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Osteoporosis is a common disease that increases the risk of fractures due to decreased bone density and weakens the bone microstructure. Preventing and diagnosing osteoporosis using the available drugs can be a costly affair with possible side effects. Therefore, natural product-derived therapeutics are promising alternatives. Our study demonstrated that the oat seedlings’ extract (OSE) inhibited the receptor activator of the nuclear factor κB ligand (RANKL)-induced osteoclastogenesis from the bone marrow-derived macrophages (BMMs). The OSE treatment significantly attenuated the RANKL-mediated induction of the tartrate-resistant acid phosphatase (TRAP) activity as well as the number of TRAP-positive (TRAP+) multinucleated cells (MNCs) counted through the TRAP staining in a dose-dependent manner. It was also confirmed that the OSE suppressed the formation of the TRAP + MNCs in the early stage of differentiation and not in the middle and late stages. The results of the real-time quantitative polymerase chain reaction (qPCR) and the western blotting showed that the OSE dramatically inhibited the mRNA and protein expressions of the osteoclastogenesis-mediated transcription factors such as the c-Fos and the nuclear factor-activated T cells c1 (NFATc1). In addition, the OSE strongly attenuated the mRNA induction of the c-Fos/NFATc1-dependent molecules such as the TRAP, the osteoclast-associatedimmunoglobulin-like receptor (OSCAR), the dendritic cell-specific transmembrane protein (DC-STAMP), and the cathepsin K. These results suggest that the naturally derived OSE may be useful for preventing bone diseases.

The physiological or dietary advantages of germinated grains have been the subject of numerous discussions over the past decade. Around 23 million tons of oats are consumed globally, making up a sizeable portion of the global grain market. Oat seedlings contain more protein, beta-glucan, free amino acids, and phenolic compounds than seeds. The progressive neurodegenerative disorder of Alzheimer’s is accompanied by worsening memory and cognitive function. A key indicator of this disorder is the unusual buildup of amyloid-beta protein (or Aβ) in human brains. In this context, oat seedling extract (OSE) has been identified as a new therapeutic candidate for AD, due to its antioxidant activity and AD-specific mechanism of action. This study directly investigated how OSE affected AD and its impacts by examining the cognitive function and exploring the inflammatory response mechanism. The dried oat seedlings were grounded finely with a grinder, inserted with 50% fermented ethanol 10 times (w/v), and extracted by stirring for 10 h at 45 °C. After filtering the extract by 0.22 um filter, some of it was used for UHPLC analysis. The results indicated that the treatment with OSE protects against Aβ25–35-induced cytotoxicity in BV2 cells. Tg-5Xfad AD mice had strong deposition of Aβ throughout their brains, while WT mice did not exhibit any such deposition within their brains. A drastic reduction was observed in terms of numbers, as well as the size, of Aβ plaques within Tg-5Xfad AD mice exposed to OSE. This study indicated OSE’s neuroprotective impacts against neurodegeneration, synaptic dysfunction, and neuroinflammation induced by amyloid-beta. Our results suggest that OSE acts as a neuroprotective agent to combat AD-specific apoptotic cell death, neuroinflammation, amyloid-beta accumulation, as well as synaptic dysfunction in AD mice’s brains. Furthermore, the study indicated that OSE treatment affects JNK/ERK/p38 MAPK signaling, with considerable inhibition in p-JNK, p-p38, and p-ERK levels seen in the brain of OSE-treated Tg-5Xfad AD mice.

Improvement of bone formation is necessary for successful treatment of the bone defects associated with osteoporosis. In this study, we sought to elucidate the osteogenic activity of peanut sprouts and their bioactive components. We found that peanut sprout water extract (PSWE) enhanced bone morphogenetic protein-2-mediated osteoblast differentiation in a dose-dependent manner by stimulating expression of runt-related transcription factor 2 (Runx2) via activation of AKT/MAP kinases. We identified a major component of PSWE, soyasaponin Bb, as the bioactive compound responsible for improvement of anabolic activity. Soyasaponin Bb from PSWE enhanced expression of the osteogenic transcription factor Runx2 and alkaline phosphatase. The soyasaponin Bb content depended on sprouting time of peanut, and the anabolic action of PSWE was dependent on soyasaponin Bb content. Thus, PSWE and soyasaponin Bb have the potential to protect against bone disorders, including osteoporosis.

