Osteoclastogenesis In Bone Marrow Macrophages Research Articles

4-Octyl itaconate promotes alveolar ridge preservation following tooth extraction.

The search for medications that can effectively reduce alveolar bone loss following tooth extraction is of great interest. This study aimed to observe the roles of 4-octyl itaconate (4-OI) in RANKL-induced osteoclastogenesis of bone marrow macrophages (BMMs) in vitro. Mandibular second molars were extracted to evaluate whether 4-OI could alleviate alveolar bone loss. 4-OI inhibited RANKL-induced osteoclastogenesis and promoted Nrf2 expression in bone marrow macrophages in vitro. Positive Nrf2 expressions were observed in inflammatory cells and osteoclasts in vivo. Treatment with 4-octyl itaconate increased Nrf2 expression, resulting in reduced inflammatory infiltration and osteoclastic activity after tooth extraction. Furthermore, increased expression of OCN and enhanced-alveolar bone healing of extraction socket were observed in the 4-OI group compared to the control group. Our results suggested that 4-OI could serve as a promising pharmacologic candidate for alveolar ridge preservation by alleviating alveolar bone loss following tooth extraction in rats.

Activating transcriptional coactivator with PDZ-binding motif by (R)-PFI-2 attenuates osteoclastogenesis and prevents ovariectomized-induced osteoporosis

Excessive osteoclast activation is a leading cause of osteoporosis. Therefore, identifying molecular targets and relevant pharmaceuticals that inhibit osteoclastogenesis is of substantial clinical importance. Prior research has indicated that transcriptional coactivator with PDZ-binding motif (TAZ) impedes the process of osteoclastogenesis by engaging the nuclear factor (NF)-κB signaling pathway, thereby suggesting TAZ activation as a potential therapeutic approach to treat osteoporosis. (R)-PFI-2 is a novel selective inhibitor of SETD7 methyltransferase activity, which prevents the nuclear translocation of YAP, a homolog of TAZ. Therefore, we hypothesized that (R)-PFI-2 could be an effective therapeutic agent in the treatment of osteoporosis. To test this hypothesis and explore the underlying mechanism, we first examined the impact of (R)-PFI-2 on osteoclastogenesis in bone marrow macrophages (BMMs) in vitro. (R)-PFI-2 treatment inhibited TAZ phosphorylation induced by NF-κB, thereby enhancing its nuclear localization, protein expression, and activation in BMMs. Moreover, (R)-PFI-2-induced TAZ activation inhibited osteoclast formation in a dose-dependent manner, which involved inhibition of osteoclastogenesis through the TAZ and downstream NF-κB pathways. Furthermore, (R)-PFI-2 inhibited osteoclastogenesis and prevented ovariectomy-induced bone loss in vivo in a mouse model. Overall, our findings suggest that TAZ activation by (R)-PFI-2 inhibits osteoclastogenesis and prevents osteoporosis, indicating an effective strategy for treating osteoclast-induced osteoporosis.

Myrislignan targets extracellular signal-regulated kinase (ERK) and modulates mitochondrial function to dampen osteoclastogenesis and ovariectomy-induced osteoporosis

BackgroundActivated osteoclasts cause excessive bone resorption, and disrupt bone homeostasis, leading to osteoporosis. The extracellular signal-regulated kinase (ERK) signaling is the classical pathway related to osteoclast differentiation, and mitochondrial reactive oxygen species are closely associated with the differentiation of osteoclasts. Myrislignan (MRL), a natural product derived from nutmeg, has multiple pharmacological activities; however, its therapeutic effect on osteoporosis is unclear. Here, we investigated whether MRL could inhibit osteoclastogenesis and bone mass loss in an ovariectomy mouse model by suppressing mitochondrial function and ERK signaling.MethodsTartrate-resistant and phosphatase (TRAP) and bone resorption assays were performed to observe the effect of MRL on osteoclastogenesis of bone marrow macrophages. MitoSOX RED and tetramethyl rhodamine methyl ester (TMRM) staining was performed to evaluate the inhibitory effect of MRL on mitochondria. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was performed to detect whether MRL suppressed the expression of osteoclast-specific genes. The impact of MRL on the protein involved in the mitogen-activated protein kinase (MAPK) and nuclear factor-κB pathways was evaluated using western blotting. In addition, a specific ERK agonist LM22B-10, was used to revalidate the inhibitory effect of MRL on ERK. Finally, we established an ovariectomy mouse model to assess the therapeutic effect of MRL on osteoporosis in vivo.ResultsMRL inhibited osteoclast differentiation and the associated bone resorption, by significantly decreasing osteoclastic gene expression. Mechanistically, MRL inhibited the phosphorylation of ERK by suppressing the mitochondrial function, thereby downregulating the nuclear factor of activated T cells 1 (NFATc1) signaling. LM22B-10 treatment further verified the targeted inhibition effect of MRL on ERK. Microscopic computed tomographic and histologic analyses of the tibial tissue sections indicated that ovariectomized mice had lower bone mass and higher expression of ERK compared with normal controls. However, MRL treatment significantly reversed these effects, indicating the anti-osteoporosis effect of MRL.ConclusionWe report for the first time that MRL inhibits ERK signaling by suppressing mitochondrial function, thereby ameliorating ovariectomy-induced osteoporosis. Our findings can provide a basis for the development of a novel therapeutic strategy for osteoporosis.

