ABSTRACT
Since increased number of osteoclasts could lead to impaired bone structure and low bone mass, which are common characteristics of bone disorders including osteoporosis, the pharmacological inhibition of osteoclast differentiation is one of therapeutic strategies for preventing and/or treating bone disorders and related facture. However, little data are available regarding the functional relevance of phosphoinositide 3-kinase (PI3K) isoforms in the osteoclast differentiation process. To elucidate the functional involvement of PI3Kδ in osteoclastogenesis, here we investigated how osteoclast differentiation was influenced by idelalisib (also called CAL-101), which is p110δ-selective inhibitor approved for the treatment of specific human B cell malignancies. Here, we found that receptor activator of nuclear factor kappa B ligand (RANKL) induced PI3Kδ protein expression, and idelalisib inhibited RANKL-induced osteoclast differentiation. Next, the inhibitory effect of idelalisib on RANKL-induced activation of the Akt-c-Fos/NFATc1 signaling cascade was confirmed by western blot analysis and real-time PCR. Finally, idelalisib inhibited pre-osteoclast migration in the last stage of osteoclast differentiation through down-regulation of the Akt-c-Fos/NFATc1 signaling cascade. It may be possible to expand the clinical use of idelalisib for controlling osteoclast differentiation. Together, the present results contribute to our understanding of the clinical value of PI3Kδ as a druggable target and the efficacy of related therapeutics including osteoclastogenesis.
Subject(s)
Cell Differentiation/drug effects , Cell Movement/drug effects , Osteoclasts/cytology , Osteoclasts/drug effects , Osteogenesis/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Purines/pharmacology , Quinazolinones/pharmacology , Signal Transduction/drug effects , Animals , Dose-Response Relationship, Drug , Male , Mice , Mice, Inbred ICR , Molecular Structure , NFATC Transcription Factors/antagonists & inhibitors , NFATC Transcription Factors/metabolism , Osteoclasts/metabolism , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-fos/antagonists & inhibitors , Proto-Oncogene Proteins c-fos/metabolism , Purines/chemistry , Quinazolinones/chemistry , Structure-Activity RelationshipABSTRACT
A new inhibitor, acredinone C (1), of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation was isolated from the culture broth of the fungus Acremonium sp. (F9A015) along with acredinones A (2) and B (3). The structure of acredinone C (1), which incorporates benzophenone and xanthone moieties, was established by the analyses of combined spectroscopic data including 1D and 2D NMR and MS. All of the acredinones studied efficiently inhibited the RANKL-induced formation of TRAP(+)-MNCs in a dose-dependent manner without any cytotoxicity up to 10 µM. Acredinone A showed dual activity in both osteoclast and osteoblast differentiation in vitro and good efficacy in an animal disease model of bone formation.
Subject(s)
Acremonium/chemistry , Benzophenones/pharmacology , Animals , Benzophenones/chemistry , Cell Differentiation , Disease Models, Animal , Mice , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Osteoclasts/drug effects , Osteogenesis/drug effects , RANK Ligand/antagonists & inhibitorsABSTRACT
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.
Subject(s)
Arginase/genetics , Cell Differentiation/genetics , Hematopoiesis/genetics , Macrophages/cytology , Osteoclasts/cytology , Animals , Arginase/metabolism , Arginine/metabolism , Bone Marrow Cells/cytology , Bone Resorption/genetics , Cells, Cultured , Down-Regulation , Male , Mice , Mice, Inbred ICR , Nitric Oxide/biosynthesis , Ornithine/biosynthesis , RANK Ligand/metabolism , RNA Interference , RNA, Small Interfering/genetics , Signal TransductionABSTRACT
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 of Alisma 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.
ABSTRACT
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.
Subject(s)
Calcium Signaling , MAP Kinase Signaling System , Osteoclasts/metabolism , Potassium Channels, Tandem Pore Domain/metabolism , Animals , Cell Differentiation , Cells, Cultured , Down-Regulation , Gene Knockdown Techniques , Mice , NFATC Transcription Factors/metabolism , Osteoclasts/cytology , Potassium Channels, Tandem Pore Domain/genetics , RANK Ligand/metabolismABSTRACT
Bone sustains its structure through dynamic interaction between osteoblastic cells and osteoclastic cells. But imbalance may lead to osteoporosis caused by overactivated osteoclast cells that have bone-resorbing function. Recently, herbs have been researched as major sources of medicines in many countries. In vitro and in vivo anti-osteoclastogenic activity of Angelica gigas NAKAI have been reported, but the biological activity of decursin, its major component in osteoclast differentiation is still unknown. Therefore, in this study, we explored whether decursin could affect RANKL-mediated osteoclastogenesis. The results showed that decursin efficiently inhibited RANKL-activated osteoclast differentiation by inhibiting transcriptional and translational expression of NFATc1, a major factor in RANKL-mediated osteoclastogenesis. Furthermore, decursin decreased fusion and migration of pre-osteoclasts by downregulating mRNA expression levels of DC-STAMP and ß3 integrin, respectively. In addition, decursin prevents lipopolysaccharide (LPS)-induced bone erosion in vivo. In summary, decursin could prevent osteoclastogenesis and inflammatory bone loss via blockage of NFATc1 activity and fusion and migration of pre-osteoclasts, and it could be developed as a potent phytochemical candidate for treating pathologies of bone diseases.
