Osteoclast-associated Receptor Research Articles

Leonurine hydrochloride inhibits osteoclastogenesis and prevents osteoporosis associated with estrogen deficiency by inhibiting the NF-κB and PI3K/Akt signaling pathways

Osteoclasts, the primary bone resorbing cells, are responsible for destructive bone diseases such as postmenopausal osteoporosis, rheumatoid arthritis, and periodontitis. Many plant-derived traditional medicines that might suppress the formation and/or function of osteoclasts are promising treatments for osteoclast-related diseases. In this study, we investigated the effects of leonurine hydrochloride (LH) on receptor activator NF-κB ligand (RANKL)-induced osteoclastogenesis and ovariectomy-induced bone loss. LH is a synthetic chemical compound based on the structure of leonurine, which is found in motherwort and has been reported to exhibit phytoestrogenic activity. In RAW 264.7 cells and mouse bone marrow monocytes (BMMs), LH suppressed RANKL-induced osteoclastogenesis and actin ring formation in a dose-dependent manner. LH targeted RANKL-induced osteoclastogenesis and bone resorption at an early stage. Molecular analysis demonstrated that LH attenuated RANKL-induced NF-κB signaling by inhibiting the phosphorylation and degradation of IκBα and NF-κB p65 nuclear translocation. LH inhibited the RANK-TRAF6 association triggered by RANKL binding and the phosphatidylinositol 3-kinase (PI3K)/Akt axis, without significantly affecting the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and AP-1 signaling pathways. LH attenuated the RANKL-stimulated expression of osteoclast-related genes including NFATc1, tartrate resistant acid phosphatase (TRAP), cathepsin K, and osteoclast-associated receptor (OSCAR). Consistent with the in vitro results, LH administration attenuated osteoclast activity, thus preventing bone loss caused by estrogen deficiency in mice. In this study, LH suppressed RANKL-induced osteoclastogenesis via RANK-TRAF6, NF-κB, and PI3K/Akt signaling. These data provide the first evidence that LH might be a promising therapeutic compound to treat osteoclast-related diseases, such as osteoporosis.

Osteoimmunology: Major and Costimulatory Pathway Expression Associated with Chronic Inflammatory Induced Bone Loss

The field of osteoimmunology has emerged in response to the range of evidences demonstrating the close interrelationship between the immune system and bone metabolism. This is pertinent to immune-mediated diseases, such as rheumatoid arthritis and periodontal disease, where there are chronic inflammation and local bone erosion. Periprosthetic osteolysis is another example of chronic inflammation with associated osteolysis. This may also involve immune mediation when occurring in a patient with rheumatoid arthritis (RA). Similarities in the regulation and mechanisms of bone loss are likely to be related to the inflammatory cytokines expressed in these diseases. This review highlights the role of immune-related factors influencing bone loss particularly in diseases of chronic inflammation where there is associated localized bone loss. The importance of the balance of the RANKL-RANK-OPG axis is discussed as well as the more recently appreciated role that receptors and adaptor proteins involved in the immunoreceptor tyrosine-based activation motif (ITAM) signaling pathway play. Although animal models are briefly discussed, the focus of this review is on the expression of ITAM associated molecules in relation to inflammation induced localized bone loss in RA, chronic periodontitis, and periprosthetic osteolysis, with an emphasis on the soluble and membrane bound factor osteoclast-associated receptor (OSCAR).

Inhibition of osteoclast differentiation by gold nanoparticles functionalized with cyclodextrin curcumin complexes.

