Gleditsiae fructus regulates osteoclastogenesis by inhibiting the c­Fos/NFATc1 pathway and alleviating bone loss in an ovariectomy model.

Medical and economic developments have allowed the human lifespan to extend and, as a result, the elderly population has increased worldwide. Osteoporosis is a common geriatric disease that has no symptoms and even a small impact can cause fractures in patients, leading to a serious deterioration in the quality of life. Osteoporosis treatment typically involves bisphosphonates and selective estrogen receptor modulators. However, these treatments are known to cause severe side effects, such as mandibular osteonecrosis and breast cancer, if used for an extended period of time. Therefore, it is essential to develop therapeutic agents from natural products that have fewer side effects. Gleditsiae fructus (GF) is a dried or immature fruit of Gleditsia sinensis Lam. and is composed of various triterpenoid saponins. The anti­inflammatory effect of GF has been confirmed in various diseases, and since the anti­inflammatory effect plays a major role in inhibiting osteoclast differentiation, GF was expected to be effective in osteoclast differentiation and menopausal osteoporosis; however, to the best of our knowledge, it has not yet been studied. Therefore, the present study was designed to examine the effect of GF on osteoclastogenesis and to investigate the mechanism underlying inhibition of osteoclast differentiation. The effects of GF on osteoclastogenesis were determined in vitro by tartrate­resistant acid phosphatase (TRAP) staining, pit formation assays, filamentous actin (F­actin) ring formation assays, western blotting and reverse transcription­quantitative PCR analyses. Furthermore, the administration of GF to an animal model exhibiting menopausal osteoporosis allowed for the analysis of alterations in the bone microstructure of the femur using micro­CT. Additionally, assessments of femoral tissue and serum were conducted. The present study revealed that the administration of GF resulted in a reduction in osteoclast levels, F­actin rings, TRAP activity and pit area. Furthermore, GF showed a dose­dependent suppression of nuclear factor of activated T­cells cytoplasmic, c­Fos and other osteoclastogenesis­related markers.

Suppressive effects of (-)-tubaic acid on RANKL-induced osteoclast differentiation and bone resorption.

Regulation of osteoclastogenesis and bone-resorbing activity can be an efficacious strategy for treating bone loss diseases because excessive osteoclastic bone resorption leads to the development of such diseases. Here, we investigated the role of (-)-tubaic acid, a thermal degradation product of rotenone, in osteoclast formation and function in an attempt to identify alternative natural compounds. (-)-Tubaic acid significantly inhibited receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclast differentiation at both the early and late stages, suggesting that (-)-tubaic acid affects the commitment and differentiation of osteoclast progenitors as well as the cell-cell fusion of mononuclear osteoclasts. (-)-Tubaic acid attenuated the activation of extracellular signal-regulated kinase (ERK) and expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and its target genes in response to RANKL. Furthermore, a pit-formation assay revealed that (-)-tubaic acid significantly impaired the bone-resorbing activity of osteoclasts. Our results demonstrated that (-)-tubaic acid exhibits anti-osteoclastogenic and anti-resorptive effects, indicating its therapeutic potential in the management of osteoclast-related bone diseases.

Non-polar lipid from greenshell mussel (Perna canaliculus) inhibits osteoclast differentiation.

The osteoclast-dependent bone resorption process is a crucial part of the bone regulatory system. The excessive function of osteoclasts can cause diseases of bone, joint, and other tissues such as osteoporosis and osteoarthritis. Greenshell mussel oil (GSM), a good source of long chain omega-3 polyunsaturated fatty acids (LCn-3PUFAs), was fractionated into total lipid, polar lipid, and non-polar lipid components and their anti-osteoclastogenic activity tested in RAW 264.7 cell cultures. Osteoclast differentiation process was achieved after 5 days of incubation with RANKL in 24-well culture plates. Introducing the non-polar lipid fraction into the culture caused a lack of cell differentiation, and a reduction in tartrate-resistant acid phosphatase (TRAP) activity and TRAP cell numbers in a dose-dependent manner (50% reduction at the concentration of 20 µg/mL, p < 0.001). Moreover, actin ring formation was significantly diminished by non-polar lipids at 10-20 µg/mL. The bone digestive enzymes released by osteoclasts into the pit formation were also compromised by downregulating gene expression of cathepsin K, carbonic anhydrase II (CA II), matrix metalloproteinase 9 (MMP-9), and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1). This study revealed that the non-polar lipid fraction of GSM oil contains bioactive substances which possess potent anti-osteoclastogenic activity.

