Bone-resorbing Activity Of Mature Osteoclasts Research Articles

Monascin abrogates RANKL-mediated osteoclastogenesis in RAW264.7 cells via regulating MAPKs signaling pathways.

Osteoclasts (OCs) are multinucleated cells that play a major role in osteolytic diseases such as osteoporosis. Monascin (Ms) is one of the active substances in the traditional Chinese medicine red yeast rice. Studies have found that red yeast rice can maintain bone health. In this study, the anti-osteoclastogenesis effects of Ms on RANKL-induced RAW264.7 cells were assessed, and the underlying mechanism was investigated. Ms exhibited inhibitory effects on OC differentiation and formation in a dose-dependent manner and suppressed the bone-resorbing activity of mature OCs. Ms blocked OCs-typical genes (c-Fos, NFATc1, CSTK, MMP-9, TRAP, ITG-β3, OSCAR and DC-STAMP). Furthermore, Ms treatment considerably inhibited the activation of MAPKs, JNK and p38. Taken together, Ms suppresses RANKL-induced osteoclastogenesis of RAW264.7 cells by restraining MAPKs signaling pathways and is a potential therapeutic option as a novel OC inhibitor to mitigate bone erosion.

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.

Pantoprazole (PPZ) Inhibits RANKL-Induced Osteoclast Formation and Function In Vitro and Prevents Lipopolysaccharide- (LPS-) Induced Inflammatory Calvarial Bone Loss In Vivo.

Bone remodeling is a process delicately balanced between osteoclastic bone resorption and osteoblastic bone formation. Osteoclasts (OCs) are multinucleated giant cells formed through the fusion of monocytic precursors of the hematopoietic stem cells lineage. OCs are the exclusive cells responsible for the resorption and degradation of the mineralized bone matrix. Pantoprazole (PPZ), a proton pump inhibitor (PPI), is commonly prescribed to reduce excess gastric acid production for conditions such as gastroesophageal reflux disease and peptic ulcer disease. Studies have found contradictory effects of PPI therapy on bone metabolism due to the lack of understanding of the exact underlying mechanism. In this study, we found that PPZ inhibits receptor activator of nuclear factor-κB (NF-κB) ligand- (RANKL-) induced osteoclastogenesis from bone marrow monocytic/macrophage (BMMs) precursors and the bone-resorbing activity of mature OCs. Correspondingly, the expression of OC marker genes was also attenuated. At the molecular level, PPZ treatment was associated with reduced activation of the ERK MAPK signaling pathways crucial to OC differentiation. Additionally, the in vivo administration of PPZ protected mice against lipopolysaccharide- (LPS-) induced inflammatory calvarial bone erosion, as a result of the reduced number and activity of OCs on the calvarial bone surface. Although PPI use is associated with increased risk of osteoporosis and bone fractures, our study provides evidence for the direct inhibitory effect of PPZ on OC formation and bone resorption in vitro and in vivo, suggesting a potential therapeutic use of PPZ in the treatment of osteolytic disease with localized bone destruction.

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.

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.

Nicotinamide phosphoribosyltransferase inhibits receptor activator of nuclear factor-κB ligand-induced osteoclast differentiation invitro.

The adipokine nicotinamide phosphoribosyltransferase (Nampt), also known as pre-B-cell colony-enhancing factor or the insulin-mimetic hormone visfatin, has a crucial role in the conversion of nicotinamide to nicotinamide mononucleotide during biosynthesis of the coenzyme nicotinamide adenine dinucleotide. Previous reports have demonstrated the inhibitory effects of Nampt on osteoclast formation from human peripheral blood mononuclear cells and CD14+ monocytes. However, the effects of Nampt on bone marrow macrophage (BMM)-derived osteoclastogenesis and its precise role in the process remain unclear. The present in vitro study used recombinant Nampt and BMMs as osteoclast precursors demonstrated that Nampt suppresses receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis by decreasing the phosphorylation of various early signal transducers, including c-Jun N-terminal kinase, Akt, glycogen synthase kinase-3 β, Bruton's tyrosine kinase and phospholipase C γ-2. In addition, western blotting and reverse transcription-quantitative polymerase chain reaction analysis indicated that Nampt downregulates the mRNA and protein expression levels of c-Fos and nuclear factor of activated T cells, cytoplasmic 1, leading to a decrease in the expression of osteoclast-specific genes including tartrate-resistant acid phosphatase, osteoclast-associated receptor and cathepsin K. However, the bone-resorbing activity of mature osteoclasts treated with Nampt was similar to untreated control osteoclasts. This finding indicates that Nampt exerts its anti-osteoclastogenic activity by targeting osteoclast precursor cells rather than mature osteoclasts. Consequently, the present study demonstrated that Nampt acts as a negative regulator of RANKL-mediated differentiation of BMMs into osteoclasts, suggesting the potential therapeutic targets to treat bone-related disorders such as osteoporosis.