Gold nanoparticles (GNPs) have been previously reported to inhibit osteoclast (OC) formation. However, previous research only confirmed the osteoclastogenesis inhibitory effect under in vitro conditions. The aim of this study was to develop a therapeutic agent for osteoporosis based on the utilization of GNPs and confirm their effect both in vitro and in vivo. We prepared β-cyclodextrin (CD) conjugated GNPs (CGNPs), which can form inclusion complexes with curcumin (CUR-CGNPs), and used these to investigate their inhibitory effects on receptor activator of nuclear factor-κb ligand (RANKL)-induced osteoclastogenesis in bone marrow-derived macrophages (BMMs). The CUR-CGNPs significantly inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells in BMMs without inducing cytotoxicity. The mRNA expressions of genetic markers of OC differentiation including c-Fos, nuclear factor of activated T cells 1 (NFATc1), TRAP, and osteoclast associated receptor (OSCAR) were significantly decreased in the presence of CUR-CGNPs. In addition, the CUR-CGNPs inhibited OC differentiation of BMMs through suppression of the RANKL-induced signaling pathway. Additionally, CUR-CGNPs caused a decrease in RANKL-induced actin ring formation, which is an essential morphological characteristic of OC formation allowing them to carry out bone resorption activity. Furthermore, the in vivo results of an ovariectomy (OVX)-induced osteoporosis model showed that CUR-CGNPs significantly improved bone density and prevented bone loss. Therefore, CUR-CGNPs may prove to be useful as therapeutic agents for preventing and treating osteoporosis.

Ampelopsis brevipedunculata extract prevents bone loss by inhibiting osteoclastogenesis in vitro and in vivo.

Osteoclasts play a critical role in bone resorbing disorders such as osteoporosis, periodontitis, and rheumatoid arthritis. Therefore, discovery of agents capable of suppressing osteoclast differentiation may aid the development of a therapeutic access for the treatment of pathological bone loss. Ampelopsis brevipedunculata has been used as herbal folk medicine to treat liver diseases and inflammation in Asia. However, its effects on osteoclast differentiation are unknown. We were aimed to investigate the anti-osteoclastogenic activity in vitro and in vivo and to elucidate the underlying mechanism of Ampelopsis brevipedunculata extract (ABE). In this study, ABE inhibited receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation, the formation of filamentous actin rings and the bone resorbing activity of mature osteoclasts. ABE inhibited RANKL-induced p38 and IκB phosphorylation and IκB degradation. Also, ABE suppressed the mRNA and protein expression of nuclear factor of activated T cells c1 (NFATc1) and c-Fos, and the mRNA expression of genes required for cell fusion and bone resorption, such as osteoclast-associated receptor (OSCAR), tartrate resistant acid phosphatase (TRAP), cathepsin K, dendritic cell-specific transmembrane protein (DC-STAMP), β3-integrin and osteoclast stimulatory transmembrane protein (OC-STAMP). Furthermore, results of micro-CT and histologic analysis indicated that ABE remarkably prevented lipopolysaccharide (LPS)-induced bone erosion. These results demonstrate that ABE prevents LPS-induced bone erosion through inhibition of osteoclast differentiation and function, suggesting the promise of ABE as a potential cure for various osteoclast-associated bone diseases.

NADPH oxidase 4 represents a potential target for the treatment of osteoporosis

NADPH oxidase 4 represents a potential target for the treatment of osteoporosis

Runx1-Mediated Regulation of Osteoclast Differentiation and Function

Excessive bone resorption is the cause of several metabolic bone diseases including osteoporosis. Thus, identifying factors that can inhibit osteoclast formation and/or activity may define new drug targets that can be used to develop novel therapies for these conditions. Emerging evidence demonstrates that the master regulator of hematopoiesis, Runx1, is expressed in preosteoclasts and may influence skeletal health. To examine the potential role of Runx1 in osteoclast formation and function, we deleted its expression in myeloid osteoclast precursors by crossing Runx1 floxed mice (Runx1(F/F)) with CD11b-Cre transgenic mice. Mice lacking Runx1 in preosteoclasts (CD11b-Cre;Runx1(F/F)) exhibited significant loss of femoral trabecular and cortical bone mass compared with that in Cre-negative mice. In addition, serum levels of collagen type 1 cross-linked C-telopeptide, a biomarker of osteoclast-mediated bone resorption, were significantly elevated in CD11b-Cre;Runx1(F/F) mice compared with those in Runx1(F/F) mice. Tartrate-resistant acid phosphatase-positive osteoclasts that differentiated from bone marrow cells of CD11b-Cre;Runx1(F/F) mice in vitro were larger, were found in greater numbers, and had increased bone resorbing activity than similarly cultured cells from Runx1(F/F) mice. CD11b-Cre;Runx1(F/F) bone marrow cells that were differentiated into osteoclasts in vitro also had elevated mRNA levels of osteoclast-related genes including vacuolar ATPase D2, cathepsin K, matrix metalloproteinase 9, calcitonin receptor, osteoclast-associated receptor, nuclear factor of activated T cells cytoplasmic 1, and cFos. These data indicate that Runx1 expression in preosteoclasts negatively regulates osteoclast formation and activity and contributes to overall bone mass.