It has been reported that cyanidin 3-O-glucoside (C3G) is an inhibitor of Helicobacter pylori toxin secretion. C3G is classified as an anthocyanin and is a major component of black rice extract (BRE). The present study aimed to identify a new functional food material to prevent H. pylori infection in Mongolian gerbil model. Toxicity in the liver and kidney were not detected after BRE administration (10 or 50 mg/kg). BRE treatment reduced bacterial colonization in animal gastric tissue, as well as infection signs as observed on the analysis of the hematological data. It was also found that the relative mRNA levels of the inflammatory cytokines were reduced in BRE-treated groups. These findings suggest that BRE acts as a potent inhibitor of H. pylori infection and pathogenesis in Mongolian gerbils. We propose that BRE may be used to manage gastroduodenal diseases caused by H. pylori infection.

Soybeans are used worldwide as food and as a healthy ingredient. Specifically, soy germ (SG) has received considerable attention owing to its abundant nutritional and biological components. This study aimed to elucidate the contents of isoflavone and soyasaponin of SG in 24 Korean soybean cultivars and the osteogenic activity of individual compounds. The isoflavone content in the SG ranged from 1110.9 to 3131.1 mg/100 g, and the soyasaponin content in SG ranged from 1173.5 to 3582.3 mg/100 g. The isoflavone and soyasaponin content depended on soybean cultivars. All isoflavone and soyasaponin compounds enhanced bone morphogenetic protein-2-mediated osteoblast differentiation in a dose-dependent manner, especially soyasaponin Ab. In conclusion, our results suggest that Seonpung cultivar with high soyasaponin Ab is beneficial for developing functional materials.

Seedlings of natural crops are valuable sources of pharmacologically active phytochemicals. In this study, we aimed to identify new active secondary metabolites in Avena sativa L. (oat) seedlings. Two new compounds, avenafuranol (1) and diosgenoside (2), along with eight known compounds (3–10) were isolated from the A. sativa L. seedlings. Their chemical structures were elucidated via 1D and 2D NMR spectroscopy, high-resolution ESIMS, IR spectroscopy, optical rotation analysis, and comparisons with the reported literature. The effect of each isolated compound on alkaline phosphatase (ALP) activity for osteoblast differentiation induced by bone morphogenetic protein-2 (BMP-2) was investigated using the C2C12 immortal mouse myoblast cell line. Compounds 1, 4, 6, 8, and 9 induced dose-dependent increases in ALP expression relative to ALP expression in cells treated with only BMP-2, and no cytotoxicity was observed. These results suggest that A. sativa L. seedlings are a natural source of compounds that may be useful for preventing bone disorders.

This is the first study to investigate antioxidant capacities of isoflavones prepared using microwave-assisted hydrolysis method from different parts (seeds, leaves, leafstalks, pods, stems and roots) of soybean at growth stages. In addition, the fluctuations in the isoflavone, protein, fatty acid, and oil contents in R6-R8 (R6: beginning; R7: beginning maturity; R8: full maturity) seeds were confirmed. The R7 seeds exhibited the most predominant contents of isoflavones (1218.1±7.3 μg/g) in the following order: daidzein (48%)>genistein (35%)>glycitein (17%). The second highest isoflavone content was found in the leaves (1052.1±10.4 μg/g), followed by R8 seeds>roots>R6 seeds>leafstalks> pods; the stems exhibited the lowest isoflavone content (57.2±1.7 μg/g). Interestingly, daidzein showed the highest individual isoflavone content with remarkable variations (57.2-766.8 μg/g), representing 46-100% of the total isoflavone content. R8 exhibited higher protein, fatty acid, and oil contents than R6 or R7. Moreover, the antioxidant capacities against two radicals in different parts of soybean plant showed considerable differences depending upon the isoflavone content. Our results suggested that soybean leaves and seeds might be useful materials for functional foods.

Obesity is a worldwide public health concern requiring safe and effective strategies. Recent studies suggest that bioactive compounds from soybeans have beneficial effects on weight loss and reducing fat accumulation. However, despite the biochemical and nutritional changes during germination, the biological effects of germinated soy germ have not been fully investigated. In this article, germinated soy germ extract (GSGE) was evaluated as a potential treatment option for obesity using 3T3-L1 pre-adipocyte and high-fat diet (HFD)-induced obese mice. In vitro studies demonstrated that GSGE suppressed the differentiation of 3T3-L1 cells into mature adipocytes, along with reductions in lipid accumulation and lipid droplet formation. In vivo studies also showed that a daily dose of 1 mg kg-1 of GSGE reduced weight gain, adipocyte area, serum triglyceride, and LDL-cholesterol in HFD-fed mice. The GSGE treatment promoted browning, which was associated with increased UCP1 expression in vitro and in vivo. In addition, GSGE treatment induced beige fat activation by upregulation of lipolysis and beta-oxidation. Furthermore, gene and protein expression levels of endocannabinoid system-related factors such as NAPE-PLD, FAAH, DAGL-α, and CB2 were altered along with browning and beige fat activation by GSGE. The present study indicates that GSGE effectively inhibits lipid accumulation and promotes beige fat transition and activation. Therefore, we suggest that GSGE treatment could be a promising strategy for the prevention of obesity by promoting weight loss, reducing fat accumulation, and improving obesity-related metabolic disorders.

