RANKL-induced Osteoclast Differentiation In RAW264 Research Articles

Study on the Mechanism of Action of the Traditional Chinese Medical Prescription Gushukang in Treating Osteoporosis Based on Network Pharmacology and Experimental Verification.

Gushukang (GSK), a traditional Chinese medical prescription, has made a great and extensive contribution to the treatment of different forms of osteoporosis, but polypharmacology studies of its mechanism of action are lacking. This study investigates the pharmacological mechanism of osteoporosis using network pharmacology and molecular docking. Experimental verification was carried out to confirm the efficacy of GSK on RANKLinduced osteoclast differentiation in RAW264.7 cells to verify the network pharmacology studies. The effective chemical components and corresponding targets of osteoporosis with oral bioavailability of more than 30% and drug-like properties greater than 0.18 were searched in the TCMSP and TCM-ID databases. DrugBank, GeneCards, OMIM, TTD, and other databases were examined for targets related to osteoporosis. Using Cytoscape software, a network of possible TCM-active ingredient-osteoporosis targets was created. STRING software was used to create the networks of protein-protein interactions. The DAVID program was carried out to conduct GO and KEGG pathway enrichment analyses of the targets. Molecular docking and pattern of action analysis were carried out using software like AutoDock Vina and Discovery Studio Visualizer. The growth media for RAW264.7 cells contained varying doses of GSK serum and 50 ng/mL RANKL. The activity of TRAP was altered. Additionally, genes related to osteoclasts were examined using an RT-PCR assay. Network pharmacological analysis revealed that the primary efficacy targets of osteoporosis were PTGS2, PTGS1, HSP90AA1, NCOA2, ADRB2, ESR1, NCOA1, and AR. The pharmacological targets of osteoporosis may be mediated by substances including quercetin, kaempferol, luteolin, naringenin, icariin, anthocyanin, tanshinone IIA, and cryptotanshinone. GSK markedly inhibited RANKL-induced TRAP activity. qRT-PCR results revealed decreased expression of the PTGS2 and ADRB2 genes upon GSK treatment. The findings of network pharmacology, molecular docking, as well as experimental verification provide a new further study for elucidating the pharmacodynamic substance basis and polypharmacology mechanism of GSK in treating osteoporosis.

Ctdnep1 phosphatase is required for negative regulation of RANKL-induced osteoclast differentiation in RAW264.7 cells

Osteoclasts are multinucleated cells with bone resorption activity. Excessive osteoclast activity has been implicated in osteoporosis, rheumatoid arthritis, and bone destruction due to bone metastases from cancer, making osteoclasts essential target cells in bone and joint diseases. C-terminal domain nuclear envelope phosphatase 1 (Ctdnep1, formerly Dullard) is a negative regulator of transforming growth factor (TGF)-β superfamily signaling and regulates endochondral ossification in mesenchymal cells during skeletal development. In this study, we investigated the role of Ctdnep1 in the Receptor activator of nuclear factor-kappa B ligand (RANKL)-induced RAW264.7 osteoclast differentiation. Expression of Ctdnep1 did not change during osteoclast differentiation; Ctdnep1 protein localized to the cytoplasm before and after osteoclast differentiation. Small interfering RNA-mediated knockdown of Ctdnep1 increased tartrate-resistant acid phosphatase-positive multinucleated osteoclasts and the expression of osteoclast marker genes, including Acp5, Ctsk, and Nfatc1. Interestingly, the knockdown of Ctdnep1 increased the protein level of Nfatc1 in cells unstimulated with RANKL. Knockdown of Ctdnep1 also enhanced calcium-resorbing activity. Mechanistically, the knockdown of Ctdnep1 increased the phosphorylation of RANKL signaling components. These results suggest that Ctdnep1 negatively regulates osteoclast differentiation by suppressing the RANKL signaling pathway.

Regulation of osteoclast differentiation and inflammatory signaling by TCF8 in periodontitis.

