RANKL-stimulated Osteoclastogenesis Research Articles

Toosendanin inhibits osteoclast formation and alleviate postmenopausal osteoporosis by regulating the p38 signaling pathway

Disruption of the balance between osteoclasts and osteoblasts could lead to bone diseases including osteoporosis. It’s well known that RANKL-RANK signaling plays a vital role in activating osteoclasts. Herein, we explored the therapeutic effects of toosendanin (TSN) in osteoporosis, showing that TSN attenuated RANKL-stimulated osteoclastogenesis and osteoclast-specific gene expression in vitro. Bioinformatics predicted that TSN could interfere p38 subunits and regulate the MAPK cascade, and we further verified and demonstrated that TSN significantly inhibited RANKL-induced p38 signaling through western blot. In ovariectomized mouse model, TSN effectively inhibited the formation of TRAP-positive osteoclasts and exhibited protective effect against bone loss. Altogether, these data indicate that TSN targeted p38 activation to inhibit osteoclastogenesis, suggesting the possible therapeutic use of TSN in osteoporosis in the future.

Bushen huoxue decoction inhibits RANKL-stimulated osteoclastogenesis and glucocorticoid-induced bone loss by modulating the NF-κB, ERK, and JNK signaling pathways.

Glucocorticoid-induced osteoporosis (GIOP) is the most common form of secondary osteoporosis, which is caused by a disorder in bone metabolism due to excessive activation of osteoclasts. Bushen Huoxue decoction (BHD) is an herbal formula with multiple pharmacological effects, including anti-inflammatory, antioxidant activity and stem cell migration promotion. However, the effect of BHD on osteoclastogenesis has not been reported. In this study, we aimed to elucidate the effect of BHD on RANKL-stimulated osteoclastogenesis and explored its underlying mechanisms of action in vitro. Our results show that BHD had no effect on BMMs and RAW264.7 cells viability, but inhibited RANKL-induced osteoclast formation in vitro. Furthermore, BHD attenuated RANKL-induced NF-κB, ERK, and JNK signaling. The attenuation of NF-κB, ERK, and JNK activation were enough to impede downstream expression of c-fos and NFATc1 and related specific genes. Meanwhile, we investigated the therapeutic effect of BHD on glucocorticoid-induced osteoporosis (GIOP) mice. The result indicated that BHD prevents glucocorticoid-induced osteoporosis and preserves bone volume by repressing osteoclast activity. Collectively, BHD shows significant osteoclast inhibition and holds great promise in the treatment of osteoporosis.

Boric Acid Inhibits RANKL-Stimulated Osteoclastogenesis In Vitro and Attenuates LPS-Induced Bone Loss In Vivo.

Boron and boric acid (BA) can promote osteogenic differentiation and reduce bone resorption, which controls bone growth and maintenance of bone tissue. It has been reported that BA activates PERK-eIF2α signaling to induce cytoplasmic stress granules and cell senescence in human prostate DU-145 cells. However, whether BA can affect osteoclasts formation and LPS-induced inflammatory bone loss, and the role of the PERK-eIF2α pathway in the process, remains unknown. In vitro, RAW264.7 cells were pre-treated with boric acid (BA, 1, 10, 100μmol/L) for 4h, and then incubated with receptor activator of nuclear factor-kappaB ligand (RANKL, 50ng/mL) in the presence or absence of BA for 5days. CCK-8 and tartrate-resistant acid phosphatase (TRAP) were used to examine cell viability, osteoclastogenesis, and bone resorption; quantitative real-time PCR was performed to examine mRNA levels of c-Fos, nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), TRAP, and cathepsin K; western blotting was used to examine protein expressions of glucose-regulated protein 78 (GRP78), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), phosphorylated PERK (p-PERK), eukaryotic initiation factor 2α (eIF2α), and phosphorylated eIF2α (p-eIF2α). In vivo, lipopolysaccharide (LPS)-induced bone loss model in mice was established, and micro-computed tomography (micro-CT) scanning, bone biochemical analysis, and osteoclastogenic cytokines were detected to evaluate the effect of BA on LPS-induced bone loss. In our vitro results showed that BA treatment for 5days inhibited osteoclasts formation as well as osteoclastic bone resorption in a dose-dependent manner. The expression of osteoclasts marker genes c-Fos, NFATc1, TRAP, and cathepsin K were attenuated by BA. Immunoblotting analysis demonstrated that BA attenuated RANKL-induced PERK-eIF2α pathway activation. The in vivo data indicated that BA significantly prevented lipopolysaccharide (LPS)-induced bone loss. Our findings strongly suggest that BA may be a promising agent for the treatment of bone destructive diseases caused by excessive osteoclastogenesis.

