Osteoclastogenesis In Bone Marrow-derived Macrophages Research Articles

Isobavachin attenuates osteoclastogenesis and periodontitis-induced bone loss by inhibiting cellular iron accumulation and mitochondrial biogenesis

As bone-resorbing cells rich in mitochondria, osteoclasts require high iron uptake to promote mitochondrial biogenesis and maintain a high-energy metabolic state for active bone resorption. Given that abnormal osteoclast formation and activation leads to imbalanced bone remodeling and osteolytic bone loss, osteoclasts may be crucial targets for treating osteolytic diseases such as periodontitis. Isobavachin (IBA), a natural flavonoid compound, has been confirmed to be an inhibitor of receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation from bone marrow-derived macrophages (BMMs). However, its effects on periodontitis-induced bone loss and the potential mechanism of its anti-osteoclastogenesis effect remain unclear. Our study demonstrated that IBA suppressed RANKL-induced osteoclastogenesis in BMMs and RAW264.7 cells and inhibited osteoclast-mediated bone resorption in vitro. Transcriptomic analysis indicated that iron homeostasis and reactive oxygen species (ROS) metabolic process were enriched among the differentially expressed genes following IBA treatment. IBA exerted its anti-osteoclastogenesis effect by inhibiting iron accumulation in osteoclasts. Mechanistically, IBA attenuated iron accumulation in RANKL-induced osteoclasts by inhibiting the mitogen-activated protein kinase (MAPK) pathway to upregulate ferroportin1 (Fpn1) expression and promote Fpn1-mediated intracellular iron efflux. We also found that IBA inhibited mitochondrial biogenesis and function, and reduced RANKL-induced ROS generation in osteoclasts. Furthermore, IBA attenuated periodontitis-induced bone loss by reducing osteoclastogenesis in vivo. Overall, these results suggest that IBA may serve as a promising therapeutic strategy for bone diseases characterized by osteoclastic bone resorption.

Protaetia brevitarsis Extract Attenuates RANKL-Induced Osteoclastogenesis by Inhibiting the JNK/NF-κB/PLCγ2 Signaling Pathway.

Protaetia brevitarsis (PB)-derived bioactive substances have been used as food and medicine in many Asian countries because of their antioxidant, antidiabetic, anti-cancer, and hepatoprotective properties. However, the effect of PB extracts (PBE) on osteoclast differentiation is unclear. In this study, we investigated the effect of PBE on RANKL-induced osteoclastogenesis in mouse bone marrow-derived macrophages (BMMs). To investigate the cytotoxicity of PBE, the viability of BMMs was confirmed via MTT assay. Tartrate-resistant acid phosphatase (TRAP) staining and pit assays were performed to confirm the inhibitory effect of PBE on osteoclast differentiation and bone resorption. The expression levels of osteoclast differentiation-related genes and proteins were evaluated using quantitative real-time PCR and Western blotting. PBE attenuated osteoclastogenesis in BMMs in TRAP and pit assays without cytotoxicity. The expression levels of osteoclast marker genes and proteins induced by RANKL were decreased after PBE treatment. PBE suppressed osteoclastogenesis by inhibiting the RANKL-induced activated JNK/NF-κB/PLCγ2 signaling pathway and the expression of NFATc1 and c-Fos. Collectively, these results suggest that PBE could be a potential therapeutic strategy or functional product for osteoclast-related bone disease.

A Vitronectin-Derived Peptide Restores Ovariectomy-Induced Bone Loss by Dual Regulation of Bone Remodeling.