The aim of this study was to explore the potential role of zinc-finger homeodomain transcription factor (TCF8) in osteoclastogenesis and inflammation during periodontitis. Rats with periodontitis were induced via Porphyromonas gingivalis-lipopolysaccharide (Pg-LPS) injection. The recombinant lentivirus delivering short hairpin RNA (shRNA) against TCF8 was used to downregulate TCF8 in vivo. Alveolar bone loss in rats was determined by micro-computed tomography (Micro-CT). Typical pathological changes, periodontal tissue inflammation, and osteoclastogenesis were evaluated via histological analyses. The RAW264.7-derived osteoclasts were induced by RANKL stimulation. TCF8 downregulation in vitro was achieved by lentivirus infection. The osteoclast differentiation and inflammatory signaling in RANKL-induced cells were measured via immunofluorescence methods and molecular biology approaches. Porphyromonas gingivalis-lipopolysaccharide induced rats exhibited overexpressed TCF8 in their periodontal tissues, while TCF8 knockdown attenuated the bone loss, tissue inflammation, and osteoclastogenesis in LPS-induced rats. Besides, TCF8 silencing inhibited RANKL-induced osteoclast differentiation in RAW264.7 cells, as evidenced by the reduced numbers of TRAP-positive osteoclasts, less formation of F-actin rings, and downregulated expressions of osteoclast-specific markers. It also exerted an inhibitory effect on the NF-κB signaling in RANKL-induced cells via blocking NF-κB p65 phosphorylation and nuclear translocation. TCF8 silencing inhibited alveolar bone loss, osteoclast differentiation, and inflammation in periodontitis.

Effects of urolithin A on osteoclast differentiation induced by receptor activator of nuclear factor-κB ligand via bone morphogenic protein 2

ABSTRACT Urolithin A (UA) is an intestinal microbial metabolite derived from ellagitannins and a promising agent for treating osteoarthritis. However, its effects on osteoporosis are unclear. This study explored the effects of urolithin A (UA) on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclasts and its underlying molecular mechanisms. RANKL treatment significantly increased tartrate-resistant acid phosphatase (TRACP) or osteoclast marker levels (P < 0.05), while adding UA decreased the RANKL-induced levels (P < 0.05) in RAW264.7 cells. Total RNA isolated from RANKL- or RANKL + UA-treated cells was sequenced, and the obtained transcriptome dataset revealed 2,399 differentially expressed genes. They were enriched in multiple pathways involved in rheumatoid arthritis, ERK1 and ERK2 cascade, regulation of inflammatory response, ECM-receptor interactions, and TNF signaling. Scanning electron microscopy showed that RANKL promoted bone resorption pits in bone biopsy specimens, whereas UA inhibited their formation. When bone morphogenic protein 2 (BMP2) was shRNA-silenced, the bone resorption pits were restored. Moreover, while RANKL significantly enhanced the levels of p-ERK2/ERK2, p-p38/p38, p-Akt1/Akt1, p-ERK1/ERK1, and osteoclast-related proteins (P < 0.05), UA reduced them. BMP2 silencing also reversed the UA inhibitory effect. Thus, UA represses the RANKL-induced osteoclast differentiation of RAW264.7 cells by regulating Akt1, p38, and ERK1/2 signaling, and BMP2 likely reverses the UA inhibitory effect via these pathways. We propose BMP2 as a potential drug target for treating bone metabolic diseases, such as osteoporosis.

Epstein-Barr Virus Promotes the Production of Inflammatory Cytokines in Gingival Fibroblasts and RANKL-Induced Osteoclast Differentiation in RAW264.7 Cells.