A Nitrobenzoyl Sesquiterpenoid Insulicolide A Prevents Osteoclast Formation via Suppressing c-Fos-NFATc1 Signaling Pathway.

It is a viable strategy to inhibit osteoclast differentiation for the treatment of osteolytic diseases such as osteoporosis, rheumatoid arthritis and tumor bone metastases. Here we assessed the effects of insulicolide A, a natural nitrobenzoyl sesquiterpenoid derived from marine fungus, on receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclastogenesis in vitro and its protective effects on LPS-induced osteolysis mice model in vivo. The results demonstrated that insulicolide A inhibited osteoclastogenesis from 1 μM in vitro. Insulicolide A could prevent c-Fos and nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) nuclear translocation and attenuate the expression levels of osteoclast-related genes and DC-STAMP during RANKL-stimulated osteoclastogenesis but have no effects on NF-κB and MAPKs. Insulicolide A can also protect the mice from LPS-induced osteolysis. Our research provides the first evidence that insulicolide A may inhibit osteoclastogenesis both in vitro and in vivo, and indicates that it may have potential for the treatment of osteoclast-related diseases.

Effects of aloe-emodin on alveolar bone in Porphyromonas gingivalis-induced periodontitis rat model: a pilot study.

Aloe-emodin (AE), a natural anthraquinone abundant in aloe plants and rhubarb (Rheum rhabarbarum), has long been used to treat chronic inflammatory diseases. However, AE's underlying mechanisms in periodontal inflammation have not been fully elucidated. Acidic mammalian chitinase (AMCase) is a potential biomarker involved in bone remodeling. This study aimed to evaluate AE's effect on periodontitis in rats and investigate AMCase expression. Eighteen Sprague-Dawley rats were separated into the following groups: healthy (group 1), disease (group 2), vehicle (group 3), AE high-dose (group 4), and AE low-dose (group 5). Porphyromonas gingivalis ligatures were placed in rats (groups 2-5) for 7 days. Groups 4 and 5 were then treated with AE for an additional 14 days. Saliva was collected from all groups, and probing pocket depth was measured in succession. Periodontal pocket tissues were subjected to histomorphometric analysis after the rats were sacrificed. Bone marrow-derived macrophages and murine macrophages were stimulated with receptor activator of nuclear factor-κB ligand (RANKL) and treated with different concentrations of AE. AMCase expression was detected from the analysis of saliva, periodontal pocket tissues, and differentiated osteoclasts. Among rats with P. gingivalis-induced periodontitis, the alveolar bone resorption levels and periodontal pocket depth were significantly reduced after treatment with AE. AMCase protein expression was significantly higher in the disease group than in the healthy control (P<0.05). However, AE inhibited periodontal inflammation by downregulating AMCase expression in saliva and periodontal pocket tissue. AE significantly reduced RANKL-stimulated osteoclastogenesis by modulating AMCase (P<0.05). AE decreases alveolar bone loss and periodontal inflammation, suggesting that this natural anthraquinone has potential value as a novel therapeutic agent against periodontal disease.

Tyloxapol inhibits RANKL-stimulated osteoclastogenesis and ovariectomized-induced bone loss by restraining NF-κB and MAPK activation.