Bone remodeling is tightly regulated through bone resorption and bone formation; imbalances in bone remodeling can cause various pathological conditions such as osteoporosis. Antiresorptive agents commonly used for treating osteoporosis do not substantially reverse osteoporotic bone loss. We evaluated the effects of the RVYFFKGKQYWE motif (residues 270-281; VnP-16) of human vitronectin on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and osteoclastogenesis of bone marrow-derived macrophages. The effects of VnP-16 were also assessed in a mouse model of estrogen deficiency-induced osteoporosis (ovariectomized female C57BL/6mice). To assay whether VnP-16 can reverse ovariectomy-induced bone loss, synthetic peptides or vehicle were subcutaneously injected into ovariectomized mice once a week for 4weeks (n = 10/group). To evaluate the bone restorative effects of VnP-16, in-vivo micro-computed tomography analysis and histological staining were performed. VnP-16 induced osteogenic differentiation of hMSCs and inhibited the RANKL-RANK-TRAF6 axis in the osteoclastogenesis signaling pathway. Furthermore, systemic administration of VnP-16 reversed ovariectomy-induced bone loss in the femoral neck, distal femur and lumbar spine by increasing osteoblast differentiation and promoting bone formation, and concomitantly decreasing osteoclastogenesis and inhibiting bone resorption. The bone restorative effect of VnP-16 was observed one week after subcutaneous administration, and although the timing of the effect differed according to bone location, it persisted for at least 3weeks. Our findings suggest that VnP-16 is a potential therapeutic agent for treating osteoporosis that mediates its effects through dual regulation of bone remodeling.

The Dibenzyl Isoquinoline Alkaloid Berbamine Ameliorates Osteoporosis by Inhibiting Bone Resorption.

Postmenopausal osteoporosis (PMOP) is a kind of primary osteoporosis that is characterized by decreased bone density and strength. Berbamine is a nonbasic quaternary benzylisoquinoline plant alkaloid that has been widely used in the clinic to treat leukopenia in China. We found that berbamine inhibited RANKL-induced osteoclastogenesis of bone marrow-derived macrophages (BMMs) in vitro, which mainly occurred in the middle phase and late phase. The gene and protein expression levels of osteoclast-related molecules, including CTSK, MMP-9, NFATc1, CD44 and DC-STAMP, were also downregulated by berbamine. In vivo, we treated PMOP mice with berbamine for 8 weeks and found that the extent of osteoporosis was alleviated significantly according to micro-CT scanning, hematoxylin-eosin staining, DC-STAMP immunohistochemical staining and TRAP immunohistochemical staining in the distal femurs of the mice. Our findings demonstrate that berbamine has an inhibitory effect on the osteoclastogenesis of BMMs and can prevent bone loss after ovariectomy in vivo. This study provides evidence that berbamine is a potential drug for the prevention and treatment of PMOP.

Macrophages in epididymal adipose tissue secrete osteopontin to regulate bone homeostasis

Epididymal white adipose tissue (eWAT) secretes an array of cytokines to regulate the metabolism of organs and tissues in high-fat diet (HFD)-induced obesity, but its effects on bone metabolism are not well understood. Here, we report that macrophages in eWAT are a main source of osteopontin, which selectively circulates to the bone marrow and promotes the degradation of the bone matrix by activating osteoclasts, as well as modulating bone marrow-derived macrophages (BMDMs) to engulf the lipid droplets released from adipocytes in the bone marrow of mice. However, the lactate accumulation induced by osteopontin regulation blocks both lipolysis and osteoclastogenesis in BMDMs by limiting the energy regeneration by ATP6V0d2 in lysosomes. Both surgical removal of eWAT and local injection of either clodronate liposomes (for depleting macrophages) or osteopontin-neutralizing antibody show comparable amelioration of HFD-induced bone loss in mice. These results provide an avenue for developing therapeutic strategies to mitigate obesity-related bone disorders.

Galangin suppresses RANKL-induced osteoclastogenesis via inhibiting MAPK and NF-κB signalling pathways.

Osteoclasts play a critical role in osteoporosis; thus, inhibiting osteoclastogenesis is a therapeutic strategy for osteoporosis. Galangin, a natural bioflavonoid extracted from a traditional Chinese herb, possesses a variety of biological activities, including anti‐inflammation and anti‐oxidation. However, its effects on osteoporosis have not been elucidated. In this study, we found that galangin treatment dose‐dependently decreased osteoclastogenesis in bone marrow–derived macrophages (BMMs). Moreover, during osteoclastogenesis, osteoclast‐specific genes, such as tartrate‐resistant acid phosphatase (TRAP), cathepsin K (CtsK), ATPase, H + transporting, lysosomal V0 subunit D2 (V‐ATPase d2) and dendritic cell–specific transmembrane protein (DC‐STAMP), were down‐regulated by galangin treatment. Furthermore, the results of the pit formation assay and F‐actin ring staining revealed impaired osteoclastic bone resorption in the galangin‐treated group compared with that in the control group. Additionally, galangin treatment also inhibited the phosphorylation of p38 and ERK of MAPK signalling pathway, as well as downstream factors of NFATc1, C‐Jun and C‐Fos. Consistent with our in vitro results, galangin suppressed lipopolysaccharide (LPS)‐induced bone resorption via inhibition of osteoclastogenesis. Taken together, our findings provide evidence that galangin is a promising natural compound for the treatment of osteoporosis and may be associated with the inhibition of MAPK and NF‐κB signalling pathways.

