Osteoclast Formation In RAW264 Research Articles

Lipid mediators obtained from docosahexaenoic acid by soybean lipoxygenase attenuate RANKL-induced osteoclast differentiation and rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic immune-mediated inflammatory disease characterized by persistent inflammation and joint destruction. A lipid mediator (LM, namely, 17S-monohydroxy docosahexaenoic acid, resolvin D5, and protectin DX in a ratio of 3:47:50) produced by soybean lipoxygenase from DHA, exhibits anti-inflammatory activity. In this study, we determined the effect of LM on collagen antibody-induced arthritis (CAIA) in mice and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation in RAW264.7 cells. LM effectively downregulated the expression of tartrate-resistant acid phosphatase (TRAP) and cathepsin K, inhibited osteoclast formation, and suppressed the NF-κB signaling pathway in vitro. In vivo, LM at 10 μg/kg/day significantly decreased paw swelling and inhibited progression of arthritis in CAIA mice. Moreover, proinflammatory cytokine (tumor necrosis factor-α, interleukin (IL)-6, IL-1β, IL-17, and interferon-γ) levels in serum were decreased, whereas IL-10 levels were increased following LM treatment. Furthermore, LM alleviated joint inflammation, cartilage erosion, and bone destruction in the ankles, which may be related to matrix metalloproteinase and Janus kinase (JAK)–signal transducer and activators of transcription (STAT) signaling pathway. Our findings suggest that LM attenuates arthritis severity, restores serum imbalances, and modifies joint damage. Thus, LM represents a promising therapy for relieving RA symptoms.

Sucrosephenylpropanoid esters and isoflavonoids isolated from Belamcanda chinensis roots and their potential anti-osteoclastogenic activity

Repeated chromatography of the CH2Cl2 and EtOAc soluble fractions from the methanol extract of Belamcanda chinensis root yielded six new sucrosephenylpropanoid esters (1–6) and twenty-one known compounds (7–27). The structures of 1–6 were elucidated using diverse nuclear magnetic resonance (NMR) techniques and high-resolution mass spectrometry (HRMS) data analysis, together with chemical methods. All the twenty-seven isolated compounds were evaluated for their anti-osteoclastogenic activities. Preliminary screening results revealed that compounds 1 and 19 exhibited strong effects against RANKL-induced osteoclast formation in RAW264.7 cells. In addition, the treatment of mouse bone marrow macrophages (BMMs) with compounds 1 and 19 significantly decreased RANKL-induced TRAP-positive multinucleated osteoclast formation in a concentration-dependent manner without affecting cell viability. Further bioassay investigation showed that compounds 1 and 19 inhibited the expression of some osteoclast-specific marker genes and the transcription factor nuclear factor of activated T cells cytoplasmic 1 (NFATc1) in response to RANKL. To the best of our knowledge, this is the first investigation of anti-osteoclastogenic activity for compounds isolated from B. chinensis.

Evaluation of culture conditions for osteoclastogenesis in RAW264.7 cells.

Osteoclasts are the only multinucleated cells in vivo responsible for bone resorption and are vital for regulating bone remodeling and maintaining bone mass. The RAW264.7 cell line is widely used to study osteoclastic differentiation and biological molecular mechanism. However, protocols for inducing osteoclast formation in RAW264.7 cells vary considerably between laboratories, hindering the replication of results. Therefore, we tested the influence of culture conditions on osteoclast differentiation, including cell density and receptor activator of nuclear factor kappa-B ligand (RANKL) concentrations with or without macrophage colony-stimulating factors (M-CSF). Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the morphology of osteoclasts. qPCR was used to detect gene expression of osteoclast-specific gene marker cathepsin K (CTSK), osteoclast transcription factors c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1). The bone resorption function was evaluated by a scanning electron microscope (SEM). RANKL treatment increased multinucleated osteoclasts formation and increased CTSK, c-Fos and NFATc1 gene expression. Compared with RANKL treatment, M-CSF significantly decreased multinucleated osteoclasts formation, reduced CTSK gene expression and had little effect on c-Fos and NFATc1 gene expression. Concerning bone resorption activity, RANKL treatment increased bone resorption pits on bovine bone slices. Significantly higher levels of osteoclastogenesis were observed with RAW264.7-cell density of 2×104 cells/well in 24-well plates. Our results suggest that the addition of 50 ng/ml M-CSF has no positive effect on osteoclastogenesis. RANKL treatment and cell density contribute to osteoclast formation, and the optimal conditions are beneficial when exploring osteoclast function and mechanism.