Subject(s)
Benzopyrans/metabolism , Butyrates/metabolism , Down-Regulation/physiology , NFATC Transcription Factors/antagonists & inhibitors , NFATC Transcription Factors/metabolism , Osteoclasts/metabolism , Osteogenesis/physiology , Animals , Benzopyrans/pharmacology , Butyrates/pharmacology , Cell Fusion , Cell Movement/drug effects , Cell Movement/physiology , Cells, Cultured , Dose-Response Relationship, Drug , Down-Regulation/drug effects , Mice , Mice, Inbred ICR , Osteogenesis/drug effectsABSTRACT
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.
Subject(s)
Calmodulin/metabolism , Coumarins/pharmacology , Osteoclasts/drug effects , Coumarins/chemistry , Humans , In Vitro Techniques , Lipopolysaccharides/pharmacology , Molecular Structure , Osteoclasts/metabolism , Phosphorylation , RANK Ligand/drug effects , Signal Transduction/drug effectsABSTRACT
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.
Subject(s)
Bone Marrow Cells/enzymology , Bone Resorption/enzymology , Cell Differentiation , Osteoclasts/enzymology , p21-Activated Kinases/metabolism , Animals , Bone Marrow Cells/pathology , Bone Resorption/chemically induced , Bone Resorption/genetics , Bone Resorption/pathology , Disease Models, Animal , Humans , Lipopolysaccharides/toxicity , Male , Mice , Mice, Inbred ICR , Osteoclasts/pathology , Pyrazoles/pharmacology , Pyrroles/pharmacology , p21-Activated Kinases/antagonists & inhibitors , p21-Activated Kinases/geneticsABSTRACT
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.
Subject(s)
Calcium/metabolism , Focal Adhesion Kinase 2/metabolism , NFATC Transcription Factors/metabolism , Osteoclasts/metabolism , Polyamines/metabolism , Adenosine Triphosphate/metabolism , Animals , Bone Resorption , Cell Differentiation , Cell Movement , Dose-Response Relationship, Drug , Male , Mice , Mice, Inbred ICR , NF-kappa B/metabolism , Osteoclasts/cytology , Phenotype , RANK Ligand/metabolism , Signal Transduction , Spermine/metabolismABSTRACT
A new inhibitor, placotylene A (1), of the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation, and a regioisomer of placotylene A, placotylene B (2), were isolated from a Korean marine sponge Placospongia sp. The chemical structures of placotylenes A and B were elucidated on the basis of 1D and 2D NMR, along with MS spectral analysis and revealed as an iodinated polyacetylene class of natural products. Placotylene A (1) displayed inhibitory activity against RANKL-induced osteoclast differentiation at 10 µM while placotylene B (2) did not show any significant activity up to 100 µM, respectively.