ObjectiveTyloxapol is a non-ionic surfactant with diverse pharmacological effects including anti-inflammatory, anti-malignant tumor and antioxidant activities. However, the effect of tyloxapol on osteoclastogenesis has not been elucidated. In this study, we intended to clarify the effect of tyloxapol on RANKL-stimulated osteoclastogenesis and the molecular mechanism both ex vivo and in vivo.MethodsIn vitro osteoclastogenesis assay was performed in BMMs and Raw 264.7 cells. The mature osteoclasts were visualized by TRAP staining. The osteoblsats were visualized by alkaline phosphatase (ALP) staining and Von Kossa staining. To assess whether tyloxapol inhibited the function of mature osteoclasts, F-actin belts and pit formation assays were carried out in BMMs. To evaluate the effect of tyloxapol on post-menopausal osteoporosis, the OVX mouse model were utilized. The bone tissue TRAP staining was used to evaluate the osteoclast activity in vivo. The von kossa staining and micro computed tomography were used to evaluate the histomorphometric parameters. The Goldner's staining was used to evaluate the osteoblast activity. The expression of osteoclastogenesis-associated markers were evaluated by Real-time PCR. The NF-κB and NFATc1 transcriptional activities were illustrated utilizing the assay of luciferase reporter. The effect of tyloxapol pretreatment on IκBa degradation and p65 phosphorylation was evaluated using Western bloting assay. The effect of tyloxapol pretreatment on p65 nuclear translocation was evaluated utilizing immunofluorescence. The effect of tyloxapol pretreatment on the phosphorylatio of ERK, p38 and JNK was examined utilizing Western bloting assay.ResultsIn our research, we found that tyloxapol suppresses RANKL-stimulated osteoclastogenesis in a dose dependent manner and in the initial stage of osteoclastogenesis. Through F-actin belts and pit formation assays, we found that tyloxapol had the ability to inhibit the function of mature osteoclasts in vitro. The results of animal experiments demonstrated that tyloxapol inhibits OVX-induced bone mass loss by inhibiting the activity of osteoclasts but had a limited effect on osteoblastic differentiation and mineralization. Molecularly, we found that tyloxapol suppresses RANKL-stimulated NF-κB activation through suppressing degradation of IκBα, phosphorylation and nuclear translocation of p65. At last, MAPK signaling pathway was also suppressed by tyloxapol in dose and time-dependent manners.ConclusionOur research illustrated that tyloxapol was able to suppress osteoclastogenesis in vitro and ovariectomized-induced bone loss in vivo by restraining NF-κB and MAPK activation. This is pioneer research could pave the way for the development of tyloxapol as a potential therapeutic treatment for osteoporosis.The translational potential of this articleThis study explores that tyloxapol, also known as Triton WR-1339, may be a drug candidate for osteoclastogenic sicknesses like osteoporosis. Our study may also extend the clinical therapeutic spectrum of tyloxapol.

Ethyl acetate extract of the Musa nana flower inhibits osteoclastogenesis and suppresses NF-κB and MAPK pathways.

Banana flowers are consumed as a vegetable and traditionally used for managing several health problems including joint pain, a symptom of bone loss. Osteoclasts are key effector cells responsible for bone loss. Some flavonoids in banana flowers, such as quercetin and quercitrin, have been shown to be able to inhibit osteoclastogenesis. Whether banana flowers can inhibit osteoclast formation is unknown. In this study, we prepared the ethyl acetate fraction (FFE-EA) of an ethanolic extract of fresh flowers of Musa nana. Using UPLC-MS/MS analyses, 76 polyphenols were identified in FFE-EA. In RANKL-stimulated RAW264.7 macrophages, FFE-EA inhibited osteoclastogenesis and osteoclastic bone resorption. Mechanistic studies revealed that FFE-EA suppressed NF-κB and MAPK pathways, and lowered mRNA levels of osteoclast formation/function-related genes. These findings suggest that flowers of M. nana could be a source for formulating functional food that benefits bone health.