Marein Prevented LPS-Induced Osteoclastogenesis by Regulating the NF-κB Pathway In Vitro.

Many bone diseases such as osteolysis, osteomyelitis, and septic arthritis are caused by gram-negative bacterial infection, and lipopolysaccharide (LPS), a bacterial product, plays an essential role in this process. Drugs that inhibit LPS-induced osteoclastogenesis are urgently needed to prevent bone destruction in infective bone diseases. Marein, a major bioactive compound of Coreopsis tinctoria, possesses anti-oxidative, anti-inflammatory, anti-hypertensive, anti-hyperlipidemic, and anti-diabetic effects. In this study, we measured the effect of marein on RAW264.7 cells by CCK-8 assay and used TRAP staining to determine osteoclastogenesis. The levels of osteoclast-related genes and NF-κB-related proteins were then analyzed by western blot, and the levels of pro-inflammatory cytokines were quantified by ELISA. Our results showed that marein inhibited LPS-induced osteoclast formation by osteoclast precursor RAW264.7 cells. The effect of marein was related to its inhibitory function on expressions of pro-inflammatory cytokines and osteoclast-related genes containing RANK, TRAF6, MMP-9, CK, and CAII. Additionally, marein leads to markedly inhibited NF-κB signaling pathway activation in LPS-induced RAW264.7 cells. Concurrently, when the NF-κB signaling pathway was inhibited, osteoclast formation and pro-inflammatory cytokine expression were decreased. Collectively, marein could inhibit LPS-induced osteoclast formation in RAW264.7 cells via regulating the NF-κB signaling pathway. Our data demonstrate that marein might be a potential drug for bacteria-induced bone destruction disease. Our findings provide new insights into LPS-induced bone disease.

Delineating role of NF‐κB and interacting cytokines during prostate cancer‐induced osteoclastogenesis

Prostate cancer (PCa) frequently metastasizes to the bone leading to devastating complications such as severe pain and fracture. However, the mechanisms by which PCa cells cause bone loss remain less understood. We investigated the role and mechanisms by which PCa cells induce osteoclastogenesis using cultured monocytic osteoclast precursors. Treatment of RAW264.7 cells with PCa cell lines: DU145, LNCaP, PC-3, or their conditioned media led to the formation of distinct multinucleated, TRAP+ osteoclasts. This phenomenon was associated with the increased activation of transcription factor nuclear factor-kB (NF-κB). High transcript level of receptor activator of nuclear factor-kB ligand (RANKL), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were detected in PCa cells. TNF-α and LT-α augmented, whereas IL-6 reduced the RANKL-induced osteoclast formation in RAW264.7 cultures. Our results also demonstrated that PCa cells-induced osteoclastogenesis involved the activation of the TRAF6-IKK-p65-NF-κB signaling cascade. Together, our study demonstrates that PCa cells produce RANKL and several other pro-inflammatory cytokines known to influence osteoclastogenesis, by targeting the NF-κB signaling pathway.

Abstract 3002: Bone-protective curcumin circulates as a pro-drug conjugate that is activated in bone by β-glucuronidase