Differential Roles of MAPK Kinases MKK3 and MKK6 in Osteoclastogenesis and Bone Loss

Bone mass is maintained by osteoclasts that resorb bone and osteoblasts that promote matrix deposition and mineralization. Bone homeostasis is altered in chronic inflammation as well as in post-menopausal loss of estrogen, which favors osteoclast activity that leads to osteoporosis. The MAPK p38α is a key regulator of bone loss and p38 inhibitors preserve bone mass by inhibiting osteoclastogenesis. p38 function is regulated by two upstream MAPK kinases, namely MKK3 and MKK6. The goal of this study was to assess the effect of MKK3- or MKK6-deficiency on osteoclastogenesis in vitro and on bone loss in ovariectomy-induced osteoporosis in mice. We demonstrated that MKK3 but not MKK6, regulates osteoclast differentiation from bone marrow cells in vitro. Expression of NFATc1, a master transcription factor in osteoclastogenesis, is decreased in cells lacking MKK3 but not MKK6. Expression of osteoclast-specific genes Cathepsin K, osteoclast-associated receptor and MMP9, was inhibited in MKK3−/− cells. The effect of MKK-deficiency on ovariectomy-induced bone loss was then evaluated in female WT, MKK3−/− and MKK6−/− mice by micro-CT analysis. Bone loss was partially inhibited in MKK3−/− as well as MKK6−/− mice, despite normal osteoclastogenesis in MKK6−/− cells. This correlated with the lower osteoclast numbers in the MKK-deficient ovariectomized mice. These studies suggest that MKK3 and MKK6 differentially regulate bone loss due to estrogen withdrawal. MKK3 directly mediates osteoclastogenesis while MKK6 likely contributes to pro-inflammatory cytokine production that promotes osteoclast formation.

Inhibition Effect of Taxilli Ramulus Extract on Osteoclast Differentiation and Bone Resorption

Bone homeostasis is maintained by co-ordination of bone-resorbing osteoclasts and bone-forming osteoblasts. Imbalance between osteoclasts and osteoblasts leads to many bone diseases such as osteoporosis, rheumatoid arthritis. Taxillus chinensis is a herb that has been widely used to improve bone health. However, the effect and mechanism of Taxillus chinensis extract on osteoclast differentiation and bone resportion has been unknown. Thus, We investigated the effect of Taxillus chinensis on expression of receptor activator of nuclear factor-kB ligand (RANKL)-induced osteoclast differentiation and bone resorption. Also, the action of Taxillus chinensis on mechanisms relating to osteoclast differentiation was studied. In this results, we identified that Taxillus chinensis significantly inhibited RANKL-induced osteoclast differentiation and bone resportion. Moreover, Taxillus chinensis was suppressed the activation of NF-kB in bone marrow macrophage treated RANKL and M-CSF. Taxillus chinensis was down-regulated the mRNA expression of c-Fos, nuclear factor of activated T-cells (NFAT)c1, osteoclast-associated receptor (OSCAR), tartrate-resistant acid phosphatase (TRAP). The cell adhesion-related molecules such as integrin av and integrin b3, and the filamentous actin (F-actin) rings of mature osteoclasts-related molecules such as dendritic cell-specific transmembrane preotein (DC-STAMP) and cathepsin K are also suppressed. Taken together, these results indicated that Taxillus chinensis will be a good candidate to treat osteoclast-mediated bone diseases.

