Inhibitory Effects of KP-A159, a Thiazolopyridine Derivative,on Osteoclast Differentiation,Function, and Inflammatory Bone Loss via Suppression of RANKL-Induced MAP Kinase Signaling Pathway

Abnormally elevated formation and activation of osteoclasts are primary causes for a majority of skeletal diseases. In this study, we found that KP-A159, a newly synthesized thiazolopyridine derivative, inhibited osteoclast differentiation and function in vitro, and inflammatory bone loss in vivo. KP-A159 did not cause a cytotoxic response in bone marrow macrophages (BMMs), but significantly inhibited the formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). KP-A159 also dramatically inhibited the expression of marker genes related to osteoclast differentiation, including TRAP (Acp5), cathepsin K (Ctsk), dendritic cell-specific transmembrane protein (Dcstamp), matrix metallopeptidase 9 (Mmp9), and nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1). Moreover, actin ring and resorption pit formation were inhibited by KP-A159. Analysis of the signaling pathway involved showed that KP-A159 inhibited RANKL-induced activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and mitogen-activated protein kinase kinase1/2 (MEK1/2). In a mouse inflammatory bone loss model, KP-A159 significantly rescued lipopolysaccharide (LPS)-induced bone loss by suppressing osteoclast numbers. Therefore, KP-A159 targets osteoclasts, and may be a potential candidate compound for prevention and/or treatment of inflammatory bone loss.     

Bone Morphogenetic Proteins Signal Via SMAD and Mitogen-activated Protein (MAP) Kinase Pathways at Distinct Times during Osteoclastogenesis

To investigate the role of bone morphogenetic protein (BMP) signaling in osteoclastogenesis in vivo, we eliminated BMPRII in osteoclasts by creating a BMPRII^fl/fl;lysM-Cre mouse strain. Conditional knock-out (cKO) mice are osteopetrotic when compared with WT controls due to a decrease in osteoclast activity. Bone marrow macrophages (BMMs) isolated from cKO mice are severely inhibited in their capacity to differentiate into mature osteoclasts in the presence of M-CSF and receptor activator of NF-κB (RANK) ligand. We also show that BMP noncanonical (MAPK) and canonical (SMAD) pathways are utilized at different stages of osteoclast differentiation. BMP2 induces p38 phosphorylation in pre-fusion osteoclasts and increases SMAD phosphorylation around osteoclast precursor fusion. Phosphorylation of MAPKs was decreased in differentiated BMMs from cKO animals. Treating BMMs with the SMAD inhibitor dorsomorphin confirms the requirement for the canonical pathway around the time of fusion. These results demonstrate the requirement for BMP signaling in osteoclasts for proper bone homeostasis and also explore the complex signaling mechanisms employed by BMP signaling during osteoclast differentiation.     

Osteoclast formation is strongly reduced both in vivo and in vitro in the absence of CD47/SIRPalpha-interaction

Physical interaction between the cell surface receptors CD47 and signal regulatory protein alpha (SIRPalpha) was reported to regulate cell migration, phagocytosis, cytokine production, and macrophage fusion. However, it is unclear if the CD47/SIRPalpha-interaction can also regulate macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-kappaB ligand (RANKL)-stimulated formation of osteoclasts. Here, we show that functional blocking antibodies to either CD47 or SIRPalpha strongly reduced formation of multinucleated tartrate-resistant acid phosphatase (TRAP)+ osteoclasts in cultures of murine hematopoietic cells, stimulated in vitro by M-CSF and RANKL. In addition, the numbers of osteoclasts formed in M-CSF/RANKL-stimulated bone marrow macrophage cultures from CD47-/- mice were strongly reduced, and bones of CD47-/- mice exhibited significantly reduced osteoclast numbers, as compared with wild-type controls. We conclude that the CD47/SIRPalpha interaction is important for M-CSF/RANKL-stimulated osteoclast formation both in vivo and in vitro, and that absence of CD47 results in decreased numbers of osteoclasts in CD47-/- mice.     