Abstract Curcumin (CURC), a turmeric-derived dietary polyphenol, prevents osteoclast formation and bone resorption in murine models of arthritis, post-menopausal osteoporosis, and bone metastatic breast cancer despite being rapidly conjugated upon ingestion in both rodents and humans to form curcumin-glucuronide (G-CURC). However, recent studies from our laboratory demonstrate that aglycone CURC is enriched in bone relative to the circulation. Studies were therefore undertaken to compare the anti-resorptive effects of aglycone CURC vs G-CURC and to elucidate mechanisms responsible for enrichment of aglycone CURC in bone, including clinically relevant influences of age, sex, and reproductive status. CURC dose-dependently inhibited RANKL-stimulated osteoclast formation in RAW264.7 cell cultures, while G-CURC was without effect. Likewise, CURC dose-dependently inhibited TGFβ-stimulated PTHrP secretion from MDA-MB-231 breast cancer cells, which readily form PTHrP-driven osteolytic bone metastases in vivo. In wild type (WT) C57BL/6J mice, heparanase (HPSE) and β-glucuronidase (GUSB) were the only enzymes with reported deglucuronidation activity expressed in bone (Western). However, only GUSB was able to recognize G-CURC as a substrate, deconjugating G-CURC to form CURC (LC/MS). Consistent with these findings, tissue-specific levels of CURC were highest in bone (vs. heart, muscle, or kidney) and proportional to tissue specific GUSB enzyme activity. To verify the role of GUSB in CURC bone enrichment, CURC metabolism and GUSB enzyme expression and activity were compared among mice with normal levels of GUSB (C57BL/6J) vs. partial (C3H/HeJ) or complete (mucopolysaccharidosis VII (mps/mps]) deficiencies in GUSB expression. As expected, partial (55% of WT) or complete deficiencies in GUSB expression significantly reduced CURC enrichment in bone. While GUSB can be positively regulated by pharmacologic doses of androgens, neither male sex nor ovariectomy (OVX) altered GUSB activity (per protein) in bone relative to intact females, although a small but statistically significant increase in free curcumin was demonstrated in males (17%) and OVX females (10%). Age did not significantly change the enrichment of CURC in bone of 4 vs 28-week mice. These findings suggest bio-inactive G-CURC acts as pro-drug that is converted to active CURC within bone, independent of sex, age or reproductive status, where it can inhibit the formation of bone resorbing osteoclasts or secretion of osteolytic PTHrP from breast cancer bone metastases. Citation Format: Andrew Kunihiro, Julia A. Brickey, jennifer B. Frye, Paula B. Luis, Claus Schneider, Janet L. Funk. Bone-protective curcumin circulates as a pro-drug conjugate that is activated in bone by β-glucuronidase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3002.

Serpina3n, Dominantly Expressed in Female Osteoblasts, Suppresses the Phenotypes of Differentiated Osteoblasts in Mice.

It is well known that sex differences exist concerning the severity of osteoporosis and bone metabolism, suggesting that factors other than sex hormones might be responsible for sex differences of bone metabolism. We therefore examined sex differences of osteoblast phenotypes of mouse osteoblasts and then performed comparative gene expression analyses using a comprehensive DNA microarray between female and male osteoblasts. Alkaline phosphatase (ALP) activity, mineralization, and the expression of Osterix, ALP, and bone sialoprotein were significantly lower in mouse female osteoblasts compared with male osteoblasts. We identified Serpina3n, a novel serine protease inhibitor, as the gene whose expression has the highest ratio of females to males. A reduction in endogenous levels of Serpina3n by small interfering RNA significantly enhanced the mRNA levels of Runx2, ALP, osteocalcin, and type I collagen (Col1a1) in both male and female osteoblasts. Moreover, Serpina3n overexpression significantly suppressed the mRNA levels of Osterix, ALP, osteocalcin, and Col1a1 in MC3T3-E1 cells. Serpina3n overexpression did not affect Osterix, ALP, and osteocalcin mRNA levels enhanced by bone morphogenetic protein (BMP)-2 in ST2 cells, adipogenic differentiation in ST2 and 3T3-L1 cells, and receptor activator of nuclear factor κB ligand-induced osteoclast formation in RAW264.7 cells, although it significantly suppressed mineralization in ST2 cells differentiated into osteoblasts by BMP-2. In conclusion, we found Serpina3n as the most female osteoblast-dominant gene. Serpina3n exerts a suppression of the osteoblast phenotypes such as Col1a1 expression and ALP activity in differentiated osteoblasts, which might partly explain sex differences of the osteoblast phenotypes in mice.

Palmitoleic Acid Inhibits RANKL-Induced Osteoclastogenesis and Bone Resorption by Suppressing NF-κB and MAPK Signalling Pathways.