Loss of myocyte enhancer factor 2 expression in osteoclasts leads to opposing skeletal phenotypes.

Osteoclasts are multinuclear cells that resorb bone. Osteoclast differentiation is regulated by multiple transcription factors which may be acting in a single or multiple factor complex to regulate gene expression. Myocyte enhancer factor 2 (MEF2) is a family of transcription factors whose role during osteoclast differentiation has not been well characterized. Because MEF2A and MEF2D are the family members most highly expressed during osteoclast differentiation, we created conditional knockout mice models for MEF2A and/or MEF2D. In vitro cultures of A- and D-KO osteoclasts were smaller and less numerous than wild type cultures, while AD-KO osteoclasts were almost completely devoid of TRAP positive mononuclear cells. Female A-KO mice are osteopetrotic while male A- and D-KO mice of either sex had no significant in vivo skeletal phenotype, suggesting a sex-specific regulation of osteoclasts by MEF2A. Lastly, in vivo male AD-KO mice are osteopenic, indicating while MEF2 is required for M-CSF and RANKL-stimulated osteoclastogenesis in vitro, osteoclasts can form in the absence of MEF2 in vivo via a RANKL-alternative pathway.

GSH attenuates RANKL-induced osteoclast formation in vitro and LPS-induced bone loss in vivo

Osteoclasts are capable of adhering the bone matrix, then secrete acid and lytic enzymes to resorb it. Reactive oxygen species (ROS), as a signaling messenger, plays an important role in the receptor activator nuclear factor κB ligand (RANKL) signal pathway during osteoclast differentiation. Glutathione (GSH) is known to be a powerful antioxidant which can scavenge intracellular ROS. This study aimed to investigate whether GSH can as a protective agent against the RANKL-stimulated osteoclastogenesis by suppressing intracellular ROS. Here, we showed that GSH markedly restricted RNAKL-induced differentiation of bone marrow-derived macrophages (BMMs) to form osteoclasts. GSH suppressed RANKL-induced ROS generation and subsequent ROS-induced NF-κB signaling pathways within BMMs during osteoclastogenesis. Further, GSH acted to significantly downregulate the osteoclastogenic genes expression of nuclear factor in activated T cells, cytoplasmic1 (NFATc1), C-fos, the tartrate-resistant acid phosphatase (TRAP), and osteoclast-associated immunoglobulin-like receptor (OSCAR). Our results suggested that GSH inhibits intracellular ROS-mediated NF-κB signal pathway involved in osteoclast differentiation. These findings might form the basis of a new strategy for treating bone disease associated with excessive bone resorption.

Fucoidan Prevents RANKL-Stimulated Osteoclastogenesis and LPS-Induced Inflammatory Bone Loss via Regulation of Akt/GSK3β/PTEN/NFATc1 Signaling Pathway and Calcineurin Activity.

Excessive osteoclast differentiation and/or function plays a pivotal role in the pathogenesis of bone diseases such as osteoporosis and rheumatoid arthritis. Here, we examined whether fucoidan, a sulfated polysaccharide present in brown algae, attenuates receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclastogenesis in vitro and lipopolysaccharide (LPS)-induced bone resorption in vivo, and investigated the molecular mechanisms involved. Our results indicated that fucoidan significantly inhibited osteoclast differentiation in RANKL-stimulated macrophages and the bone resorbing activity of osteoclasts. The effects of fucoidan may be mediated by regulation of Akt/GSK3β/PTEN signaling and suppression of the increase in intracellular Ca2+ level and calcineurin activity, thereby inhibiting the translocation of nuclear factor-activated T cells c1 (NFATc1) into the nucleus. However, fucoidan-mediated NFATc1 inactivation was greatly reversed by kenpaullone, a GSK3β inhibitor. In addition, using microcomputer tomography (micro-CT) scanning and bone histomorphometry, we found that fucoidan treatment markedly prevented LPS-induced bone erosion in mice. Collectively, we demonstrated that fucoidan was capable of inhibiting osteoclast differentiation and inflammatory bone loss, which may be modulated by regulation of Akt/GSK3β/PTEN/NFATc1 and Ca2+/calcineurin signaling cascades. These findings suggest that fucoidan may be a potential agent for the treatment of osteoclast-related bone diseases.