Solanum nigrum Line inhibits osteoclast differentiation and suppresses bone mineral density reduction in the ovariectomy­induced osteoporosis model.

Bone homeostasis is maintained by osteoclasts that absorb bone and osteoblasts that form bone tissue. Menopausal osteoporosis is a disease associated with aging and hormonal changes due to menopause causing abnormal activation of osteoclasts, resulting in a decrease in bone density. Existing treatments for osteoporosis have been reported to have serious side effects, such as jawbone necrosis and breast and uterine cancer; therefore, their use by patients is decreasing, whilst studies focusing on alternative treatments are increasingly popular. Solanum nigrum Line (SL) has been used as a medicinal plant that possesses several pharmacological effects, such as anti­inflammatory and hepatotoxic protective effects. To the best of our knowledge, however, its effects on osteoporosis and osteoclasts have not been demonstrated previously. In the present study, the anti­osteoporotic effect of SL was investigated using a postmenopausal model of osteoporosis in which Sprague­Dawley rat ovaries were extracted. In addition, the inhibitory effects on osteoclast differentiation and function of SL was confirmed using an osteoclast model treated with receptor activator of NF­κB ligand (RANKL) on murine RAW 264.7 macrophages. In vivo experiments showed that SL reduced the decrease in bone mineral density and improved changes in the morphological index of bone microstructure, such as trabecular number and separation. In addition, the number of tartrate resistant acid phosphatase­positive cells in the femur and the expression levels of nuclear factor of activated T­cells cytoplasmic 1 (NFATc1) and cathepsin K protein were inhibited. In vitro, SL suppressed RANKL­induced osteoclast differentiation and bone resorption ability; this was mediated by NFATc1/c­Fos, a key transcription factor involved in osteoclast differentiation, ultimately inhibiting expression of various osteoclast­associated genes. These experimental results show that SL may be an alternative treatment for osteoporosis caused by abnormal activation of osteoclasts in the future.

Role of vasodilator-stimulated phosphoprotein in RANKL-differentiated murine macrophage RAW264.7 cells: Modulation of NF-κB, c-Fos and NFATc1 transcription factors.

Vasodilator-stimulated phosphoprotein (VASP) is essential for osteoclast differentiation, and reduced VASP expression results in depressed osteoclast differentiation. Previously, we demonstrated the importance of VASP and Ras-related C3 botulinum toxin substrate 1 interactions in osteosarcoma cell migration and metastasis using Mg-63 and Saos2 cells. However, the molecular details of the functional role of VASP in cell motility and migration remain to be elucidated. The present study demonstrated that VASP affects the expression of αV-integrin, tartrate-resistant acid phosphatase (TRAP) and lamellipodia protrusion in RAW 264.7 murine macrophage cells. The RAW 264.7 mouse monocyte macrophage cell line was used as an osteoclast precursor. RAW 264.7 cells were treated with 50 ng/ml of receptor activator of nuclear factor κ-Β ligand (RANKL) in order to induce cell differentiation (osteoclastogenesis). Small interfering RNA (siRNA) was used to silence VASP, and RT-PCR and western blotting were used to determine the expression for genes and proteins, respectively. TRAP staining as a histochemical marker for osteoclast and fluorescent microscopy for lamellipodia protrusion was performed. RANKL treatment significantly increased the gene and protein expression of VASP, αV-integrin and TRAP in RAW 264.7 cells. Silencing of VASP significantly reduced the RANKL-induced expression of αV-integrin, TRAP and lamellipodia protrusion. In addition, knockdown of VASP attenuated RANKL-stimulated activation of NF-κB, c-Fos and nuclear factor of activated T cells cytoplasmic 1 transcription factors, and the phosphorylation of the p65 and IκBα. These results suggest the critical role of VASP in regulating osteoclast differentiation, which should be further explored in osteosarcoma research.

