular number to some extent in eeRc-treated mice. In addition, eeRc slightly decreased the serum levels of C-terminal telopeptide fragments of type I collagen, the collagen-breakdown product generated by osteoclasts. Collectively, our results indicate that eeRc has the potential to inhibit bone loss by blocking osteoclast differentiation and could therefore be a promising natural product for the prevention and/or treatment of inflammatory bone loss.

The Murine Osteoclasts Lacking Transferrin Receptor 1 have Altered Mitochondrial Metabolism, Cytoskeletal Organization and Increased Trabecular Bone Mass

Bone is a highly dynamic tissue, which is continuously remodeled by osteoclastic breakdown and osteoblastic rebuilding. Osteoclastogenesis and bone resorbing activity of mature osteoclasts (OC) are high energy demanding processes, thus requiring increased levels of ATP generated via mitochondrial metabolism. Iron plays an important role in the assembly of the mitochondrial electron transport chain. Respiratory complexes depend on heme and Fe-S clusters for correct assembly, folding, and function. Mammalian cells acquire iron through the uptake of transferrin (Tf), heme, and ferritins. The expression of Tf-dependent pathway proteins of iron uptake and processing during OC differentiation are up-regulated, whereas transporters of heme and ferritins remain unchanged, suggesting TfR1 receptor mediated iron uptake is the dominant pathway of iron entry into these cells. To elucidate the role of cellular Tf-dependent iron transport and homeostasis in OCs, we generated TfR1 myeloid and mature OC deficient mice in C57BL6/129 mixed background by crossing TfR1-floxed mice with LysM-Cre (TfR1f/f;LysM-Cre) and Cathepsin K-Cre (TfR1f/f;CTSK-Cre) mice, respectively. Both strains of mice had an over two-fold increase in trabecular bone mass in femurs of male and female mice. In vitro studies revealed that loss of TfR1 totally abolished Tf-dependent iron uptake in monocytes and mature OCs. Cellular bioenergetics studies using the Seahorse Extracellular Flux analyzer demonstrated a significant decrease in reserved respiratory capacity, a parameter reflecting the difference between maximum and basal respiration, in TfR1-deficient OC precursors compared to control cells. They also demonstrated less mitosox oxidation suggesting steady-state levels of mitochondrial superoxide is decreased when Tf-dependent iron uptake is impaired. TfR1-null monocytes differentiated OC normally in vitro. TfR1-deficient mature OCs, however, exhibited defective podosome-belt and actin-ring formation. From these results we conclude that TfR1-dependent iron uptake is critical for OC energy metabolism and cytoskeleton organization, but not for OC differentiation; and it is needed for OC bone resorption and bone homeostasis.

Water extract of Uncaria sinensis suppresses RANKL-induced bone loss by attenuating osteoclast differentiation and bone resorption

BackgroundThe hooks and stems of Uncaria sinensis have been used to mitigate cardiovascular and central nervous system disorders in Asia traditional medicine. Regulation of osteoclast differentiation and activity is a major target for preventing and treating pathological bone diseases. MethodsTartrate-resistant acid phosphatase (TRAP) activity and the number of TRAP-stained multinucleated cells were used to examine receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. The activation of RANKL-induced signaling pathways and the expression of transcription factors were investigated by western blot analysis and quantitative real-time polymerase chain reaction. The bone resorption activity of osteoclast was studied using a plate coated with hydroxyl-apatite. Trabecular bone destruction was investigated using a RANKL-induced trabecular bone loss mouse model. ResultsWe found that water extract of the hooks and stems of U. sinensis (WEUS) inhibits RANKL-induced differentiation of murine bone marrow macrophages and RAW264.7 cells into osteoclasts. WEUS inhibited the activation of NF-κB and the expression of nuclear factor of activated T-cells, cytoplasmic 1. In addition, WEUS suppressed the bone resorbing activity of mature osteoclasts without affecting their survival. Furthermore, oral administration of WEUS suppressed RANKL-induced bone loss with a significant amelioration of trabecular bone micro-structures. WEUS also reduced RANKL-induced increase in serum TRAP5b activity and C-terminal cross-linked telopeptide of type I collagen levels. ConclusionThe present study demonstrates that WEUS has a pharmacological activity that inhibits osteoclast-mediated bone destruction by suppressing osteoclast differentiation and function. These results suggest that U. sinensis could be a promising herbal candidate for preventing and treating bone diseases such as osteoporosis.