Osteoclast Bone Resorption Activity Research Articles

Effects of cabozantinib on bone turnover markers in patients with metastatic renal cell carcinoma.

638 Background: In-vitro studies have shown that cabozantinib has a strong inhibitory action on osteoclast differentiation and bone-resorption activity. The aim of this analysis was to investigate the effects of cabozantinib on bone turnover markers in patients (pts) with metastatic renal cell carcinoma (mRCC). Methods: We included mRCC pts treated with cabozantinib within the Italian Managed Access Program (MAP) at the Istituto Nazionale Tumori of Milan (Italy). Plasma samples from every patient were collected at baseline and after 3 and 6 months of treatment. The bone resorption markers C-terminal telopeptide of type I collagen (CTx) and tartrate-resistant acid phosphatase isoenzyme 5b (TRACP 5b), the osteoclastogenesis markers osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL), vitamin D and parathormone (PTH) were measured by immunometric assay techniques. Data were analyzed using the RM One-Way ANOVA test with Greenhouse-Geisser correction followed by uncorrected Fisher’s LSD multiple comparison tests with individual variances computed for each comparison. Statistical tests were performed using the program GraphPad Prism (San Diego, CA). Results: Twenty-two pts have been treated with cabozantinib. Mature data were available for 11 pts. Analysis of TRACP 5b, OPG, RANKL and vitamin D did not show any significant variations during treatment with cabozantinib. CTx showed a significant reduction after 6 months of treatment (MV 171.5 pg/mL, STD 208.4) from baseline (mean value [MV] 352.2 pg/mL, standard deviation [STD] 210.1) (p = 0.0439) in the whole study population. PTH levels significantly increased after 3 months of treatment (MV 86.79 pg/mL, STD 34.99) from baseline (MV 30.77 pg/mL, STD 12.64) (p = 0.0003), while significantly decreased after 6 months (MV 75.01 pg/mL, STD 53.1) (p = 0.0174). Conclusions: Our data suggested that cabozantinib significantly reduced bone resorption as demonstrated by the decrease of CTx; it was also associated with an asymptomatic transient secondary increase of parathormone. This is the first clinical evidence on effects of cabozantinib on bone metabolism in a small population of mRCC pts.

The Actin-Binding Protein PPP1r18 Regulates Maturation, Actin Organization, and Bone Resorption Activity of Osteoclasts.

Osteoclasts resorb bone by attaching on the bone matrix and forming a sealing zone. In Src-deficient mice, osteoclasts cannot form the actin ring, a characteristic actin structure that seals the resorbed area, and resorb hardly any bone as a result. However, the molecular mechanism underlying the role of Src in the regulation and organization of the actin ring is still unclear. We identified an actin-regulatory protein, protein phosphatase 1 regulatory subunit 18 (PPP1r18), as an Src-binding protein in an Src-, Yes-, and Fyn-deficient fibroblast (SYF) cell line overexpressing a constitutively active form of Src. PPP1r18 was localized in the nucleus and actin ring. PPP1r18 overexpression in osteoclasts inhibited terminal differentiation, actin ring formation, and bone-resorbing activity. A mutation of the protein phosphatase 1 (PP1)-binding domain of PPP1r18 rescued these phenotypes. In contrast, PPP1r18 knockdown promoted terminal differentiation and actin ring formation. In summary, we showed that PPP1r18 likely plays a role in podosome organization and bone resorption.

Umbilical cord extracts improve osteoporotic abnormalities of bone marrow-derived mesenchymal stem cells and promote their therapeutic effects on ovariectomised rats