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.

Osthole inhibits osteoclasts formation and bone resorption by regulating NF-κB signaling and NFATc1 activations stimulated by RANKL.

Chinese herbal medicine Fructus Cnidii has an outstanding effect on chronic lumbar pain and impotence, also has been used against osteoporosis with high frequency. Yet, the mechanisms of osthole, a derivative of Fructus Cnidii, on osteoclasts remains barely known. In this study, it was found out that osthole (10-6 mol/L, 10-5 mol/L) had the influence of inhibiting osteoclast formation and bone resorptive activities induced by receptor activator of nuclear factor κB ligand (RANKL), rather than affecting the viability of osteoclast-like cells. Furthermore, osthole could also inhibit the messenger RNA expressions of c-Src, tartrate-resistant acid phosphatase, ß3-Integrin, matrix metallopeptidase 9, and cathepsin K. The results of the mechanistic study indicated that osthole regulated the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and nuclear factor-κB (NF-κB) activations following the RANKL stimulation. These findings suggested that the inhibitory effects of osthole were associated with restraining the activations of NFATc1 and NF-κB induced by RANKL. Thus osthole can be used as a potential treatment for abnormal bone-resorption related diseases.

Stimuli and Relevant Signaling Cascades for NFATc1 in Bone Cell Homeostasis: Friend or Foe?

Bone homeostasis is strictly regulated by balanced activity of bone-forming osteoblasts and bone-resorbing osteoclasts.Disruption of the balance of activity between osteoblasts and osteoclasts leads to various metabolic bone diseases. Osteoclasts are cells of hematopoietic origin that they are large, multinucleated cells formed by the fusion of precursor cells of monocyte/macrophage lineage, they are unique cells that degrade the bone matrix, activation of transcription factors nuclear factoractivated T cells c1 (NFATc1) is required for sufficient osteoclast differentiation and it plays the role of a master transcription regulator of osteoclast differentiation, meanwhile, NFATc1 could be employed to elicit anabolic effects on bone. In this review, we have summarized the various mechanisms that control NFATc1 regulation during osteoclast and osteoblast differentiation as well as a new strategy for promoting bone regeneration in osteopenic disease.

MiRNA-199a-5p positively regulated RANKL-induced osteoclast differentiation by target Mafb protein.

MicroRNAs are involved in osteoclast differentiation. Although miR-199a-5p plays an important role in many different systems and diseases, its function during osteoclastogenesis remains unclear. In this study, we investigated the function and the target gene of miR-199a-5p in osteoclast differentiation. The in vitro data showed that miR-199a-5p was significantly upregulated after the stimulation by receptor activator of nuclear factor kappa-B ligand in macrophages and RAW 264.7 cells. After transfection of miR-199a-5p mimic, the messenger RNA expression level of nuclear factor of activated T-cells cytoplasmic 1, tartrate-resistant acid phosphatase (TRAP), and receptor activator of nuclear factor kappa-B was significantly increased in RAW 264.7 cells and the number of TRAP-positive cells was also increased. MiR-199a-5p inhibitor showed the complete opposite outcome which brought additional proof to our finding. Overexpression of miR-199a-5p led to downregulation of Mafb protein. The luciferase activity was obviously repressed when WT-pGL3-Mafb and miR-199a-5p mimics were cotransfected into 293 T cells and the inhibitors cotransfected demonstrated reverse result. MiR-199a-5p overexpressed during osteoclast differentiation and positively regulated osteoclast formation in vitro by target Mafb.

Ameloblastin attenuates RANKL-mediated osteoclastogenesis by suppressing activation of nuclear factor of activated T-cell cytoplasmic 1 (NFATc1).