Periodontitis is an inflammatory condition that causes the destruction of the supporting tissues of teeth and is a major public health problem affecting more than half of the adult population worldwide. Recently, members of the herpes virus family, such as the Epstein–Barr virus (EBV), have been suggested to be involved in the etiology of periodontitis because bacterial activity alone does not adequately explain the clinical characteristics of periodontitis. However, the role of EBV in the etiology of periodontitis is unknown. This study aimed to examine the effect of inactivated EBV on the expression of inflammatory cytokines in human gingival fibroblasts (HGFs) and the induction of osteoclast differentiation. We found that extremely high levels of interleukin (IL)-6 and IL-8 were induced by inactivated EBV in a copy-dependent manner in HGFs. The levels of IL-6 and IL-8 in HGFs were higher when the cells were treated with EBV than when treated with lipopolysaccharide and lipoteichoic acid. EBV induced IκBα degradation, NF-κB transcription, and RAW264.7 cell differentiation into osteoclast-like cells. These findings suggest that even without infecting the cells, EBV contributes to inflammatory cytokine production and osteoclast differentiation by contact with oral cells or macrophage lineage, resulting in periodontitis onset and progression.

Mitigating RANKL-induced cholesterol overload in macrophages with β-cyclodextrin-threaded polyrotaxanes suppresses osteoclastogenesis.

Free cholesterol acts as an endogenous agonist for estrogen-related receptor α (ERRα), a nuclear receptor that regulates osteoclastogenesis. Because stimulation of macrophages with receptor activator of nuclear factor κB ligand (RANKL) induces an overload of free cholesterol and activates ERRα, we hypothesized that direct removal of cellular cholesterol would suppress osteoclastogenesis. In this study, the effect of 2-hydroxypropyl β-cyclodextrin (HP-β-CD), a highly water-soluble cyclic glucopyranose, and β-CD-threaded polyrotaxanes (PRXs), supramolecular polymers designed to release threaded β-CDs in acidic lysosomes, on RANKL-induced cholesterol overload and osteoclast differentiation of murine macrophage-like RAW264.7 cells were investigated. PRXs suppressed RANKL-induced cholesterol overload. Additionally, RANKL-induced osteoclast differentiation of RAW264.7 cells was inhibited by PRXs. In contrast, HP-β-CD did not reduce cholesterol levels or inhibit osteoclast differentiation in RAW264.7 cells. Gene expression analysis of osteoclast markers suggested that PRXs suppress only the early stage of osteoclast differentiation, as PRXs cannot be internalized into multinucleated osteoclasts. However, modification of PRXs with cell-penetrating peptides facilitated their cellular uptake into multinucleated osteoclasts and inhibited osteoclast maturation. Thus, PRXs are promising candidates for inhibiting osteoclast differentiation by suppressing cholesterol overload and may be useful for treating osteoporosis or other bone defects caused by the overactivity of osteoclasts.

2E-Decene-4,6-diyn-1-ol-acetate inhibits osteoclastogenesis through mitogen-activated protein kinase-c-Fos-NFATc1 signalling pathways.

An imbalance of osteoclasts and osteoblasts can result in a variety of bone-related diseases, including osteoporosis. Thus, decreasing the activity of osteoclastic bone resorption is the main therapeutic method for treating osteoporosis. 2E-Decene-4, 6-diyn-1-ol-acetate (DDA) is a natural bioactive compound with anti-inflammatory and anti-cancer properties. However, its effects on osteoclastogenesis are unknown. Murine bone marrow-derived macrophages (BMMs) or RAW264.7 cells were treated with DDA, followed by evaluation of cell viability, RANKL-induced osteoclast differentiation, and pit formation assay. Effects of DDA on RANKL-induced phosphorylation of MAPKs were assayed by western blot analysis. Expression of osteoclast-specific genes was examined with reverse transcription-PCR (RT-PCR) and western blot analysis. In this study, DDA significantly inhibited RANKL-induced osteoclast differentiation in RAW264.7 cells as well as in BMMs without cytotoxicity. DDA also strongly blocked the resorbing capacity of BMM on calcium phosphate-coated plates. DDA inhibited RANKL-induced phosphorylation of ERK, JNK and p38MAPKs, as well as expression of c-Fos and NFATc1, which are essential transcription factors for osteoclastogenesis. In addition, DDA decreased expression levels of osteoclastogenesis-specific genes, including matrix metalloproteinase-9 (MMP-9), tartrate-resistant acid phosphatase (TRAP), and receptor activator of NF-κB (RANK) in RANKL-induced RAW264.7 cells. Collectively, these findings indicated that DDA attenuates RANKL-induced osteoclast formation by suppressing the MAPKs-c-Fos-NFATc1signalling pathway and osteoclast-specific genes. These results indicate that DDA may be a potential candidate for bone diseases associated with abnormal osteoclast formation and function.