Elevated Expression of Cathepsin K in Periodontal Ligament Fibroblast by Inflammatory Cytokines Accelerates Osteoclastogenesis via Paracrine Mechanism in Periodontal Disease.

Cathepsin K (CTSK) is a cysteine protease that is mainly produced from mature osteoclasts and contributes to the destruction of connective tissues and mineralized matrix as a consequence of periodontal disease (PD). However, few studies have reported its regulatory role in osteoclastogenesis-supporting cells in inflammatory conditions. Here, we investigated the role of CTSK in osteoclastogenesis-supporting cells, focusing on the modulation of paracrine function. Microarray data showed that CTSK was upregulated in PD patients compared with healthy individuals, which was further supported by immunohistochemistry and qPCR analyses performed with human gingival tissues. The expression of CTSK in the osteoclastogenesis-supporting cells, including dental pulp stem cells, gingival fibroblasts, and periodontal ligament fibroblasts (PDLFs) was significantly elevated by treatment with inflammatory cytokines such as TNFα and IL-1β. Moreover, TNFα stimulation potentiated the PDLF-mediated osteoclastogenesis of bone marrow-derived macrophages. Interestingly, small interfering RNA-mediated silencing of CTSK in PDLF noticeably attenuated the TNFα-triggered upregulation of receptor activator of nuclear factor kappa-B ligand (RANKL), macrophage colony-stimulating factor, and RANKL/osteoprotegerin ratio, thereby abrogating the enhanced osteoclastogenesis-supporting activity of PDLF. Collectively, these results suggest a novel role of CTSK in the paracrine function of osteoclastogenesis-supporting cells in periodontal disease.

RANKL-induced osteoclastogenesis in bone marrow-derived macrophages is suppressed by cisapride

Cisapride is a selective 5-hydroxytryptamine (5-HT)4 receptor (5-HT4R) agonist. However, the effects of cisapride on osteoclasts and osteoblasts have not been fully explored. We therefore examined the effects of cisapride on osteoclastogenesis in cultured primary bone marrow-derived macrophages (BMMs), and on osteoblast differentiation in cultured primary calvarial pre-osteoblasts. Cisapride significantly inhibited tartrate-resistant acid phosphatase (TRAP)-positive multinuclear osteoclasts and F-actin ring formation during receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis. No cytotoxicity was observed. Cisapride also suppressed RANKL-induced expression of NF-ATc1, TRAP, and cathepsin K genes in BMMs. In addition, cisapride significantly stimulated apoptotic cell death of osteoclasts. However, cisapride had no effect on osteoblast differentiation, as assessed by alkaline phosphatase (ALP) activity and mineralized nodule formation in the cultured primary pre-osteoblasts. Overall, our findings suggest that cisapride may improve the clinical management of metabolic bone diseases such as osteoporosis.

CYTL1 regulates bone homeostasis in mice by modulating osteogenesis of mesenchymal stem cells and osteoclastogenesis of bone marrow-derived macrophages

