Osteopontin (OPN) is a non-collagenous extracellular sialylated glycoprotein located in bone. It is believed to be one of the key components in osteoclast attachment to bone during resorption. In this study, we characterized OPN and other glycoproteins found in the resorption lacunae to confirm the role of osteoclasts in OPN secretion using electron microscopy and mass spectrometry. Additionally, we examined the glycan epitopes of resorption pits and the effects of different glycan epitopes on the differentiation and function of osteoclasts. Osteoarthritic femoral heads were examined by immunohistochemistry to reveal the presence of OPN in areas of increased bone metabolism in vivo. Our results demonstrate that human osteoclasts secrete OPN into resorption lacunae on native human bone and on carbonated hydroxyapatite devoid of natural OPN. OPN is associated with an elevated bone turnover in osteoarthritic bone under experimental conditions. Our data further confirm that osteoclasts secrete OPN into the resorption pit where it may function as a chemokine for subsequent bone formation. We show that α2,3- and α2,6-linked sialic acids have a role in the process of osteoclast differentiation. OPN is one of the proteins that has both of the above sialic residues, hence we propose that de-sialylation can effect osteoclast differentiation in bone.

Inhibition of sympathetic signaling to bone reduces bone resorption in rodents. In contrast, we show that pharmacological reduction of the sympathetic tone increases bone resorption in humans in vivo. This effect does not appear to be mediated via a direct pharmacological effect on the osteoclast.

Pycnodysostosis, a rare autosomal recessive skeletal dysplasia, is caused by a deficiency of cathepsin K. Patients have impaired bone resorption in the presence of normal or increased numbers of multinucleated, but dysfunctional, osteoclasts. Cathepsin K degrades collagen type I and generates N-telopeptide (NTX) and the C-telopeptide (CTX) that can be quantified. Levels of these telopeptides are increased in lactating women and are associated with increased bone resorption. Nothing is known about the consequences of cathepsin K deficiency in lactating women. Here we present for the first time normalized blood and CTX measurements in a patient with pycnodysostosis, exclusively related to the lactation period. In vitro studies using osteoclasts derived from blood monocytes during lactation and after weaning further show consistent bone resorption before and after lactation. Increased expression of cathepsins L and S in osteoclasts derived from the lactating patient suggests that other proteinases could compensate for the lack of cathepsin K during the lactation period of pycnodysostosis patients.

The peroxisome proliferator-activated receptor gamma (PPARγ) regulates osteoblast and osteoclast differentiation, and is the molecular target of thiazolidinediones (TZDs), insulin sensitizers that enhance glucose utilization and adipocyte differentiation. However, clinical use of TZDs has been limited by side effects including a higher risk of fractures and bone loss. Here we demonstrate that the same post-translational modifications at S112 and S273, which influence PPARγ pro-adipocytic and insulin sensitizing activities, also determine PPARγ osteoblastic (pS112) and osteoclastic (pS273) activities. Treatment of either hyperglycemic or normoglycemic animals with SR10171, an inverse agonist that blocks pS273 but not pS112, increased trabecular and cortical bone while normalizing metabolic parameters. Additionally, SR10171 treatment modulated osteocyte, osteoblast, and osteoclast activities, and decreased marrow adiposity. These data demonstrate that regulation of bone mass and energy metabolism shares similar mechanisms suggesting that one pharmacologic agent could be developed to treat both diabetes and metabolic bone disease.

Osteoporosis is an enormous health problem caused by the imbalance between bone resorption and bone formation. The current therapeutic strategies for osteoporosis still have some limitations. Boldine, an alkaloid isolated from Peumus boldus, has been shown to have antioxidant and anti-inflammatory effects in vivo. For the first time, we discover that boldine has a protective effect for the estrogen deficiency-induced bone loss in mice. According to the Micro-CT and histomorphometry assays, boldine conducts this protective effect through inhibiting bone resorption without affecting bone formation in vivo. Moreover, we showed that boldine can inhibit receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation via impairing the AKT signaling pathways, while SC79 (an AKT agonist) partially rescue this effect. In conclusion, our results suggest that boldine can prevent estrogen deficiency-induced osteoporosis by inhibiting osteoclastogenesis. Thus, boldine may be served as a novel therapeutic agent for anti-osteoporotic therapy.

To determine the relationship between maternal bone resorption and bone development in fetuses.