Connective tissue growth factor (CCN2) is a matricellular preproprotein controlled by proteolytic activation

Connective tissue growth factor (CTGF; now often referred to as CCN2) is a secreted protein predominantly expressed during development, in various pathological conditions that involve enhanced fibrogenesis and tissue fibrosis, and in several cancers and is currently an emerging target in several early-phase clinical trials. Tissues containing high CCN2 activities often display smaller degradation products of full-length CCN2 (FL-CCN2). Interpretation of these observations is complicated by the fact that a uniform protein structure that defines biologically active CCN2 has not yet been resolved. Here, using DG44 CHO cells engineered to produce and secrete FL-CCN2 and cell signaling and cell physiological activity assays, we demonstrate that FL-CCN2 is itself an inactive precursor and that a proteolytic fragment comprising domains III (thrombospondin type 1 repeat) and IV (cystine knot) appears to convey all biologically relevant activities of CCN2. In congruence with these findings, purified FL-CCN2 could be cleaved and activated following incubation with matrix metalloproteinase activities. Furthermore, the C-terminal fragment of CCN2 (domains III and IV) also formed homodimers that were ∼20-fold more potent than the monomeric form in activating intracellular phosphokinase cascades. The homodimer elicited activation of fibroblast migration, stimulated assembly of focal adhesion complexes, enhanced RANKL-induced osteoclast differentiation of RAW264.7 cells, and promoted mammosphere formation of MCF-7 mammary cancer cells. In conclusion, CCN2 is synthesized and secreted as a preproprotein that is autoinhibited by its two N-terminal domains and requires proteolytic processing and homodimerization to become fully biologically active.

Cynaropicrin from Cynara scolymus L. suppresses Porphyromonas gingivalis LPS-induced production of inflammatory cytokines in human gingival fibroblasts and RANKL-induced osteoclast differentiation in RAW264.7 cells.

Periodontal diseases are a major public health problem affecting over half of the adult population worldwide. Lipopolysaccharide (LPS) produced by the periodontopathic bacterium Porphyromonas gingivalis induces the expression of inflammatory cytokines that promote inflammatory bone destruction. Mounting evidence supports that periodontal diseases are involved in the onset and progression of several systemic diseases, such as aspiration pneumonia and diabetes. Although treatment of periodontal diseases by removing the periodontopathic bacteria by brushing is a standard practice, it has limitations and is not effective in all cases. Therefore, a new method to replace or complement brushing is needed for the treatment of periodontal diseases. In this study, we investigated the anti-inflammatory effects of an extract from Cynara scolymus L. and its pharmacologically effective compound cynaropicrin, a sesquiterpene lactone, on human gingival fibroblasts (HGFs) stimulated by LPS and the potential anti-osteoclastogenic effects on RAW264.7 cells induced by receptor activator of NF-κB ligand (RANKL). We found that cynaropicrin inhibited IL-8 and IL-6 mRNA and protein synthesis in LPS-stimulated HGFs in a dose-dependent manner. P. gingivalis LPS-induced degradation of IκBα and phosphorylation of NF-κB p65 were also suppressed by cynaropicrin, as was LPS-stimulated NF-κB transactivation. Thus, cynaropicrin's inhibition of P. gingivalis LPS-induced IL-8 and IL-6 expression may be due to the inhibition of the NF-κB pathway. Furthermore, we showed that cynaropicrin dramatically reduced RANKL-induced osteoclast differentiation. These results suggest that cynaropicrin may be useful for preventing periodontal diseases and could prove valuable in the development of more effective preventative approaches for periodontal diseases.

Anti-inflammatory and Antiosteoporosis Flavonoids from the Rhizomes of Helminthostachys zeylanica.