Subject(s)
Diynes/pharmacology , Fatty Alcohols/pharmacology , Osteoclasts/drug effects , Porifera/chemistry , Animals , Cell Differentiation/drug effects , Diynes/chemistry , Diynes/isolation & purification , Dose-Response Relationship, Drug , Fatty Alcohols/chemistry , Fatty Alcohols/isolation & purification , Magnetic Resonance Spectroscopy , Mass Spectrometry , Mice , Mice, Inbred ICR , Osteoclasts/metabolism , RANK Ligand/metabolism , Republic of Korea , StereoisomerismABSTRACT
BACKGROUND: A decrease of bone mass is a major risk factor for fracture. Several natural products have traditionally been used as herbal medicines to prevent and/or treat bone disorders including osteoporosis. Praeruptorin A is isolated from the dry root extract of Peucedanum praeruptorum Dunn and has several biological activities, but its anti-osteoporotic activity has not been studied yet. MATERIALS AND METHODS: The effect of praeruptorin A on the differentiation of bone marrow-derived macrophages into osteoclasts was examined by phenotype assay and confirmed by real-time PCR and immunoblotting. The involvement of NFATc1 in the anti-osteoclastogenic action of praeruptorin A was evaluated by its lentiviral ectopic expression. Intracellular Ca(2+) levels were also measured. RESULTS: Praeruptorin A inhibited the RANKL-stimulated osteoclast differentiation accompanied by inhibition of p38 and Akt signaling, which could be the reason for praeruptorin A-downregulated expression levels of c-Fos and NFATc1, transcription factors that regulate osteoclast-specific genes, as well as osteoclast fusion-related molecules. The anti-osteoclastogenic effect of praeruptorin A was rescued by overexpression of NFATc1. Praeruptorin A strongly prevented the RANKL-induced Ca(2+) oscillation without any changes in the phosphorylation of PLCγ. CONCLUSION: Praeruptorin A could exhibit its anti-osteoclastogenic activity by inhibiting p38/Akt-c-Fos-NFATc1 signaling and PLCγ-independent Ca(2+) oscillation.
Subject(s)
Cell Differentiation/drug effects , Coumarins/pharmacology , MAP Kinase Signaling System/drug effects , Macrophages/cytology , Osteoclasts/cytology , Osteoporosis/prevention & control , Acid Phosphatase , Calcium/metabolism , Coumarins/chemistry , DNA Primers/genetics , Humans , Immunoblotting , Isoenzymes , Macrophages/drug effects , Molecular Structure , NFATC Transcription Factors/metabolism , Oncogene Protein v-akt/metabolism , Osteoclasts/drug effects , Phospholipase C gamma/metabolism , Proto-Oncogene Proteins c-fos/metabolism , Real-Time Polymerase Chain Reaction , Tartrate-Resistant Acid PhosphataseABSTRACT
BACKGROUND: Matairesinol is a plant lignan present in a wide variety of foodstuffs such as seeds, vegetables and fruits. It has various biological functions including anti-angiogenic, anti-cancer and anti-fungal activities, but its anti-osteoporotic activity, if any, is unknown. METHODS: For osteoclast differentiation, primary mouse bone marrow-derived macrophage cells (BMMs) were cultured for 4 days in the presence of RANKL and M-CSF with the vehicle (DMSO) or matairesinol. Cell cytotoxicity was examined by CCK-8 assay. Gene expression of NFATc1, TRAP, OSCAR, v-ATPasev0d2 were observed in the presence or absence of matairesinol (10 µM) for the indicated times. For evaluating the involvement of NFATc1 in the anti-osteoclastogenic action of matairesinol, BMMs were infected with pMX-IRES-GFP or pMX-IRES-CA-NFATc1-GFP for 8 h with polybrene, and then infected BMMs were cultured with M-CSF and RANKL for 4 days in the presence or absence of matairesinol (10 µM). MAPK signaling activation was examined by immunoblotting. For measuring the resorptive activity of mature osteoclasts, osteoclasts and osteoblasts were co-cultured on BioCoat Osteologic MultiTest slides, and treated with matairesinol for 24 h. RESULT: Here we show that matairesinol dose-dependently inhibited the RANKL-induced differentiation of BMMs into osteoclasts by downregulating RANKL-induced expression and activity of NFATc1. Ectopic overexpression of NFATc1 blunted the anti-osteoclastogenic effect of matairesinol implicating NFATc1 in the action of matairesinol. Additionally, matairesinol blocked the RANKL-induced activation of p38 and ERK in BMMs, but had no effect on bone resorption activity in mature osteoclasts. CONCLUSION: Taken together, our results suggest that the anti-osteoporotic activity of matairesinol could arise from its anti-osteoclastogenic potential via p38/ERK-NFATc1 signaling, but not by way of anti-resorptive action.
Subject(s)
Bone Resorption/prevention & control , Furans/pharmacology , Lignans/pharmacology , Macrophages/drug effects , Osteoclasts/drug effects , Osteoporosis/metabolism , Plant Extracts/pharmacology , Animals , Bone Resorption/metabolism , Cell Differentiation/drug effects , Down-Regulation/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Furans/therapeutic use , Lignans/therapeutic use , Macrophage Colony-Stimulating Factor/genetics , Macrophages/metabolism , Male , Mice, Inbred ICR , NFATC Transcription Factors/metabolism , Osteoblasts/drug effects , Osteoblasts/metabolism , Osteoclasts/metabolism , Osteoporosis/prevention & control , Phosphorylation , Phytotherapy , Plant Extracts/therapeutic use , Plants, Edible/chemistry , RANK Ligand/metabolism , Signal Transduction/drug effects , Transcription Factors/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
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 ß-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.