Osteoclasts are large, multinucleated cells that are responsible for the breakdown or resorption of bone during bone remodelling. Studies have shown that certain fatty acids (FAs) can increase bone formation, reduce bone loss, and influence total bone mass. Palmitoleic acid (PLA) is a 16-carbon, monounsaturated FA that has shown anti-inflammatory properties similar to other FAs. The effects of PLA in bone remain unexplored. Here we investigated the effects of PLA on receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL)-induced osteoclast formation and bone resorption in RAW264.7 murine macrophages. PLA decreased the number of large, multinucleated tartrate resistant acid phosphatase (TRAP) positive osteoclasts and furthermore, suppressed the osteolytic capability of these osteoclasts. This was accompanied by a decrease in expression of resorption markers (Trap, matrix metalloproteinase 9 (Mmp9), cathepsin K (Ctsk)). PLA further decreased the expression of genes involved in the formation and function of osteoclasts. Additionally, PLA inhibited NF-κB activity and the activation of mitogen activated protein kinases (MAPK), c-Jun N-terminal kinase (JNK) and extracellular signal–regulated kinase (ERK). Moreover, PLA induced apoptosis in mature osteoclasts. This study reveals that PLA inhibits RANKL-induced osteoclast formation in RAW264.7 murine macrophages through suppression of NF-κB and MAPK signalling pathways. This may indicate that PLA has potential as a therapeutic for bone diseases characterized by excessive osteoclast formation.

Carvacrol Inhibits Osteoclastogenesis and Negatively Regulates the Survival of Mature Osteoclasts.

Bone is a dynamic tissue that undergoes continuous remodeling coupled with the action of osteoblasts and osteoclasts. Osteoclast activity is elevated during osteoporosis and periodontitis resulting in excessive loss of trabecular and alveolar bone. Osteoclasts are formed in an inflammatory response to cytokine production receptor activator of nuclear factor-kappaB (NF-κB) ligand (RANKL) and bacterial challenge lipopolysaccharide (LPS). Carvacrol, a monoterpenic phenol present in Origanum vulgare and Thymus vulgaris, is a natural compound with known medicinal properties. We investigated the effects of carvacrol on osteoclast formation induced by RANKL and LPS. Carvacrol suppressed RANKL-induced formation of tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells in RAW264.7 macrophages and human CD14(+) monocytes. Furthermore, carvacrol inhibited LPS-induced osteoclast formation in RAW264.7 macrophages. Investigation of the underlying molecular mechanisms revealed that carvacrol downregulated RANKL-induced NF-κB activation in a dose-dependent manner. Furthermore, the suppression of NF-κB activation correlated with inhibition of inhibitor of kappaB (IκB) kinase (IKK) activation and attenuation of inhibitor of NF-κB (IκBa) degradation. Carvacrol potentiated apoptosis in mature osteoclasts by caspase-3 activation and DNA fragmentation. Moreover, carvacrol did not affect the viability of proliferating MC3T3-E1 osteoblast-like cells. Collectively, these results demonstrate that carvacrol mitigates osteoclastogenesis by impairing the NF-κB pathway and induction of apoptosis in mature osteoclasts.

WSS25, a sulfated polysaccharide, inhibits RANKL-induced mouse osteoclast formation by blocking SMAD/ID1 signaling.

WSS25 is a sulfated polysaccharide extracted from the rhizome of Gastrodia elata BI, which has been found to bind to bone morphogenetic protein 2 (BMP-2) in hepatocellular cancer cells. Since BMP-2 may regulate both osteoclasts and osteoblasts, here we investigated the effects of WSS25 on osteoclastogenesis in vitro and bone loss in ovariectomized mice. RAW264.7 cells or mouse bone marrow macrophages (BMMs) were treated with RANKL to induce osteoclastogenesis, which was assessed using TRAP staining, actin ring formation and pit formation assays, as well as bone resorption assay. Cell viability was detected with MTT assay. The mRNA levels of osteoclastogenesis-related genetic markers (TRAP, NFATc1, MMP-9 and cathepsin K) were detected using RT-PCR, while the protein levels of p-Smad1/5/8 and Id1 were measure with Western blotting. WSS25 was administered to ovariectomized mice (100 mg·kg(-1)·d(-1), po) for 3 months. After the mice were euthanized, total bone mineral density and cortical bone density were measured. In RAW264.7 cells and BMMs, WSS25 (2.5, 5, 10 μg/mL) did not affect the cell viability, but dose-dependently inhibited RANKL-induced osteoclastogenesis. Furthermore, WSS25 potently suppressed RANKL-induced expression of TRAP, NFATc1, MMP-9 and cathepsin K in RAW264.7 cells. Treatment of RAW264.7 cells with RANKL increased BMP-2 expression, Smad1/5/8 phosphorylation and Id1 expression, which triggered osteoclast differentiation, whereas co-treatment with WSS25 or the endogenous BMP-2 antagonist noggin suppressed the BMP-2/Smad/Id1 signaling pathway. In RAW264.7 cells, knockdown of Id1 attenuated RANKL-induced osteoclast differentiation, which was partially rescued by Id1 overexpression. In conformity to the in vitro experiments, chronic administration of WSS25 significantly reduced the bone loss in ovariectomized mice. WSS25 inhibits RANKL-induced osteoclast formation in RAW264.7 cells and BMMs by blocking the BMP-2/Smad/Id1 signaling pathway. WSS25 administration reduces bone loss in ovariectomized mice, suggesting that it may be a promising therapeutic agent for osteoporosis.