Chemical investigation of the rhizomes of Helminthostachys zeylanica led to the isolation of eight new flavonoids including six cyclized geranylflavonoids, ugonins V-X (1-3), (10R,11S)-ugonin N (4), (10R,11S)-ugonin S (5), and ugonin Y (6), as well as two quercetin glucosides, quercetin-4'-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (7) and quercetin-3-O-β-d-glucopyranosyl-4'-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (8). The structures of these compounds were established by spectroscopic analyses and acid hydrolysis of the sugar moiety. Among the isolated compounds, 1, 2, 5, 6, ugonins J-S (9-13), ugonstilbene A (14), and ugonin L (23) were evaluated for their anti-inflammatory activity on lipopolysaccharide-induced nitric oxide (NO) production in microglial cells. Except for 1, 5, and 13, all other compounds inhibited NO production with IC50 values of 6.2-10.1 μM and were more potent than the positive control, pyrrolidine dithiocarbamate. Compounds 1, 2, 5, 6, and 10-13 were tested for antiosteoporotic activities, and ugonin K (10) exhibited the highest inhibitory activity against RANKL-induced osteoclast differentiation in RAW264.7 cells with an IC50 value of 1.8 ± 0.2 μM.

Ellagic acid inhibits RANKL-induced osteoclast differentiation by suppressing the p38 MAP kinase pathway.

Bone undergoes continuous remodeling by a coupled action between osteoblasts and osteoclasts. During osteoporosis, osteoclast activity is often elevated leading to increased bone destruction. Hence, osteoclasts are deemed as potential therapeutic targets to alleviate bone loss. Ellagic acid (EA) is a polyphenol reported to possess anticancer, antioxidant and anti-inflammatory properties. However, its effects on osteoclast formation and function have not yet been examined. Here, we explored the effects of EA on RANKL-induced osteoclast differentiation in RAW264.7 murine macrophages (in vitro) and human CD14+monocytes (ex vivo). EA dose-dependently attenuated RANKL-induced TRAP+ osteoclast formation in osteoclast progenitors with maximal inhibition seen at 1µM concentration without cytotoxicity. Moreover, owing to perturbed osteoclastogenesis, EA disrupted actin ring formation and bone resorptive function of osteoclasts. Analysis of the underlying molecular mechanisms revealed that EA suppressed the phosphorylation and activation of the p38 MAP kinase pathway which subsequently impaired the RANKL-induceddifferentiation of osteoclast progenitors. Taken together, these novel results indicate that EA alleviates osteoclastogenesis by suppressing the p38 signaling pathway downstream of RANKL and exerts inhibitory effects on bone resorption and actin ring formation.

Aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing NF-κB and NFATc1 activation and DC-STAMP expression.

Aconiti Lateralis Radix Preparata is a traditional Chinese medicine used to treat chronic arthritis and is highly effective against rheumatoid arthritis. However, the effects of aconine, a derivative of aconitum alkaloids, on osteoclasts, which can absorb bone, remain unknown. Here, we investigated the effects of aconine on osteoclast differentiation and bone resorption in vitro. The viability of mouse leukemic monocyte/macrophage cell line RAW264.7 was measured using CCK-8 assays. Osteoclast differentiation was induced by incubation of RAW264.7 cells in the presence of RANKL, and assessed with TRAP staining assay. Bone resorption was examined with bone resorption pits assay. The expression of relevant genes and proteins was analyzed using RT-PCR and Western blots. The activation of NF-κB and nuclear factor of activated T-cells (NFAT) was examined using stable NF-κB and NFATc1 luciferase reporter gene systems, RT-PCR and Western blot analysis. Aconine (0.125, 0.25 μmol/L) did not affect the viability of RAW264.7 cells, but dose-dependently inhibited RANKL-induced osteoclast formation and bone resorptive activity. Furthermore, aconine dose-dependently inhibited the RANKL-induced activation of NF-κB and NFATc1 in RAW264.7 cells, and subsequently reduced the expression of osteoclast-specific genes (c-Src, β3-Integrin, cathepsin K and MMP-9) and the expression of dendritic cell-specific transmembrane protein (DC-STAMP), which played an important role in cell-cell fusion. These findings suggest that aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing the activation of NF-κB and NFATc1 and the expression of the cell-cell fusion molecule DC-STAMP.