Osteoporosis is a common health problem worldwide caused by an imbalance of bone formation vs. bone resorption. However, current therapeutic approaches aimed at enhancing bone formation or suppressing bone resorption still have some limitations. In this study, we demonstrated for the first time that cepharanthine (CEP, derived from Stephania cepharantha Hayata) exerted a protective effect on estrogen deficiency-induced bone loss. This protective effect was confirmed to be achieved through inhibition of bone resorption in vivo, rather than through enhancement of bone formation in vivo. Furthermore, the in vitro study revealed that CEP attenuated receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast formation, and suppressed bone resorption by impairing the c-Jun N-terminal kinase (JNK) and phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathways. The inhibitory effect of CEP could be partly reversed by treatment with anisomycin (a JNK and p38 agonist) and/or SC79 (an AKT agonist) in vitro. Our results thus indicated that CEP could prevent estrogen deficiency-induced bone loss by inhibiting osteoclastogenesis. Hence, CEP might be a novel therapeutic agent for anti-osteoporosis therapy.

Septins are known to play key roles in supporting cytoskeletal stability, vesicular transport, endo-/exocytosis, stabilizing cellular membranes and forming diffusion barriers. Their function in mammalian cells is poorly investigated. The osteoclast offers an interesting tool to investigate septins because all cellular activities septins were reported to be involved in are critical for osteoclasts. However, the existence of septins in osteoclasts has not even been reported. Here we show that the SEPT9 gene and Septin 9 (SEPT9) protein are expressed and synthesized during differentiation of human osteoclasts. Pharmacological stabilization of septin filaments dose dependently inhibits bone resorption of human osteoclasts in vitro suggesting a role for septins in bone resorption. Attesting to this, conditional deletion of Sept9 in mice leads to elevated levels of trabecular bone and diminished femoral growth in vivo. Finally, systematic interrogation of the spatial organization of SEPT9 by confocal microscopy reveals that SEPT9 is closely associated to the structures known to be critical for osteoclast activity. We propose that septins in general and SEPT9 in particular play a previously unappreciated role in osteoclastic bone resorption.

Activation of osteoclasts during orthodontic tooth treatment is a prerequisite for alveolar bone resorption and tooth movement. However, the key regulatory molecules involved in osteoclastogenesis during this process remain unclear. Long noncoding RNAs (lncRNAs) are a newly identified class of functional RNAs that regulate cellular processes, such as gene expression and translation regulation. Recently, lncRNAs have been reported to be involved in osteogenesis and bone formation. However, as the most abundant noncoding RNAs in vivo, the potential regulatory role of lncRNAs in osteoclast formation and bone resorption urgently needs to be clarified. We recently found that the lncRNA Nron (long noncoding RNA repressor of the nuclear factor of activated T cells) is highly expressed in osteoclast precursors. Nron is downregulated during osteoclastogenesis and bone ageing. To further determine whether Nron regulates osteoclast activity during orthodontic treatment, osteoclastic Nron transgenic (Nron cTG) and osteoclastic knockout (Nron CKO) mouse models were generated. When Nron was overexpressed, the orthodontic tooth movement rate was reduced. In addition, the number of osteoclasts decreased, and the activity of osteoclasts was inhibited. Mechanistically, Nron controlled the maturation of osteoclasts by regulating NFATc1 nuclear translocation. In contrast, by deleting Nron specifically in osteoclasts, tooth movement speed increased in Nron CKO mice. These results indicate that lncRNAs could be potential targets to regulate osteoclastogenesis and orthodontic tooth movement speed in the clinic in the future.

Coccidiosis is an economically significant disease in the global poultry industry, but little is known about the mechanisms of bone defects caused by coccidiosis; thus, the study focused on effects of coccidiosis on the bone homeostasis of young broiler chickens. A total of 480 male Cobb500 broilers were randomly allocated into four treatment groups, including an uninfected control consuming diet ad libitum, two infected groups were orally gavaged with two different concentrations of sporulated Eimeria oocysts, and an uninfected pair-fed group fed the same amount of feed as the high Eimeria-infected group consumed. Growth performance and feed intake were recorded, and samples were collected on 6 days post infection. Results indicated that coccidiosis increased systemic oxidative status and elevated immune response in bone marrow, suppressing bone growth rate (P < 0.05) and increasing bone resorption (P < 0.05) which led to lower bone mineral density (P < 0.05) and mineral content (P < 0.05) under Eimeria infection. With the same amount of feed intake, the uninfected pair-fed group showed a distinguished bone formation rate and bone resorption level compared with the Eimeria infected groups. In conclusion, inflammatory immune response and oxidative stress in broilers after Eimeria infection were closely associated with altered bone homeostasis, highlighting the role of inflammation and oxidative stress in broiler bone homeostasis during coccidiosis.