Ameloblastin (Ambn) is an extracellular matrix protein and member of the family of enamel-related gene products. Like amelogenin, Ambn is mainly associated with tooth development, especially biomineralization of enamel. Previous studies have shown reductions in the skeletal dimensions of Ambn-deficient mice, suggesting that the protein also has effects on the differentiation of osteoblasts and/or osteoclasts. However, the specific pathways used by Ambn to influence osteoclast differentiation have yet to be identified. In the present study, two cellular models, one based on bone marrow cells and another on RAW264.7 cells, were used to examine the effects of Ambn on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis. The results showed that Ambn suppresses osteoclast differentiation, cytoskeletal organization, and osteoclast function by the downregulation of the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts, actin ring formation, and areas of pit resorption. The expression of the osteoclast-specific genes TRAP, MMP9, cathepsin K, and osteoclast stimulatory transmembrane protein (OC-STAMP) was abolished in the presence of Ambn, while that of nuclear factor of activated T cells cytoplasmic 1 (NFATc1), the master regulatory factor of osteoclastogenesis, was also attenuated by the downregulation of c-Fos expression. In Ambn-induced RAW264.7 cells, phosphorylation of cAMP-response element-binding protein (CREB), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK), but not extracellular signal-regulated kinase 1/2 (ERK1/2), was reduced. Calcium oscillation was also decreased in the presence of Ambn, suggesting its involvement in both RANKL-induced osteoclastogenesis and costimulatory signaling. B-lymphocyte-induced maturation protein-1 (Blimp1), a transcriptional repressor of negative regulators of osteoclastogenesis, was also downregulated by Ambn, resulting in the elevated expression of v-maf musculoaponeurotic fibrosarcoma oncogene family, protein B (MafB), B-cell lymphoma 6 (Bcl6), and interferon regulatory factor-8 (Irf8). Taken together, these findings suggest that Ambn suppresses RANKL-induced osteoclastogenesis by modulating the NFATc1 axis.

Effect of CaMKIIγ RNA interference on the expression of NFATc1,TRAP and c-Src genes in osteoclast differentiation / 实用肿瘤学杂志

Objective The aim of this study was to investigate the effect of Ca2+ /calmodulin - dependent kinase II (CaMKII)γ RNA interference on the expression of nuclear factor of activated T-cells cytoplasmic 1(NFATc1),tyrosine kinase(c-Src)and tartrate resistant acid phosphatase(TRAP)genes,and its role and molecular mechanism in osteoclast differentiation. Methods The CaMKII γ RNA interference vector was constructed by lentivirus and transfected into RAW264. 7 cells. The experiment was di-vided into three groups:A,B and C,which were the control group,negative vector group and interference vector group. After transfec-tion for 12 hours,osteoclasts induced by 50 ng/mL RANKL and the cells were harvested after induction for 5 days. Real-time quanti-tative PCR,Western blot and immunofluorescence were used to detect the expression of NFATc1,TRAP and c-Src genes in three groups. Results The mRNA levels of NFATc1,TRAP and c-Src in the group C decreased by 49. 86% ,43. 65% and 53. 57% ,re-spectively(P<0. 001),and the protein levels decreased by 54. 22% ,46. 75% and 45. 86% ,respectively(P<0. 001). There was no significant difference between the A and the B groups(P>0. 05). The fluorescence intensity of the above genes in the group C was significantly weaker than that in the A and B groups,and the formation of osteoclasts was significantly less than that in the A and B groups. Conclusion CaMKIIγ RNA interference significantly inhibited the expression of NFATc1,TRAP and c-Src genes,sugges-ting that CaMKIIγ plays a key regulatory role in osteoclast differentiation.

Pine needles attenuate receptor activator for nuclear factor-B ligand (RANKL)-induced trabecular bone loss by inhibiting osteoclast differentiation.