Inhibition of andrographolide in RAW 264.7 murine macrophage osteoclastogenesis by downregulating the nuclear factor-kappaB signaling pathway.

This study aims to investigate the effects of andrographolide (AGP) on osteoclast formation in RAW 264.7 murine macrophage cells. The effects of AGP on cell viability were determined in RAW 264.7 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of AGP on osteoclast formation were tested by osteoclast staining with tartrate-resistant acid phosphatase (TRAP). The effects of AGP on receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL)-induced, NF-kappaB-dependent transcription in RAW 264.7 cells were assessed using luciferase reporter assays. The results demonstrated that the viability of osteoclast precursor RAW 264.7 cells was not affected by AGP treatment at a concentration of 0.4 to 10 μM. Additionally, the number of TRAP-positive osteoclasts was significantly reduced by the same concentrations of AGP treatment. AGP also inhibited RANKL-induced NF-kappaB activation in a dose-dependent fashion as evidenced by luciferase reporter assays. In summary, this study demonstrates that AGP inhibits osteoclastogenesis in RAW 264.7 murine macrophage cells through downregulation of the NF-kappaB signaling pathway.

Tartrate-resistant acid phosphatase 5b is a marker of osteoclast number and volume in RAW 264.7 cells treated with receptor-activated nuclear κB ligand.

Tartrate-resistant acid phosphatase 5b (TRACP 5b) has been used as a biomarker of bone resorption and cancer metastasis. TRACP 5b has also been suggested to be a reliable marker of osteoclast number. In this study, the correlation of TRACP 5b level and osteoclast-like cell number was investigated in RAW 264.7 cells treated with receptor-activated nuclear factor κB ligand (RANKL). RAW 264.7 cells were cultured with α-MEM containing RANKL (40 ng/ml) for 3, 5 and 7 days. Osteoclast formation and TRACP 5b levels were determined by TRACP staining, scanning electron microscopy and enzyme-linked immunosorbent assay. The RAW 264.7 cells that were not exposed to RANKL did not secrete TRACP 5b. RANKL induced the RAW 264.7 cells to differentiate into osteoclasts and to secrete TRACP 5b. The TRACP 5b level in the RAW 264.7 cells treated with RANKL was significantly correlated with the number and volume of osteoclasts (r=0.95 and r=0.92, respectively; P<0.0001). TRACP 5b is a good marker of RANKL-induced osteoclast formation in RAW 264.7 cells. TRACP 5b analysis may be used as an alternative to osteoclast counting in vitro.

BMP-2 and titanium particles synergistically activate osteoclast formation.

A previous study showed that BMP-2 (bone morphogenetic protein-2) and wear debris can separately support osteoclast formation induced by the receptor activator of NF-κB ligand (RANKL). However, the effect of BMP-2 on wear debris-induced osteoclast formation is unclear. In this study, we show that neither titanium particles nor BMP-2 can induce osteoclast formation in RAW 264.7 mouse leukemic monocyte macrophage cells but that BMP-2 synergizes with titanium particles to enhance osteoclast formation in the presence of RANKL, and that at a low concentration, BMP-2 has an optimal effect to stimulate the size and number of multinuclear osteoclasts, expression of osteoclast genes, and resorption area. Our data also clarify that the effects caused by the increase in BMP-2 on phosphorylated SMAD levels such as c-Fos expression increased throughout the early stages of osteoclastogenesis. BMP-2 and titanium particles stimulate the expression of p-JNK, p-P38, p-IkB, and P50 compared with the titanium group. These data suggested that BMP-2 may be a crucial factor in titanium particle-mediated osteoclast formation.