We previously showed that mice with knockout of Cytl1, a functionally uncharacterized cytokine candidate, exhibit normal endochondral ossification and long-bone development. Here, we investigated the potential functions of CYTL1 in bone homeostasis. We found that Cytl1−/− mice exhibited higher bone mass than wild-type littermates and resisted ovariectomy-induced bone resorption. This led us to investigate the functions of CYTL1 in the osteogenesis and osteoclastogenesis of bone marrow-derived stem cells. CYTL1 was down-regulated during the osteogenesis of human mesenchymal stem cells (hMSCs). The osteogenesis of hMSCs was inhibited by overexpression or exogenous treatment of CYTL1, but enhanced by CYTL1 knockdown. CYTL1 decreased osteogenesis by inhibiting RUNX2 and promoted proliferation among undifferentiated hMSCs, but stimulated apoptosis among osteogenically differentiating cells. Finally, Cytl1−/− mice exhibited inhibition of osteoclast activity and the osteoclastogenesis of bone marrow-derived macrophages. Our results collectively suggest that CYTL1 negatively regulates the osteogenesis of MSCs and positively regulates osteoclastogenesis to modulate bone mass in mice.

Sirtuin 1 inhibits TNF-α-mediated osteoclastogenesis of bone marrow-derived macrophages through both ROS generation and TRPV1 activation.

Sirtuin 1 (SIRT1), also known as NAD-dependent deacetylase, has been reported to increase in vivo osteoclast-mediated bone resorption. However, its effects on osteoclastogenesis or bone loss in vitro have not been widely examined. Therefore, the effects and underlying mechanism of SIRT1 on osteoclast differentiation in mice in vitro were studied. During RANKL-induced osteoclastogenesis in differentiated bone marrow-derived macrophages (BMMs), SIRT1 downregulation was observed. The use of resveratrol (SIRT1 activator) and SIRT1 overexpression was found to inhibit osteoclastogenesis, which was confirmed by TRAP staining and activity loss, reduced expression of osteoclast markers and related genes, and a decrease in the number of multinuclear cells. In contrast, treatment with EX-527 (SIRT1 inhibitor) as well as SIRT1 silencing promoted osteoclastogenesis. Furthermore, the tumor necrosis factor (TNF)-α level was reduced by resveratrol treatment and SIRT1 overexpression but increased following EX-527 incubation and SIRT1 depletion. TNF-α silencing blocked the osteoclastogenesis of BMMs promoted by SIRT1 depletion. Moreover, transient receptor potential vanilloid 1 (TRPV1) channel activation and reactive oxygen species (ROS) production, which are associated with osteoclastogenesis, were impaired by TNF-α silencing. These data demonstrate that SIRT1 directly inhibits osteoclastogenesis by inhibiting ROS generation and TRPV1 channel activation under mediation of TNF-α.

Methylsulfonylmethane Inhibits RANKL-Induced Osteoclastogenesis in BMMs by Suppressing NF-κB and STAT3 Activities.

Osteoclast differentiation is dependent on the activities of receptor activator NF-kB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Given that RANKL plays a critical role in osteoclast formation and bone resorption, any new compounds found to alter its activity would be predicted to have therapeutic potential for disorders associated with bone loss. Methylsulfonylmethane (MSM) is a naturally occurring sulfur compound with well-documented anti-oxidant and anti-inflammatory properties; currently its effects on osteoclast differentiation are unknown. We sought to investigate whether MSM could regulate osteoclastogenesis, and if so, its mechanism of action. In this study, we investigated the effects of MSM on RANKL-induced osteoclast differentiation, together with STAT3’s involvement in the expression of osteoclastic gene markers. These experiments were conducted using bone marrow derived macrophages (BMMs) and cell line material, together with analyses that interrogated both protein and mRNA levels, as well as signaling pathway activity. Although MSM was not toxic to osteoclast precursors, MSM markedly inhibited RANKL-induced TRAP activity, multinucleated osteoclast formation, and bone resorptive activity. Additionally, the expression of several osteoclastogenesis-related marker genes, including TRAF6, c-Fos, NFATc1, cathepsin K, and OSCAR were suppressed by MSM. MSM mediated suppression of RANKL-induced osteoclastogenesis involved inhibition of ITAM signaling effectors such as PLCγ and Syk, with a blockade of NF-kB rather than MAPK activity. Furthermore, MSM inhibited RANKL-induced phosphorylation of STAT3 Ser727. Knockdown of STAT3 using shRNAs resulted in reduced RANKL-mediated phosphorylation of Ser727 STAT3, and TRAF6 in cells for which depletion of STAT3 was confirmed. Additionally, the expression of RANKL-induced osteoclastogenic marker genes were significantly decreased by MSM and STAT3 knockdown. Taken together, these results indicate that STAT3 plays a pivotal role in RANKL-induced osteoclast formation, and that MSM can attenuate RANKL-induced osteoclastogenesis by blocking both NF-kB and STAT3 activity.