Resistant dextrin protects against pathological bone loss in ovariectomized rats and inhibits RANKL-induced osteoclastogenesis.

Osteoporosis is a common disease in postmenopausal women characterized by systemic bone mass loss, microstructure fragility and increased incidence of fractures. Resistant dextrin (RD) is a soluble fiber with beneficial metabolic effects. However, the beneficial effect of RD in osteoporosis remains to be determined. In this study, we investigated the effect of dietary RD supplement on osteoporosis in ovariectomized (OVX) rats. Both the control (sham) and OVX group rats were gavaged with RD (10 g/kg/d) or equal amount of saline for 12 weeks, and histological and biomechanical analyses were conducted to evaluate bone microstructure and strength. Furthermore, we also evaluated the effects of RD on osteoclastogenesis in bone marrow macrophages (BMMs) by detecting the expression of osteoclast-related genes using qRT-PCR and Western blot analysis. The results showed that in OVX rats the bone strength and microstructure characteristics were significantly improved with RD supplement for 12 weeks. Additionally, the mRNA and protein expression of osteoclast markers, such as CTSK, NF-κB and NFATC1, were significantly down-regulated in BMMs isolated from RD supplement group. RD also suppressed RANKL-induced osteoclastogenesis in BMMs. These findings suggest that RD ameliorates osteoporosis in OVX rats by inhibiting osteoclast differentiation. RD suppresses RANKL-induced osteoclastogenesis possibly through modulating Akt and NF-κB signaling pathways. These data indicate that a dietary supplement of RD might serve as an intervention strategy for menopausal osteoporosis.

Gut Metabolite Urolithin A Inhibits Osteoclastogenesis and Senile Osteoporosis by Enhancing the Autophagy Capacity of Bone Marrow Macrophages

Senile osteoporosis (SOP) is a systemic bone disease that is significantly associated with age and eventually leads to deteriorated bone strength and increased fracture risk. Urolithin A (Uro-A) is a gut microbiome-derived compound that is mainly produced from pomegranates and some nuts. Uro-A has attracted great attention in recent years in view of its protective effects on aging-related diseases, including muscle dysfunction, kidney disease and knee injury. However, its protective influence and possible mechanisms in senile osteoporosis remain unclear. Our study describes the beneficial effect of Uro-A on bone marrow macrophages (BMMs). The in vitro results demonstrated that Uro-A inhibited receptor activator for nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis in BMMs in a concentration-dependent manner. Uro-A significantly reduced the expression of osteoclast-related genes and bone resorption. Mechanistically, we found that the autophagy ability of BMMs was significantly enhanced in the early stage of Uro-A treatment, accompanied by the activation of LC3 and Beclin 1. At the same time, this enhanced autophagy activity was maintained until the later stage after stimulation with RANKL. Furthermore, we found that the MARK signaling pathway was blocked by Uro-A treatment. In a mouse model of aging, Uro-A effectively inhibited bone loss in the proximal femur, spine and tibia of aging mice. These results indicated that Uro-A is a robust and effective treatment for preventing senile osteoporosis bone loss.

Diverse Sesquiterpenoids and Polyacetylenes from Atractylodes lancea and Their Anti-Osteoclastogenesis Activity.