Treatment with hydrogen molecules prevents RANKL-induced osteoclast differentiation associated with inhibition of ROS formation and inactivation of MAPK, AKT and NF-kappa B pathways in murine RAW264.7 cells

The bone protective effects of the hydrogen molecule (H2) have been demonstrated in several osteoporosis models while the underlying molecular mechanism has remained unclear. Osteoclast differentiation is an important factor related to the pathogenesis of bone-loss related diseases. In this work, we evaluated the effects of incubation with H2 on receptor activator of NFκB ligand (RANKL)-induced osteoclast differentiation. We found that treatment with H2 prevented RANKL-induced osteoclast differentiation in RAW264.7 cells and BMMs. Treatment with H2 inhibits the ability to form resorption pits of BMMs stimulated by RANKL. Treatment with H2 reduced mRNA levels of osteoclast-specific markers including tartrate resistant acid phosphatase, calcitonin receptor, cathepsin K, metalloproteinase-9, carbonic anhydrase typeII, and vacuolar-type H(+)-ATPase. Treatment with H2 decreased intracellular reactive oxygen species (ROS) formation, suppressed NADPH oxidase activity, down-regulated Rac1 activity and Nox1 expression, reduced mitochondrial ROS formation, and enhanced nuclear factor E2-related factor 2 nuclear translocation and heme oxygenase-1 activity. In addition, treatment with H2 suppressed RANKL-induced expression of nuclear factor of activated T cells c1 and c-Fos. Furthermore, treatment with H2 suppressed NF-κB activation and reduced phosphorylation of p38, extracellular signal-regulated kinase, c-Jun-N-terminal kinase, and protein kinases B (AKT) stimulated with RANKL. In conclusion, hydrogen molecules prevented RANKL-induced osteoclast differentiation associated with inhibition of reactive oxygen species formation and inactivation of NF-κB, mitogen-activated protein kinase and AKT pathways.

Low-Magnitude High-Frequency Vibration Inhibits RANKL-Induced Osteoclast Differentiation of RAW264.7 Cells

Osteoclasts are the key participants in regulation of bone mass. Low-magnitude high-frequency vibration (LMHFV) has been found to be anabolic to bone in vivo. This study aimed to investigate the effect of LMHFV on osteoclast differentiation in vitro. Murine monocyte cell line RAW264.7 cells in the presence of receptor activator of nuclear factor-kappaB ligand (RANKL) were treated with or without LMHFV at 45 Hz (0.3 g) for 15 min day−1. Tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) and actin ring formation were evaluated. Expression of the osteoclast-specific genes, such as cathepsin K, matrix metallopeptidase-9 (MMP-9) and TRAP, were analyzed using real time-PCR. c-Fos, an osteoclast-specific transcription factor, was determined using Western blot. We found that LMHFV significantly decreased the number of RANKL-induced TRAP-positive MNCs (P<0.01), and inhibited the actin ring formation. The mRNA expression of the cathepsin K, MMP-9 and TRAP were down-regulated by LMHFV intervention (all P<0.001). Furthermore, LMHFV also inhibited the expression of c-Fos protein in the RANKL-treated RAW264.7 cells (P<0.05). Our results suggest that LMHFV can inhibit the RANKL-induced osteoclast differentiation of RAW264.7 cells, which give some new insight into the anabolic effects of LMHFV on bone.