CD200R/CD200 Inhibits Osteoclastogenesis: New Mechanism of Osteoclast Control by Mesenchymal Stem Cells in Human

Bone homeostasis is maintained by the balance between bone-forming osteoblasts and bone-degrading osteoclasts. Osteoblasts have a mesenchymal origin whereas osteoclasts belong to the myeloid lineage. Osteoclast and osteoblast communication occurs through soluble factors secretion, cell-bone interaction and cell–cell contact, which modulate their activities. CD200 is an immunoglobulin superfamilly member expressed on various types of cells including mesenchymal stem cells (MSCs). CD200 receptor (CD200R) is expressed on myeloid cells such as monocytes/macrophages. We assume that CD200 could be a new molecule involved in the control of osteoclastogenesis and could play a role in MSC–osteoclast communication in humans. In this study, we demonstrated that soluble CD200 inhibited the differentiation of osteoclast precursors as well as their maturation in bone-resorbing cells in vitro. Soluble CD200 did not modify the monocyte phenotype but inhibited the receptor activator of nuclear factor kappa-B ligand (RANKL) signaling pathway as well as the gene expression of osteoclast markers such as osteoclast-associated receptor (OSCAR) and nuclear factor of activated T cells cytoplasmic 1 (NFATc1). Moreover, MSCs inhibited osteoclast formation, which depended on cell–cell contact and was associated with CD200 expression on the MSC surface. Our results clearly demonstrate that MSCs, through the expression of CD200, play a major role in the regulation of bone resorption and bone physiology and that the CD200-CD200R couple could be a new target to control bone diseases.

Monocytic expression of osteoclast-associated receptor (OSCAR) is induced in atherosclerotic mice and regulated by oxidized low-density lipoprotein in vitro

The osteoclast-associated receptor (OSCAR), primarily described as a co-stimulatory regulator of osteoclast differentiation, represents a potential link between bone metabolism and vascular biology. Previously, we identified OSCAR as an endothelial cell-derived target of the proatherogenic factor oxidized low density lipoprotein (oxLDL). Since monocytes play an important role in the progression of atherosclerosis, we assessed whether atherogenic stimuli also regulate the expression of OSCAR on monocytes. Four-week-old male wild-type (WT), apolipoprotein e knockout (apoe KO), and LDL receptor knockout (ldlr KO) mice were fed a high-fat diet or normal chow for 6weeks. Peripheral blood mononuclear cells (PBMCs) isolated from the spleen were stained with antibodies against CD14 and OSCAR for subsequent flow cytometric analysis. OSCAR surface expression on CD14-positive monocytes increased 2-fold in PBMCs from apoe KO mice compared to WT mice. Feeding a high-fat diet further increased OSCAR surface expression 1.5-fold in apoe KO mice compared to normal diet. Moreover, OSCAR-positive macrophages were detected in atherosclerotic plaques of apoe KO mice. Interestingly, monocytic OSCAR expression was not altered in ldlr KO mice. In the murine macrophage cell line RAW 264.7, TNFα and oxLDL induced OSCAR mRNA expression by 2-fold and 5-fold (p<0.01), respectively. Blocking the oxLDL receptor LOX-1 and inhibiting the NF-κB pathway prevented OSCAR induction. In conclusion, OSCAR expression in monocytic cells is regulated by proatherogenic stimuli further pointing towards a role in vascular inflammation or plaque vulnerability during atherosclerosis.

Effects of Osteochondrin S and select connective tissue ribonucleinate components on human osteoclasts in vitro

Osteochondrin S, a natural product derived from connective tissues and yeast, is used to treat osteoarthritis. The aim of this study was to determine the effect of Osteochondrin S on human osteoclast activity in vitro. Osteoclasts were derived from human peripheral blood mononuclear cells stimulated with macrophage colony-stimulating factor and receptor activator of nuclear factor kappa B (RANK) ligand. Cells were treated with 23.5-587.2 ng/ml Osteochondrin S or 0.2-5 mg/ml of RNA components (synovia, placenta, intervertebral disc or cartilage). The effects on osteoclast formation and resorptive activity were assessed. Real-time polymerase chain reaction was conducted to assess the expression of key osteoclast genes. Osteochondrin S and the individual RNA extracts resulted in a concentration-dependent inhibition of human osteoclast activity. Osteochondrin S did not affect RANK, nuclear factor of activated T cells (NFATc1), osteoclast-associated receptor or cathepsin K expression. However, there was a significant (P < 0.05) reduction in mRNA expression of calcitonin receptor. Osteochondrin S treatment also significantly increased the expression of osteoclast inhibitory factor interferon-β and, interestingly, increased the expression of tumour necrosis-α-like weak inducer of apoptosis (TWEAK). Osteochondrin S inhibited the resorptive ability of osteoclasts. These actions are likely to occur at a late stage during osteoclast formation, downstream of NFATc1. Overall, the findings show that Osteochondrin S inhibition of osteoclast activity may be responsible for its beneficial effects on diseases such as osteoarthritis.