Tetraspanin 7 regulates sealing zone formation and the bone-resorbing activity of osteoclasts

Tetraspanin family proteins regulate morphology, motility, fusion, and signaling in various cell types. We investigated the role of the tetraspanin 7 (Tspan7) isoform in the differentiation and function of osteoclasts. Tspan7 was up-regulated during osteoclastogenesis. When Tspan7 expression was reduced in primary precursor cells by siRNA-mediated gene knock-down, the generation of multinuclear osteoclasts was not affected. However, a striking cytoskeletal abnormality was observed: the formation of the podosome belt structure was inhibited and the microtubular network were disrupted by Tspan7 knock-down. Decreases in acetylated microtubules and levels of phosphorylated Src and Pyk2 in Tspan7 knock-down cells supported the involvement of Tspan7 in cytoskeletal rearrangement signaling in osteoclasts. This cytoskeletal defect interfered with sealing zone formation and subsequently the bone-resorbing activity of mature osteoclasts on dentin surfaces. Our results suggest that Tspan7 plays an important role in cytoskeletal organization required for the bone-resorbing function of osteoclasts by regulating signaling to Src, Pyk2, and microtubules.

Protocatechuic Acid Attenuates Osteoclastogenesis by Downregulating JNK/c-Fos/NFATc1 Signaling and Prevents Inflammatory Bone Loss in Mice.

Protocatechuic acid (PCA) plays a critical role in nutritional metabolism; it is a major metabolite of anthocyanins, which are flavonoids with a range of health benefits. PCA has a variety of biological activities including anti-oxidant, antiinflammatory, anti-apoptosis, and anti-microbial activities. However, the pharmacological effect of PCA, especially on osteoclastogenesis, remains unknown. We examined the effect of PCA on receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation and bone resorption. PCA dose-dependently inhibited RANKL-induced osteoclast differentiation in mouse bone marrow macrophages (BMMs) and suppressed the bone-resorbing activity of mature osteoclasts. At the molecular level, PCA suppressed RANKL-induced phosphorylation of JNK among MAPKs only, without significantly affecting the early signaling pathway. PCA also suppressed RANKL-stimulated expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1) at the mRNA and protein levels, without altering c-Fos mRNA expression. Additionally, PCA down-regulated the expression of downstream osteoclastogenesis-related genes including β3-integrin, DC-STAMP, OC-STAMP, Atp6v0d2, CTR, and CtsK. Mice treated with PCA efficiently recovered from lipopolysaccharide-induced bone loss in vivo. Thus, PCA inhibits RANKL-induced osteoclast differentiation and function by suppressing JNK signaling, c-Fos stability, and expression of osteoclastic marker genes. These results suggest that PCA could be useful in treatment of inflammatory bone disorders.