This study was performed to verify the possibility of manufacturing a germinated aromatic rice tea, which was roasted at 200, 250, and 300℃ each for 10, 20, and 30 min. The roasted aromatic rice was analysed physicochemical properties, sensory characteristics and aromatic compounds. The total polyphenol content and DPPH radical scavenging activities of the germinated aromatic rice increased as the roasting temperature and time increased. Total soluble solid contents, turbidity and browning index of the germinated aromatic rice tea also increased was the roasting temperature and roasting time increased. The pH did not change by roasting. The main aromatic components in roasted germinated aromatic rice tea were 2-methyl butanal, 3-methyl butanal, benzaldehyde and nonanal, which increased according to increasing temperature and time. However, those favorable aroma components were decreased at more than 300℃ of roasting temperature. In addition, methyl benzene, pentanol were increased which affect odor aroma. The sensory score of germinated aromatic rice tea also increased with high roasting temperature and time. However, aromatic rice roasted at a higher temperature (300℃) showed lower sensory score. Therefore roasting temperature and time must be controlled for manufactureing high quality of germinated aromatic rice tea, and the optimun roasting conditions were 250℃ and 30 min, which provide best physicochemical characteristics of aromatic rice tea.

The changes in phenolic and antioxidant capacities of seedlings of various barley cultivars through four different harvest times were investigated. Ten phenolics were characterised as hydroxycinnamic acid, orientin, isoorientin, and isovitexin derivatives using UPLC-PDA-ESI/MS and NMR. In particular, lutonarin (2) and saponarin (3) were the predominant constituents (71–75%) with significant differences in cultivars and harvest times. The highest average total phenolic content was found as 2671.8 mg/100 g on 23 days after the sowing, whereas the lowest was 1400.8 mg/100 g on 56 days. The radical scavenging abilities also exhibited considerable differences in 80% of methanol extracts (40 µg/mL), depending upon the phenolic contents. Interestingly, isoorientin (5) and orientin (6) possessed potent antioxidant effects with IC50 values of 20.7 ± 1.1 and 27.5 ± 7.3 µM (DPPH) and 5.7 ± 0.3 and 8.2 ± 0.3 µM (ABTS), respectively. These results may be contributed to enhancing the value of barley as a functional food ingredient and dietary supplement.

Hypoxia-inducible factor (HIF)-1α is a key regulator associated with tumorigenesis, angiogenesis, and metastasis. HIF-1α regulation under hypoxia has been highlighted as a promising therapeutic target in angiogenesis-related diseases. Here, we demonstrate that diacetyl atractylodiol (DAA) from Atractylodes japonica (A. japonica) is a potent HIF-1α inhibitor that inhibits the Akt signaling pathway. DAA dose-dependently inhibited hypoxia-induced HIF-1α and downregulated Akt signaling without affecting the stability of HIF-1α protein. Furthermore, DAA prevented hypoxia-mediated angiogenesis based on in vitro tube formation and in vivo chorioallantoic membrane (CAM) assays. Therefore, DAA might be useful for treatment of hypoxia-related tumorigenesis, including angiogenesis. [BMB Reports 2016; 49(9): 508-513].

Multifunctional zinc oxide (ZnO) has been generated as nanoparticles or nanorods and applied to various medical purposes since it exhibits several biological actions including anticancer activity. Especially, due to antibacterial activity and effects on bone regeneration, ZnO is widely used in implants and scaffolds in the orthopedic and dental fields. However, concerns over side effects have been raised recently in the clinical use of ZnO, and it is necessary to assess the safety of ZnO regarding its inflammatory potential in the bone environment. This made us hypothesize that the inflammatory activity of zinc ions released from ZnO NPs could be harmful to induce bone resorption but that their cytotoxicity would be beneficial to kill osteosarcoma. To clarify this hypothesis, in the present work, the effects of ZnO on bone matrix and abnormal bone environments were investigated quantitatively using ZnO chips, filter paper, or glass slides coated with thin films of ZnO grown via atomic layer deposition (ALD). ALD-grown ZnO thin films exhibit thickness with atomic precision, which enables the quantitative analysis of the effects of ZnO.