Siglec-15 Regulates Osteoclast Differentiation by Modulating RANKL-Induced Phosphatidylinositol 3-Kinase/Akt and Erk Pathways in Association With Signaling Adaptor DAP12

Siglecs are a family of sialic acid-binding immunoglobulin-like lectins that regulate the functions of cells in the innate and adaptive immune systems through glycan recognition. Here we show that Siglec-15 regulates osteoclast development and bone resorption by modulating receptor activator of nuclear factor κB ligand (RANKL) signaling in association with DNAX-activating protein 12 kDa (DAP12), an adaptor protein bearing an immunoreceptor tyrosine-based activation motif (ITAM). Among the known Siglecs expressed in myeloid lineage cells, only Siglec-15 was upregulated by RANKL in mouse primary bone marrow macrophages. Siglec-15-deficient mice exhibit mild osteopetrosis resulting from impaired osteoclast development. Consistently, cells lacking Siglec-15 exhibit defective osteoclast development and resorptive activity in vitro. RANKL-induced activation of phosphatidylinositol 3-kinase (PI3K)/Akt and Erk pathways were impaired in Siglec-15-deficient cells. Retroviral transduction of Siglec-15-null osteoclast precursors with wild-type Siglec-15 or mutant Siglec-15 revealed that the association of Siglec-15 with DAP12 is involved in the downstream signal transduction of RANK. Furthermore, we found that the ability of osteoclast formation is preserved in the region adjacent to the growth plate in Siglec-15-deficient mice, indicating that there is a compensatory mechanism for Siglec-15-mediated osteoclastogenesis in the primary spongiosa. To clarify the mechanism of this compensation, we examined whether osteoclast-associated receptor (OSCAR)/Fc receptor common γ (FcRγ) signaling, an alternative ITAM-mediated signaling pathway to DAP12, rescues impaired osteoclastogenesis in Siglec-15-deficient cells. The ligands in type II collagen activate OSCAR and rescue impaired osteoclastogenesis in Siglec-15-deficient cells when cultured on bone slices, indicating that Siglec-15-mediated signaling can be compensated for by signaling activated by type II collagen and other bone matrix components in the primary spongiosa. Our findings indicate that Siglec-15 plays an important role in physiologic bone remodeling by modulating RANKL signaling, especially in the secondary spongiosa.

Monocytic expression of osteoclast-associated receptor is induced in atherosclerotic mice and regulated by oxidized low-density lipoprotein in vitro

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

Deletion of the receptor tyrosine kinase Tyro3 inhibits synovial hyperplasia and bone damage in arthritis

ObjectiveTo test whether the tyrosine kinase Tyro3 affects arthritis. Tyro3, the ligand of growth arrest–specific protein 6 (GAS6) is a receptor tyrosine kinase involved in cell survival. Tyro3 and GAS6...

Coenzyme Q10 Regulates Osteoclast and Osteoblast Differentiation

Coenzyme Q10 (CoQ10), a powerful antioxidant, is a key component in mitochondrial bioenergy transfer, generating energy in the form of ATP. Many studies suggest that antioxidants act as inhibitors of osteoclastogenesis and we also have previously demonstrated the inhibitory effect of CoQ10 on osteoclast differentiation. Despite the significance of this effect, the molecular mechanism when CoQ10 is present at high concentrations in bone remodeling still remains to be elucidated. In this study, we investigated the inhibitory effect of CoQ10 on osteoclastogenesis and its impact on osteoblastogenesis at concentrations ranging from 10 to 100 μM. We found that nontoxic CoQ10 markedly attenuated the formation of receptor activator of nuclear factor κB ligand (RANKL)-induced tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in both bone-marrow-derived monocytes (BMMs) and RAW 264.7 cells. Osteoclastogenesis with CoQ10 was significantly suppressed the gene expression of NFATc1, TRAP, and osteoclast-associated immunoglobulin-like receptor, which are genetic markers of osteoclast differentiation and scavenged intracellular reactive oxygen species, an osteoclast precursor, in a dose-dependent manner. Furthermore, CoQ10 strongly suppressed H2 O2 -induced IκBα, p38 signaling pathways for osteoclastogenesis. In bone formation study, CoQ10 acted to enhance the induction of osteoblastogenic biomarkers including alkaline phosphatase, type 1 collagen, bone sialoprotein, osteoblast-specific transcription factor Osterix, and Runt-related transcription factor 2 and, also promoted matrix mineralization by enhancing bone nodule formation in a dose-dependent manner. Together, CoQ10 acts as an inhibitor of RANKL-induced osteoclast differentiation and an enhancer of bone-forming osteoblast differentiation. These findings highlight the potential therapeutic applications of CoQ10 for the treatment of bone disease.