BACKGROUND: The leaf of Pinus densiflora known as pine needles has been used to treat vascular disease, gastrointestinal diseases, and urinary diseases in traditional medicine. We evaluated anti-osteoporotic effect of water extract of Pinus densiflora (WEPN) on acute bone loss and osteoclastogenesis induced by receptor activator for nuclear factor-κB ligand (RANKL). METHODS: After oral administration of WEPN (0.25 g/kg) for 5 days, femora were collected, and bone parameter [trabecular bone volume/tissue volume (BV/TV), trabecular thickness (Tb. Th), trabecular separation (Tb. Sp), trabecular number (Tb. N), and bone mineral density (BMD)] were analyzed by micro-CT analysis. Anti-osteoclastic effect of WEPN was examined using tartrate-resistant acid phosphatase activity and activation of RANKL signaling pathway. RESULTS: We found that WEPN significantly attenuated RANKL-induced decrease of BV/TV, Tb.Th., Tb.N, and BMD but increase of Tb. Sp in femora. WEPN dose-dependently decreased osteoclastogenesis accompanied by inhibiting the activation of RANKL signaling components (JNK, p38, and p65) and mRNA expression level of osteoclast specific genes (NFATc1, c-Fos, TRAP, cathepsin K, DC-STAMP, and carbonic anhydrate). CONCLUSION: WEPN inhibition on osteoclastogenesis could contribute to attenuate RANKL-induced trabecular bone loss in vivo. Therefore, it might suggest that WEPN could be prescribed in traditional medicine or used in health functional food to prevent or treat osteoporotic bone diseases.

Exercise induces muscle fiber type switching via transient receptor potential melastatin 2-dependent Ca2+ signaling.

The aim of the present study was to examine whether transient receptor potential melastatin 2 (TRPM2) plays a role in muscle fiber-type transition during exercise. Mice were trained at a speed of 12 m/min at a slope of 0° for 60 min for 5 consecutive days/wk for 4 wk. Exhaustion tests were performed on the treadmill (the speed was set at 6 m/min at a slope of 0° and increased at a rate of 1 m/min every 6 min). Isolated primary skeletal muscle cells from TRPM2-knockout (KO) mice showed lower amplitudes of electrical stimuli (ES)-induced Ca2+ signals when compared with wild-type (WT) mice due to a defect in Ca2+ influx. Moreover, TRPM2-KO mice had a higher proportion of fast-twitch skeletal muscle fibers and a lower proportion of slow-twitch muscle fibers before exercise than WT mice. After exercise, the expression of slow-twitch skeletal muscle fibers was increased only in WT mice but not in TRPM2-KO mice. ES-induced nuclear translocation of the Ca2+-dependent transcription factor NFATc1 was significantly lower in TRPM2-KO mice than in WT mice. TRPM2-KO mice also showed decreased mitochondrial Ca2+ and membrane potential. Lactate levels were higher in the skeletal muscle cells of TRPM2-KO mice before and after ES compared with WT mice. Collectively, these data indicate that TRPM2-mediated Ca2+ signaling plays a critical role in the regulation of fiber-type switching and mitochondrial function in skeletal muscle. NEW & NOTEWORTHY TRPM2 has been shown to play an important role in a variety of cellular functions. However, the role of TRPM2 in skeletal muscle remains poorly understood. Here, we provide evidence that TRPM2-mediated Ca2+ signaling is required for training-induced improvement in skeletal muscle mitochondrial function and fiber type transition.

Regulation of osteoclast differentiation by CaMK Ⅱδ mediated through CREB and ERK1/2 signal pathways / 解放军医学杂志

Objective To investigate the function of cAMP response element binding protein (CREB) and extracellular signal-regulated kinase (ERK1/2) in osteoclast differentiation mediated by Ca2+/calmodulin-dependent kinase Ⅱ (CaMK Ⅱ)δ,and elucidate the molecular mechanism thereof.Methods CaMK Ⅱδ RNA interference lentivirus vector was constructed and mouse RAW264.7 cells were transfected with the virus to determine the interference efficiency.After virus transfection,RAW264.7 cells were treated with 50ng/ml receptor activator of nuclear factor κB ligand (RANKL) and the phosphorylation levels of CREB and ERK1/2 were detected at different time points.The cells were also treated with PD98059,an ERK1/2 inhibitor,to determine the effect of ERK1/2 signal blocking on the expression of nuclear factor activated T-cells cytoplasmic 1 (NFATc1) and osteoclast differentiation.Results Interference efficiency of recombinant CaMK Ⅱδ virus vector was 77.2％ at mRNA level and 70.2％ at protein level.CaMK Ⅱδ RNA interference significantly suppressed phosphorylation of CREB and ERK1/2,and the levels ofp-CREB and p-ERK1/2 were down-regulated by 21％-55％ and 55％-64％,respectively.ERK1/2 inhibitor significantly down-regulated the protein expression of NFATc1,and the number of osteoclast,the number and size of bone resorption lacunae decreased by 39.3％,50.0％ and 52.3％,respectively.Conclusion CaMK Ⅱδ RNA interference may significantly suppress the phosphorylation of CREB and ERK1/2,and CREB and ERK1/2 have mediated the CaMK Ⅱδ-induced osteoclast differentiation.