Subject(s)
Cell Movement/drug effects , Cyclic AMP Response Element-Binding Protein/genetics , Lung Neoplasms/drug therapy , NF-kappa B/genetics , Pyrazoles/pharmacology , Pyrroles/pharmacology , beta Catenin/genetics , p21-Activated Kinases/antagonists & inhibitors , Cell Line, Tumor , Cell Movement/genetics , Down-Regulation/drug effects , Down-Regulation/genetics , Extracellular Matrix/genetics , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/genetics , Humans , Lung Neoplasms/genetics , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 9/genetics , Neoplasm Invasiveness/genetics , Signal Transduction/drug effects , Signal Transduction/geneticsABSTRACT
Receptor activator of NF-κB ligand (RANKL) triggers the differentiation of bone marrow-derived monocyte/macrophage precursor cells (BMMs) of hematopoietic origin into osteoclasts through the activation of mitogen-activated protein (MAP) kinases and transcription factors. Recently, reactive oxygen species (ROS) and antioxidant enzymes were shown to be closely associated with RANKL-mediated osteoclast differentiation. Although glutaredoxin2 (Glrx2) plays a role in cellular redox homeostasis, its role in RANKL-mediated osteoclastogenesis is unclear. We found that Glrx2 isoform b (Glrx2b) expression is induced during RANKLmediated osteoclastogenesis. Over-expression of Glrx2b strongly enhanced RANKL- mediated osteoclastogenesis. In addition, Glrx2b-transduced BMMs enhanced the expression of key transcription factors c-Fos and NFATc1, but pre-treatment with SB203580, a p38-specific inhibitor, completely blocked this enhancement. Conversely, down-regulation of Glrx2b decreased RANKL- mediated osteoclastogenesis and the expression of c-Fos and NFATc1 proteins. Also, Glrx2b down-regulation attenuated the RANKL-induced activation of p38. Taken together, these results suggest that Glrx2b enhances RANKL-induced osteoclastogenesis via p38 activation.
Subject(s)
Glutaredoxins/metabolism , Osteoclasts/cytology , Osteoclasts/metabolism , RANK Ligand/metabolism , Signal Transduction , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Cells, Cultured , Isoenzymes/metabolism , Male , Mice , Mice, Inbred ICRABSTRACT
Glutaredoxin 5 (Grx5) is a monothiol member of the Grx family that comprises two dithiol and three monothiol members. Using a yeast two-hybrid system, we isolated a Grx5-binding protein, SPT10, which has been previously suggested to act as a global transcriptional regulator of specific histone genes. We find that among the five members of the Grx family and two members of the thioredoxin (Trx) family (Trx1 and Trx2), Grx5 alone interacts with SPT10 via an intermolecular disulfide linkage between Cys60 of Grx5 and Cys385 of SPT10. To evaluate the physiological function of the Grx5/SPT10 interaction, we investigated the phenotypes of three null mutant strains (Grx5Δ, SPT10Δ, and Grx5ΔSPT10Δ). Taken together, the results show that all of these phenotypes are probably a consequence of the disruption of the interaction between Grx5 and SPT10. From this study, we suggest an interaction between Grx5 and SPT10 via intermolecular disulfide linkage and propose a model for a role of Grx5 in the regulation of protein expression under the control of SPT10.
Subject(s)
Glutaredoxins/metabolism , Histone Acetyltransferases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Transcription Factors/metabolism , Base Sequence , DNA Primers , Disulfides/metabolism , Electrophoresis, Gel, Two-Dimensional , Microscopy, Confocal , Microscopy, Fluorescence , Plasmids , Polymerase Chain Reaction , Proteomics , Two-Hybrid System TechniquesABSTRACT
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-Ca(2+)-NFAT signaling. The involvement of PLCγ2-Ca(2+)-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-Ca(2+)-NFAT signaling.
