Relevance of an in vitro osteoclastogenesis system to study receptor activator of NF-kB ligand and osteoprotegerin biological activities

Receptor activator of NF-kB Ligand (RANKL) is an essential requirement for osteoclastogenesis and its activity is neutralized by binding to the soluble decoy receptor osteoprotegerin (OPG). The purpose of this work was to study the effects of RANKL and OPG during osteoclastogenesis using the murine monocytic cell line RAW 264.7 that can differentiate into osteoclasts in vitro. RAW 264.7 cells plated at 10(4) cells/cm(2) and cultured for 4 days in the presence of RANKL represent the optimal culture conditions for osteoclast differentiation, with an up-regulation of all parameters related to bone resorption: tartrate resistant acid phosphatase (TRAP), calcitonin receptor (CTR), RANK, cathepsin K, matrix metalloproteinase (MMP)-9 mRNA expressions. RANKL and OPG biological effects vary according to the differentiation state of the cells: in undifferentiated RAW 264.7 cells, TRAP expression was decreased by OPG and RANKL, RANK expression was inhibited by OPG, while MMP-9 and cathepsin K mRNA expressions were not modulated. In differentiated RAW 264.7 cells, RANKL and OPG both exert an overall inhibitory effect on the expression of all the parameters studied. In these experimental conditions, OPG-induced MMP-9 inhibition was abrogated in the presence of a blocking anti-RANKL antibody, suggesting that part of OPG effects are RANKL-dependent.     

Aminothiazoles inhibit RANKL‐ and LPS‐mediated osteoclastogenesis and PGE2 production in RAW 264.7 cells

Periodontitis is characterized by chronic inflammation and osteoclast‐mediated bone loss regulated by the receptor activator of nuclear factor‐κB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG). The aim of this study was to investigate the effect of aminothiazoles targeting prostaglandin E synthase‐1 (mPGES‐1) on RANKL‐ and lipopolysaccharide (LPS)‐mediated osteoclastogenesis and prostaglandin E₂ (PGE₂) production in vitro using the osteoclast precursor RAW 264.7 cells. RAW 264.7 cells were treated with RANKL or LPS alone or in combination with the aminothiazoles 4‐([4‐(2‐naphthyl)‐1,3‐thiazol‐2‐yl]amino)phenol (TH‐848) or 4‐(3‐fluoro‐4‐methoxyphenyl)‐N‐(4‐phenoxyphenyl)‐1,3‐thiazol‐2‐amine (TH‐644). Aminothiazoles significantly decreased the number of multinucleated tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclast‐like cells in cultures of RANKL‐ and LPS‐stimulated RAW 264.7 cells, as well as reduced the production of PGE₂ in culture supernatants. LPS‐treatment induced mPGES‐1 mRNA expression at 16 hrs and the subsequent PGE₂ production at 72 hrs. Conversely, RANKL did not affect PGE₂ secretion but markedly reduced mPGES‐1 at mRNA level. Furthermore, mRNA expression of TRAP and cathepsin K (CTSK) was reduced by aminothiazoles in RAW 264.7 cells activated by LPS, whereas RANK, OPG or tumour necrosis factor α mRNA expression was not significantly affected. In RANKL‐activated RAW 264.7 cells, TH‐848 and TH‐644 down‐regulated CTSK but not TRAP mRNA expression. Moreover, the inhibitory effect of aminothiazoles on PGE₂ production was also confirmed in LPS‐stimulated human peripheral blood mononuclear cell cultures. In conclusion, the aminothiazoles reduced both LPS‐ and RANKL‐mediated osteoclastogenesis and PGE₂ production in RAW 264.7 cells, suggesting these compounds as potential inhibitors for treatment of chronic inflammatory bone resorption, such as periodontitis.     

Osteoclastogenic activity and RANKL expression are inhibited in osteoblastic cells expressing constitutively active Gα12 or constitutively active RhoA