Bone remodeling is precisely coordinated by bone resorption and formation. Apoptotic osteoclasts generate large amounts of apoptotic bodies (ABs) marking the end of the bone resorption phase, whereas the functions of osteoclast-derived ABs remain largely unknown. Here, we identified the molecular profile of ABs derived from osteoclasts at distinct differentiation stages and investigated their corresponding functions. ABs were isolated from apoptotic bone marrow macrophages, preosteoclasts, and mature osteoclasts induced by staurosporine. Proteomic signature analysis with liquid chromatography-tandem mass spectrometry suggested marked protein cargo differences among the different ABs. Further bioinformatic analysis showed that the proteomic signatures of the ABs were highly similar to those of their parental cells. Functionally, pOC-ABs induced endothelial progenitor cell differentiation and increased CD31hiEmcnhi endothelial cell formation in a murine bone defect model via their PDGF-BB cargo. mOC-ABs induced osteogenic differentiation of mesenchymal stem cells and facilitated osteogenesis via RANKL reverse signaling. In summary, we mapped the detailed proteomic landscapes of ABs derived from osteoclasts and showed that their potential biological roles are important in coupling bone formation with resorption during bone remodeling.

To date, mesenchymal cells have only been associated with bone resorption indirectly, and it has been hypothesized that the degradation of bone is associated exclusively with specific functions of osteoclasts. Here we show, in aseptic prosthesis loosening, that aggressive fibroblasts at the bone surface actively contribute to bone resorption and that this is independent of osteoclasts. In two separate models (a severe combined immunodeficient mouse coimplantation model and a dentin pit formation assay), these cells produce signs of bone resorption that are similar to those in early osteoclastic resorption. In an animal model of aseptic prosthesis loosening (i.e. intracranially self-stimulated rats), it is shown that these fibroblasts acquire their ability to degrade bone early on in their differentiation. Upon stimulation, such fibroblasts readily release acidic components that lower the pH of their pericellular milieu. Through the use of specific inhibitors, pericellular acidification is shown to involve the action of vacuolar type ATPases. Although fibroblasts, as mesenchymal derived cells, are thought to be incapable of resorbing bone, the present study provides the first evidence to challenge this widely held belief. It is demonstrated that fibroblast-like cells, under pathological conditions, may not only enhance but also actively contribute to bone resorption. These cells should therefore be considered novel therapeutic targets in the treatment of bone destructive disorders.

Normal bone homeostasis, which is regulated by bone-resorbing osteoclasts and bone-forming osteoblasts is perturbed by inflammation. In chronic inflammatory disease with disturbed bone remodelling, e.g. rheumatoid arthritis, patients show increased serum levels of the chemokine eotaxin-1 (CCL11). Herein, we demonstrate an inflammatory driven expression of CCL11 in bone tissue and a novel role of CCL11 in osteoclast migration and resorption. Using an inflammatory bone lesion model and primary cell cultures, we discovered that osteoblasts express CCL11 in vivo and in vitro and that expression increased during inflammatory conditions. Osteoclasts did not express CCL11, but the high affinity receptor CCR3 was significantly upregulated during osteoclast differentiation and found to colocalise with CCL11. Exogenous CCL11 was internalised in osteoclast and stimulated the migration of pre-osteoclast and concomitant increase in bone resorption. Our data pinpoints that the CCL11/CCR3 pathway could be a new target for treatment of inflammatory bone resorption.

Autoantibodies are detected in most patients with rheumatoid arthritis (RA) and can be of the IgM, IgG or IgA subclass. Correlations between IgA autoantibodies and more severe disease activity have been previously reported, but the functional role of IgA autoantibodies in the pathogenesis of RA is ill understood. In this study, we explored the effect of IgA immune complexes on osteoclast mediated bone resorption.

Osteoporosis is one of the major health problems characterized by decreased bone density and increased risk of fractures. Nowadays, the treating strategies against osteoporosis are efficient, but still have some drawbacks. Micheliolide, a guaianolide sesquiterpene lactone isolated from Michelia compressa and Michelia champac, has been reported to have anti-inflammatory effects. Here, our data suggest that Micheliolide could protect mice from ovariectomy induced bone loss. According to the Micro-CT scan and histomorphometry quantification data, Micheliolide treatment inhibits excessive osteoclast bone resorption without affecting bone formation in estrogen deficiency mice. Consistently, our data suggest that Micheliolide could inhibit osteoclastogenesis in vitro. Additionally, we confirmed that Micheliolide inhibits osteoclasts formation via inhibiting P38 MAPK signaling pathway, and P79350 (a P38 agonist) could rescue this effect. In summary, our data suggest that Micheliolide could ameliorate estrogen deficiency-induced bone loss via attenuating osteoclastogenesis. Hence, Micheliolide could be used as a novel anti-resorptive agent against osteoporosis.