The effect of rotative stress on CAII, FAS, FASL, OSCAR, and TRAP gene expression in osteoclasts

This study was designed to explore the effects of rotative stress on carbonic anhydrase II (CAII), TNF receptor superfamily member 6 (FAS), FAS ligand (FASL), osteoclast-associated receptor (OSCAR), and tartrate-resistant acid phosphatase (TRAP) gene expression in osteoclasts. Osteoclasts were induced from RAW264.7 cells cultured in medium containing recombinant murine soluble receptor activator of NF-Kβ ligand (sRANKL). The mRNA and protein expression of CAII, FAS, FASL, OSCAR, and TRAP genes in osteoclasts was detected by RT-PCR and Western blot, respectively, after osteoclasts were loaded at various rotative stress strengths and times. No significant differences in mRNA and protein expression were observed between any of the control groups (P > 0.05). Importantly, rotative stress had a significant effect on the mRNA and protein expression of these genes (P < 0.05). We found a negative relationship between rotative stress strength and prolonged loading time and the expression of FAS/FASL genes in osteoclasts. In addition, there was a positive relationship between rotative stress strength and prolonged loading time and the expression of CAII, OSCAR, or TRAP genes in osteoclasts. Based on these results, rotative stress has a significant effect on CAII, FAS, FASL, OSCAR, and TRAP gene expression in osteoclasts.

Protein phosphatase 2A Cα is involved in osteoclastogenesis by regulating RANKL and OPG expression in osteoblasts

We examined whether alteration of PP2A Cα expression in osteoblasts is involved in osteoclast differentiation. Reduction of PP2A Cα in MC3T3-E1 cells (shPP2A) decreased receptor activator of nuclear factor κB ligand (RANKL) expression and increased osteoprotegerin (OPG) expression. The conditioned medium from shPP2A cells failed to induce NFATc1 as well as the expression of osteoclast marker genes cathepsin K and osteoclast-associated receptor (OSCAR) in bone marrow macrophage cells. Treatment of bone marrow macrophage cells with the conditioned medium from shPP2A cells impaired osteoclastogenesis. These results suggest that alteration of PP2A Cα expression in osteoblasts modulates the expressions of RANKL and OPG, which are involved in osteoclastogenesis via the NFATc1 transcription factor.

Regulation of ITAM adaptor molecules and their receptors by inhibition of calcineurin-NFAT signalling during late stage osteoclast differentiation

Osteoclasts are specialised bone resorptive cells responsible for both physiological and pathological bone loss. Osteoclast differentiation and activity is dependent upon receptor activator NF-kappa-B ligand (RANKL) interacting with its receptor RANK to induce the transcription factor, nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). The immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway has been identified as a co-stimulatory pathway in osteoclasts. Osteoclast-associated receptor (OSCAR) and triggering receptor expressed in myeloid cells (TREM2) are essential receptors that pair with adaptor molecules Fc receptor common gamma chain (FcRγ) and DNAX-activating protein 12kDa (DAP12) respectively to induce calcium signalling. Treatment with calcineurin-NFAT inhibitors, Tacrolimus (FK506) and the 11R-VIVIT (VIVIT) peptide, reduces NFATc1 expression consistent with a reduction in osteoclast differentiation and activity. This study aimed to investigate the effects of inhibiting calcineurin-NFAT signalling on the expression of ITAM factors and late stage osteoclast genes including cathepsin K (CathK), Beta 3 integrin (β3) and Annexin VIII (AnnVIII). Human peripheral blood mononuclear cells (PBMCs) were differentiated with RANKL and macrophage-colony stimulating factor (M-CSF) over 10days in the presence or absence of FK506 or VIVIT. Osteoclast formation (as assessed by tartrate resistant acid phosphatase (TRAP)) and activity (assessed by dentine pit resorption) were significantly reduced with treatment. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis demonstrated that FK506 treatment significantly (p<0.05) reduced the expression of NFATc1, CathK, OSCAR, FcRγ, TREM2 and DAP12 during the terminal stage of osteoclast formation. VIVIT treatment significantly (p<0.05) decreased CathK, OSCAR, FcRγ, and AnnVIII, gene expression. This data suggest FK506 and VIVIT act differently in targeting the calcineurin-NFAT signalling cascade to suppress key mediators of the ITAM pathway during late stage osteoclast differentiation and this is associated with a reduction in both osteoclast differentiation and activity.

