Osteoclast Differentiation In RAW264 Research Articles

The liver X receptor promotes macrophage differentiation and suppresses osteoclast formation in mouse RAW264.7 promyelocytic leukemia cells exposed to bacterial lipopolysaccharide

Lipopolysaccharide (LPS), the principal component of Gram-negative bacterial cell walls, is a stimulator of osteoclastogenesis and thus a key factor in inflammatory bone loss. We have recently reported that the important cholesterol and inflammatory regulator, liver X receptor (LXRα/β), can potently inhibit osteoclast formation from bone marrow-derived osteoclast precursors in a bacterial/LPS environment. In this manuscript, we further studied the effect of the LXR agonist GW3965 on osteoclast differentiation in RAW264.7 promyelocytic leukemia cells exposed to LPS. We found that not only did activation of the LXR potently inhibit the formation of TRAP-positive osteoclast-like cells, but promoted a population of TRAP-negative mononuclear cells with high phagocytic activity. We observed reduced expression of the osteoclast markers TRAP/Acp5, Ctsk, Calcr and Oscar after 3–4days of GW3965 treatment, coinciding with an increase in the expression of the anti-osteoclastogenic factor Irf8. Expression of the macrophage/phagocytic marker Cd68 was increased, however the “classical” macrophage markers F4/80 and Cd14 and the “alternatively” activated macrophage markers Tgfβ and Il10 were not altered. Further, activation of LXR increased the expression of the macrophage survival gene AIM/SPα, a known LXR target gene, and osteoclast/macrophage-related markers (Mitf, Pu.1, Usf1/2, Ostm1 and Mfr). Although Akt phosphorylation was reduced, GW3965 seemed to act independently of MAPKs (p38, ERK, JNK) and NFκB, and had no inhibitory effect on cytokine expression (Tnfα, Il6, or Il1β). Our results indicate that activation of the LXR not only inhibits the differentiation of osteoclast-like cells from RAW264.7 cells in a bacterial/LPS environment, but is also involved in the fate determination of myeloid progenitor cells into macrophages with high phagocytic capacity.

Silibinin inhibits prostate cancer cells‐ and RANKL‐induced osteoclastogenesis by targeting NFATc1, NF‐κB, and AP‐1 activation in RAW264.7 cells

Currently, there are limited therapeutic options against bone metastatic prostate cancer (PCA), which is primarily responsible for high mortality and morbidity in PCA patients. Enhanced osteoclastogenesis is an essential feature associated with metastatic PCA in the bone microenvironment. Silibinin, an effective chemopreventive agent, is in phase II clinical trials in PCA patients but its efficacy against PCA cells-induced osteoclastogenesis is largely unknown. Accordingly, here we examined silibinin effect on PCA cells-induced osteoclastogenesis employing human PCA (PC3MM2, PC3, and C4-2B) and murine macrophage RAW264.7 cells. We also assessed silibinin effect on receptor activator of nuclear factor κB ligand (RANKL)-induced signaling associated with osteoclast differentiation in RAW264.7 cells. Further, we analyzed silibinin effect on osteomimicry biomarkers in PCA cells. Results revealed that silibinin (30-90 μM) inhibits PCA cells-induced osteoclast activity and differentiation in RAW264.7 cells via modulating expression of several cytokines (IGF-1, TGF-β, TNF-α, I-TAC, M-CSF, G-CSF, GM-CSF, etc.) that are important in osteoclastogenesis. Additionally, in RAW264.7 cells, silibinin decreased the RANKL-induced expression and nuclear localization of NFATc1, which is considered the master regulator of osteoclastogenesis. Furthermore, silibinin decreased the RANKL-induced DNA binding activity of NFATc1 and its regulators NF-κB and AP1, and the protein expression of osteoclast specific markers (TRAP, OSCAR, and cathepsin K). Importantly, silibinin also decreased the expression of osteomimicry biomarkers (RANKL, Runx2, osteocalcin, and PTHrP) in cell culture (PC3 and C4-2B cells) and/or in PC3 tumors. Together, our findings showing that silibinin inhibits PCA cells-induced osteoclastogenesis, suggest that silibinin could be useful clinically against bone metastatic PCA.