We report anti-cancer activity of ZnO thin-film-coated chips by sustained release of zinc ions. ZnO chips were fabricated by precisely tuning ZnO thickness using atomic layer deposition, and their potential to release zinc ions relative to the number of deposition cycles was evaluated. ZnO chips exhibited selective cytotoxicity in human B lymphocyte Raji cells while having no effect on human peripheral blood mononuclear cells. Of importance, the half-maximal inhibitory concentration of the ZnO chip on the viability of Raji cells was 121.5 cycles, which was comparable to 65.7 nM of daunorubicin, an anti-cancer drug for leukemia. Molecular analysis of cells treated with ZnO chips revealed that zinc ions released from the chips increased cellular levels of reactive oxygen species, including hydrogen peroxide, which led to the down-regulation of anti-apoptotic molecules (such as HIF-1a, survivin, cIAP-2, claspin, p-53, and XIAP) and caspase-dependent apoptosis. Because the anti-cancer activity of ZnO chips and the mode of action were comparable to those of daunorubicin, the development and optimization of ZnO chips that gradually release zinc ions might have clinical anti-cancer potential. A further understanding of the biological action of ZnO-related products is crucial for designing safe biomaterials with applications in disease treatment. (C) 2016 The Authors. Published by Elsevier Ireland Ltd.

The number of patients with bone metabolic disorders including osteoporosis is increasing worldwide. These disorders often facilitate bone fractures, which seriously impact the patient&rsquo, s quality of life and could lead to further health complications. Bone homeostasis is tightly regulated to balance bone resorption and formation. However, many anti-osteoporotic agents are broadly categorized as either bone forming or anti-resorptive, and their therapeutic use is often limited due to unwanted side effects. Therefore, safe and effective therapeutic agents are needed for osteoporosis. This study aims to clarify the bone protecting effects of oat bran water extract (OBWE) and its mode of action. OBWE inhibited RANKL (receptor activator of nuclear factor-&kappa, B ligand)-induced osteoclast differentiation by blocking c-Fos/NFATc1 through the alteration of I-&kappa, B. Furthermore, we found that OBWE enhanced BMP-2-stimulated osteoblast differentiation by the induction of Runx2 via Smad signaling molecules. In addition, the anti-osteoporotic activity of OBWE was also evaluated using an in vivo model. OBWE significantly restored ovariectomy-induced bone loss. These in vitro and in vivo results showed that OBWE has the potential to prevent and treat bone metabolic disorders including osteoporosis.

Osteoporosis is frequently induced following menopause, and bone fractures result in serious problems including skeletal deformity, pain, and increased mortality. Therefore, safe and effective therapeutic agents are needed for osteoporosis. This study aimed to clarify the bone protecting effects of germinated soy germ extracts (GSGE) and their mode of action. GSGE increased expression of alkaline phosphatase (ALP) and osteocalcin (OCL) by stimulating the expression of runt-related transcription factor 2 (Runx2) and osterix (Osx) through activation of Smad signaling molecules. Furthermore, germination of soy germ increased levels of nutritional components, especially soyasaponin Ab. The anabolic activity of soyasaponin Ab in GSGE was also evaluated. GSGE and soyasaponin Ab significantly protected against ovariectomy (OVX)-induced bone loss and improved bone-specific alkaline phosphatase (BALP) level in mouse serum. These in vitro and in vivo study results demonstrated that GSGE and soyasaponin Ab have potential as therapeutic candidate agents for bone protection in postmenopausal osteoporosis.

MicroRNA (miR)-150 has been shown to control B and T cell differentiation in the bone marrow. The regulation of B and T cells is directly or systematically associated with bone remodeling cells such as osteoclasts; however, the functional role of miR-150 in bone homeostasis has not been well studied. Here, we observed down-regulation of miR-150 during in vitro osteoclast differentiation and, furthermore, that miR-150 knockout mice exhibit decreased bone mass and an increased number of osteoclasts. miR-150 deficiency did not affect osteoclast differentiation, but miR150 knockout mice had significantly lower osteoprotegrin (OPG) serum levels, suggesting that the reduction of serum OPG level in miR-150 knockout mice might induce B cell expansion and subsequently increase serum levels of immunoglobulins for activating osteoclast differentiation., Highlights • miR-150 knockout mice exhibited decreased bone mass and an increased number of osteoclasts. • miR-150 expression gradually decreased during in vitro osteoclast differentiation. • Osteoclast differentiation of BMMs isolated from miR-150 knockout mice was similar to that from wild type. • miR-150 knockout mice exhibited significantly lower OPG serum levels.

Novel oxadiazoles bearing 5-phenyl-tetrazole (5a–k) were designed and efficiently synthesized by treating 2-(5-phenyl-2H-tetrazole-2-yl)acetohydrazide (4) with aromatic carboxylic acids in POCl3 , and their in vitro anti-osteoclastogenic activities were evaluated. In the cell-based osteoclast differentiation model, all compounds (5a–k) inhibited the formation of osteoclasts. In addition, the potential target molecules of compound 5 analogs were predicted with their chemical substructures via a web-based interface, and some of them were found to be related to osteoclast differentiation. Consequently, the scaffold containing oxadiazole–tetrazole in a single molecule and their analogs are of potential use in the design of novel anti-osteoclastogenic therapeutics.