Kruppel-like factor 4 attenuates osteoblast formation,function, and cross talk with osteoclasts

Osteoblasts not only control bone formation but also support osteoclast differentiation. Here we show the involvement of Kruppel-like factor 4 (KLF4) in the differentiation of osteoclasts and osteoblasts. KLF4 was down-regulated by 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) in osteoblasts. Overexpression of KLF4 in osteoblasts attenuated 1,25(OH)₂D₃-induced osteoclast differentiation in co-culture of mouse bone marrow cells and osteoblasts through the down-regulation of receptor activator of nuclear factor κB ligand (RANKL) expression. Direct binding of KLF4 to the RANKL promoter repressed 1,25(OH)₂D₃-induced RANKL expression by preventing vitamin D receptor from binding to the RANKL promoter region. In contrast, ectopic overexpression of KLF4 in osteoblasts attenuated osteoblast differentiation and mineralization. KLF4 interacted directly with Runx2 and inhibited the expression of its target genes. Moreover, mice with conditional knockout of KLF4 in osteoblasts showed markedly increased bone mass caused by enhanced bone formation despite increased osteoclast activity. Thus, our data suggest that KLF4 controls bone homeostasis by negatively regulating both osteoclast and osteoblast differentiation.     

High mobility group box 1 protein regulates osteoclastogenesis through direct actions on osteocytes and osteoclasts in vitro

Old age and Cx43 deletion in osteocytes are associated with increased osteocyte apoptosis and osteoclastogenesis. We previously demonstrated that apoptotic osteocytes release elevated concentrations of the proinflammatory cytokine, high mobility group box 1 protein (HMGB1) and apoptotic osteocyte conditioned media (CM) promotes osteoclast differentiation. Further, prevention of osteocyte apoptosis blocks osteoclast differentiation and attenuates the extracellular release of HMGB1 and RANKL. Moreover, sequestration of HMGB1, in turn, reduces RANKL production/release by MLO-Y4 osteocytic cells silenced for Cx43 (Cx43^def), highlighting the possibility that HMGB1 promotes apoptotic osteocyte-induced osteoclastogenesis. However, the role of HMGB1 signaling in osteocytes has not been well studied. Further, the mechanisms underlying its release and the receptor(s) responsible for its actions is not clear. We now report that a neutralizing HMGB1 antibody reduces osteoclast formation in RANKL/M-CSF treated bone marrow cells. In bone marrow macrophages (BMMs), toll-like receptor 4 (TLR4) inhibition with LPS-RS, but not receptor for advanced glycation end products (RAGE) inhibition with Azeliragon attenuated osteoclast differentiation. Further, inhibition of RAGE but not of TLR4 in osteoclast precursors reduced osteoclast number, suggesting that HGMB1 produced by osteoclasts directly affects differentiation by activating TLR4 in BMMs and RAGE in preosteoclasts. Our findings also suggest that increased osteoclastogenesis induced by apoptotic osteocytes CM is not mediated through HMGB1/RAGE activation and that direct HMGB1 actions in osteocytes stimulate pro-osteoclastogenic signal release from Cx43^def osteocytes. Based on these findings, we propose that HMGB1 exerts dual effects on osteoclasts, directly by inducing differentiation through TLR4 and RAGE activation and indirectly by increasing pro-osteoclastogenic cytokine secretion from osteocytes.     

Osteoclastic activity induces osteomodulin expression in osteoblasts

Bone resorption by osteoclasts stimulates bone formation by osteoblasts. To isolate osteoblastic factors coupled with osteoclast activity, we performed microarray and cluster analysis of 8 tissues including bone, and found that among 10,490 genes, osteomodulin (OMD), an extracellular matrix keratan sulfate proteoglycan, was simultaneously induced with osteoclast-specific markers such as MMP9 and Acp5. OMD expression was detected in osteoblasts and upregulated during osteoblast maturation. OMD expression in osteoblasts was also detected immunohistochemically using a specific antibody against OMD. The immunoreactivity against OMD decreased in op/op mice, which lack functional macrophage colony stimulating factor (M-CSF) and are therefore defective in osteoclast formation, when compared to wild-type littermates. OMD expression in op/op mice was upregulated by M-CSF treatment. Since the M-CSF receptor c-Fms was not expressed in osteoblasts, it is likely that OMD is an osteoblast maturation marker that is induced by osteoclast activity.     

Macrophage colony-stimulating factor suppresses osteoblast formation.