Water extract of Rumex crispus prevents bone loss by inhibiting osteoclastogenesis and inducing osteoblast mineralization.

BACKGROUND: Rumex crispus root has traditionally been used in Asian medicine for the treatment of hemorrhage and dermatolosis. The aim of this study was to explore the pharmaceutical effects of water extract of Rumex crispus (WERC) on osteoblast and osteoclast differentiation. We also studied the effect of WERC on the receptor activator of nuclear factor kappa-B ligand (RANKL)-induced trabecular bone destruction mice model. METHODS: High performance liquid chromatography analysis was used to identify three compounds (emodin, chrysophanol, and physcion) of WERC. The in vivo effect of WERC was examined using an administration of WERC or vehicle on the ICR mice with bone loss induced by intraperitoneal RANKL injection on day 0 and 1. All mice were sacrificed by cervical dislocation at day 7 and the femurs of mice were isolated for soft X-ray and Micro-CT analysis. The in vitro effect of WERC on osteoblast mineralization or osteoclast differentiation was examined by alizarin red S staining or by tartrate-resistant acid phosphatase staining and assay. To determine the transcription level of osteoblast or osteoclast-specific genes, real-time quantitative polymerase chain reaction was used. Western blot analysis was performed to study the effect of WERC on mitogen-activated protein kinases (MAPK) or nuclear factor-κB (NF-κB) signaling molecules. RESULTS: The presence of three compounds in WERC was determined. WERC significantly suppressed RANKL-induced trabecular bone loss by preventing microstructural deterioration. In vitro, WERC increased osteoblast mineralization by enhancing the transcription of runt-related transcription factor 2 and its transcriptional coactivators, and by stimulating extracellular signal-regulated kinase phosphorylation. Furthermore, WERC significantly inhibited osteoclast differentiation by suppressing the activation of the RANKL signalings (MAPK and NF-κB) and the increasing inhibitory factors of nuclear factor of activated T cells cytoplasmic 1. CONCLUSION: This study showed that WERC could protect against osteoporosis and suggested that the possible mechanism of WERC might be related to increased osteoblast differentiation by activating Runx2 signaling and inhibition of osteoclast differentiation by suppression of RANKL signaling.

Isoproterenol Increases RANKL Expression in a ATF4/NFATc1-Dependent Manner in Mouse Osteoblastic Cells.

Sympathetic nervous system stimulation-induced ß-adrenergic signal transduction is known to induce bone loss and increase of osteoclast activity. Although isoproterenol, a nonspecific ß-adrenergic receptor agonist, has been shown to increase receptor activator of NF-κB ligand (RANKL), the details of the regulatory mechanisms remain unclear. In the present study, we investigated the role of the nuclear factor of activated T-cells (NFAT) in isoproterenol-induced RANKL expression in C2C12 and in primary cultured mouse calvarial cells. Isoproterenol increased nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and RANKL expressions at both mRNA and protein levels and increased NFAT reporter activity. NFATc1 knockdown blocked isoproterenol-mediated RANKL expression. Isoproterenol also promoted cAMP response element-binding protein 1 (CREB1) and activating transcription factor 4 (ATF4) phosphorylation. Isoproterenol-mediated transcriptional activation of NFAT was blocked by protein kinase A (PKA) inhibitor H89. Isoproterenol-induced CREB1, ATF4, NFATc1, and RANKL expressions were suppressed by H89. Mutations in cAMP response element-like or NFAT-binding element suppressed isoproterenol-induced RANKL promoter activity. Chromatin immunoprecipitation analysis demonstrated that isoproterenol increased NFAT-binding and ATF4-binding activities on the mouse RANKL promoter, but did not increase CREB1-binding activity. Association of NFATc1 and ATF4 was not observed in a co-immunoprecipitation study. ATF4 knockdown suppressed isoproterenol-induced NFAT binding to the RANKL promoter, whereas NFATc1 knockdown did not suppress isoproterenol-induced ATF4 binding to the RANKL promoter. ATF4 knockdown suppressed isoproterenol-induced expressions of NFATc1 and RANKL. These results suggest that isoproterenol increases RANKL expression in an ATF4/NFATc1-dependent manner.