Local delivery of alendronate eluting chitosan scaffold can effectively increase osteoblast functions and inhibit osteoclast differentiation

The aim of this study was to investigate the effect of alendronate released from chitosan scaffolds on enhancement of osteoblast functions and inhibition of osteoclast differentiation in vitro. The surface and cell morphologies of chitosan scaffolds and alendronate-loaded chitosan scaffolds were characterized by variable pressure field emission scanning electron microscope (VP-FE-SEM). Alendronate was released in a sustained manner. For evaluating osteoblast functions in MG-63 cells, we investigated cell proliferation, alkaline phosphatase (ALP) activity, and calcium deposition. Furthermore, for evaluating inhibition of osteoclast differentiation in RAW 264.7 cells, we investigated tartrate-resistant acid phosphatase (TRAP) activity, TRAP staining, and gene expressions. The in vitro studies revealed that osteoblasts grown on alendronate-loaded chitosan scaffold showed a significant increment in cell proliferation, ALP activity, and calcium deposition as compared to those grown on chitosan scaffolds. In addition, the in vitro study showed that osteoclast differentiation in RAW 264.7 cells cultured on alendronate-loaded chitosan scaffolds was greatly inhibited as compared to those cultured on chitosan scaffolds by the results of TRAP activity, TRAP staining, and gene expressions. Taken together, alendronate-loaded chitosan scaffolds could achieve the dual functions of improvement in osteoblast functions and inhibition of osteoclast differentiation. Thus, alendronate-eluting chitosan substrates are promising materials for enhancing osteoblast functions and inhibiting osteoclast differentiation in orthopedic and dental fields.

Effects of veraguensin and galgravin on osteoclast differentiation and function

The dried flower buds of Magnolia sp. are widely used as herbal medicines because of their anti-inflammatory, anti-malarial and anti-platelet activities. Here, we found that veraguensin and galgravin, lignan compounds derived from Magnolia sp., dose-dependently inhibited osteoclast formation in co-cultures of bone marrow cells and osteoblastic cells. These compounds also inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation in RAW264.7 cells and bone marrow macrophages. In the RANKL-induced signaling pathway, veraguensin and galgravin reduced p38 phosphorylation and suppressed the expression of c-Fos, a key transcription factor for osteoclastogenesis. Veraguensin and galgravin also inhibited osteoclastic pit formation, which was accompanied by decreased mature osteoclast viability. In conclusion, these results indicate that veraguensin and galgravin can inhibit bone resorption and may offer novel compounds for the development of drugs to treat bone-destructive diseases such as osteoporosis.

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.

EphB4/ephrinB2 Reverse Signaling Regulates Expression Levels of PDZ-domain Proteins During Osteoclast Differentiation of RAW264.7 Cells*

Syntenin and Pick1(PDZ-domain proteins) have been reported to bind to ephrinB ligands.However,there is no data related to whether ephrinB ligands,located on the membrance of osteoclasts,regulate expression levels of syntenin and Pick1,following activation by EphB receptors.RAW264.7 cell line was used as osteoclast precursors,which can differentiate into osteoclast induced by RANKL.Western blot analysis and/or immunofluorescence staining revealed that not only syntenin and Pick1,but also ephrinB2 were prominently expressed during RANKL-induced osteoclast differentiation of RAW264.7 cells,thus furtherly illustrating that the model of RANKL-induced osteoclast differentiation of RAW264.7 cells can be used for further investigation.In order to study the effects of reverse signaling on the expression levels of PDZ-domain proteins,soluble EphB4-Fc protein was used to stimulate ephrinB2 because EphB4 exclusively interacts with ephrinB2.In contrast to the similar expression level of syntenin between EphB4-Fc and Fc treated group,the protein and mRNA expression levels of Pick1 were obviously enhanced in EphB4-Fc treated group compared with Fc treated group.However,co-immunoprecipitation results showed that there were no direct interactions between ephrinB2 and endogenously expressed syntenin and Pick1 in the RANKL-induced osteoclasts of RAW264.7 cells in vitro.In summary,it was demonstrated that both syntenin and Pick1 were expressed during RANKL-induced osteoclast differentiation of RAW264.7 cells,and EphB4/ephrinB2 reverse signaling regulates the expression levels of Pick1,but not of syntenin.These data help to preliminarily explore the potential PDZ-domain proteins involved in the downstream of ephrinB2 during the osteoclast differentiation of RAW264.7 cells in vitro.