We provide the first evidence that the bone marrow-derived cytokine, macrophage colony-stimulating factor (M-CSF), inhibits the formation of bone-forming osteoblasts. We examined both osteoclast and osteoblast formation in primary rat bone marrow cultures. As expected, M-CSF together with osteoprotegerin ligand (OPGL) markedly accelerated osteoclastogenesis. In contrast, treatment with M-CSF alone yielded no osteoclasts at any time. The most striking and novel observation was that M-CSF with or without OPGL dramatically suppressed osteoblast formation. In separate experiments, estradiol markedly suppressed osteoclast formation in the M-CSF/OPGL-treated cultures independently of osteoblasts. Consistent with this was the expression of estrogen receptor-alpha (ERalpha) and ERbeta mRNA in osteoclast precursors. We therefore conclude that in addition to the well-known action of M-CSF to modulate osteoclastogenesis, this cytokine may also regulate osteoblast formation.     

Receptor Activator of NF-κB (RANK) Cytoplasmic IVVY535–538 Motif Plays an Essential Role in Tumor Necrosis Factor-α (TNF)-mediated Osteoclastogenesis

Tumor necrosis factor-α (TNF) enhances osteoclast formation and activity leading to bone loss in various pathological conditions, but its precise role in osteoclastogenesis remains controversial. Although several groups showed that TNF can promote osteoclastogenesis independently of the receptor activator of NF-κB (RANK) ligand (RANKL), others demonstrated that TNF-mediated osteoclastogenesis needs permissive levels of RANKL. Here, we independently reveal that although TNF cannot stimulate osteoclastogenesis on bone slices, it can induce the formation of functional osteoclasts on bone slices in the presence of permissive levels of RANKL or from bone marrow macrophages (BMMs) pretreated by RANKL. TNF can still promote the formation of functional osteoclasts 2 days after transient RANKL pretreatment. These data have confirmed that TNF-mediated osteoclastogenesis requires priming of BMMs by RANKL. Moreover, we investigated the molecular mechanism underlying the dependence of TNF-mediated osteoclastogenesis on RANKL. RANK, the receptor for RANKL, contains an IVVY^535–538 motif that has been shown to play a vital role in osteoclastogenesis by committing BMMs to the osteoclast lineage. We show that TNF-induced osteoclastogenesis depends on RANKL to commit BMMs to the osteoclast lineage and RANKL regulates the lineage commitment through the IVVY motif. Mechanistically, the IVVY motif controls the lineage commitment by reprogramming osteoclast genes into an inducible state in which they can be activated by TNF. Our findings not only provide important mechanistic insights into the action of RANKL in TNF-mediated osteoclastogenesis but also establish that the IVVY motif may serve as an attractive therapeutic target for bone loss in various bone disorders.     

Kirenol inhibits RANKL-induced osteoclastogenesis and prevents ovariectomized-induced osteoporosis via suppressing the Ca2+-NFATc1 and Cav-1 signaling pathways

BackgroundOsteoporosis is a threat to aged people who have excessive osteoclast activation and bone resorption, subsequently causing fracture and even disability. Inhibiting osteoclast differentiation and absorptive functions has become an efficient approach to treat osteoporosis, but osteoclast-targeting inhibitors available clinically remain rare. Kirenol (Kir), a bioactive diterpenoid derived from an antirheumatic Chinese herbal medicine Herba Siegesbeckiae, can treat collagen-induced arthritis in vivo and promote osteoblast differentiation in vitro, while the effects of Kir on osteoclasts are still unclear.PurposeWe explore the role of Kir on RANKL-induced osteoclastogenesis in vitro and bone loss in vivo.MethodsThe in vitro effects of Kir on osteoclast differentiation, bone resorption and the underlying mechanisms were evaluated with bone marrow-derived macrophages (BMMs). In vivo experiments were performed using an ovariectomy (OVX)-induced osteoporosis model.ResultsWe found that Kir remarkably inhibited osteoclast generation and bone resorption in vitro. Mechanistically, Kir significantly inhibited F-actinring formation and repressed RANKL-induced NF-κB p65 activation and p-p38, p-ERK and c-Fos expression. Moreover, Kir inhibited both the expression and nuclear translocation of NFATc1. Ca²⁺ oscillation and caveolin-1 (Cav-1) were also reduced by Kir during osteoclastogenesis in vitro. Consistent with these findings, 2–10 mg/kg Kir attenuated OVX-induced osteoporosis in vivo as evidenced by decreased osteoclast numbers and downregulated Cav-1 and NFATc1 expression.ConclusionsKir suppresses osteoclastogenesis and the Cav-1/NFATc1 signaling pathway both in vitro and in vivo and protects against OVX-induced osteoporosis. Our findings reveal Kir as a potential safe oral treatment for osteoporosis.     