Twenty-two sesquiterpenoids (1-22) and 11 polyacetylenes (23-33) were obtained from the rhizomes of Atractylodes lancea. Among them, 11 compounds (1-5, 11, 12, 23, 24, 30, and 31) are new. The scaffolds represented by the isolates of sesquiterpenoids were found to be varied and included two rare rearranged spirovetivane sesquiterpenoids with a spiro [4,4] skeleton, eight spirovetivanes, three guaianes, eight eudesmanes, and one eremophilane. Their planar structures and relative configurations were elucidated by UV, IR, 1D and 2D NMR, and HRESIMS data analysis. The absolute configurations of the new sesquiterpenoids were determined using X-ray diffraction analysis and by comparison of the calculated and experimental electronic circular dichroism and optical rotation data, as well as chemical transformations. All the isolated compounds (1-33) were evaluated for their activity against RANKL-induced osteoclastogenesis in bone marrow macrophages. Two polyacetylene-type compounds, 25 and 32, showed potent activity with IC50 values of 1.3 and 0.64 μM, respectively. Rearranged spirovetivane sesquiterpenoids with a spiro [4,4] skeleton are reported herein from the genus Atractylodes for the first time. Polyacetylenes were demonstrated as the main active constituents of A. lancea with osteoclastogenesis inhibitory activity.

Wnt7b Inhibits Osteoclastogenesis via AKT Activation and Glucose Metabolic Rewiring.

The imbalance between bone formation and bone resorption causes osteoporosis, which leads to severe bone fractures. It is known that increases in osteoclast numbers and activities are the main reasons for increasing bone resorption. Although extensive studies have investigated the regulation of osteoclastogenesis of bone marrow macrophages (BMMs), new pharmacological avenues still need to be unveiled for clinical purpose. Wnt ligands have been widely demonstrated as stimulators of bone formation; however, the inhibitory effect of the Wnt pathway in osteoclastogenesis is largely unknown. Here, we demonstrate that Wnt7b, a potent Wnt ligand that enhances bone formation and increases bone mass, also abolishes osteoclastogenesis in vitro. Importantly, enforced expression of Wnt in bone marrow macrophage lineage cells significantly disrupts osteoclast formation and activity, which leads to a dramatic increase in bone mass. Mechanistically, Wnt7b impacts the glucose metabolic process and AKT activation during osteoclastogenesis. Thus, we demonstrate that Wnt7b diminishes osteoclast formation, which will be beneficial for osteoporosis therapy in the future.

Hypoxia-inducible factor-2\u03b1 mediates senescence-associated intrinsic mechanisms of age-related bone loss

Aging is associated with cellular senescence followed by bone loss leading to bone fragility in humans. However, the regulators associated with cellular senescence in aged bones need to be identified. Hypoxia-inducible factor (HIF)−2α regulates bone remodeling via the differentiation of osteoblasts and osteoclasts. Here, we report that HIF-2α expression was highly upregulated in aged bones. HIF-2α depletion in male mice reversed age-induced bone loss, as evidenced by an increase in the number of osteoblasts and a decrease in the number of osteoclasts. In an in vitro model of doxorubicin-mediated senescence, the expression of Hif-2α and p21, a senescence marker gene, was enhanced, and osteoblastic differentiation of primary mouse calvarial preosteoblast cells was inhibited. Inhibition of senescence-induced upregulation of HIF-2α expression during matrix maturation, but not during the proliferation stage of osteoblast differentiation, reversed the age-related decrease in Runx2 and Ocn expression. However, HIF-2α knockdown did not affect p21 expression or senescence progression, indicating that HIF-2α expression upregulation in senescent osteoblasts may be a result of aging rather than a cause of cellular senescence. Osteoclasts are known to induce a senescent phenotype during in vitro osteoclastogenesis. Consistent with increased HIF-2α expression, the expression of p16 and p21 was upregulated during osteoclastogenesis of bone marrow macrophages. ChIP following overexpression or knockdown of HIF-2α using adenovirus revealed that p16 and p21 are direct targets of HIF-2α in osteoclasts. Osteoblast-specific (Hif-2αfl/fl;Col1a1-Cre) or osteoclast-specific (Hif-2αfl/fl;Ctsk-Cre) conditional knockout of HIF-2α in male mice reversed age-related bone loss. Collectively, our results suggest that HIF-2α acts as a senescence-related intrinsic factor in age-related dysfunction of bone homeostasis.