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.

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.

Erythrina variegata extract exerts osteoprotective effects by suppression of the process of bone resorption

Our previous study showed that Erythrina variegata L. (EV) inhibited bone loss and improved bone properties in ovariectomised rats. The purpose of the present study is to investigate the potential mechanism involved in mediating the osteoprotective actions of EV. Female Sprague-Dawley rats were fed a phyto-oestrogen-free diet and subjected to either ovariectomy or a sham operation. Ovariectomised rats were treated with genistein (40 mg/kg) as well as low (200 mg/kg), medium (500 mg/kg) or high (1000 mg/kg) doses of EV extract. Bone properties and mRNA expressions were evaluated by micro-computed tomography and quantitative RT-PCR, respectively. Osteoclast differentiation in RAW 264.7 cells was studied by tartrate-resistant acid phosphatase (TRAP) staining. High doses of EV could decrease urinary Ca and P excretion, maintain serum Ca and P level, and exert beneficial effects on the micro-structure and morphology of trabecular bone and cortical bone in ovariectomised rats. EV suppressed the up-regulation of cathepsin K mRNA and the down-regulation of osteoprotegrin mRNA in the tibia of ovariectomised rats. TRAP-positive cell numbers were significantly decreased in receptor activator of nuclear factor-κB ligand (RANKL)-induced RAW 264.7 cells when co-cultured with EV extracts. The present study indicated that the protective effects of EV on bone properties in ovariectomised rats are likely to be mediated by its inhibitory actions on the process of bone resorption via the suppression of osteoclast differentiation and maturation.

TRP14 attenuates osteoclast differentiation by inhibiting NF‐κB and MAPK signaling

TRP14 is a disulfide reductase containing the CXXC motif which regulates NF‐κB and mitogen‐activated protein kinases (MAPKs) stimulated by TNFα. Given that NF‐κB and MAPK signaling play an important role in osteoclastogenesis, it was examined whether TRP14 can inhibit osteoclast differentiation. Osteoclast differentiation of RAW264.7 macrophages by receptor activator of NF‐κB ligand (RANKL) was enhanced by depletion of TRP14, while it was inhibited by TRP14 overexpression, as revealed by tartrate‐resistant acid phosphatase (TRAP) staining and TRAP activity assay. When RAW264.7 cells was exposed to RANKL, depletion of TRP14 augmented phosphorylation and degradation of IκBα, and activation of c‐Jun NH2‐terminal kinase (JNK) and p38 as well as consequent induction of nuclear factor of activated T cell, cytoplasmic 1 (NFATc1) that is a pivotal determinant of osteoclastogenesis. In addition depletion of TRP14 strongly expanded actin ring formation that is a prerequisite for osteoclast bone resorption, and significantly increased RANKL‐mediated ROS production. Taken together TRP14 attenuates osteoclast differentiation by inhibiting NF‐κB, JNK and p38 activated by RANKL.