Inhibition of Osteoclast Differentiation and Bone Resorption by Bisphosphonate-conjugated Gold Nanoparticles.

In recent years, gold nanoparticles (GNPs) have been reported to affect the regeneration of bone tissue. The goal of this study was to improve bone tissue regeneration by using targeted GNPs. We fabricated a functionalized GNPs conjugated with alendronate (ALD), of the bisphosphonate group. Subsequently, the ALD, GNPs, and ALD conjugated GNPs (GNPs-ALD) were analyzed by ultraviolet-visible absorbance (UV-vis) spectrophotometer, Attenuated total reflectance Fourier transform infrared spectrometer (ATR-FTIR), and thermo gravimetric analysis (TGA). The prepared GNPs-ALD were used to investigate their inhibitory effects on the receptor activator of nuclear factor- κb ligand (RANKL)-induced osteoclastogenesis in bone marrow-derived macrophages (BMMs). Additionally, the GNPs-ALD were applied to ovariectomy (OVX)-induced osteoporotic mice and the experiments were evaluated. ALD was found to be successfully conjugated to the GNPs surface, and it displayed significant adhesion onto the bone surface. The in-vitro study indicated that the GNPs, ALD and GNPs-ALD suppressed osteoclast formation in a dose-dependent manner. Furthermore, in the OVX mouse model, the mice treated GNPs-ALD had higher bone density as compared to other OVX mice groups. The results from these tests indicated that GNPs-ALD can be useful agents for preventing and treating osteoporosis.

The Sirt1 Activators SRT2183 and SRT3025 Inhibit RANKL-Induced Osteoclastogenesis in Bone Marrow-Derived Macrophages and Down-Regulate Sirt3 in Sirt1 Null Cells.

Increased osteoclast-mediated bone resorption is characteristic of osteoporosis, malignant bone disease and inflammatory arthritis. Targeted deletion of Sirtuin1 (Sirt1), a key player in aging and metabolism, in osteoclasts results in increased osteoclast-mediated bone resorption in vivo, making it a potential novel therapeutic target to block bone resorption. Sirt1 activating compounds (STACs) were generated and were investigated in animal disease models and in humans however their mechanism of action was a source of controversy. We studied the effect of SRT2183 and SRT3025 on osteoclastogenesis in bone-marrow derived macrophages (BMMs) in vitro, and discovered that these STACs inhibit RANKL-induced osteoclast differentiation, fusion and resorptive capacity without affecting osteoclast survival. SRT2183 and SRT3025 activated AMPK, increased Sirt1 expression and decreased RelA/p65 lysine310 acetylation, critical for NF-κB activation, and an established Sirt1 target. However, inhibition of osteoclastogenesis by these STACs was also observed in BMMs derived from sirt1 knock out (sirt1-/-) mice lacking the Sirt1 protein, in which neither AMPK nor RelA/p65 lysine 310 acetylation was affected, confirming that these effects require Sirt1, but suggesting that Sirt1 is not essential for inhibition of osteoclastogenesis by these STACs under these conditions. In sirt1 null osteoclasts treated with SRT2183 or SRT3025 Sirt3 was found to be down-regulated. Our findings suggest that SRT2183 and SRT3025 activate Sirt1 and inhibit RANKL-induced osteoclastogenesis in vitro however under conditions of Sirt1 deficiency can affect Sirt3. As aging is associated with reduced Sirt1 level and activity, the influence of STACs on Sirt3 needs to be investigated in vivo in animal and human disease models of aging and osteoporosis.