Diverse diterpenoids and sesquiterpenoids from Siegesbeckia pubescens and their activity against RANKL-induced osteoclastogenesis

Phytochemical investigation of the aerial parts of Siegesbeckia pubescens led to seventeen diterpenoids (1–17) and twelve sesquiterpenoids (18–29). Their structures were varied including twelve ent-pimarane (1–12), three ent-kaurane (13–15), two acyclic diterpenoids (16–17), ten germacrene (18–27), one guaiane (28), and one caryolane (29) sesquiterpenoids. Eight of twenty-nine were new ones (1, 3, 4, 16–18, 23, and 28). Their structures were elucidated by extensive spectroscopic analysis. The absolute configurations of compounds 1 and 2 were identified using X-ray diffraction analysis, and of compounds 18, 23, and 28 were elucidated by the experimental and calculated electronic circular dichroism (ECD) spectra. All the isolated compounds (1–29) were assayed for their inhibition of RANKL-induced osteoclastogenesis in bone marrow macrophages (BMMs). Four sesquiterpenoids 18, 25, 26, and 27 exhibited potent inhibition of osteoclastogenesis with IC50 value of 0.51, 0.80, 0.50, and 0.83 μM, respectively. Here we demonstrated that S. pubescens may be a resource for discovery of anti-osteoporosis agents.

Association of high HIF-1α levels in serous periodontitis with external root resorption by the NFATc1 pathway.

Whether external root resorption is associated with hypoxia in the periodontal ligaments of teeth with severe periodontitis remains unclear. Hypoxia inducible factor-1α (HIF-1α) expression and external resorption sites in the periodontal ligaments of these teeth were observed to elaborate upon the relationship between hypoxia and external root resorption in severe periodontitis. Histological analysis was performed to observe external root resorption. The expressions of HIF-1α and Nuclear factor-activated T cells c1 (NFATc1) in the periodontal ligaments were detected by immunofluorescence, western blotting and real-time PCR. Bone marrow macrophages (BMMs) were stimulated by Porphyromonas gingivalis lipopolysaccharide (Pg.LPS) and cultured under hypoxia in vitro. High levels of HIF-1α and NFATc1 were detected in severe periodontitis. HIF-1α positive-cells were observed in the external resorption sites. Hypoxia promoted Pg.LPS-stimulated osteoclastogenesis of BMMs and bone resorption by the NFATc1 pathway. Increased HIF-1α in severe periodontitis are associated with external root resorption by the NFATc1 pathway.

Niclosamide and its derivative DK-520 inhibit RANKL-induced osteoclastogenesis.

Niclosamide is a potent inhibitor of osteoclastogenesis and bone remodeling. DK-520 is an acyl derivative of Niclosamide and significantly increased both the plasma concentration and the duration of exposure of Niclosamide when dosed orally. However, at present the effect of DK-520 on osteoclastogenesis has not been reported. Here, we investigated whether DK-520 can regulate receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis of bone marrow macrophages (BMMs) invitro. Following induction of BMMs with RANKL for three days, we detected differentiated osteoclasts with typical morphology and high levels of tartrate-resistant acid phosphatase (TRAP), RANKL, and cathepsin K (CTSK) expression. Treatment with either Niclosamide or DK-520 did not affect the viability of osteoclast precursors (OCPs), but significantly inhibited RANKL-induced transdifferentiation of macrophages into OCPs, particularly in the early stage of osteoclastogenesis. Both Niclosamide and DK-520 significantly decreased the relative levels of transcription factor PU.1 mRNA transcripts and dendritic cell-specific transmembrane protein (DC-STAMP), but not v-ATPasev0 d2 protein expression in OCPs. In addition, the inhibitory effect of DK-520 on osteoclastogenesis is realized through impairment of the NF-kB (nuclear factor-κB) and MAPK (mitogen-activated protein kinase) signaling pathways. These results demonstrate that DK-520, like Niclosamide, effectively inhibits the early stage of osteoclastogenesis. The findings presented here, together with its increased oral plasma concentrations and bioavailability, suggest that DK-520 may be a promising drug candidate for treatment of osteoclast-related diseases.

Bergapten suppresses RANKL-induced osteoclastogenesis and ovariectomy-induced osteoporosis via suppression of NF-κB and JNK signaling pathways

Bergapten (BP), derived from Cnidium monnieri (L.) Cusson, is an ingredient widely used in traditional Chinese medicine and has important biological and pharmacological activities. However, the effect of BP on ovariectomy-induced osteoporosis and the underlying mechanism are not entirely clear. In this study, we investigated the effects of BP on ovariectomy-induced osteoporosis and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis in vivo and in vitro, and explored the underlying mechanism. We found that BP treatment exerted beneficial effects on ovariectomy-induced osteoporosis in vivo. Further, BP attenuated osteoclastogenesis in bone marrow macrophages (BMMs) and RAW264.7 cells without any cytotoxicity. Additionally, BP specifically inhibited RANKL-induced NF-κB and JNK signaling,but did not suppress p38 and ERK. At the mRNA level, BP inhibits the OC-associated transcription factor NFATc1 and c-fos, thereby affecting the expression of OC differentiation-related genes. Moreover, BP disrupted the formation of F-actin rings, which are important for bone-resorbing activity, and impairs OC bone resorption. Therefore, BP may be a useful alternative therapy for post-menopausal osteoporosis.