Ostericum koreanum Reduces LPS-Induced Bone Loss Through Inhibition of Osteoclastogenesis

The roots of Ostericum koreanum (OK) Maximowicz have traditionally been used to produce an herbal medicine reported to possess anti-inflammatory, anti-oxidant, antimicrobial, and antitumor activities; however, its effect on bone metabolism has not yet been reported. The present study examined the effects of OK extract on lipopolysaccharide (LPS)-induced bone loss in mice by investigating bone structure and the levels of the receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) in serum and bone marrow fluid (BMF). The effects of OK extract on osteoclastogenesis were also investigated in mouse bone marrow macrophages by examining the formation of tartrate-resistant acid phosphatase (TRAP)-positive cells, the actin ring, and bone resorption activity. OK reduced LPS-induced bone destruction in vivo via a decrease in the RANKL/OPG ratio. Furthermore, it suppressed the formation of TRAP-positive cells and the actin ring, and reduced the bone-resorbing activity of mature osteoclasts. OK also significantly down-regulated the expression of various osteoclast-specific genes. However, it did not affect osteoblast differentiation, or the expression of genes involved in this process. These results demonstrated that OK prevented LPS-induced bone loss by decreasing the RANKL/OPG ratio in serum and BMF, and inhibited osteoclast differentiation and function, suggesting that OK represents a potential therapeutic drug for the treatment of osteoclast-associated bone diseases.

Aconitum pseudo-laeve var. erectum inhibits receptor activator of nuclear factor kappa-B ligand-induced osteoclastogenesis via the c-Fos/nuclear factor of activated T-cells, cytoplasmic 1 signaling pathway and prevents lipopolysaccharide-induced bone loss in mice.

Aconitum pseudo-laeve var. erectum (APE) has been widely shown in herbal medicine to have a therapeutic effect on inflammatory conditions. However, there has been no evidence on whether the extract of APE is involved in the biological bone metabolism process, particularly osteoclast-mediated bone resorption. In this study, we confirmed that the administration of APE could restore normal skeletal conditions in a murine model of lipopolysaccharide (LPS)-induced bone loss via a decrease in the receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) ratio and osteoclast number. We then investigated the effect of APE on the RANKL-induced formation and function of osteoclasts to elucidate its underlying molecular mechanisms. APE suppressed the formation of tartrate-resistant acid phosphatase (TRAP)-positive cells, as well as the bone-resorbing activity of mature osteoclasts. Furthermore, APE attenuated nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and c-Fos without affecting any early signal pathway of osteoclastogenesis. Subsequently, APE significantly downregulated the expression of various genes exclusively expressed in osteoclasts. These results demonstrate that APE restores LPS-induced bone loss through a decrease of the serum RANKL/OPG ratio, and inhibits osteoclast differentiation and function, suggesting the promise of APE as a potential cure for various osteoclast-associated bone diseases.

Hypericin suppresses osteoclast formation and wear particle-induced osteolysis via modulating ERK signalling pathway

Osteoclast-induced bone resorption and wear-particle-induced osteolysis leads to prosthetic loosening, one of the most common causes of joint implant failure, resulting in revision surgery. Thus, inhibition of osteoclastic bone resorption, which further prevents wear particle-induced osteolysis, is a potential treatment strategy for prosthetic loosening. Here, we examined the therapeutic effect of hypericin (HP), which was photosensitive, on osteoclastogenesis and wear particle-induced osteolysis in the absence of visible light. HP inhibited RANKL-induced osteoclast differentiation in bone marrow macrophages (BMMs) and RAW264.7 cell line without any evidence of cytotoxicity. The bone-resorbing activity of mature osteoclasts was significantly inhibited by HP. As HP has been previously reported to inhibit signalling pathway such as ERK and NF-κB in other cells, which is also important in osteoclast differentiation. We thus examined the molecular mechanism and showed that HP significantly inhibited the ERK/mitogen-activated protein kinase (MAPK) signalling pathway without affecting nuclear factor kappaB (NF-κB), c-Jun N-terminal kinase (JNK) and p38 signalling in RANKL-stimulated BMMs. Further in vivo studies revealed HP attenuated osteoclast formation and subsequently prevented wear particle-induced bone erosion. Taken together, the results suggest that HP inhibits RANKL-mediated osteoclastogenesis via affecting ERK signalling in vitro and suppresses wear particle-induced osteolysis in vivo. We therefore conclude that HP may be an innovative and safe alternative treatment for osteoclast-related prosthetic loosening.