Subject(s)
Calcium/metabolism , Cell Differentiation , Membrane Proteins/metabolism , NFATC Transcription Factors/metabolism , Osteoclasts/cytology , Phospholipase C gamma/metabolism , Signal Transduction , Cell Differentiation/drug effects , Down-Regulation/drug effects , Humans , Membrane Proteins/genetics , Osteoclasts/drug effects , Osteoclasts/metabolism , RANK Ligand/pharmacology , Signal Transduction/drug effects , Vesicular Transport ProteinsABSTRACT
Interleukin-3 (IL-3) is produced under various pathological conditions and is thought to be involved in the pathogenesis of inflammatory diseases; however, its function in bone homeostasis under normal conditions or nature of the downstream molecular targets remains unknown. Here we examined the effect of IL-3 on osteoclast differentiation from mouse and human bone marrow-derived macrophages (BMMs). Although IL-3 can induce osteoclast differentiation of multiple myeloma bone marrow cells, IL-3 greatly inhibited osteoclast differentiation of human BMMs isolated from healthy donors. These inhibitory effects of IL-3 were only observed at early time points (days 0 and 1). IL-3 inhibited the expression of c-Fos and NFATc1 in BMMs treated with RANKL. However, IL-3-mediated inhibition of osteoclast differentiation was not completely reversed by ectopic expression of c-Fos or NFATc1. Importantly, IL-3 induced inhibitor of DNA binding/differentiation (Id)1 in hBMMs, while Id2 were sustained during osteoclast differentiation of mBMMs treated with IL-3. Ectopic expression of NFATc1 in Id2-deficient BMMs completely reversed the inhibitory effect of IL-3 on osteoclast differentiation. Furthermore, inflammation-induced bone erosion was markedly inhibited by IL-3 administration. Taken together, our results suggest that IL-3 plays an inhibitory role in osteoclast differentiation by regulating c-Fos and Ids, and also exerts anti-bone erosion effects.
Subject(s)
Cell Differentiation/physiology , Inhibitor of Differentiation Protein 1/metabolism , Interleukin-3/pharmacology , Macrophages/physiology , Osteoclasts/physiology , Proto-Oncogene Proteins c-fos/metabolism , Adult , Aged , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/physiology , Bone and Bones/cytology , Bone and Bones/drug effects , Bone and Bones/pathology , Cells, Cultured , Female , Gene Expression/drug effects , Humans , Inhibitor of Differentiation Protein 1/genetics , Inhibitor of Differentiation Protein 2/genetics , Inhibitor of Differentiation Protein 2/metabolism , Macrophage Colony-Stimulating Factor/pharmacology , Macrophages/cytology , Male , Mice , Microphthalmia-Associated Transcription Factor/genetics , Microphthalmia-Associated Transcription Factor/metabolism , Middle Aged , NFATC Transcription Factors/genetics , NFATC Transcription Factors/metabolism , Osteoclasts/cytology , Proto-Oncogene Proteins c-fos/genetics , RANK Ligand/pharmacology , Random AllocationABSTRACT
Osteoclasts are responsible for bone erosion in diseases as diverse as osteoporosis, periodontitis, and rheumatoid arthritis. Natural plant-derived products have received recent attention as potential therapeutic and preventative drugs in human disease. The effect of rotenone in RANKL-induced osteoclast differentiation was examined in this study. Rotenone inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages (BMMs) in a dose-dependent manner without any evidence of cytotoxicity. The mRNA expression of c-Fos, NFATc1, TRAP, and OSCAR in RANKL-treated BMMs was inhibited by rotenone treatment. Rotenone strongly inhibited p38 and ERK phosphorylation and I-kappaB degradation in RANKL-stimulated BMMs, and did not inhibit JNK phosphorylation. Further, RANKL-induced c-Fos and NFATc1 protein expression was suppressed by rotenone. Rotenone additionally inhibited the bone resorptive activity of differentiated osteoclasts. A lipopolysaccharide (LPS)-induced bone erosion study was also performed to assess the effects of rotenone in vivo. Mice treated with rotenone demonstrated marked attenuation of bone erosion based on Micro CT and histologic analysis of femurs. These results collectively suggested that rotenone demonstrated inhibitory effects on osteoclast differentiation in vitro and suppressed inflammatory bone loss in vivo. Rotenone may therefore serve as a useful drug in the prevention of bone loss.
Subject(s)
Bone Resorption/prevention & control , Down-Regulation/drug effects , Growth Inhibitors/pharmacology , NFATC Transcription Factors/antagonists & inhibitors , Osteoclasts/drug effects , Proto-Oncogene Proteins c-fos/antagonists & inhibitors , RANK Ligand/antagonists & inhibitors , Rotenone/pharmacology , Animals , Animals, Newborn , Bone Morphogenetic Proteins/physiology , Bone Resorption/metabolism , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Line , Cells, Cultured , Down-Regulation/physiology , Insecticides/pharmacology , Male , Mice , Mice, Inbred ICR , NFATC Transcription Factors/biosynthesis , Osteoclasts/cytology , Proto-Oncogene Proteins c-fos/biosynthesis , RANK Ligand/genetics , RANK Ligand/physiologyABSTRACT
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-alpha 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.