Prohibitin-1 (PHB) regulates diverse cellular processes by controlling several signaling pathways. In this study, we investigated the functional involvement of PHB in osteoclast differentiation. PHB expression was time-dependently increased by RANKL in BMMs. However, the retroviral over-expression of PHB strongly inhibited the expression of c-Fos and NFATc1, and activation of p38-Elk-1-SRE signaling pathway. Anti-osteoclastogenic action of PHB was significantly inhibited by constitutively active forms of MKK6, but not Elk-1. Collectively, PHB negatively regulates the formation of mature osteoclasts via inhibition of MKK6 activity that affects the activation of the p38-Elk-1 signaling axis required for the expression of c-Fos and NFATc1.

Drug repositioning is a rational approach for expanding the use of existing drugs or candidate drugs to treat additional disorders. Here we investigated the possibility of using the anticancer p21-activated kinase 4 (PAK4)-targeted inhibitor PF-3758309 to treat osteoclast-mediated disorders. PAK4 was highly expressed in bone marrow cells and was phosphorylated during their differentiation into osteoclasts, and osteoclast differentiation was significantly inhibited by the dominant negative form of PAK4 and by PF-3758309. Specifically, PF-3758309 significantly inhibited the fusion of preosteoclasts, the podosome formation, and the migration of preosteoclasts. PF-3758309 also had in vivo antiresorptive activity in a lipopolysaccharide-induced bone erosion model and in vitro antiosteoclastogenic activity in the differentiation of human bone marrow-derived cells and peripheral blood mononuclear cells into osteoclasts. These data demonstrate the relevance of PAK4 in osteoclast differentiation and the potential of PAK4 inhibitors for treating osteoclast-related disorders.

Bone is maintained by osteoclast-mediated resorption and osteoblast-mediated formation. Recently, anti-osteoporotic activity of Saururus chinensis extract (SCE) and anti-osteoclastogenic activity of its components have been reported, but the effect of SCE on bone formation has not been studied well. Therefore, in this study, we investigated whether Saururus chinensis SCE exhibits in vitro osteogenic and in vivo bone-forming activity. extract strongly enhanced the bone morphogenetic protein (BMP)-2-stimulated induction of alkaline phosphatase, an early phase biomarker of osteoblast differentiation, in bi-potential mesenchymal progenitor C2C12 cells. In vitro osteogenic activity of SCE was accompanied by enhanced expression of BMP-2, BMP-4, BMP-7 and BMP-9 mRNA. In addition, a pharmacological inhibition study suggested the involvement of p38 activation in the osteogenic action of SCE. Moreover, the BMP dependency and the involvement of p38 activation in the osteogenic action of SCE were confirmed by the treatment of noggin, an antagonist of BMP. Saururus chinensis extract also exhibited to induce runt-related transcription factor 2 activation at the high concentration. Furthermore, the in vivo osteogenic activity of SCE was confirmed in zebrafish and mouse calvarial bone formation models, suggesting the possibility of its use for bone formation. In conclusion, we suggested that in vivo anti-osteoporotic activity of SCE could be because of its dual action in bone, anti-osteoclastogenic and anabolic activity.

Excessive activity and/or increased number of osteoclasts lead to bone resorption-related disorders. Here, we investigated the potential of praeruptorin A to inhibit migration/fusion of preosteoclasts in vitro and bone erosion in vivo. Praeruptorin A inhibited the RANKL-induced migration/fusion of preosteoclasts accompanied by the nuclear translocation of NFATc1, a master regulator of osteoclast differentiation. Antimigration/fusion activity of praeruptorin A was also confirmed by evaluating the mRNA expression of fusion-mediating molecules. In silico binding studies and several biochemical assays further revealed the potential of praeruptorin A to bind with Ca(2+)/calmodulin and inhibit its downstream signaling pathways, including the Ca(2+)/calmodulin-CaMKIV-CREB and Ca(2+)/calmodulin-calcineurin signaling axis responsible for controlling NFATc1. In vivo application of praeruptorin A significantly reduced lipopolysaccharide-induced bone erosion, indicating its possible use to treat bone resorption-related disorders. In conclusion, praeruptorin A has the potential to inhibit migration/fusion of preosteoclasts in vitro and bone erosion in vivo by targeting calmodulin and inhibiting the Ca(2+)/calmodulin-CaMKIV-CREB-NFATc1 and/or Ca(2+)/calmodulin-calcineurin-NFATc1 signaling axis.