Dihydroartemisinin represses osteoclastogenesis of bone marrow macrophages through reduced NFATc1 expression and impaired phosphorylation of IκBα.

Osteoclasts are multinucleated bone resorbing cells whose differentiation is regulated by several important signaling pathways. Several lines of evidence indicate that dihydroartemisinin (DHA), an anti-malarial drug, inhibits osteoclast differentiation with little cytotoxicity. However, the detailed inhibitory mechanisms of DHA on osteoclastogenesis from native cells remain to be elucidated. In this study, we investigated the effects of DHA on the differentiation of bone marrow-derived macrophages into osteoclasts. DHA inhibited receptor activator of nuclear factor κ-B ligand (RANKL)-induced osteoclast formation and its bone resorbing activity. Mechanistically, DHA treatment markedly abolished phosphorylation of IκBα, and slightly affected a p38 MAPK dependent pathway. Moreover, DHA treatment induced down-regulation of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), a master regulator for osteoclast differentiation and its target proteins, such as Src and cathepsin K. These results indicate that DHA represses RANKL-induced osteoclastogenesis of bone marrow macrophages through reduced NFATc1 expression and impaired phosphorylation of IκBα.

The systemic bone protective effects of Gushukang granules in ovariectomized mice by inhibiting osteoclastogenesis and stimulating osteoblastogenesis

Primary osteoporosis (POP), which is caused by unbalanced bone remodeling, leads to significant economic and societal burdens globally. Gushukang (GSK) granule serves as one commonly used prescription for POP in Traditional Chinese Medicine (TCM). The present study aimed to clarify the exact roles of GSK in bone remodeling with in vivo and in vitro assays. Here we showed that GSK prevented bone loss and the alternations of osteoporotic bone parameters as well as the decreased density of osteoclast in ovariectomized (OVX) mice. GSK inhibited receptor activator for nuclear factor-κ B Ligand (RANKL)-activated osteoclastogenesis in bone marrow macrophages (BMMs). At the molecular levels, GSK inhibited the expression of nuclear factor of activated T cells cytoplasm 1(NFATc1) and c-Fos, two master regulators of osteoclastogenesis. GSK also inhibited bone resorbed genetic expression of matrix metalloproteinase 9 (MMP9), cathepsin K (Ctsk), TRAP and carbonic anhydrase II (Car2). Meanwhile, GSK stimulated osteoblastogenesis from bone primary mesenchymal stem cells (MSCs) and enhanced the expression of Osteirx, and Runx2. GSK also stimulated the expression of Col-1, Osteocalcein and alkaline phosphatase (ALP). Our investigation established the systemic bone protective effects of GSK by suppressing osteoclastogenesis and stimulating osteoblastogenesis and laid bases for new drugs discovery in treating POP.

Sappanone A inhibits RANKL-induced osteoclastogenesis in BMMs and prevents inflammation-mediated bone loss

Receptor activator of nuclear factor-kB ligand (RANKL) is a key factor in the differentiation and activation of osteoclasts. Suppressing osteoclastogenesis is considered an effective therapeutic approach for bone-destructive diseases, such as osteoporosis and rheumatoid arthritis. Sappanone A (SPNA), a homoisoflavanone compound isolated from the heartwood of Caesalpinia sappan, has been reported to exert anti-inflammatory effects; however, the effects of SPNA on osteoclastogenesis have not been investigated. In the present study, we describe for the first time that SPNA inhibits RANKL-induced osteoclastogenesis in mouse bone marrow macrophages (BMMs) and suppresses inflammation-induced bone loss in a mouse model. SPNA inhibited the formation of osteoclasts from BMMs, osteoclast actin-ring formation, and bone resorption in a concentration-dependent manner. At the molecular level, SPNA significantly inhibited RANKL-induced activation of the AKT/glycogen synthase kinase-3β (GSK-3β) signaling pathway without affecting its activation of the mitogen-activated protein kinases (MAPKs) JNK, p38, and ERK. In addition, SPNA suppressed the induction of nuclear factor of activated T cells cytoplasmic 1 (NFATc1), which is a crucial transcription factor in osteoclast differentiation. As a result, SPNA decreased osteoclastogenesis-related marker gene expression, including CtsK, TRAP, dendritic cell-specific transmembrane protein (DC-STAMP), MMP-9 and osteoclast-associated receptor (OSCAR). In a mouse inflammatory bone loss model, SPNA significantly inhibited lipopolysaccharide (LPS)-induced bone loss by suppressing the number of osteoclasts. Taken together, these findings suggest that SPNA inhibits osteoclastogenesis and bone resorption by inhibiting the AKT/GSK-3β signaling pathway and may be a potential candidate compound for the prevention and/or treatment of inflammatory bone loss.