MicroRNA-183 increases osteoclastogenesis by repressing heme oxygenase-1

Emerging evidence suggests that microRNAs (miRs) influence skeletal structure by modulating osteoclastogenesis and bone resorption. We have demonstrated previously that the up-regulation of heme oxygenase-1 (HO-1) attenuated osteoclastogenesis in bone marrow-derived macrophages (BMMs). RANKL-induced osteoclastogenesis elevates microRNA-183 (miR-183) in BMM. We show here that HO-1 is a target gene of miR-183 and that this miRNA binds to the 3′-UTR of HO-1. We find that a synthetic inhibitor that binds to miR-183 decreases osteoclast (OC) differentiation and increases the expression of HO-1, while a mimic of endogenous mature miR-183 has the opposite effect. Moreover, the HO-1 inducers, resveratrol and piceatannol decrease the expression of miR-183, resulting in attenuated osteoclastogenesis. Our findings reveal how miR-183 affects OC formation.

Type I saikosaponins a and d inhibit osteoclastogenesis in bone marrow-derived macrophages and osteolytic activity of metastatic breast cancer cells.

Many osteopenic disorders, including a postmenopausal osteoporosis and lytic bone metastasis in breast and prostate cancers, are linked with a hyperosteoclast activity due to increased receptor activator of nuclear factor kappa-B ligand (RANKL) expression in osteoblastic/stromal cells. Therefore, inhibition of RANKL-induced osteoclastogenesis and osteoclast-induced bone resorption is an important approach in controlling pathophysiology of these skeletal diseases. We found that, of seven type I, II, and III saikosaponins isolated from Bupleurum falcatum, saikosaponins A and D, type I saikosaponins with an allyl oxide linkage between position 13 and 28 and two carbohydrate chains that are directly attached to the hydroxyl groups in position 3, exhibited the most potent inhibition on RANKL-induced osteoclast formation at noncytotoxic concentrations. The stereochemistry of the hydroxyl group at C16 did not affect their activity. Saikosaponins A and D inhibited the formation of resorptive pits by reducing the secreted levels of matrix metalloproteinase- (MMP-) 2, MMP-9, and cathepsin K in RANKL-induced osteoclasts. Additionally, saikosaponins A and D inhibited mRNA expression of parathyroid hormone-related protein as well as cell viability and invasion in metastatic human breast cancer cells. Thus, saikosaponins A and D can serve as a beneficial agent for the prevention and treatment of osteoporosis and cancer-induced bone loss.

Inhibition of osteoclast differentiation by gold nanoparticles functionalized with cyclodextrin curcumin complexes.

Gold nanoparticles (GNPs) have been previously reported to inhibit osteoclast (OC) formation. However, previous research only confirmed the osteoclastogenesis inhibitory effect under in vitro conditions. The aim of this study was to develop a therapeutic agent for osteoporosis based on the utilization of GNPs and confirm their effect both in vitro and in vivo. We prepared β-cyclodextrin (CD) conjugated GNPs (CGNPs), which can form inclusion complexes with curcumin (CUR-CGNPs), and used these to investigate their inhibitory effects on receptor activator of nuclear factor-κb ligand (RANKL)-induced osteoclastogenesis in bone marrow-derived macrophages (BMMs). The CUR-CGNPs significantly inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells in BMMs without inducing cytotoxicity. The mRNA expressions of genetic markers of OC differentiation including c-Fos, nuclear factor of activated T cells 1 (NFATc1), TRAP, and osteoclast associated receptor (OSCAR) were significantly decreased in the presence of CUR-CGNPs. In addition, the CUR-CGNPs inhibited OC differentiation of BMMs through suppression of the RANKL-induced signaling pathway. Additionally, CUR-CGNPs caused a decrease in RANKL-induced actin ring formation, which is an essential morphological characteristic of OC formation allowing them to carry out bone resorption activity. Furthermore, the in vivo results of an ovariectomy (OVX)-induced osteoporosis model showed that CUR-CGNPs significantly improved bone density and prevented bone loss. Therefore, CUR-CGNPs may prove to be useful as therapeutic agents for preventing and treating osteoporosis.