The number of patients with osteoporosis is increasing worldwide, and a decrease in bone mass is a main risk factor for fracture. The prevention of bone loss is critical for improving the quality of life for patients. However, the long-term use of antiosteoporotic agents is limited due to their side effects. Barley has been traditionally ingested for thousands of years as a safe, natural food with pharmaceutical properties, and its seedling can enhance the biological activity of the medicinal components found in food. This study aimed to clarify the antiresorptive activity of barley seedling and its mode of action. Barley seedling extracts (BSE) dose-dependently inhibited RANKL-induced osteoclast differentiation with alteration of IκB degradation, c-Fos, and NFATc1 molecules in the early-to-middle stages of osteoclastogenesis. In the late phase of osteoclastogenesis, BSE also prevented DC-STAMP and cathepsin K, which are required for cell fusion and bone degradation, such as osteoclast function. In conclusion, barley seedling from natural foods may provide long-term safety and be useful for the prevention or treatment of osteoclast-mediated bone metabolic diseases, including osteoporosis.

Translationally-controlled tumor protein (TCTP) plays a role in a number of cellular processes, but there is limited information about its function in cell differentiation. Previous observations of a twofold induction of TCTP mRNA during osteoclast differentiation prompted us to investigate its involvement in osteoclast differentiation. The osteoclastogenicity of TCTP gradually expressed during osteoclast differentiation was confirmed in mouse and human cells using loss-of-function studies and TCTP heterogeneous mice and transgenic mice. Higher expression ratios of TCTP to p21 could represent TCTP-mediated phenotypic induction of osteoclast differentiation accompanied by p21 down-regulation, attenuating the proliferation of osteoclast precursor cells.

Natural polyamines have numerous biological activities. Several studies have reported their beneficial role in bone metabolism, but their mode of action is not fully understood. Bone diseases such as osteoporosis, which is characterized by impaired bone structure and low bone mass, are caused by an increased number of osteoclasts and/or overactivation of osteoclastogenesis. Osteoclast differentiation is a multi-complex procedure involving the following sequential steps: differentiation-migration-fusion-resorption. In this study, we found that putrescine, spermidine or spermine inhibited the RANKL-mediated migration of preosteoclasts. Furthermore, the RANKL-mediated activation of the Src-PYK2 signaling axis and of transcription factors such as NF-κB and NFATc1 was prevented by each polyamine. Anti-osteoclastogenic and anti-migration activities of polyamines were confirmed by evaluating their potential to downregulate the mRNA expression levels of osteoclastogenesis-related genes such as OSCAR, TRAP, cathepsin K and c-Src, and genes related to fusion and/or migration of preosteoclasts. Moreover, ATP-mediated elevation of cytosolic free Ca(2+) concentration ([Ca(2+)]i) was strongly inhibited by each polyamine, indicating the involvement of [Ca(2+)]i in the anti-fusion activities of polyamines. In conclusion, polyamines could exhibit anti-osteoclastogenic activity by inhibiting the migration of preosteoclasts via the Ca(2+)-PYK2-Src-NFATc1 signaling axis.

Selective inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) have considerable potential as treatment for osteoporosis as well as metabolic syndrome including type 2 diabetes mellitus. Here, we investigated the anti-diabetic, anti-adipogenic, and anti-osteoporotic activity of KR-67500, as a novel selective 11β-HSD1 inhibitor. Cellular 11β-HSD1 activity was tested based on a homogeneous time-resolved fluorescence method. Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) levels were measured in diet-induced obese (DIO)-C57BL/6 mice administered KR-67500 (50 mg/kg per day, p.o.) for 28 days and, additionally, its anti-diabetic effect was evaluated by OGTT and ITT. Thein vitroanti-adipogenic effect of KR-67500 was determined by Oil Red O Staining. Thein vitroanti-osteoporotic activity of KR-67500 was evaluated using bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation model systems. KR-67500 improved thein vivoglucose tolerance and insulin sensitivity in DIO-C57BL/6 mice. KR-67500 suppressed cortisone-induced differentiation of 3T3-L1 cells into adipocytes. KR-67500 enhanced BMP2-induced osteoblastogenesis in C2C12 cells and inhibited RANKL-induced osteoclastogenesis in mouse bone marrow-derived macrophages. KR-67500, a new selective 11β-HSD1 inhibitor, may provide a new therapeutic window in the prevention and/or treatment of type 2 diabetes, obesity, and/or osteoporosis.