Fibrodysplasia Ossificans Progressiva-related Activated Activin-like Kinase Signaling Enhances Osteoclast Formation during Heterotopic Ossification in Muscle Tissues

Fibrodysplasia ossificans progressiva is characterized by extensive ossification within muscle tissues, and its molecular pathogenesis is responsible for the constitutively activating mutation (R206H) of the bone morphogenetic protein type 1 receptor, activin-like kinase 2 (ALK2). In this study, we investigated the effects of implanting ALK2 (R206H)-transfected myoblastic C2C12 cells into nude mice on osteoclast formation during heterotopic ossification in muscle and subcutaneous tissues. The implantation of ALK2 (R206H)-transfected C2C12 cells with BMP-2 in nude mice induced robust heterotopic ossification with an increase in the formation of osteoclasts in muscle tissues but not in subcutaneous tissues. The implantation of ALK2 (R206H)-transfected C2C12 cells in muscle induced heterotopic ossification more effectively than that of empty vector-transfected cells. A co-culture of ALK2 (R206H)-transfected C2C12 cells as well as the conditioned medium from ALK2 (R206H)-transfected C2C12 cells enhanced osteoclast formation in Raw264.7 cells more effectively than those with empty vector-transfected cells. The transfection of ALK2 (R206H) into C2C12 cells elevated the expression of transforming growth factor (TGF)-β, whereas the inhibition of TGF-β signaling suppressed the enhanced formation of osteoclasts in the co-culture with ALK2 (R206H)-transfected C2C12 cells and their conditioned medium. In conclusion, this study demonstrated that the causal mutation transfection of fibrodysplasia ossificans progressiva in myoblasts enhanced the formation of osteoclasts from its precursor through TGF-β in muscle tissues.

Inhibition of receptor activator of nuclear factor-κB ligand- or lipopolysaccharide-induced osteoclast formation by conophylline through downregulation of CREB

The effect of conophylline (CNP) on the receptor activator of nuclear factor-κB ligand (RANKL) or lipopolysaccharide (LPS)-induced osteoclast formation was studied in vitro using bone marrow-derived macrophages (BMMs) or the mouse macrophage-like cell line RAW 264.7. CNP inhibited RANKL-induced formation of osteoclasts identified as tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in a culture of BMMs. It also inhibited RANKL- or LPS-induced osteoclast formation in RAW 264.7 cells. CNP lowered the osteoclast maturation markers such as calcitonin receptor, MMP9 and cathepsin K in BMMs, suggesting that CNP would inhibit the process of osteoclast differentiation. CNP inhibited the RANKL-induced expressions of c-Fos and nuclear factor of activated T cells (NFATc1), key transcription factors for osteoclastogenesis. On the other hand, CNP did not inhibit the signaling pathway of NF-κB and mitogen-activated protein kinases (MAPKs) in RANKL-stimulated BMMs. Interestingly, CNP inhibited RANKL-induced CREB activation that can mediate c-Fos and NFATc1. CNP also inhibited RANKL- or LPS-induced CREB, c-Fos and NFATc1 activation in RAW 264.7 cells. We have previously found that CNP directly binds to ADP-ribosylation-like factor-6 interacting protein (ARL6ip), although its role in osteoclastogenesis is not clear. Gene knockdown of ARL6ip by siRNA inhibited RANKL-induced c-Fos expression, suggesting that inactivation of ARL6ip may be involved in an inhibitory effect of CNP. Taken together, CNP was shown to inhibit osteoclast formation possibly via CREB inactivation following a decrease in c-Fos and NFATc1 expression.