Mechanical stress directly suppresses osteoclast differentiation in RAW264.7 cells

Although it is known that mechanical stress to osteoblast and periodontal ligament cells suppresses osteoclast differentiation, little is known about the direct effect of mechanical stress on osteoclast differentiation. In this study, we examined the role of mechanical stress on osteoclast differentiation using murine pre-osteoclastic RAW264.7 cells treated with receptor activator of nuclear factor-kappaB ligand (RANKL). RAW cells were cultured with RANKL, and mechanical stress was applied for a given period. We counted the number of osteoclast cells which were tartrate-resistant acid phosphatase (TRAP)-positive and multinucleated (2 nuclei or more), and measured mRNA by RT-PCR. There was a decrease in the number of osteoclasts under mechanical stress compared with the number under no mechanical stress. The number of nuclei per osteoclast also decreased compared to the number of nuclei per osteoclast cultured with the application of mechanical stress. As the cells were cultured for a period of 1-7 days and/or for different periods of mechanical stress application, osteoclast differentiation decreased with mechanical stress and increased after removing mechanical stress. Expression of mRNA for the osteoclast-specific genes, TRAP, matrix metalloproteinase-9, cathepsin-K and calcitonin receptor, decreased with mechanical stress and was associated with the number of osteoclasts. Inducible nitric oxide synthase mRNA which inhibits osteoclast differentiation, increased with mechanical stress. In spite of the decrease in osteoclast number with mechanical stress, nuclear factor of activated T cell cytoplasmic 1 (NFATc1) and NFATc2 mRNA expression increased with mechanical stress. These findings indicate that mechanical stress directly suppresses osteoclast differentiation and increases NFATc1 and NFATc2 suggesting delayed differentiation.

Docosahexaenoic acid is more potent inhibitor of osteoclast differentiation in RAW 264.7 cells than eicosapentaenoic acid

Fish oil rich in n-3 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) protects inflammation induced bone loss in chronic inflammatory diseases like rheumatoid arthritis, periodontitis, and osteoporosis. EPA and DHA differentially regulate functional parameters and gene expression in different cell types. One of the risk factors for bone loss in inflammatory bone diseases is the elevation of bone-resorbing osteoclasts and a very few studies so far have indicated that attenuation of osteoclastogenesis might be one of the mechanisms by which n-3 PUFA exert its effect on bone loss protection. However, the precise mechanism underlying this process remains unclear. Receptor activator of NF-kappaB ligand (RANKL) is known to be the most critical mediator of osteoclastogenesis. Therefore, in this study, we examined the differential effect of EPA and DHA on RANKL-stimulated osteoclastogenesis and RANKL signaling using a murine monocytic cell line RAW 264.7. DHA was found to inhibit osteoclast differentiation, activation and function more potently than EPA. The differential potential also closely correlated with the inhibition of osteoclast-specific genes like tartrate resistant acid phosphatase, cathepsin K, calcitonin receptor, matrix metalloproteinase-9 expression and osteoclast-specific transcription factor, c-Fos, as well as osteotropic proinflammatory cytokine, TNF-alpha to a greater extent with DHA than EPA. Further, pretreatment of RAW 264.7 cells with DHA also showed significantly reduced activation of NF-kappaB and p38MAPK than EPA. Our findings suggest that DHA may be much more effective than EPA in alleviating RANKL induced proinflammatory cytokine production, intracellular signaling activation, thereby decreasing osteoclast activation and bone resorption.

Inhibitory Effects of Platelet-rich Plasma on Osteoclast Differentiation in RAW264.7 Cells

Platelet-rich plasma (PRP) is an autologous source of platelet-derived growth factors, and has been used successfully in oral surgery repair and in the placement of osseointegrated implants. However, little is known about the underlying release mechanisms of these factors or the manner in which inhibition of osteoclastic cells is regulated. The present study investigated the efficacy of PRP as an inhibitor of receptor activator of NF-kappaB ligand (RANKL)-induced TRAP-positive multinucleated cell formation in RAW264.7 cells. RANKL-induced formation of osteoclasts was significantly inhibited by treatment with PRP, while washed platelet treatment was more effective in inhibiting osteoclast formation. These results suggest that PRP and washed platelets are potent inhibitors of osteoclast differentiation.