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are the most valuable source of autologous cells for transplantation and tissue regeneration to treat osteoporosis. Although BM-MSCs are the primary cells responsible for maintaining bone metabolism and homeostasis, their regenerative ability may be attenuated in postmenopausal osteoporosis patients. Therefore, we first examined potential abnormalities of BM-MSCs in an oestrogen-deficient rat model constructed by ovariectomy (OVX-MSCs). Cell proliferation, mobilisation, and regulation of osteoclasts were downregulated in OVX-MSCs. Moreover, therapeutic effects of OVX-MSCs were decreased in OVX rats. Accordingly, we developed a new activator for BM-MSCs using human umbilical cord extracts, Wharton’s jelly extract supernatant (WJS), which improved cell proliferation, mobilisation and suppressive effects on activated osteoclasts in OVX-MSCs. Bone volume, RANK and TRACP expression of osteoclasts, as well as proinflammatory cytokine expression in bone tissues, were ameliorated by OVX-MSCs activated with WJS (OVX-MSCs-WJ) in OVX rats. Fusion and bone resorption activity of osteoclasts were suppressed in macrophage-induced and primary mouse bone marrow cell-induced osteoclasts via suppression of osteoclast-specific genes, such as Nfatc1, Clcn7, Atp6i and Dc-stamp, by co-culture with OVX-MSCs-WJ in vitro. In this study, we developed a new activator, WJS, which improved the functional abnormalities and therapeutic effects of BM-MSCs on postmenopausal osteoporosis.

Methods to Investigate the Role of Rho GTPases in Osteoclast Function.

The actin cytoskeleton is essential for the biology of osteoclasts, in particular during bone resorption. As key regulators of actin dynamics, the small GTPases of the Rho family are very important in the control of osteoclast activity. The study of Rho GTPase signaling pathways is essential to uncover the mechanisms of bone resorption and can have interesting applications for the treatment of osteolytic diseases. In this chapter, we describe various techniques to obtain primary osteoclasts from murine bone marrow cells, to measure Rho GTPase activation levels, to monitor bone resorption activity of osteoclasts and to introduce the expression of proteins of interest using a retroviral approach. We illustrate the different methods with experimental examples of the effect of Rac1 activation by the exchange factor Dock5 on bone resorption by osteoclasts.

Interferon β protects against avascular osteonecrosis through interleukin 6 inhibition and silent information regulator transcript-1 upregulation

Synovitis of the affected joint is a common in avascular osteonecrosis (AVN). Increased levels of pro-inflammatory cytokine interleukin-6 (IL-6) have been reported in AVN, but the mechanism of this increase remains unclear. Silent information regulator transcript-1 (SIRT1), an NAD-dependent deacetylase, inhibits the release of inflammatory cytokines. Interferon β (IFN-β) has clear anti-inflammatory properties. We sought to investigate the effects of IFN-β treatment on AVN and to evaluate the specific signal pathway relating to IL-6 and SIRT1 affected during AVN. Using a dissection microscope, AVN was surgically induced in the distal femurs of mice. Exogenous IFN-β was administered to the model mice. The effects of exogenous IFN-β on AVN model mice were assessed using hematoxylin eosin and safranin-O staining, and bone resorption activity was measured using tartrate-resistant acid phosphatase (TRAP) and CD68 staining. Western blots, real-time RT-PCR, and immunohistochemical staining were performed to evaluate the production of SIRT1 and IL-6 in tissues. The RAW 264.7 cell line and bone marrow derived osteoclasts treated with exogenous IFN-β. Histological findings indicated well preserved trabecular bone and decreased osteoclast bone resorption activity in IFN-β treated mice compared with mice in the AVN group. Treatment with IFN-β increased SIRT1 expression and inhibited secretion of IL-6 in this AVN mouse model. IFN-β decreased IL-6 secretion by activating SIRT1 in the RAW 264.7 cell and bone marrow derived osteoclasts. Our work suggests that IFN-β could be used to treat AVN and that both SIRT1 and IL-6 are useful targets for treating patients with AVN.

Graphene-Based MicroRNA Transfection Blocks Preosteoclast Fusion to Increase Bone Formation and Vascularization.

The objective of this study is to design a graphene‐based miRNA transfection drug delivery system for antiresorptive therapy. An efficient nonviral gene delivery system is developed using polyethylenimine (PEI) functionalized graphene oxide (GO) complex loaded with miR‐7b overexpression plasmid. GO‐PEI complex exhibits excellent transfection efficiency within the acceptable range of cytotoxicity. The overexpression of miR‐7b after GO‐PEI‐miR‐7b transfection significantly abrogates osteoclast (OC) fusion and bone resorption activity by hampering the expression of an essential fusogenic molecule dendritic cell‐specific transmembrane protein. However, osteoclastogenesis occurs without cell–cell fusion and preosteoclast (POC) is preserved. Through preservation of POC, GO‐PEI‐miR‐7b transfection promotes mesenchymal stem cell osteogenesis and endothelial progenitor cells angiogenesis in the coculture system. Platelet‐derived growth factor‐BB secreted by POC is increased by GO‐PEI‐miR‐7b both in vitro and in vivo. In treating osteoporotic ovariectomized mice, GO‐PEI‐miR‐7b significantly enhances bone mineral density, bone volume as well as bone vascularization through increasing CD31hiEmcnhi cell number. This study provides a cell–cell fusion targeted miRNA transfection drug delivery strategy in treating bone disorders with excessive osteoclastic bone resorption.