Defective Entry into Mitosis 1 (Dim1) Negatively Regulates Osteoclastogenesis by Inhibiting the Expression of Nuclear Factor of Activated T-cells, Cytoplasmic, Calcineurin-dependent 1 (NFATc1)

Bone remodeling is a continuous process of osteoblastic bone formation and osteoclastic bone resorption to maintain normal bone mass. NFATc1 is the master regulator of osteoclastogensis and transcriptionally activated by c-Fos and NF-κB in response to receptor activator of NF-κB ligand (RANKL) treatment. Defective entry into mitosis 1 (Dim1) is a nuclear protein that is implicated in pre-mRNA splicing and cell cycle progression, but the possible role of Dim1 in regulating other cellular processes remains unknown. Here, we demonstrate that Dim1 attenuates RANKL-induced osteoclastogenesis by targeting NFATc1 signaling pathway. Expression levels of Dim1 and NFATc1 are significantly increased during the formation of multinucleated osteoclasts. RNAi-mediated knockdown of Dim1 markedly enhances the expression of NFATc1 and its target genes, leading to the increase of RANKL-induced osteoclastogenesis in bone marrow-derived macrophages. Conversely, ectopic expression of Dim1 decreases RANKL-induced osteoclast differentiation by silencing NFATc1 and its target genes, further linking Dim1 to the dynamic regulation of osteoclastogenesis. Consistent with this notion, ChIP and interaction studies show that Dim1 directly associates with c-Fos and prevents c-Fos from binding to the NFATc1 promoter, resulting in targeted inactivation of the NFATc1 gene. Therefore, our studies reveal an unrecognized role for Dim1 as a master modulator of osteoclast differentiation, as well as the molecular mechanism underlying its repressive action toward osteoclastogensis.

A medium-chain fatty acid, capric acid, inhibits RANKL-induced osteoclast differentiation via the suppression of NF-κB signaling and blocks cytoskeletal organization and survival in mature osteoclasts.

Fatty acids, important components of a normal diet, have been reported to play a role in bone metabolism. Osteoclasts are bone-resorbing cells that are responsible for many bone-destructive diseases such as osteoporosis. In this study, we investigated the impact of a medium-chain fatty acid, capric acid, on the osteoclast differentiation, function, and survival induced by receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (MCSF). Capric acid inhibited RANKL-mediated osteoclastogenesis in bone marrow-derived macrophages and suppressed RANKL-induced IκBα phosphorylation, p65 nuclear translocation, and NF-κB transcriptional activity. Capric acid further blocked the RANKL-stimulated activation of ERK without affecting JNK or p38. The induction of NFATc1 in response to RANKL was also attenuated by capric acid. In addition, capric acid abrogated M-CSF and RANKL-mediated cytoskeleton reorganization, which is crucial for the efficient bone resorption of osteoclasts. Capric acid also increased apoptosis in mature osteoclasts through the induction of Bim expression and the suppression of ERK activation by M-CSF. Together, our results reveal that capric acid has inhibitory effects on osteoclast development. We therefore suggest that capric acid may have potential therapeutic implications for the treatment of bone resorption-associated disorders.

The Inhibitory Effect of Roasted Licorice Extract on Human Metastatic Breast Cancer Cell‐Induced Bone Destruction

The aim of this study was to determine whether the ethanol extract of roasted licorice (rLE) could inhibit breast cancer-mediated bone destruction. rLE treatment reduced the viability of MDA-MB-231 human metastatic breast cancer cells but did not show any cytotoxicity in hFOB1.19 human osteoblastic cells and murine bone marrow-derived macrophages (BMMs). rLE inhibited expression and secretion of receptor activator of nuclear factor κB ligand (RANKL) as well as the mRNA and protein expression of cyclooxygenase-2 in osteoblastic cells exposed to the conditioned medium of breast cancer cells. rLE dramatically inhibited RANKL-induced osteoclastogenesis in BMMs, thereby reducing osteoclast-mediated pit formation. Moreover, treatment with licochalcone A and isoliquiritigenin as the active components, whose contents are increased by the roasting process, remarkably suppressed RANKL-induced osteoclast formation in BMMs, respectively. Furthermore, orally administered rLE substantially blocked tumor growth and bone destruction in mice inoculated with breast cancer cells in the tibiae. Serum levels of tartrate-resistant acid phosphatase and C-terminal cross-linking telopeptide of type I collagen and trabecular bone morphometric parameters were reversed to almost the same levels as the control mice by the rLE treatment. In conclusion, rLE may be a beneficial agent for preventing and treating bone destruction in patients with breast cancer.