Effects of Hachimi-jio-gan Extract on Intestinal Absorption of Calcium in Ovariectomized Mice and Stimulation of RANKL-induced Osteoclast Differentiation of Raw264.7 Cells by Lipopolysaccharide

Osteoporosis is a common metabolic bone disease often affecting postmenopausal women, and various medicines are available that attempt to treat and/or prevent this disease. Hachimi-jio-gan is a traditional oriental (Kampo) medicine used clinically, but it's mode of action is still unknown. In this study, the effects of Hachimi-jio-gan extract on intestinal calcium absorption were evaluated in an osteoporosis model using ovariectomized (OVX) and sham-operated (SHAM) mice by investigating pharmacokinetic parameters in these animals. The ability of Hachimi-jio-gan extract to inhibit lipopolysaccharide-mediated stimulation of osteoclasts in bones using receptor activator of the NF-kB ligand (RANKL)-induced osteoclast differentiation of RAW264.7 cells was also investigated. The metabolic behavior of calcium did not differ between OVX and SHAM mice as the pharmacokinetics of calcium was equivalent after intravenous administration. The absolute bioavailability of calcium indicated that the extent of intestinal calcium absorption in the OVX (10.3%) and SHAM (10.7%) mice was at similar low levels. Hachimi-jio-gan extract potentially improved the intestinal calcium absorption by 1.96- and 1.86-fold, respectively. Hachimi-jio-gan extract further suppressed the potent stimulation of RANKL-induced osteoclast differentiation in RAW264.7 cells. These results suggest that Hachimi-jio-gan extract may be suitable for treatment and prevention of osteoporosis through its ability to increase intestinal calcium absorption and suppress osteoclast differentiation.

In vitro에서 polycalcium 복합조성물이 파골세포와 조골세포에 미치는 영향

The present study evaluated the beneficial effects of polycalcium (a mixture of Polycan and calcium lactate-gluconate 1:9 [g/g]) on osteoporosis using in vitro assays. Cell proliferation and alkaline phosphatase activities of osteoblasts (human primary osteoblasts) and osteoclast differentiation of RAW264.7 cells (murine osteoclast progenitor cells) treated with different concentrations of polycalcium for various periods were assessed. Osteoblast proliferation was stimulated and prevented RANKL-induced osteoclast differentiation of RAW264.7 cells. These results support the development of ideal anti-osteoporotic agents, such as polycalcium, that exhibit properties that accelerate bone formation and inhibit bone resorption.

Effect of amorphous silica nanoparticles on in vitro RANKL-induced osteoclast differentiation in murine macrophages

Amorphous silica nanoparticles (nSP) have been used as a polishing agent and/or as a remineralization promoter for teeth in the oral care field. The present study investigates the effects of nSP on osteoclast differentiation and the relationship between particle size and these effects. Our results revealed that nSP exerted higher cytotoxicity in macrophage cells compared with submicron-sized silica particles. However, tartrate-resistant acid phosphatase (TRAP) activity and the number of osteoclast cells (TRAP-positive multinucleated cells) were not changed by nSP treatment in the presence of receptor activator of nuclear factor κB ligand (RANKL) at doses that did not induce cytotoxicity by silica particles. These results indicated that nSP did not cause differentiation of osteoclasts. Collectively, the results suggested that nanosilica exerts no effect on RANKL-induced osteoclast differentiation of RAW264.7 cells, although a detailed mechanistic examination of the nSP70-mediated cytotoxic effect is needed.

Samul-tang suppresses RANKL-induced osteoclast differentiation in RAW264.7 cells

Samul-tang (SMT) is a traditional herbal medicine used for the treatment of ischemic brain and heart diseases. In this study, we investigated the molecular mechanism underlying the inhibitory effects of SMT on osteoclast differentiation induced by the receptor activator for nuclear factor κB ligand (RANKL) in RAW264.7 cells. SMT dose-dependently and significantly inhibited the RANKL-induced tartrate-resistant acid phosphatase (TRAP) activity and the formation of multi-nucleated osteoclasts. In addition, SMT significantly reduced the RANKL-induced mRNA expression of osteoclast differentiation-related genes that encode TRAP, c-src, matrix metalloproteinase (MMP)-9, cathepsin K, and the d2 isoform of vacuolar ATPase V(0) domain (ATPv0d2). SMT also inhibited the RANKL-induced activation of MAP kinases and NF-κB p65, and mRNA expression of transcription factors (Fra-2 and NFATc1). In conclusion, these results suggested that SMT suppresses the RANKL-induced activation of signaling molecules and transcription factors, which consequently inhibits osteoclast differentiation.