The HIV co-receptor CCR5 regulates osteoclast function

C–C chemokine receptor 5 (CCR5) is a co-receptor of HIV. Epidemiological findings suggest that the functional loss of CCR5 is correlated with a lower incidence of bone-destructive diseases as well as of HIV transmission. However, it is not clear whether CCR5 is involved in regulation of the function of bone cells, in addition to that of immune cells. Here we show that blockade of CCR5 using specific antibodies impairs human osteoclast function in vitro. Ccr5-deficient (Ccr5−/−) mice presented with dysfunctional osteoclasts and were resistant to osteoporosis induced by receptor activator of nuclear factor kappa-B ligand (RANKL), which triggers osteoporosis independently of inflammatory and immunomodulatory pathways. Furthermore, Ccr5 deficiency impairs the cellular locomotion and bone-resorption activity of osteoclasts, which is associated with the disarrangement of podosomes and adhesion complex molecules including Pyk2. Overall, the data provides evidence that CCR5 has an essential role in bone-destructive conditions through the functional regulation of osteoclasts.

The effect of icariin on bone metabolism and its potential clinical application.

Osteoporosis is a bone disease characterized by reduced bone mass, which leads to increased risk of bone fractures, and poses a significant risk to public health, especially in the elderly population. The traditional Chinese medicinal herb Epimedii has been utilized for centuries to treat bone fracture and bone loss. Icariin is a prenylated flavonol glycoside isolated from Epimedium herb, and has been shown to be the main bioactive component. This review provides a comprehensive survey of previous studies on icariin, including its structure and function, effect on bone metabolism, and potential for clinical application. These studies show that icariin promotes bone formation by stimulating osteogenic differentiation of BMSCs (bone marrow-derived mesenchymal stem cells), while inhibiting osteoclastogenic differentiation and the bone resorption activity of osteoclasts. Furthermore, icariin has been shown to be more potent than other flavonoid compounds in promoting osteogenic differentiation and maturation of osteoblasts. A 24-month randomized double-blind placebo-controlled clinical trial reported that icariin was effective in preventing postmenopausal osteoporosis with relatively low side effects. In conclusion, icariin may represent a class of flavonoids with bone-promoting activity, which could be used as potential treatment of postmenopausal osteoporosis.

DIHYDROTESTOSTERONE DOWNREGULATES BONE RESORPTION ACTIVITY OF OSTEOCLASTS IN DOSE DEPENDENT MANNER: AN IN VITRO MODEL USING RAW 264.7 CELLS

Objective: Numerous studies have evidenced the bone regulatory potential of dihydrotestosterone in androgen-deficient osteoporosis. The present study was thus aimed to explore the translational mechanism of dihydrotestosterone to down-regulate the bone resorption activity of osteoclasts using RAW 264.7 cells as in vitro model.Methods: Prior to analyze the efficacy of dihydrotestosterone (5α-DHT) to alleviate osteoclastic differentiation, their cell viability and cell proliferative ability was assessed using lactate dehydrogenase (LDH) and MTS assays. The osteoclastic differentiation capacity of dihydrotestosterone was evaluated by measuring TRAP activity and the expression of bone resorption-related proteins such as matrix metallopeptidase-9 (MMP-9), cathepsin-K, tartrate-resistant acid phosphatase (TRAP) and NFATc1. Moreover, the effects of dihydrotestosterone were also evaluated on superoxide (free radicals) generation and superoxide dismutase (SOD) activity in RANKL-induced osteoclasts.Results: Dihydrotestosterone showed no toxicity towards RAW 264.7 cells and significantly enhanced their proliferation and growth rates in a dose-dependent fashion. It was also observed that dihydrotestosterone exhibits a remarkable inhibitory effect on differentiation, maturation and activation of osteoclasts. The marked inhibition of differentiation and activation of osteoclasts caused by 5α-DHT was due to down-regulation of the expression of MMP-9, cathepsin-K, TRAP, NFATc1, generation of superoxide and up-regulation of SOD activity in the RAW 264.7 cells.Conclusion: Resulting data provided substantially in vitroevidence for the pronounced anti-osteoclastogenetic activity of dihydrotestosterone and its therapeutic value in treating osteoporosis and other bone-erosive disorders.