Beta-Glucuronidase Catalyzes Deconjugation and Activation of Curcumin-Glucuronide in Bone.

The biological basis for documented in vivo bone-protective effects of turmeric-derived curcumin is unclear since curcumin is barely detectable in serum, being rapidly conjugated to form what is thought to be an inactive glucuronide. Studies were therefore undertaken to test the postulate that antiresorptive effects of curcumin require deconjugation within bone to form the bioactive aglycone and that β-glucuronidase (GUSB), a deconjugating enzyme expressed by hematopoietic marrow cells, facilitates this site-specific transformation. Consistent with this postulate, aglycone, but not glucuronidated, curcumin inhibited RANKL-stimulated osteoclastogenesis, a key curcumin target in bone. Aglycone curcumin, expressed relative to total curcumin, was higher in bone marrow than in serum of curcumin-treated C57BL/6J mice, while remaining a minor component. Ex vivo, under conditions preventing further metabolism of the unstable aglycone, the majority of curcumin-glucuronide delivered to marrow in vivo was hydrolyzed to the aglycone, a process that was inhibited by treatment with saccharolactone, a GUSB inhibitor, or in mice having reduced (C3H/HeJ) or absent (mps/mps) GUSB activity. These findings suggest that curcumin, despite low systemic bioavailability, may be enzymatically activated (deconjugated) within GUSB-enriched bone to exert protective effects, a metabolic process that could also contribute to bone-protective effects of other highly glucuronidated dietary polyphenols.

Inhibition effects of a natural inhibitor on RANKL downstream cellular signalling cascades cross-talking.

Myricitrin is a natural occurring flavonoid glycoside that possesses effects on inhibiting nitric oxide (NO) transmission and preventing inflammatory reaction. Although previous study showed the myricitrin possesses antibone loss effects via reducing the expression of IL‐6 and partially suppressing reactive oxygen species (ROS) production. However, the effects of myricitrin on nuclear factor‐kappaB ligand (RANKL)‐stimulated osteoclastogenesis have not yet been further investigated. The current study was aimed to demonstrating the inhibitory effects of myricitrin on RANKL‐stimulated osteoclastogenesis and relevant mechanisms. We found myricitrin significantly suppressed osteoclastogenesis suggesting that it may acts on RANKL/RANK induced downstream signal cross cascading in osteoclast precursors. In that, our Western blotting results showed myricitrin significantly attenuated RNAKL/MAPKs (phosphorylation of p38, ERK, JNK) and AKT signal cascading. Complementing previous study, our results suggesting as a natural inhibitor, myricitrin possesses the potential therapeutic effects on inflammatory osteolysis.

Combined treatment with Dendrobium candidum and black tea extract promotes osteoprotective activity in ovariectomized estrogen deficient rats and osteoclast formation