Migration and invasion comprise key steps in cancer metastasis. Through the migration and invasion into and out of lymphatic and/or blood vessels, cancer cells can be spread out into the tissues in remote site from the origin. Degradation of extracellular matrix (ECM) must be preceded prior to the metastasis of cancer cells. Matrix metalloproteinases (MMP) can degrade ECM, thus allow cells to migrate from the original site. Among MMPs, two gelatinase MMP-2 and MMP-9 play particularly important roles in ECM degradation. Here, we report that recently developed p21-activated kinase 4 inhibitor PF-3758309 shows anti-metastatic effect in A549 human lung cancer cell. PF-3758309 suppresses CREB, NF-κB, and β-catenin pathways, which are well known to be closely related with cell migration. This leads to the downregulation of MMP-2/MMP-9 expressions and the inhibition of A549 lung cancer metastasis.

This is the first study to investigate antioxidant capacities of isoflavones prepared using microwave-assisted hydrolysis method from different parts (seeds, leaves, leafstalks, pods, stems and roots) of soybean at growth stages. In addition, the fluctuations in the isoflavone, protein, fatty acid, and oil contents in R6-R8 (R6: beginning; R7: beginning maturity; R8: full maturity) seeds were confirmed. The R7 seeds exhibited the most predominant contents of isoflavones (1218.1±7.3 μg/g) in the following order: daidzein (48%)>genistein (35%)>glycitein (17%). The second highest isoflavone content was found in the leaves (1052.1±10.4 μg/g), followed by R8 seeds>roots>R6 seeds>leafstalks> pods; the stems exhibited the lowest isoflavone content (57.2±1.7 μg/g). Interestingly, daidzein showed the highest individual isoflavone content with remarkable variations (57.2-766.8 μg/g), representing 46-100% of the total isoflavone content. R8 exhibited higher protein, fatty acid, and oil contents than R6 or R7. Moreover, the antioxidant capacities against two radicals in different parts of soybean plant showed considerable differences depending upon the isoflavone content. Our results suggested that soybean leaves and seeds might be useful materials for functional foods.

VapB has been shown to regulate calcium homeostasis in amyotrophic lateral sclerosis. Calcium signaling is also important in metabolic bone diseases, but the role of VapB in the generation of osteoclasts for bone resorption during osteoclastogenesis is not known. Therefore, we investigated the role of VapB in RANKL-induced osteoclast differentiation. Interestingly, VapB is induced during osteoclastogenesis, and regulates osteoclast differentiation by modulating NFATc1. The results also suggest that VapB regulates osteoclastogenesis via PLCγ2-Ca2+-NFAT signaling. The involvement of PLCγ2-Ca2+-NFAT signaling in VapB-regulated osteoclastogenesis was confirmed by a pharmacological study. Taken together, the results indicate that VapB positively regulates RANKL-mediated osteoclastogenesis via PLCγ2-Ca2+-NFAT signaling.

Osteoclasts are multinucleated cells that are formed by the fusion of mononuclear osteoclasts, which is an essential process in bone resorption leading to bone remodeling. Herein we show that GM-CSF promoted the fusion of prefusion osteoclasts (pOCs). The expression of GM-CSF receptor-α was significantly up-regulated at the fusion stage of pOCs induced by RANKL. GM-CSF induced the expression of dendritic cell-specific transmembrane protein (DC-STAMP), which was mediated by inducing NFATc1 via induction of c-Fos. The expression of c-Fos and NFATc1 was regulated by the ERK signaling pathway. Inhibition of ERK and NFATc1 suppressed the expression of DC-STAMP and led to the fusion inhibition of pOC. However, retrovirus-mediated expression of NFATc1 in pOCs rescued the defect in pOC fusion, despite the presence of U0126 and cyclosporin A. GM-CSF-stimulated pOCs had an intact actin ring and could resorb bone. Importantly, pOCs infected with constitutively active MEK adenovirus expressed c-Fos and NFATc1, followed by the binding of NFATc1 to the DC-STAMP promoter, which enables its transcription and expression. Constitutively active MEK-infected pOCs are able to resorb bone by undergoing cell-cell fusion. Taken together, our results demonstrated that GM-CSF induced fusion of pOCs to form multinucleated osteoclasts, making the osteoclast capable of bone resorption.

KCNK1 (K(+) channel, subfamily K, member 1) is a member of the inwardly rectifying K(+) channel family, which drives the membrane potential towards the K(+) balance potential. Here, we investigated its functional relevance during osteoclast differentiation. KCNK1 was significantly induced during osteoclast differentiation, but its functional overexpression significantly inhibited osteoclast differentiation induced by RANKL (also known as TNFSF11), which was accompanied by the attenuation of the RANKL-induced Ca(2+) oscillation, JNK activation and NFATc1 expression. In contrast, KCNK1 knockdown enhanced the RANKL-induced osteoclast differentiation, JNK activation and NFATc1 expression. In conclusion, we suggest that KCNK1 is a negative regulator of osteoclast differentiation; the increase of K(+) influx by its functional blockade might inhibit osteoclast differentiation by inhibiting Ca(2+) oscillation and the JNK-NFATc1 signaling axis. Together with the increased attention on the pharmacological possibilities of using channel inhibition in the treatment of osteoclast-related disorders, further understanding of the functional roles and mechanisms of K(+) channels underlying osteoclast-related diseases could be helpful in developing relevant therapeutic strategies.