Water extract of Uncaria sinensis suppresses RANKL-induced bone loss by attenuating osteoclast differentiation and bone resorption

BackgroundThe hooks and stems of Uncaria sinensis have been used to mitigate cardiovascular and central nervous system disorders in Asia traditional medicine. Regulation of osteoclast differentiation and activity is a major target for preventing and treating pathological bone diseases. MethodsTartrate-resistant acid phosphatase (TRAP) activity and the number of TRAP-stained multinucleated cells were used to examine receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. The activation of RANKL-induced signaling pathways and the expression of transcription factors were investigated by western blot analysis and quantitative real-time polymerase chain reaction. The bone resorption activity of osteoclast was studied using a plate coated with hydroxyl-apatite. Trabecular bone destruction was investigated using a RANKL-induced trabecular bone loss mouse model. ResultsWe found that water extract of the hooks and stems of U. sinensis (WEUS) inhibits RANKL-induced differentiation of murine bone marrow macrophages and RAW264.7 cells into osteoclasts. WEUS inhibited the activation of NF-κB and the expression of nuclear factor of activated T-cells, cytoplasmic 1. In addition, WEUS suppressed the bone resorbing activity of mature osteoclasts without affecting their survival. Furthermore, oral administration of WEUS suppressed RANKL-induced bone loss with a significant amelioration of trabecular bone micro-structures. WEUS also reduced RANKL-induced increase in serum TRAP5b activity and C-terminal cross-linked telopeptide of type I collagen levels. ConclusionThe present study demonstrates that WEUS has a pharmacological activity that inhibits osteoclast-mediated bone destruction by suppressing osteoclast differentiation and function. These results suggest that U. sinensis could be a promising herbal candidate for preventing and treating bone diseases such as osteoporosis.

Effect of OPG gene mutation on protein expression and biological activity in osteoporosis.

The effect of an osteoprotegerin (OPG) gene mutation was investigated on its protein expression and biological activity in osteoporosis. The pcDNA3.0-OPG plasmid or wild-type plasmid were transfected into HEK293 cells. Osteoclast tartrate-resistant acid phosphatase (TRAP) staining and counting were then performed, and the expression of genes related to osteoclast differentiation and activation were measured by RT-PCR. The wild-type or mutant-type OPG at concentrations of 0, 10, 20, 50 and 100 ng/ml were added respectively to RAW264.7 cells and incubated for 24 h. The viability of cells treated with the wild-type and mutant-type OPG at a concentration of 100 ng/ml was still over 99%, which indicated that the wild-type and mutant-type OPG at this concentration had no cytotoxic effect on RAW264.7 cells. The number of TRAP-positive cells decreased with increasing concentration of wild-type or mutant-type OPG. At the concentrations of 20, 50 and 100 ng/ml, the inhibitory effect of wild-type OPG was significantly higher than that of mutant-type OPG (p<0.05). Both mutant-type and wild-type OPG inhibited the bone resorption activity of osteoclasts, and the inhibitory effect of wild-type OPG was significantly higher than that of mutant-type OPG at the concentrations of 20, 50 and 100 ng/ml (p<0.05). The levels of TRAP and RANK mRNA in the wild-type OPG treatment group were significantly lower than those in the control group, while the levels of TRAP and RANK mRNA in the mutant-type OPG treatment group were significantly lower than those in the wild-type group (p<0.05). The genetic mutation did not affect the protein expression levels of OPG, but inhibited the normal activity of OPG.