AimsDendrobium candidum (DC) and black tea, are traditional chinese drinks, which contain multiple active ingredients. However, whether or not the combination of these two ingredients can improve osteoporosis remains unknown. This study therefore aimed to examine the effects of the combination of DC and black tea extract (BTE) on osteoporosis. Main methodsOvariectomy (OVX)-induced osteoporosis in vivo as well as receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis in vitro was selected. Key findingsResults showed that OVX rats that were treated orally with a DC and BTE combination for 12 weeks maintained their calcium (Ca) and phosphorus (P) homeostasis and exhibited significantly enhanced estradiol (E2) and OPG levels. This combination treatment also simultaneously reduced levels of interleukin (IL)-1β, IL-6 and improved the organ coefficients of the uterus and femur as well as BMD and BMC in OVX rats. In addition, this DC and BTE combination suppressed osteoclast differentiation in the RANKL-stimulated osteoclastogenesis of RAW 264.7 cells and effectively inhibited the expression of osteoclast-associated genes and proteins. The results of this study further highlight the fact that a combination of DC and BTE improved ovariectomy-induced osteoporosis in rats and suppressed RANKL-stimulated osteoclastogenesis in RAW 264.7 cells. SignificanceThis combination also significantly alleviated osteoporosis when compared to the alternative sole treatments above, due to synergistic effects among components. One partial mechanism of this combination might be the inhibition of osteoclast proliferation and the regulation of NFATC1/c-Fos expression.

Regulation of RANKL-induced osteoclastogenesis by RING finger protein RNF114.

Normal bone remodeling is a continuous process orchestrated by bone-resorbing osteoclasts and bone-forming osteoblasts, which an imbalance in bone remodeling results in metabolic bone diseases. RANKL, a member of the TNF cytokine family, functions as a key stimulator for osteoclast differentiation and maturation. Here, we report that RNF114, previously identified as a psoriasis susceptibility gene, plays a regulatory role in the RANKL/RANK/TRAF6 signaling pathway that mediates osteoclastogenesis. Our results demonstrated that RNF114 expression was significantly down-regulated in mouse osteoclast precursor cells undergoing RANKL-induced osteoclast differentiation. RNF114 knockout did not affect development or viability of the subpopulation of bone marrow macrophages capable of differentiating into osteoclasts in culture. However, in the presence of RANKL, RNF114 knockout bone marrow macrophages exhibited enhanced cell proliferation and augmented osteoclast differentiation, as shown by an increased expression of mature osteoclast markers, increased osteoclastic TRAP activity and bone resorption. Conversely, ectopic expression of RNF114 inhibited CTSK expression, TRAP activity, and bone resorption in RANKL-treated pre-osteoclasts. RNF114 also suppressed RANKL-activated NFATc1 expression and NFAT-regulated promoter activity. RNF114 suppressed TRAF6-, but not TAK1/TAB2-mediated NF-κB activation downstream of RANKL/RANK. In particular, TRAF6 protein levels were down-regulated by RNF114, possibly via K48-mediated proteasome-dependent degradation. These data suggested that RNF114's inhibitory effect on RANKL-stimulated osteoclastogenesis was mediated by blocking RANK/TRAF6/NF-κB signal transduction. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:159-166, 2018.

A pterostilbene derivative suppresses osteoclastogenesis by regulating RANKL-mediated NFκB and MAPK signaling in RAW264.7 cells.

A dysfunctional osteoclast activity is often the cause of bone destructive diseases, such as osteoporosis, periodontitis, erosive arthritis, and cancer. The NFκB ligand (RANKL) has been identified as a major mediator of bone resorption. Agents that suppress RANKL signaling have the potential to inhibit bone resorption or osteoclastogenesis. The present study aimed to determine the effect of a pterostilbene derivative (PTERC-T) for suppressing RANKL or tumor cells-induced osteoclastogenesis in RAW264.7 murine macrophages. Cytotoxicity was measured by MTT assay and inhibitory effect on osteoclastogenesis was analyzed by counting the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and measuring the expression levels of the osteoclast-specific genes. The reactive oxygen species (ROS) generation was detected by FACS. Further, signaling pathways were analyzed by immunofluorescence and immunoblot analyses. PTERC-T suppressed the differentiation of monocytes to osteoclasts in a dose and time-dependent manner. The expression of osteoclast marker genes like TRAP, cathepsin K (CTSK), matrix metalloproteinase 9 (MMP9) and transcription factors c-Fos, and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) were also diminished by PTERC-T. PTERC-T scavenged intracellular ROS generation within osteoclast precursors during RANKL-stimulated osteoclastogenesis. Mechanistically, PTERC-T abrogated the phosphorylation of MAPKs (ERK and JNK) and inhibited RANKL-induced activation of NFκB by suppressing IκBα phosphorylation and preventing NFκB/p65 nuclear translocation. This study thus identifies PTERC-T as an inhibitor of osteoclast formation and provides evidence for its role in preventing osteoporosis and other bone related disorders. However, further studies are needed to establish its efficacy in vivo.