A review of denosumab for the treatment of osteoporosis.

Osteoporosis is an age-related systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility. Bone remodeling involves two types of cells: osteoblasts and osteoclasts. Receptor activator of nuclear factor-κB ligand (RANKL) is a key regulator of the formation and function of bone-resorbing osteoclasts, and its cell surface receptor, receptor activator of nuclear factor-κB (RANK), is expressed by both osteoclast precursors and mature osteoclasts. Denosumab is a fully human monoclonal anti-RANKL antibody that inhibits the binding of RANKL to RANK, thereby decreasing osteoclastogenesis and bone-resorbing activity of mature osteoclasts. Although there are many medications available for the treatment of osteoporosis, inhibition of RANKL by denosumab has been shown to significantly affect bone metabolism. Denosumab appears to be a promising, highly effective, and safe parenteral therapy with good adherence for osteoporosis. Moreover, denosumab may be cost-effective therapy compared with existing alternatives. Therefore, in this review, we focus on studies of denosumab and the risks and benefits identified for this type of treatment for osteoporosis.

Emodin regulates bone remodeling by inhibiting osteoclastogenesis and stimulating osteoblast formation.

Bone remodeling, a physiological process in which new bone is formed by osteoblasts and the preexisting bone matrix is resorbed by osteoclasts, is vital for the maintenance of healthy bone tissue in adult humans. Imbalances in this process can cause various pathological conditions, including osteoporosis. Emodin, a naturally occurring anthraquinone derivative found in Asian herbal medicines, has numerous beneficial pharmacologic effects, including anticancer and antidiabetic activities. However, the effect of emodin on the regulation of osteoblast and osteoclast activity has not yet been investigated. We show here that emodin is a potential target for osteoporosis therapeutics, as treatment with this agent enhances osteoblast differentiation and bone growth and suppresses osteoclast differentiation and bone resorption. In this study, emodin suppressed receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast differentiation of bone marrow macrophages (BMMs) and the bone-resorbing activity of mature osteoclasts by inhibiting RANKL-induced NF-κB, c-Fos, and NFATc1 expression. Emodin also increased ALP, Alizarin Red-mineralization activity, and the expression of osteoblastogenic gene markers, such as Runx2, osteocalcin (OCN), and ALP in mouse calvarial primary osteoblasts, as well as activated the p38-Runx2 pathway, which enhanced osteoblast differentiation. Moreover, mice treated with emodin showed marked attenuation of lipopolysaccharide (LPS)-induced bone erosion and increased bone-forming activity in a mouse calvarial bone formation model based on micro-computed tomography and histologic analysis of femurs. Our findings reveal a novel function for emodin in bone remodeling, and highlight its potential for use as a therapeutic agent in the treatment of osteoporosis that promotes bone anabolic activity and inhibits osteoclast differentiation.

Dual Effect ofChrysanthemum indicumExtract to Stimulate Osteoblast Differentiation and Inhibit Osteoclast Formation and ResorptionIn Vitro

The risk of bone-related diseases increases due to the imbalance between bone resorption and bone formation by osteoclasts and osteoblasts, respectively. The goal in the development of antiosteoporotic treatments is an agent that will improve bone through simultaneous osteoblast stimulation and osteoclast inhibition without undesirable side effects. To achieve this goal, numerous studies have been performed to identify novel approaches using natural oriental herbs to treat bone metabolic diseases. In the present study, we investigated the effect of Chrysanthemum indicum extract (CIE) on the differentiation of osteoclastic and osteoblastic cells. CIE inhibited the formation of TRAP-positive mature osteoclasts and of filamentous-actin rings and disrupted the bone-resorbing activity of mature osteoclasts in a dose-dependent manner. CIE strongly inhibited Akt, GSK3β, and IκB phosphorylation in RANKL-stimulated bone marrow macrophages and did not show any effects on MAP kinases, including p38, ERK, and JNK. Interestingly, CIE also enhanced primary osteoblast differentiation via upregulation of the expression of alkaline phosphatase and the level of extracellular calcium concentrations during the early and terminal stages of differentiation, respectively. Our results revealed that CIE could have a potential therapeutic role in bone-related disorders through its dual effects on osteoclast and osteoblast differentiation.