Downregulated fat mass and obesity-associated protein inhibits bone resorption and osteoclastogenesis by nuclear factor-kappa B inactivation

During osteoporosis, fat mass and obesity-associated protein (FTO) promotes the shift of bone marrow mesenchymal stem cells to adipocytes and represses osteoblast activity. However, the role and mechanisms of FTO on osteoclast formation and bone resorption remain unknown. In this study, we investigated the effect of FTO on RAW264.7 cells and bone marrow monocytes (BMMs)-derived osteoclasts in vitro and observed the influence of FTO on ovariectomized (OVX) mice model to mimic postmenopausal osteoporosis in vivo. Results found that FTO was up-regulated in BMMs from OVX mice. Double immunofluorescence assay showed co-localization of FTO with tartrate-resistant acid phosphatase (TRAP) in femurs of OVX mice. FTO overexpression enhanced TRAP-positive osteoclasts and F-actin ring formation in RAW264.7 cells upon RANKL stimulation. The expression of osteoclast differentiation-related genes, including nuclear factor of activated T cells c1 (NFATc1) and c-FOS, was upregulated in BMMs and RAW264.7 cells after FTO overexpression. FTO overexpression induced the phosphorylation and nuclear translocation of factor-kappa B (NF-κB) p65 in BMMs and RAW264.7 cells exposed to RANKL. ChIP and dual-luciferase assays revealed that FTO overexpression contributed to RANKL-induced binding of NF-κB to NFATc1 promoter. Rescue experiments suggested that FTO overexpression-mediated osteoclast differentiation was suppressed after intervention with a NF-κB inhibitor pyrrolidine dithiocarbamate. Further in vivo evidence revealed that FTO knockdown increased bone trabecula and bone mineral density, inhibited bone resorption and osteoclastogenesis in osteoporotic mice. Collectively, our research demonstrates that downregulated FTO inhibits bone resorption and osteoclastogenesis through NF-κB inactivation, which provides a novel reference for osteoporosis treatment.     

Carboxypeptidase E Is a Novel Modulator of RANKL-Induced Osteoclast Differentiation

Osteoclasts are large polykaryons that have the unique capacity to degrade bone and are generated by the differentiation of myeloid lineage progenitors. To identify the genes involved in osteoclast development, we performed microarray analysis, and we found that carboxypeptidase E (CPE), a prohormone processing enzyme, was highly upregulated in osteoclasts compared with their precursors, bone marrow-derived macrophages (BMMs). Here, we demonstrate a novel role for CPE in receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation. The overexpression of CPE in BMMs increases the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear osteoclasts and the expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1), which are key regulators in osteoclastogenesis. Furthermore, employing CPE knockout mice, we show that CPE deficiency attenuates osteoclast formation. Together, our data suggest that CPE might be an important modulator of RANKL-induced osteoclast differentiation.     

Pyrroloquinoline Quinine Inhibits RANKL-Mediated Expression of NFATc1 in Part via Suppression of c-Fos in Mouse Bone Marrow Cells and Inhibits Wear Particle-Induced Osteolysis in Mice

The effects of pyrroloquinoline quinine (PQQ) on RANKL-induced osteoclast differentiation and on wear particle-induced osteolysis were examined in this study. PQQ inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages (BMMs) in a dose-dependent manner without any evidence of cytotoxicity. The mRNA expression of c-Fos, NFATc1, and TRAP in RANKL-treated BMMs was inhibited by PQQ treatment. Moreover, RANKL-induced c-Fos and NFATc1 protein expression was suppressed by PQQ. PQQ additionally inhibited the bone resorptive activity of differentiated osteoclasts. Further a UHMWPE-induced murine calvaria erosion model study was performed to assess the effects of PQQ on wear particle-induced osteolysis in vivo. Mice treated with PQQ demonstrated marked attenuation of bone erosion based on Micro-CT and histologic analysis of calvaria. These results collectively suggested that PQQ demonstrated inhibitory effects on osteoclast differentiation in vitro and may suppress wear particle-induced osteolysis in vivo, indicating that PQQ may therefore serve as a useful drug in the prevention of bone loss.     