The vitamin D analogue ED71 but Not 1,25(OH)2D3 targets HIF1α protein in osteoclasts.

Although both an active form of the vitamin D metabolite, 1,25(OH)2D3, and the vitamin D analogue, ED71 have been used to treat osteoporosis, anti-bone resorbing activity is reportedly seen only in ED71- but not in 1,25(OH)2D3 -treated patients. In addition, how ED71 inhibits osteoclast activity in patients has not been fully characterized. Recently, HIF1α expression in osteoclasts was demonstrated to be required for development of post-menopausal osteoporosis. Here we show that ED71 but not 1,25(OH)2D3, suppress HIF1α protein expression in osteoclasts in vitro. We found that 1,25(OH)2D3 or ED71 function in osteoclasts requires the vitamin D receptor (VDR). ED71 was significantly less effective in inhibiting M-CSF and RANKL-stimulated osteoclastogenesis than was 1,25(OH)2D3 in vitro. Downregulation of c-Fos protein and induction of Ifnβ mRNA in osteoclasts, both of which reportedly block osteoclastogenesis induced by 1,25(OH)2D3 in vitro, were both significantly higher following treatment with 1,25(OH)2D3 than with ED71. Thus, suppression of HIF1α protein activity in osteoclasts in vitro, which is more efficiently achieved by ED71 rather than by 1,25(OH)2D3, could be a reliable read-out in either developing or screening reagents targeting osteoporosis.

Ethanol Extracts of FreshDavallia formosana(WL1101) Inhibit Osteoclast Differentiation by Suppressing RANKL-Induced Nuclear Factor-κB Activation

The rhizome of Davallia formosana is commonly used to treat bone disease including bone fracture, arthritis, and osteoporosis in Chinese herbal medicine. Here, we report the effects of WL1101, the ethanol extracts of fresh rhizomes of Davallia formosana on ovariectomy-induced osteoporosis. In addition, excess activated bone-resorbing osteoclasts play crucial roles in inflammation-induced bone loss diseases, including rheumatoid arthritis and osteoporosis. In this study, we examined the effects of WL1101 on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. Treatment with WL1101 significantly inhibited RANKL-stimulated osteoclastogenesis. Two isolated active compounds, ((−)-epicatechin) or WL14 (4-hydroxy-3-aminobenzoic acid) could also inhibit RANKL-induced osteoclastogenesis. WL1101 suppressed the RANKL-induced nuclear factor-κB (NF-κB) activation and nuclear translocation, which is the key process during osteoclastogenesis, by inhibiting the activation of IκB kinase (IKK) and IκBα. In animal model, oral administration of WL1101 (50 or 200 mg/kg/day) effectively decreased the excess bone resorption and significantly antagonized the trabecular bone loss in ovariectomized rats. Our results demonstrate that the ethanol extracts of fresh rhizomes of Davallia formosana inhibit osteoclast differentiation via the inhibition of NF-κB activation and effectively ameliorate ovariectomy-induced osteoporosis. WL1101 may thus have therapeutic potential for the treatment of diseases associated with excessive osteoclastic activity.