Current Understanding of RANK Signaling in Osteoclast Differentiation and Maturation.

Osteoclasts are bone-resorbing cells that are derived from hematopoietic precursor cells and require macrophage-colony stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL) for their survival, proliferation, differentiation, and activation. The binding of RANKL to its receptor RANK triggers osteoclast precursors to differentiate into osteoclasts. This process depends on RANKL-RANK signaling, which is temporally regulated by various adaptor proteins and kinases. Here we summarize the current understanding of the mechanisms that regulate RANK signaling during osteoclastogenesis. In the early stage, RANK signaling is mediated by recruiting adaptor molecules such as tumor necrosis factor receptor-associated factor 6 (TRAF6), which leads to the activation of mitogen-activated protein kinases (MAPKs), and the transcription factors nuclear factor-κB (NF-κB) and activator protein-1 (AP-1). Activated NF-κB induces the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), which is the key osteoclastogenesis regulator. In the intermediate stage of signaling, the co-stimulatory signal induces Ca2+ oscillation via activated phospholipase Cγ2 (PLCγ2) together with c-Fos/AP-1, wherein Ca2+ signaling facilitates the robust production of NFATc1. In the late stage of osteoclastogenesis, NFATc1 translocates into the nucleus where it induces numerous osteoclast-specific target genes that are responsible for cell fusion and function.

Peucedanum japonicum Thunb. ethanol extract suppresses RANKL-mediated osteoclastogenesis.

The constituents of Peucedanum japonicum Thunb. (PJ) exhibit biological and pharmacological activities, including anti-obesity, anti-oxidant and anti-allergic activities. The aim of the present study was to examine in vitro effects of PJ in RANKL-induced signaling pathways, which determine osteoclast differentiation. PJ ethanol extract (PEE) exhibited anti-osteoporotic activity by disrupting the phospholipase C (PLC)-Ca2+-c-Fos/cAMP response element-binding protein (CREB)-nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) signaling pathway during osteoclastogenesis. Murine bone marrow-derived macrophages (BMMs) were cultured and used to determine the effects of PJ in the receptor activator of nuclear factor κB ligand (RANKL)-mediated osteoclastogenesis. The effects of PEE in the RANKL-mediated signaling cascade were evaluated using a standard in vitro osteoclastogenesis system. PEE treatment of BMMs significantly reduced the number of RANKL-mediated tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells (P<0.05 for 5 and 10 µg/ml PEE, P<0.01 for 25 and 50 µg/ml PEE), without cytotoxic effects. Furthermore, the expression of differentiation-related marker genes, including TRAP, Oscar, Cathepsin K, dendrocyte expressed seven transmembrane protein, ATPase H+ Transporting V0 Subunit D2 and NFATc1, were markedly suppressed. PEE induced a transient increase in free cytoplasmic Ca2+ ([Ca2+]i) mobilization via voltage-gated Ca2+ channels and PLC-sensitive pathways. Transient [Ca2+]i increase consequently resulted in the suppression of c-Fos, CREB and NFATc1 activities. These findings highlight the potential use of PJ in treating bone disorders caused by osteoclast overgrowth.

Cyperus Rotundus L. extract suppresses RANKL-induced osteoclastogenesis through NFATc1/c-fos downregulation and prevent bone loss in OVX-induced osteoporosis rat.