Extracellular glutamate alters mature osteoclast and osteoblast functions

Glutamatergic intercellular communication is involved in many aspects of metabolic homeostasis in normal bone. In bone metastasis, the balance between bone formation and degradation is disrupted. Although the responsible mechanisms are not clear, we have previously identified that cancer cell lines used in bone tumour models secrete glutamate, suggesting that tumour-derived glutamate may disrupt sensitive signalling systems in bone. This study examines the role of glutamate in mature osteoclastic bone resorption, osteoblast differentiation, and bone nodule formation. Glutamate was found to have no effect on the survival or activity of mature osteoclasts, although glutamate transporter inhibition and receptor blockade increased the number of bone resorption pits. Furthermore, transporter inhibition increased the area of resorbed bone while significantly decreasing the number of osteoclasts. Alkaline phosphatase activity and extracellular matrix mineralization were used as measurements of osteoblast differentiation. Glutamate significantly increased osteoblast differentiation and mineralization, but transport inhibitors had no effect. These studies support earlier findings suggesting that glutamate may be more important for osteoclastogenesis than for osteoclast proliferation or functions. Since glutamate is capable of changing the differentiation and activities of both osteoclast and osteoblast cell types in bone, it is reasonable to postulate that tumour-derived glutamate may impact bone homeostasis in bone metastasis.     

Citrus limonoid nomilin inhibits osteoclastogenesis in vitro by suppression of NFATc1 and MAPK signaling pathways

BackgroundAnimal experiment studies have revealed a positive association between intake of citrus fruits and bone health. Nomilin, a limonoid present in citrus fruits, is reported to have many biological activities in mammalian systems, but the mechanism of nomilin on bone metabolism regulation is currently unclear.PurposeTo reveal the mechanism of nomilin on osteoclastic differentiation of mouse primary bone marrow-derived macrophages (BMMs) and the mouse RAW 264.7 macrophage cell line into osteoclasts.Study designControlled laboratory study. Effects of nomilin on osteoclastic differentiation were studied in in vitro cell cultures.MethodsCell viability of RAW 264.7 cells and BMMs was measured with the Cell Counting Kit. TRAP-positive multinucleated cells were counted as osteoclast cell numbers. The number and area of resorption pits were measured as bone-resorbing activity. Osteoclast-specific genes expression was evaluated by quantitative real-time PCR; and proteins expression was evaluated by western blot.ResultsNomilin significantly decreased TRAP-positive multinucleated cell numbers compared with the control, and exhibited no cytotoxicity. Nomilin decreased bone resorption activity. Nomilin downregulated osteoclast-specific genes, NFATc1 and TRAP mRNA levels. Furthermore, nomilin suppressed MAPK signaling pathways.ConclusionThis study demonstrates clearly that nomilin has inhibitory effects on osteoclastic differentiation in vitro. These findings indicate that nomilin-containing herbal preparations have potential utility for the prevention of bone metabolic diseases.     

Artemisinin inhibits breast cancer-induced osteolysis by inhibiting osteoclast formation and breast cancer cell proliferation

In addition to being used to treat malaria, artemisinin (Art) can be used as an anti-inflammatory and antitumor agent. In this study, we evaluated the effects of Art on osteoclast formation and activation and on the development of breast cancer cells in bone. To evaluate the effect of Art on osteoclast differentiation in vitro, we treated bone marrow-derived macrophages (BMMs) with various concentrations of Art and evaluated the expression of genes and proteins involved in osteoclast formation. We also performed cell counting kit-8 assays to evaluate the toxicity of Art in BMMs and MDA-MB-231 cells. We also performed Transwell assays, wound-healing assays, colony formation assays, and cell apoptosis assays to evaluate the effect of Art in MDA-MB-231 cells. We also evaluated the effect of Art in an in vivo osteoclast bone resorption assay using a nude mouse model. We demonstrated that Art inhibits the differentiation and establishment of osteoclasts even though Art is not toxic to osteoclasts. In addition, Art reduced expression of genes involved in osteoclast formation and inhibited osteoclast bone resorption in a concentration-dependent manner. Based on our data, we believe that Art can inhibit proliferation of breast cancer cells by activating apoptosis pathways, and inhibit osteoclast formation and differentiation by inhibiting activation of cathepsin K, ATPase H+ transporting V0 subunit D2, nuclear factor of activated T cells 1, calcitonin receptor, and tartrate-resistant acid phosphatase and by inhibiting nuclear factor-κB activation.