NFAM1 signaling enhances osteoclast formation and bone resorption activity in Paget's disease of bone

Paget's disease of bone (PDB) is marked by the focal activity of abnormal osteoclasts (OCLs) with excess bone resorption. We previously detected measles virus nucleocapsid protein (MVNP) transcripts in OCLs from patients with PDB. Also, MVNP stimulates pagetic OCL formation in vitro and in vivo. However, the mechanism by which MVNP induces excess OCLs/bone resorption activity in PDB is unclear. Microarray analysis identified MVNP induction of NFAM1 (NFAT activating protein with ITAM motif 1) expression. Therefore, we hypothesize that MVNP induction of NFAM1 enhances OCL differentiation and bone resorption in PDB. MVNP transduced normal human PBMC showed an increased NFAM1 mRNA expression without RANKL treatment. Further, bone marrow cells from patients with PDB demonstrated elevated levels of NFAM1 mRNA expression. Interestingly, shRNA suppression of NFAM1 inhibits MVNP induced OCL differentiation and bone resorption activity in mouse bone marrow cultures. Live cell widefield fluorescence microscopy analysis revealed that MVNP induced intracellular Ca2+ oscillations and levels were significantly reduced in NFAM1 suppressed preosteoclasts. Further, western blot analysis demonstrates that shRNA against NFAM1 inhibits MVNP stimulated PLCγ, calcineurin, and Syk activation in preosteoclast cells. Furthermore, NFAM1 expression controls NFATc1, a critical transcription factor expression and nuclear translocation in MVNP transuded preosteoclast cells. Thus, our results suggest that MVNP modulation of the NFAM1 signaling axis plays an essential role in pagetic OCL formation and bone resorption activity.

Human Alternative Macrophages Populate Calcified Areas of Atherosclerotic Lesions and Display Impaired RANKL-Induced Osteoclastic Bone Resorption Activity.

Vascular calcification is a process similar to bone formation leading to an inappropriate deposition of calcium phosphate minerals in advanced atherosclerotic plaques. Monocyte-derived macrophages, located in atherosclerotic lesions and presenting heterogeneous phenotypes, from classical proinflammatory M1 to alternative anti-inflammatory M2 macrophages, could potentially display osteoclast-like functions. To characterize the phenotype of macrophages located in areas surrounding the calcium deposits in human atherosclerotic plaques. Macrophages near calcium deposits display an alternative phenotype being both CD68 and mannose receptor-positive, expressing carbonic anhydrase type II, but relatively low levels of cathepsin K. In vitro interleukin-4-polarization of human primary monocytes into macrophages results in lower expression and activity of cathepsin K compared with resting unpolarized macrophages. Moreover, interleukin-4 polarization lowers expression levels of the osteoclast transcriptional activator nuclear factor of activated T cells type c-1, associated with increased gene promoter levels of the transcriptional repression mark H3K27me3 (histone 3 lysine 27 trimethylation). Despite higher expression of the receptor activator of nuclear factor κB receptor, receptor activator of nuclear factor κB ligand/macrophage colony-stimulating factor induction of nuclear factor of activated T cells type c-1 and cathepsin K expression is defective in these macrophages because of reduced Erk/c-fos-mediated downstream signaling resulting in impaired bone resorption capacity. These results indicate that macrophages surrounding calcium deposits in human atherosclerotic plaques are phenotypically defective being unable to resorb calcification.

Actin-binding protein coronin 1A controls osteoclastic bone resorption by regulating lysosomal secretion of cathepsin K

Osteoclasts degrade bone matrix proteins via the secretion of lysosomal enzymes. However, the precise mechanisms by which lysosomal components are transported and fused to the bone-apposed plasma membrane, termed ruffled border membrane, remain elusive. Here, we identified coronin 1A as a negative regulator of exocytotic release of cathepsin K, one of the most important bone-degrading enzymes in osteoclasts. The modulation of coronin 1A expression did not alter osteoclast differentiation and extracellular acidification, but strongly affected the secretion of cathepsin K and osteoclast bone-resorption activity, suggesting the coronin 1A-mediated regulation of lysosomal trafficking and protease exocytosis. Further analyses suggested that coronin 1A prevented the lipidation-mediated sorting of the autophagy-related protein LC3 to the ruffled border and attenuated lysosome–plasma membrane fusion. In this process, the interactions between coronin 1A and actin were crucial. Collectively, our findings indicate that coronin 1A is a pivotal component that regulates lysosomal fusion and the secretion pathway in osteoclast-lineage cells and may provide a novel therapeutic target for bone diseases.

3',4',7,8-Tetrahydroxyflavone inhibits RANKL-induced osteoclast formation and bone resorption.