ETHNOPHARMACOLOGICAL RELEVANCE: Cyperus Rotundus L. (CyR) has been widely used for the treatment of gynecologic disorder. Recent studies have reported that CyR can prevent the formation of cystic follicles and ovarian malfunction. However, the effects of CyR on osteoclastogenesis and postmenopausal osteoporosis remain unknown. AIM OF THE STUDY: This study was aimed to investigate the preventive effects of CyR on RANKL-induced osteoclast formation and ovariectomy (OVX)-induced bone loss. MATERIALS AND METHODS: In this in vitro study, we investigate the anti-osteoporotic effect of CyR on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis, the formation of tartrate-resistant acid phosphatase (TRAP) multinucleated cells, pit formation, transcription factors such as NFATc1 and c-Fos, and mRNA expression of osteoclast-associated genes were investigated. Forty 12-weeks female Sprague-Dawley rats for in vivo effect of CyR were used and OVX rat model was determined. The rats were randomly assigned into sham group and four OVX groups, i.e. OVX with D.W; OVX with estradiol (E2, 100µg/kg/day), OVX with CyR-L (16mg/kg/day), OVX with CyR-H (160mg/kg/day). The treatment lasted for 8weeks. RESULTS: CyR inhibited osteoclast differentiation and pit formation in the RANKL-induced osteoclastogenesis of RAW 264.7 cells. Reverse transcription polymerase chain reaction analysis also showed that CyR reduced the mRNA expression of osteoclast-associated genes such as carbonic anhydrase II, TRAP, RANK, cathepsin K, matrix metalloproteinase 9, nuclear factor of activated T cells cytoplasmic 1 (NFATc1), and c-Fos. In addition, CyR decreased protein levels of NFATc1 and c-Fos. CyR inhibited trabecular bone loss in the femur caused by OVX. CONCLUSION: The results of this study indicate that CyR inhibits the RANKL-induced osteoclast differentiation in RAW 264.7 cells and trabecular bone loss in OVX rats.

Effects of zoledronate on CaMKⅡ δ and down-stream gene expressions during osteoclast differentiation / 重庆医学

Objective To study the effect of zoledronate (ZOL) on Ca2+/calmodulin-dependent kinase Ⅱ δ (CaMK Ⅱ δ) and down-stream gene expressions during osteoclast differentiation.Methods Mouse osteoclast precursors RAW264.7 cells were divided into the control group and ZOL group.The cells in both groups were induced with 50μg/L receptor activator of nuclear factor kappa B ligand (RANKL) and were harvested on 5 d,while the cells in ZOL group were also simultaneously treated with 1 × 10-6 mol/L ZOL for 2 d.Five days later,the cells were harvested and examined osteoclastogenesis,as well as gene expressions of CaMK Ⅱ δ,nuclear factor of activated T-cells cytoplasmic 1 (NFATc1),tartrate-resistant acid phosphatase (TRAP) and cell-sarcoma receptor coactivator (c-Src).Results The number of TRAP positive multinuclear osteoclasts,number and size of dentin absorption lacunae and area in the ZOL group were (20.0±3.2),(18.0±4.2) and (6 335.3± 1 043.2)μm2 respectively,which were significantly lower than (36.0 ± 8.4),(37.2 ± 5.0) and (11 636.2 ± 3 661.1) μm2 in the control group and decreased by 44.4 ％,51.6 ％ and 45.6 ％ respectively (P＜0.01).ZOL also significantly inhibited the gene expressions of CaMK Ⅱ δ,NFATc1,TRAP and c-Src,and the mRNA levels of these genes were decreased by 44.1％,49.0％,53.8％ and 49.6％ respectively,the protein level were decreased by 43.5 ％,32.2 ％,45.5 ％ and 48.0 ％ respectively.The immunofluorescent cytochemistry detection results showed the fluorescence intensity of CaMK Ⅱ δ,NFATc1,TRAP and c-Srcin in the ZOL group was significantly weakened when compared with the control group.Conclusion ZOL could significantly inhibit the osteoclast formation and bone absorption function,and down-regulates gene expressions of CaMK Ⅱ δ,NFATc1,TRAP and c-Src in osteoclast differentiation.