RANKL Employs Distinct Binding Modes to Engage RANK and the Osteoprotegerin Decoy Receptor

Osteoprotegerin (OPG) and receptor activator of nuclear factor κB (RANK) are members of the tumor necrosis factor receptor (TNFR) superfamily that regulate osteoclast formation and function by competing for RANK ligand (RANKL). RANKL promotes osteoclast development through RANK activation, while OPG inhibits this process by sequestering RANKL. For comparison, we solved crystal structures of RANKL with RANK and RANKL with OPG. Complementary biochemical and functional studies reveal that the monomeric cytokine-binding region of OPG binds RANKL with ∼500-fold higher affinity than RANK and inhibits RANKL-stimulated osteoclastogenesis ∼150 times more effectively, in part because the binding cleft of RANKL makes unique contacts with OPG. Several side chains as well as the C-D and D-E loops of RANKL occupy different orientations when bound to OPG versus RANK. High affinity OPG binding requires a 90s loop Phe residue that is mutated in juvenile Paget's disease. These results suggest cytokine plasticity may help to fine-tune specific tumor necrosis factor (TNF)-family cytokine/receptor pair selectivity.

Plumbagin Inhibits Breast Tumor Bone Metastasis and Osteolysis by Modulating the Tumor-Bone Microenvironment

Bone metastasis is a common and serious consequence of breast cancer. Bidirectional interaction between tumor cells and the bone marrow microenvironment drives a so-called 'vicious cycle' that promotes tumor cell malignancy and stimulates osteolysis. Targeting these interactions and pathways in the tumor-bone microenvironment has been an encouraging strategy for bone metastasis therapy. In the present study, we examined the effects of plumbagin on breast cancer bone metastasis. Our data indicated that plumbagin inhibited cancer cell migration and invasion, suppressed the expression of osteoclast-activating factors, altered the cancer cell induced RANKL/OPG ratio in osteoblasts, and blocked both cancer cell- and RANKL-stimulated osteoclastogenesis. In mouse model of bone metastasis, we further demonstrated that plumbagin significantly repressed breast cancer cell metastasis and osteolysis, inhibited cancer cell induced-osteoclastogenesis and the secretion of osteoclast-activating factors in vivo. At the molecular level, we found that plumbagin abrogated RANKL-induced NF-κB and MAPK pathways by blocking RANK association with TRAF6 in osteoclastogenesis, and by inhibiting the expression of osteoclast-activating factors through the suppression of NF-κB activity in breast cancer cells. Taken together, our data demonstrate that plumbagin inhibits breast tumor bone metastasis and osteolysis by modulating the tumor-bone microenvironment and that plumbagin may serve as a novel agent in the treatment of tumor bone metastasis.

Antioxidants, like coenzyme Q10, selenite, and curcumin, inhibited osteoclast differentiation by suppressing reactive oxygen species generation

Coenzyme Q10 (CoQ10), selenium, and curcumin are known to be powerful antioxidants. Osteoclasts are capable of resorbing mineralized bone and excessive bone resorption by osteoclasts causes bone loss-related diseases. During osteoclast differentiation, the reactive oxygen species (ROS) acts as a secondary messenger on signal pathways. In this study, we investigated whether antioxidants can inhibit RANKL-induced osteoclastogenesis through suppression of ROS generation and compared the relative inhibitory activities of CoQ10, sodium selenite, and curcumin on osteoclast differentiation. We found that antioxidants markedly inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in both bone marrow-derived monocytes (BMMs) and RAW 264.7 cells. Antioxidants scavenged intracellular ROS generation within osteoclast precursors during RANKL-stimulated osteoclastogenesis. These also acted to significantly suppress the gene expression of NFATc1, TRAP, and osteoclast-associated immunoglobulin-like receptor (OSCAR), which are genetic markers of osteoclast differentiation in a dose-dependent manner. These antioxidants also suppressed ROS-induced IκBα signaling pathways for osteoclastogenesis. Specially, curcumin displayed the highest inhibitory effect on osteoclast differentiation when concentrations were held constant. Together, CoQ10, selenite, and curcumin act as inhibitors of RANKL-induced NFATc1 which is a downstream event of NF-κB signal pathway through suppression of ROS generation, thereby suggesting their potential usefulness for the treatment of bone disease associated with excessive bone resorption.