Involvement of redox balance in in vitro osteoclast formation of RAW 264.7 macrophage cells in response to LPS

Here we report that LPS induces osteoclast (OC) formation in murine RAW 264.7 macrophage cells in RPMI-1640 medium but not in α-minimum essential medium (α-MEM) as the original culture medium. LPS-induced OC formation in both media was examined to clarify the differential response. Receptor activator of NF-κB ligand induced OC formation in either α-MEM or RPMI-1640 medium. However, LPS-induced OC formation in RAW 264.7 cells maintained in RPMI-1640 medium, but not α-MEM, which was also supported by mouse bone marrow-derived macrophages, although they were less sensitive to LPS than RAW 264.7 cells. LPS augmented the expression of nuclear factor of activated T-cells (NFATc1) as a key transcription factor of osteoclastogenesis in cells maintained in RPMI-1640 medium, but reduced it in cells maintained in α-MEM. A high concentration of LPS was cytotoxic against cells maintained in α-MEM. Glutathione exclusively present in RPMI-1640 medium prevented LPS-induced cell death in α-MEM and augmented LPS-induced NFATc1 expression, followed by enhanced LPS-induced OC formation. LPS induced higher generation of reactive oxygen species in α-MEM than RPMI-1640 medium. An antioxidant enhanced LPS-induced OC formation, whereas a pro-oxidant reduced it. Taken together, redox balance in the culture condition was suggested to regulate in vitro LPS-induced OC formation.

Hyperbaric oxygen therapy suppresses osteoclast formation and bone resorption

The cellular and molecular mechanism through which hyperbaric oxygen therapy (HBO) improves osteonecrosis (ON) is unclear. The present study therefore examined the effect of HBO, pressure and hyperoxia on RANKL-induced osteoclast formation in RAW 264.7 cells and human peripheral blood monocytes (PBMC). Daily exposure to HBO (2.4 ATA, 97% O2 , 90 min), hyperbaric pressure (2.4 ATA, 8.8% O2 , 90 min) or normobaric hyperoxia (1 ATA, 95% O2 , 90 min) significantly decreased RANKL-induced osteoclast formation and bone resorption in normoxic conditions. HBO had a more pronounced anti-osteoclastic effect than hyperoxia or pressure alone and also directly inhibited osteoclast formation and resorption in hypoxic conditions a hallmark of many osteolytic skeletal disorders. The suppressive action of HBO was at least in part mediated through a reduction in RANK, NFATc1, and Dc-STAMP expression and inhibition of hypoxia-induced HIF-1α mRNA and protein expression. This data provides mechanistic evidence supporting the use of HBO as an adjunctive therapy to prevent osteoclast formation and bone loss associated with low oxygen partial pressure.

Vaspin Attenuates RANKL-Induced Osteoclast Formation in RAW264.7 Cells

Visceral adipose tissue-derived serine protease inhibitor (vaspin), an adipokine that was recently identified in a rat model of type 2 diabetes, has been suggested to have an insulin-sensitizing effect. In this study, we investigated whether vaspin inhibits receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis using two types of osteoclast precursors: RAW264.7 cells and bone marrow cells (BMCs). Vaspin inhibited RANKL-induced osteoclastogenesis in RAW264.7 cells and BMCs. Interestingly, vaspin also inhibited the RANKL-induced expression of nuclear factor of activated T cells c1 (NFATc1) in RAW264.7 cells and BMCs. Furthermore, it inhibited the RANKL-induced upregulation of matrix metalloproteinase-9 and cathepsin K in RAW264.7 cells. Thus, we suggest that vaspin downregulates osteoclastogenesis in part by inhibiting expression of the transcription factor NFATc1.

DICAM inhibits osteoclast differentiation through attenuation of the integrin αVβ3 pathway

Dual immunoglobulin (Ig) domain-containing adhesion molecule (DICAM) is involved in cell-cell adhesion through a heterophilic interaction with αVβ3 integrin, which suggests that DICAM may participate in osteoclast differentiation. DICAM was localized in the plasma membrane of RAW264.7 and THP-1 cells, and its expression gradually increased during osteoclastogenesis in mouse bone marrow-derived macrophages (BMMs) treated with receptor activator of nuclear factor κ-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Forced expression of DICAM in BMMs and RAW264.7 cells blocked the generation of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts. Conversely, knockdown of DICAM by small hairpin RNA (shRNA) increased osteoclast formation in RAW264.7 cells. DICAM-mediated suppression of osteoclast differentiation was in part due to the inhibition of the p38 mitogen-activated protein (MAP) kinase pathway, which was corroborated by a decrease in the expression of c-Fos and nuclear factor of activated T cells (NFAT)c1. Mechanistically, DICAM directly interacted with integrin β3, which inhibited heterodimerization between integrin αV and β3. Exogenous expression of integrin β3 or high-dose M-CSF rescued DICAM-mediated inhibition of osteoclastogenesis, suggesting crosstalk between the integrin β3 and c-Fms pathways. Finally, recombinant DICAM ectodomain suppressed the RANKL- and M-CSF-induced osteoclastogenesis of BMMs. Collectively, these results indicate that DICAM acts as a negative regulator of osteoclast differentiation by suppressing the integrin αVβ3 pathway.