In vitro anti-osteoclastogenic activity of p38 inhibitor doramapimod via inhibiting migration of pre-osteoclasts and NFATc1 activity

The mitogen activated protein kinase p38 plays a role in the receptor activator of NF-ĸB ligand (RANKL)-induced osteoclast differentiation. In this study, we investigated the effect of p38 inhibitor doramapimod on the osteoclast differentiation. Doramapimod significantly inhibited the osteoclastogenesis of bone marrow macrophages (BMMs) via attenuating the activation of p38 induced by M-CSF and RANKL. Importantly, doramapimod blocked the migration and fusion in pre-osteoclasts via the down-regulating NFATc1. The inhibitory effect of doramapimod on the migration/fusion of pre-osteoclasts via inhibiting NFATc1 activity were confirmed by measuring NFATc1 luciferase activity and evaluating the mRNA expression of NFATc1-responsive genes related to the osteoclastic migration/fusion. These results suggested anti-osteoclastogenic activity of doramapimod via inhibiting migration/fusion of pre-osteoclasts and NFATc1 activity.

Abstract 34: Inhibitory effects of betulinic acid isolated from Nelumbo nucifera (Lotus) on breast cancer-mediated bone destruction

Abstract Most of cancer patients die, not because of the tumor in the primary site, but because of spreading to other site. In particular, bone metastasis is relatively common in patients with breast, prostate, and lung cancer. Many breast cancer patients suffer from bone metastasis with bone pain and pathological fractures. These metastases are predominantly osteolytic and develop when tumor cells interact with bone. Tumor and bone interact in a vicious cycle, where tumor-secreted factors stimulate bone cells, which in turn release growth factors and cytokines that act back on the tumor cells. Within the vicious cycle are many potential therapeutic targets for treatment of bone metastatic disease. Nelumbo nucifera (Lotus) has been known as a medicinal herb for treatment of cough, fever and hyperdipsia. Betulinic acid (BA) is a naturally occurring triterpenoid present in lotus. In this study, we found that BA reduced mRNA expressions of osteolytic factors in MDA-MB-231 human metastatic breast cancer cells. In particular, mRNA level of parathyroid hormone-related protein (PTHrP), the major component of vicious cycle within bone, was significantly inhibited by BA. Treatment with BA decreased receptor activator of nuclear factor kappa-B ligand (RANKL) /osteoprotegerin (OPG) ratio in PTHrP-treated human osteoblasts. In addition, BA dose-dependently inhibited the RANKL-induced osteoclast differentiation and bone resorption by reducing the secretion of matrix metalloproteinases (MMPs) and cathepsin K derived from osteoclasts, without any evidence of cytotoxicity. We further examined the effect of BA on ovariectomy-induced osteoporosis in mice. Serum levels of tartrate-resistant acid phosphatase isoform 5b, alkaline phosphate, calcium, osteocalcin and C-terminal telopeptide of type I collagen were significantly increased in ovariectomized (OVX) mice than in sham mice. BA inhibited increases in the serum levels of these osteoporotic markers in OVX mice. We analyzed bone volume, trabecular bone thickness, trabecular number and trabecular separation by micro-computer tomography (μ-CT) scanning. Taken together, BA may reduce the expression of PTHrP in MDA-MB-231 human metastatic breast cancer cells, RANKL/OPG ratio in PTHrP-stimulated human osteoblasts, and RANKL-induced osteoclastogenesis in bone marrow macrophages. BA may also prevent osteoporotic bone loss in OVX mice. Thus, BA can be beneficial agents for patients with secondary bone lesions associated with breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 34. doi:1538-7445.AM2012-34