Osteoclasts, which are specialized bone multinuclear cells, are responsible for bone lytic diseases such as osteoporosis. 3',4',7,8-tetrahydroxyflavone is a flavonoid from Acacia confusa. In the present study, we found that 3',4',7,8-tetrahydroxyflavone markedly inhibited receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastic differentiation from mouse bone marrow-derived macrophages (BMMs). 3',4',7,8-tetrahydroxyflavone also reduced the mRNA expression levels of osteoclastic marker genes including the calcitonin receptor (CTR) and cathepsin K. In addition, 3',4',7,8-tetrahydroxyflavone decreased the bone resorption activity of osteoclasts on dentin slices. We found that 3',4',7,8-tetrahydroxyflavone inhibited RANKL-induced expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1), a key transcription factor of osteoclast differentiation. Furthermore, ectopic overexpression of a constitutively active form of NFATc1 completely rescued the anti-osteoclastogenic effect of 3',4',7,8-tetrahydroxyflavone, suggesting that the anti-osteoclastogenic effect was mainly attributed to the reduction in NFATc1 expression. Taken together, our data suggest that 3',4',7,8-tetrahydroxyflavone inhibits osteoclast differentiation and bone loss and may therefore be considered a promising drug candidate for treating or preventing bone-lytic diseases.

Cabozantinib targets bone microenvironment modulating human osteoclast and osteoblast functions.

Cabozantinib, a c-MET and vascular endothelial growth factor receptor 2 inhibitor, demonstrated to prolong progression free survival and improve skeletal disease-related endpoints in castration-resistant prostate cancer and in metastatic renal carcinoma. Our purpose is to investigate the direct effect of cabozantinib on bone microenvironment using a total human model of primary osteoclasts and osteoblasts.Osteoclasts were differentiated from monocytes isolated from healthy donors; osteoblasts were derived from human mesenchymal stem cells obtained from bone fragments of orthopedic surgery patients. Osteoclast activity was evaluated by tartrate resistant acid phosphatase (TRAP) staining and bone resorption assays and osteoblast differentiation was detected by alkaline phosphatase and alizarin red staining.Our results show that non-cytotoxic doses of cabozantinib significantly inhibit osteoclast differentiation (p=0.0145) and bone resorption activity (p=0.0252). Moreover, cabozantinib down-modulates the expression of osteoclast marker genes, TRAP (p=0.006), CATHEPSIN K (p=0.004) and Receptor Activator of Nuclear Factor k B (RANK) (p=0.001). Cabozantinib treatment has no effect on osteoblast viability or differentiation, but increases osteoprotegerin mRNA (p=0.015) and protein levels (p=0.004) and down-modulates Receptor Activator of Nuclear Factor k B Ligand (RANKL) at both mRNA (p<0.001) and protein levels (p=0.043). Direct cell-to-cell contact between cabozantinib pre-treated osteoblasts and untreated osteoclasts confirmed the indirect anti-resorptive effect of cabozantinib.We demonstrate that cabozantinib inhibits osteoclast functions “directly” and “indirectly” reducing the RANKL/osteoprotegerin ratio in osteoblasts.

Baicalin positively regulates osteoclast function by activating MAPK/Mitf signalling.

Activation of osteoblasts in bone formation and osteoclasts in bone resorption is important during the bone fracture healing process. There has been a long interest in identifying and developing a natural therapy for bone fracture healing. In this study, we investigated the regulation of osteoclast differentiation by baicalin, which is a natural molecule extracted from Eucommiaulmoides (small tree native to China). It was determined that baicalin enhanced osteoclast maturation and bone resorption activity in a dose‐dependent manner. Moreover, this involves the activation of MAPK, increased Mitf nuclear translocation and up‐regulation of downstream osteoclast‐related target genes expression. The baicalin‐induced effect on osteoclast differentiation can be mimicked by specific inhibitors of p‐ERK (U0126) and the Mitf‐specific siRNA, respectively. Protein–ligand docking prediction identified that baicalin might bind to RANK, which is the upstream receptor of p‐ERK/Mitf signalling in osteoclasts. This indicated that RANK might be the binding target of baicalin. In sum, our findings revealed baicalin increased osteoclast maturation and function via p‐ERK/Mitf signalling. In addition, the results suggest that baicalin can potentially be used as a natural product for the treatment of bone fracture.