The effect of hyperbaric oxygen therapy on osteoclast formation and bone resorption

Event Abstract Back to Event The effect of hyperbaric oxygen therapy on osteoclast formation and bone resorption HW Al-Hadi1*, SW Fox1 and GR Smerdon2 1 University of Plymouth, School of Biomedical and Biological Science, United Kingdom 2 the Diving Diseases Research Centre, United Kingdom Hyperbaric oxygen therapy (HBO) is the breathing of pure oxygen in a sealed chamber at greater than normal atmospheric pressure. HBO is employed as an adjunctive therapy in a number of skeletal disorders such radio and bisphosphonate induced osteonecrosis of the jaw and chronic osteomyelitis. These disorders are associated with aberrant remodelling and excessive levels of resorption leading to increased fracture risk. The purpose of the present study was to evaluate the effect of HBO, pressure and hyperoxia on RANKL-induced osteoclast formation in RAW 264.7 cells and human peripheral blood monocytes (PBMC). Daily, HBO (2.4 ATA, 97% O2, 90 minutes per day) or hyperoxia (1 ATM, 95% O2, 90 minutes per day) treatments significantly decreased the number of RANKL-induced TRAP positive mononuclear and multinuclear osteoclasts forming in RAW and PBMC cultures (p <0.05). Similarly, HBO and hyperoxia significantly reduced bone resorption as assessed by the bone slice assay. Furthermore, pressure alone (2.4 ATA, 8.8% O2, 90 minutes per day) also significantly suppressed osteoclast differentiation and bone resorption to a similar extent as HBO and hyperoxia. Quantitative PCR analysis of key regulators of osteoclast differentiation (RANK) and multinuclearity (DC-STAMP) indicated that HBO, hyperoxia and pressure all significantly suppressed RANK and DC-STAMP mRNA expression. Interestingly, intermittent HBO, hyperoxia and pressure (daily, 90 minutes per day) also significantly reduced RANKL-induced osteoclast formation in hypoxic conditions (2% O2, 22.5 hours per day). This data suggests that HBO, elevated O2 and pressure suppress osteoclast differentiation and bone resorption in mouse and human monocytes. Furthermore the data indicates that elevated pressure and O2 also directly inhibit osteoclast formation in hypoxic conditions a hallmark of many skeletal disorders. This provides evidence supporting the use of HBO as an adjunctive therapy to prevent bone loss in a range of skeletal disorders associated with low oxygen partial pressure. Keywords: Bones, Bone Research Conference: 2011 joint meeting of the Bone Research Society & the British Orthopaedic Research Society, Cambridge, United Kingdom, 27 Jun - 29 Jun, 2011. Presentation Type: Oral Poster Topic: Abstracts Citation: Al-Hadi H, Fox S and Smerdon G (2011). The effect of hyperbaric oxygen therapy on osteoclast formation and bone resorption. Front. Endocrinol. Conference Abstract: 2011 joint meeting of the Bone Research Society & the British Orthopaedic Research Society. doi: 10.3389/conf.fendo.2011.02.00002 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Sep 2011; Published Online: 30 Sep 2011. * Correspondence: Mrs. HW Al-Hadi, University of Plymouth, School of Biomedical and Biological Science, United Kingdom, hadil.al-hadi@plymouth.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers HW Al-Hadi SW Fox GR Smerdon Google HW Al-Hadi SW Fox GR Smerdon Google Scholar HW Al-Hadi SW Fox GR Smerdon PubMed HW Al-Hadi SW Fox GR Smerdon Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.