(2<i>S</i>)-2′-Methoxykurarinone Inhibits Osteoclastogenesis and Bone Resorption through Down-Regulation of RANKL Signaling

(2S)-2'-Methoxykurarinone (MK), a compound isolated from the roots of Sophora flavescens, has various physiological properties, such as anti-inflammatory, antipyretic, antidiabetic, and antineoplastic effects. However, the effect of S. flavescens-derived MK on osteoclastogenesis remains unknown. Therefore, we examined the effect and mechanism of action of MK on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and bone resorption. MK inhibited osteoclast differentiation in bone marrow cell-osteoblast cocultures but did not affect the RANKL-to-osteoprotegerin ratio induced by osteoclastogenic factors in osteoblasts. MK also inhibited RANKL-induced osteoclast differentiation from bone marrow macrophages in a dose-dependent manner, without cytotoxicity. Pretreatment with MK significantly suppressed the Akt, p38, c-Jun N terminal kinase (JNK), c-Fos, and nuclear factor of activated T cells c1 (NFATc1) pathways and inhibited the bone-resorbing activity of mature osteoclasts. These results collectively suggest that MK inhibits osteoclast differentiation and bone resorption through RANKL-induced mitogen-activated protein kinases (MAPKs) and c-Fos-NFATc1 signaling pathways.

Oleanolic acid acetate inhibits osteoclast differentiation by downregulating PLCγ2–Ca2 +-NFATc1 signaling, and suppresses bone loss in mice

Owing to their potential pharmacological activities in human disease, natural plant-derived compounds have recently become the focus of increased research interest. In this study, we first isolated oleanolic acid acetate (OAA), a triterpenoid compound, from Vigna angularis (azuki bean) to discover anti-bone resorptive agents. Many studies have identified and described the various medicinal effects of V. angularis extract. However, the pharmacological effect of OAA-derived V. angularis extract, particularly the effect on osteoclastogenesis, is not known. Therefore, we investigated the effect and mechanism of OAA in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. OAA inhibited RANKL-induced osteoclast differentiation in bone marrow macrophages (BMMs) without any evidence of cytotoxicity. Interestingly, OAA significantly inhibited Btk phosphorylation, phospholipase Cγ2 (PLCγ2) phosphorylation, calcium ion (Ca2+) oscillation, and nuclear factor of activated T cell c1 (NFATc1) expression in RANKL-stimulated BMMs, but did not affect RANKL-induced mitogen-activated protein kinase. OAA also inhibited the bone-resorbing activity of mature osteoclasts. Furthermore, mice treated with OAA demonstrated marked attenuation of lipopolysaccharide-induced bone erosion based on micro-computed tomography and histologic analysis of femurs. Taken together, the results suggested that OAA inhibited RANKL-mediated osteoclastogenesis via PLCγ2-Ca2+-NFATc1 signaling in vitro and suppressed inflammatory bone loss in vivo.

The tyrosine kinase inhibitor GNF-2 suppresses osteoclast formation and activity

GNF-2, a tyrosine kinase inhibitor, was developed to overcome imatinib-resistant mutations found in CML patients. Osteoclasts are the principal bone-resorbing cells that are responsible for bone diseases, such as osteoporosis, tumor-induced osteolysis, and metastatic cancers. In this study, we investigated the effect of GNF-2 on osteoclast development induced by RANKL and M-CSF. We found that GNF-2 inhibited osteoclast differentiation from BMMs. GNF-2 suppressed RANKL-induced NF-κB transcriptional activity and the induction of c-Fos and NFATc1, which are two key transcription factors in osteoclastogenesis. We also observed that GNF-2 dose-dependently inhibited the proliferation of osteoclast precursors through the suppression of the M-CSFR c-Fms. In addition, GNF-2 accelerated osteoclast apoptosis by inducing caspase-3 and Bim expression. Furthermore, GNF-2 interfered with actin cytoskeletal organization and subsequently blocked the bone-resorbing activity of mature osteoclasts. In agreement with its in vitro effects, GNF-2 reduced osteoclast number and bone loss in a mouse model of LPS-induced bone destruction. Taken together, our data reveal that GNF-2 possesses anti-bone-resorptive properties, suggesting that GNF-2 may have therapeutic value for the treatment of bone-destructive disorders that can occur as a result of excessive osteoclastic bone resorption.