Arginase 1 (Arg1) limits the availability of l-arginine for producing nitric oxide (NO) and ornithine, a substrate for polyamine synthesis. Anti-osteoclastogenic activities of NO and polyamines, and the involvement of Arg1 on the dendritic cell differentiation of dendritic cells have been reported, but the relevance of Arg1 to osteoclast differentiation has not been investigated. Here, we observed Arg1 down-regulation during the RANKL-induced differentiation of bone marrow-derived macrophages into osteoclasts. Arg1 overexpression significantly inhibited osteoclast differentiation with low NO production, while Arg1 knockdown enhanced osteoclast differentiation with high NO production. These results suggest that Arg1 and NO have reciprocal roles as negative and positive regulators, respectively, of osteoclast differentiation. We conclude that Arg1 is down-regulated during osteoclast differentiation and may negatively regulate osteoclast differentiation by regulating NO production.

Osteoporosis is a disease that decreases bone mass. The number of patients with osteoporosis has been increasing, including an increase in patients with bone fractures, which lead to higher medical costs. Osteoporosis treatment is all-important in preventing bone loss. One strategy for osteoporosis treatment is to inhibit osteoclastogenesis. Osteoclasts are bone-resorbing multinucleated cells, and overactive osteoclasts and/or their increased number are observed in bone disorders including osteoporosis and rheumatoid arthritis. Bioactivity-guided fractionations led to the isolation of alisol A 24-acetate from the dried tuber ofAlisma canaliculatum. Alisol A 24-acetate inhibited RANKL-mediated osteoclast differentiation by downregulating NFATc1, which plays an essential role in osteoclast differentiation. Furthermore, it inhibited the expression of DC-STAMP and cathepsin K, which are related to cell-cell fusion of osteoclasts and bone resorption, respectively. Therefore, alisol A 24-acetate could be developed as a new structural scaffold for inhibitors of osteoclast differentiation in order to develop new drugs against osteoporosis.

Ouabain, a cardiac glycoside found in plants, is primarily used in the treatment of congestive heart failure and arrhythmia because of its ability to inhibit Na+/K+-ATPase pump. Recently ouabain has been shown to exert anticancer effects but the underlying mechanism is not clear. Here, we explored the molecular mechanism by which ouabain exerts anticancer effects in human lung adenocarcinoma. Employing proteomic techniques, we found 7 proteins downregulated by ouabain in A549 including p-ezrin, a protein associated with pulmonary cancer metastasis in a dose-dependent manner. In addition, when the relative phosphorylation levels of 39 intracellular proteins were compared between control and ouabain-treated A549 cells, p-Src (Y416) was also found to be downregulated by ouabain. Furthermore, western blot revealed the ouabain-mediated downregulation of p-FAK (Y925), p-paxillin (Y118), p130CAS, and Na+/K+-ATPase subunits that have been shown to be involved in the migration of cancer cells. The inhibitory effect of ouabain and Src inhibitor PP2 on the migration of A549 cells was confirmed by Boyden chamber assay. Anticancer effects of ouabain in A549 cells appear to be related to its ability to regulate and inactivate Src-to-ezrin signaling, and proteins involved in focal adhesion such as Src, FAK, and p130CAS axis are proposed here.

Drug repositioning is a rational approach for expanding the use of existing drugs or candidate drugs to treat additional disorders. Here we investigated the possibility of using the anticancer p21-activated kinase 4 (PAK4)-targeted inhibitor PF-3758309 to treat osteoclast-mediated disorders. PAK4 was highly expressed in bone marrow cells and was phosphorylated during their differentiation into osteoclasts, and osteoclast differentiation was significantly inhibited by the dominant negative form of PAK4 and by PF-3758309. Specifically, PF-3758309 significantly inhibited the fusion of preosteoclasts, the podosome formation, and the migration of preosteoclasts. PF-3758309 also had in vivo antiresorptive activity in a lipopolysaccharide-induced bone erosion model and in vitro antiosteoclastogenic activity in the differentiation of human bone marrow-derived cells and peripheral blood mononuclear cells into osteoclasts. These data demonstrate the relevance of PAK4 in osteoclast differentiation and the potential of PAK4 inhibitors for treating osteoclast-related disorders.