Preliminary identification of potential PDZ-domain proteins downstream of ephrin B2 during osteoclast differentiation of RAW264.7 cells

The EphB4 receptor and ephrinB2 ligand were recently reported to influence the coupling between osteoclasts and osteoblasts in bone biology, but their downstream signaling pathways remain unclear. This study focuses on the preliminary identification of downstream PDZ-domain proteins involved in EphB4/ephrinB2 reverse signaling in osteoclasts. Similarly to primary osteoclast precursors isolated from the bone, we observed that the RAW264.7 cell line, a mouse monocyte/macrophage cell line that is used in conventional assays for osteoclast function, expressed ephrinB2 during RANKL-induced osteoclast differentiation, and that preclustered EphB4 inhibited this osteoclast differentiation. The results demonstrate that RAW264.7 cells provide a good model for further research of EphB4/ephrinB2 signaling in osteoclasts. Immunofluorescence staining and Western blot analysis revealed that all of the eight PDZ-domain proteins previously reported to interact with ephrinB ligands were expressed in the differentiated RAW264.7 osteoclasts. However, in a co-immunoprecipitation assay, only Dishevelled2 (Dvl2) among eight PDZ-domain proteins tested co-precipitated with ephrinB2 and vice versa, suggesting an endogenous interaction between Dvl2 and ephrinB2 in RANKL-induced osteoclasts. Furthermore, preclustered EphB4 reduced the expression level of Dvl2. Collectively, our results indicate that Dvl2 could be the potential PDZ-domain protein that acts downstream of ephrinB2 in RANKL-induced osteoclast differentiation of RAW264.7 cells, providing a potential novel therapeutic target for bone diseases.

Double stranded RNA-dependent protein kinase is involved in osteoclast differentiation of RAW264.7 cells in vitro

Double-stranded RNA-dependent protein kinase (PKR) plays a critical role in antiviral defence of the host cells. PKR is also involved in cell cycle progression, cell proliferation, cell differentiation, tumorigenesis, and apoptosis. We previously reported that PKR is required for differentiation and calcification of osteoblasts. However, it is unknown about the role of PKR in osteoclast differentiation. A dominant-negative PKR mutant cDNA, in which the amino acid lysine at 296 was replaced with arginine, was transfected into RAW264.7 cells. We have established the cell line that stably expresses the PKR mutant gene (PKR-K/R). Phosphorylation of PKR and α-subunit of eukaryotic initiation factor 2 was not stimulated by polyinosic–polycytidylic acid in the PKR-K/R cells. RANKL stimulated the formation of TRAP-positive multinuclear cells in RAW264.7 cells. However, TRAP-positive multinuclear cells were not formed in the PKR-K/R cells even when the cells were stimulated with higher doses of RANKL. A specific inhibitor of PKR, 2-aminopurine, also suppressed the RANKL-induced osteoclast differentiation in RAW264.7 cells. The expression of macrophage fusion receptor and dendritic cell-specific transmembrane protein significantly decreased in the PKR-K/R cells by real time PCR analysis. The results of RT-PCR revealed that the mRNA expression of osteoclast markers (cathepsin K and calcitonin receptor) was suppressed in the PKR-K/R cells and RAW264.7 cells treated with 2-aminopurine. Expression of NF-κB protein was suppressed in the PKR-K/R cells and 2-aminopurine-treated RAW264.7 cells. The level of STAT1 protein expression was elevated in the PKR-K/R cells compared with that of the wild-type cells. Immunohistochemical study showed that PKR was localized in osteoclasts of metatarsal bone of newborn mouse. The finding that the PKR-positive multinuclear cells should be osteoclasts was confirmed by TRAP-staining. Our present study indicates that PKR plays important roles in the differentiation of osteoclasts.