Picrasidine I from Picrasma Quassioides Suppresses Osteoclastogenesis via Inhibition of RANKL Induced Signaling Pathways and Attenuation of ROS Production

Background/Aims: Osteoporosis is a metabolic bone disorder that tortures about millions of people worldwide. Recent study demonstrated agents derived from picrasma quassioides is a promising drug for targets multiple signaling pathways. However its potential in treatment of bone loss has not been fully understood. Methods: The bone marrow macrophages (BMMs) were cultured and induced with M-CSF and RANKL followed by picrasidine I (PI) treatment. Then the effects of PI on osteoclast formation were evaluated by counting tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. Moreover, effects of PI on bone resorption activity of mature osteoclast were studied through bone resorption pit counting and actin ring structure analysis. Further, the involved potential signaling pathways cross-talking were investigated by performed Western blotting and quantitative real-time PCR examination. Results: Results demonstrated PI strongly inhibited RANKL induced osteoclast formation from its precursors. Mechanistically, the inhibitory effect of PI on osteoclast differentiation was due to the suppression of osteoclastogenic transcription factors, c-Fos and NFATc1. Moreover, PI markedly blocked the RANKL-induced osteoclastogenesis by attenuating MAPKs and NF-κB signaling pathways. In addition, PI decreased the ROS generation in osteoclast and osteoblast. Conclusion: Taken together our data demonstrate that PI has antiosteoclastogenic effect by inhibiting inflammation induced activation of MAPKs, NF-κB and ROS generation followed by suppressing the gene expression of c-Fos and NFATc1 in osteoclast precursors.

The Inactivation of ERK1/2, p38 and NF-kB Is Involved in the Down-Regulation of Osteoclastogenesis and Function by A2B Adenosine Receptor Stimulation.

A2B adenosine receptor (A2BAR) is known to be the regulator of bone homeostasis, but its regulatory mechanisms in osteoclast formation are less well-defined. Here, we demonstrate the effect of A2BAR stimulation on osteoclast differentiation and activity by RANKL. A2BAR was expressed in bone marrow-derived monocyte/macrophage (BMM) and RANKL increased A2BAR expression during osteoclastogenesis. A2BAR stimulation with its specific agonist BAY 60-6583 was sufficient to inhibit the activation of ERK1/2, p38 MAP kinases and NF-κB by RANKL as well as it abrogated cell-cell fusion in the late stage of osteoclast differentiation. Stimulation of A2BAR suppressed the expression of osteoclast marker genes, such as c-Fos, TRAP, Cathepsin-K and NFATc1, induced by RANKL, and transcriptional activity of NFATc1 was also inhibited by stimulation of A2BAR. A2BAR stimulation caused a notable reduction in the expression of Atp6v0d2 and DC-STAMP related to cell-cell fusion of osteoclasts. Especially, a decrease in bone resorption activity through suppression of actin ring formation by A2BAR stimulation was observed. Taken together, these results suggest that A2BAR stimulation inhibits the activation of ERK1/2, p38 and NF-κB by RANKL, which suppresses the induction of osteoclast marker genes, thus contributing to the decrease in osteoclast cell-cell fusion and bone resorption activity.

Crocin inhibits RANKL-induced osteoclast formation and bone resorption by suppressing NF-κB signaling pathway activation

Crocin is a dietary compound with antioxidant and anti-inflammatory properties, but its effects on bone resorption have not been well characterized. Here we address this issue by examining the direct effects of crocin on osteoclast cells in vitro. Osteoclastogenesis was induced by RANKL (receptor activator of NF-κB ligand) in mouse bone marrow-derived macrophages in the absence or presence of crocin at various concentrations. Further, the bone resorption activity of mature osteoclast treated with crocin was assessed by pit assay. Without altering cell viability, crocin was shown to inhibit the differentiation and function of osteoclast cells in a dose-dependent manner. Mechanistically, RANKL-induced NF-κB and NFATc1 activation, the critical signaling pathways for osteoclast differentiation and function, were both repressed by crocin in bone marrow-derived macrophages. Thus, crocin suppresses osteoclastogenesis through direct inhibition of intracellular molecular pathways, which may contribute to future development of anti-bone resorption treatment.