Quantitative proteomics reveals novel functions of osteoclast-associated receptor in STAT signaling and cell adhesion in human endothelial cells

Previous studies indicate a novel role for the osteoclast-associated receptor (OSCAR) in oxidative stress-mediated atherogenesis. However, the functional role of OSCAR in endothelial cells is unknown. Here we characterized OSCAR signaling in human endothelial cells using a proteomic approach. OSCAR was either overexpressed or silenced, and the functional effects were assessed by an in-depth proteomic study using stable isotope labeling with amino acids in cell culture (SILAC). Reduction of complexity using subcellular protein fractions from the membrane, the cytosol, and the nucleus of human endothelial cells enabled the detection of 4975 unique proteins. Of these proteins, OSCAR overexpression regulated 145 and OSCAR silencing regulated 110. These proteins were mainly involved in cellular proliferation, inflammatory response and cell-to-cell signaling. Interestingly, OSCAR modulation reciprocally regulated signal transducer and activator of transcription 1 (STAT1) and 3 (STAT3). Thus, STAT1 and several interferon-induced proteins showed a clear inverse correlation to OSCAR expression, which was further verified by Western blot analysis. In contrast, it was found that OSCAR overexpression activated STAT3. Furthermore, OSCAR overexpression increased proteins involved in cell adhesion, which correlated with an increased adhesion of monocytes to the endothelium after OSCAR overexpression. In conclusion, using a comprehensive proteomic approach, endothelial cell-derived OSCAR was found to be involved in the STAT signaling pathway and to affect monocyte adhesion. This indicates a novel role of OSCAR in the vascular-immune cross-talk.

Dietary emu oil supplementation suppresses 5-fluorouracil chemotherapy-induced inflammation, osteoclast formation, and bone loss

Cancer chemotherapy can cause osteopenia or osteoporosis, and yet the underlying mechanisms remain unclear, and currently, no preventative treatments are available. This study investigated damaging effects of 5-fluorouracil (5-FU) on histological, cellular, and molecular changes in the tibial metaphysis and potential protective benefits of emu oil (EO), which is known to possess a potent anti-inflammatory property. Female dark agouti rats were gavaged orally with EO or water (1 ml·day(-1)·rat(-1)) for 1 wk before a single ip injection of 5-FU (150 mg/kg) or saline (Sal) was given. The treatment groups were H(2)O + Sal, H(2)O + 5-FU, EO + 5-FU, and EO + Sal. Oral gavage was given throughout the whole period up to 1 day before euthanasia (days 3, 4, and 5 post-5-FU). Histological analysis showed that H(2)O + 5-FU significantly reduced heights of primary spongiosa on days 3 and 5 and trabecular bone volume of secondary spongiosa on days 3 and 4. It reduced density of osteoblasts slightly and caused an increase in the density of osteoclasts on trabecular bone surface on day 4. EO supplementation prevented reduction of osteoblasts and induction of osteoclasts and bone loss caused by 5-FU. Gene expression studies confirmed an inhibitory effect of EO on osteoclasts since it suppressed 5-FU-induced expression of proinflammatory and osteoclastogenic cytokine TNFα, osteoclast marker receptor activator of nuclear factor-κB, and osteoclast-associated receptor. Therefore, this study demonstrated that EO can counter 5-FU chemotherapy-induced inflammation in bone, preserve osteoblasts, suppress osteoclast formation, and potentially be useful in preventing 5-FU chemotherapy-induced bone loss.