This study aimed to investigate the correlation between bone mineral density (BMD) and the turnover rate [√(MoMf2 + MoMr2), multiple of median formation (MoMf) was calculated as bone-specific alkaline phosphatase (BAP) value/18.6 and multiple of median resorption (MoMr) as tartrate-resistant acid phosphatase 5b (TRACP-5b) value/463] and the balance (MoMf/MoMr) and to compare differences in therapeutic effects evoked by differences in previous treatments.

Calmodulin is a highly versatile protein that regulates intracellular calcium homeostasis and is involved in a variety of cellular functions including cardiac excitability, synaptic plasticity and signaling transduction. During osteoclastic bone resorption, calmodulin has been reported to concentrate at the ruffled border membrane of osteoclasts where it is thought to modulate bone resorption activity in response to calcium. Here we report an interaction between calmodulin and Rab3D, a small exocytic GTPase and established regulator osteoclastic bone resorption. Using yeast two-hybrid screening together with a series of protein-protein interaction studies, we show that calmodulin interacts with Rab3D in a calcium dependent manner. Consistently, expression of a calcium insensitive form of calmodulin (i.e. CaM1234) perturbs calmodulin-Rab3D interaction as monitored by bioluminescence resonance energy transfer (BRET) assays. In osteoclasts, calmodulin and Rab3D are constitutively co-expressed during RANKL-induced osteoclast differentiation, co-occupy plasma membrane fractions by differential gradient sedimentation assay and colocalise in the ruffled border as revealed by confocal microscopy. Further, functional blockade of calmodulin-Rab3D interaction by calmidazolium chloride coincides with an attenuation of osteoclastic bone resorption. Our data imply that calmodulin- Rab3D interaction is required for efficient bone resorption by osteoclasts in vitro.

Vav1 is a Rho/Rac guanine nucleotide exchange factor primarily expressed in hematopoietic cells. In this study, we investigated the potential role of Vav1 in osteoclast (OC) differentiation by comparing the ability of bone marrow mononuclear cells (BMMCs) obtained from Vav1-deficient (Vav1-/-) and wild-type (WT) mice to differentiate into mature OCs upon stimulation with macrophage colony stimulating factor and receptor activator of nuclear kappa B ligand in vitro. Our results suggested that Vav1 deficiency promoted the differentiation of BMMCs into OCs, as indicated by the increased expression of tartrate-resistant acid phosphatase, cathepsin K, and calcitonin receptor. Therefore, Vav1 may play a negative role in OC differentiation. This hypothesis was supported by the observation of more OCs in the femurs of Vav1-/- mice than in WT mice. Furthermore, the bone status of Vav1-/- mice was analyzed in situ and the femurs of Vav1-/- mice appeared abnormal, with poor bone density and fewer number of trabeculae. In addition, Vav1-deficient OCs showed stronger adhesion to vitronectin, an αvβ3 integrin ligand important in bone resorption. Thus, Vav1 may inhibit OC differentiation and protect against bone resorption. [BMB Reports 2019; 52(11): 659-664].

Amylin is a member of the calcitonin family of hormones cosecreted with insulin by pancreatic beta cells. Cell culture assays suggest that amylin could affect bone formation and bone resorption, this latter function after its binding to the calcitonin receptor (CALCR). Here we show that Amylin inactivation leads to a low bone mass due to an increase in bone resorption, whereas bone formation is unaffected. In vitro, amylin inhibits fusion of mononucleated osteoclast precursors into multinucleated osteoclasts in an ERK1/2-dependent manner. Although Amylin +/- mice like Amylin-deficient mice display a low bone mass phenotype and increased bone resorption, Calcr +/- mice display a high bone mass due to an increase in bone formation. Moreover, compound heterozygote mice for Calcr and Amylin inactivation displayed bone abnormalities observed in both Calcr +/- and Amylin +/- mice, thereby ruling out that amylin uses CALCR to inhibit osteoclastogenesis in vivo. Thus, amylin is a physiological regulator of bone resorption that acts through an unidentified receptor.

Osteoporosis is a metabolic bone disease. Osteoclasts are significantly involved in the pathogenesis of osteoporosis. AS-605240 (AS) is a small molecule PI3K-γ inhibitor and is less toxic compared to pan-PI3K inhibitors. AS also exerts multiple biological effects including anti-inflammatory, anti-tumor, and myocardial remodeling promotion. However, the involvement of AS in the differentiation and functions of osteoclasts and the effect of AS in treating patients with osteoporosis is still unclear.