AB0110 Iguratimod inhibits rankl-induced osteoclastogenesis in raw264.7 cells

BackgroundIguratimod, also named as T- 614, is a member of the family of methanesulfonanilide. We previously reported that Iguratimod is effective and well tolerated for treatment of patients with active...

Role of Bruton's tyrosine kinase in myeloma cell migration and induction of bone disease

Myeloma cells typically grow in bone, recruit osteoclast precursors and induce their differentiation and activity in areas adjacent to tumor foci. Bruton's tyrosine kinase (BTK), of the TEC family, is expressed in hematopoietic cells and is particularly involved in B-lymphocyte function and osteoclastogenesis. We demonstrated BTK expression in clinical myeloma plasma cells, interleukin (IL)-6- or stroma-dependent cell lines and osteoclasts. SDF-1 induced BTK activation in myeloma cells and BTK inhibition by small hairpin RNA or the small molecule inhibitor, LFM-A13, reduced their migration toward stromal cell-derived factor-1 (SDF-1). Pretreatment with LFM-A13 also reduced in vivo homing of myeloma cells to bone using bioluminescence imaging in the SCID-rab model. Enforced expression of BTK in myeloma cell line enhanced cell migration toward SDF-1 but had no effect on short-term growth. BTK expression was correlated with cell-surface CXCR4 expression in myeloma cells (n = 33, r = 0.81, P < 0.0001), and BTK gene and protein expression was more profound in cell-surface CXCR4-expressing myeloma cells. BTK was not upregulated by IL-6 while its inhibition had no effect on IL-6 signaling in myeloma cells. Human osteoclast precursors also expressed BTK and cell-surface CXCR4 and migrated toward SDF-1. LFM-A13 suppressed migration and differentiation of osteoclast precursors as well as bone-resorbing activity of mature osteoclasts. In primary myeloma-bearing SCID-rab mice, LFM-A13 inhibited osteoclast activity, prevented myeloma-induced bone resorption and moderately suppressed myeloma growth. These data demonstrate BTK and cell-surface CXCR4 association in myeloma cells and that BTK plays a role in myeloma cell homing to bone and myeloma-induced bone disease. Am. J. Hematol. 88:463-471, 2013. © 2013 Wiley Periodicals, Inc.

Regulation of bone resorption and sealing zone formation in osteoclasts occurs through protein kinase b–mediated microtubule stabilization

We investigated the role of protein kinase B (Akt), a downstream effector of phosphatidylinositol 3-kinase, in bone-resorbing activity of mature osteoclasts. Treatment with a specific Akt inhibitor disrupted sealing zone formation and decreased the bone-resorbing activity of osteoclasts. The normal microtubule structures were lost and the Akt inhibitor reduced the amount of acetylated tubulin, which reflects stabilized microtubules, whereas forced Akt activation by adenovirus vectors resulted in the opposite effect. Forced Akt activation increased the binding of the microtubule-associated protein adenomatous polyposis coli (APC), the APC-binding protein end-binding protein 1 (EB1) and dynactin, a dynein activator complex, with microtubules. Depletion of Akt1 and Akt2 resulted in a disconnection of APC/EB1 and a decrease in bone-resorbing activity along with reduced sealing zone formation, both of which were recovered upon the addition of LiCl, a glycogen synthase kinase-3β (GSK-3β) inhibitor. The Akt1 and Akt2 double-knockout mice exhibited osteosclerosis due to reduced bone resorption. These findings indicate that Akt controls the bone-resorbing activity of osteoclasts by stabilizing microtubules via a regulation of the binding of microtubule associated proteins.