Gα₁₂–RhoA signaling is a parathyroid hormone (PTH)‐stimulated pathway that mediates effects in bone and may influence genetic susceptibility to osteoporosis. To further elucidate effects of the pathway in osteoblasts, UMR‐106 osteoblastic cells were stably transfected with constitutively active (ca) Gα₁₂ or caRhoA or dominant negative (dn) RhoA and co‐cultured with RAW 264.7 cells to determine effects on hormone‐stimulated osteoclastogenesis. Whereas PTH and calcitriol‐stimulated osteoclastogenesis in co‐cultures with UMR‐106 cells expressing pcDNA or dominant negative RhoA, the osteoclastogenic effects of PTH and calcitriol were significantly attenuated when the UMR‐106 cells expressed either caRhoA or caGα₁₂. These inhibitory effects were partially reversed by the Rho kinase inhibitor Y27632. None of the constructs affected osteoclastogenesis in untreated co‐cultures, and the constructs did not inhibit the osteoclastogenic responses to receptor activator of NFκB ligand (RANKL). To investigate the mechanism of the inhibitory effects of caGα₁₂ and caRhoA, expression of RANKL, osteoprotegerin (OPG), osteopontin (OPN), and intercellular adhesion molecule‐1 (ICAM) in response to PTH or calcitriol was examined in the UMR‐106 cells. In the cells expressing pcDNA or dnRhoA, PTH and calcitriol increased RANKL mRNA and decreased OPG mRNA, whereas these effects were absent in the cells expressing caGα₁₂ or caRhoA. Basal expression of RANKL and OPG was unaffected by the constructs. The results suggest that Gα₁₂–RhoA signaling can inhibit hormone‐stimulated osteoclastogenesis by effects on expression of RANKL and OPG. Since PTH can stimulate the Gα₁₂–RhoA pathway, the current findings could represent a homeostatic mechanism for regulating osteoclastogenic action. J. Cell. Biochem. 111: 1531–1536, 2010. © 2010 Wiley‐Liss, Inc.     

mu-Calpain regulates receptor activator of NF-kappaB ligand (RANKL)-supported osteoclastogenesis via NF-kappaB activation in RAW 264.7 cells

To clarify the role of calpain in the receptor activator of NF-kappaB ligand (RANKL)-supported osteoclastogenesis, RANKL-induced calpain activation was examined by using murine RAW 264.7 cells and bone marrow-derived monocyte/macrophage progenitors. We found that calpain activity increased in response to RANKL in both cell types based on alpha-spectrinolysis and that mu-calpain, rather than m-calpain, was activated during RANKL-supported osteoclastogenesis in RAW 264.7 cells. Overexpression of mu-calpain clearly augmented RANKL-supported osteoclastogenesis in RAW 264.7 cells, thereby implicating its pivotal role in this process. Cell-permeable calpain inhibitors, including calpastatin and calpeptin, were sufficient to suppress RANKL-supported osteoclastogenesis based on decreased expression of the osteoclastogenic marker, matrix metalloproteinase 9, and the generation of tartrate-resistant acid phosphatase-positive multinucleated cells in both cell types. Calpain inhibitors suppressed NF-kappaB activation via inhibition of the cleavage of inhibitor of NF-kappaB(IkappaBalpha)in RAW 264.7 cells. Taken together, our findings suggest that mu-calpain is essential to the regulation of RANKL-supported osteoclastogenesis via NF-kappaB activation.     

Taurine inhibits osteoclastogenesis through the taurine transporter

Several studies have suggested a direct link between taurine and bone homeostasis. However, the mechanisms of taurine on the regulation of bone metabolism have not been elucidated. Using a coculture of osteoblasts and bone marrow cells as a model for the study of osteoclastogenesis, RANKL-stimulated RAW264.7 cells and M-CSF- and RANKL-induced bone marrow macrophages were investigated to elucidate the possible roles of taurine in osteoclastogenesis. Taurine inhibited osteoclastogenesis in the coculture of osteoblasts and bone marrow cells, but did not influence the expression of OPG and RANKL in osteoblasts. The taurine transporter (TAUT) expressed by RAW264.7 and bone marrow macrophages exhibited typical taurine uptake activity. Taurine directly reduced osteoclastogenesis in RANKL-stimulated RAW264.7 cells and M-CSF- and RANKL-induced bone marrow macrophages, while TAUT siRNA relieved this effect. Our study demonstrated that taurine directly inhibited osteoclastogenesis through the taurine transporter. Taken together, these data suggest that taurine plays a direct role in bone homeostasis by inhibiting osteoclastogenesis.     

Cellular activity and signaling induced by osteoprotegerin in osteoclasts: involvement of receptor activator of nuclear factor kappaB ligand and MAPK

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of nuclear factor kappaB ligand (RANKL), an inducer of osteoclastogenesis via its receptor RANK. We recently demonstrated that OPG also exerts a direct effect in osteoclasts by regulating protease expression. Herein, we showed that OPG-induced pro-matrix metalloproteinase-9 activity was abolished by ras/MAPK inhibitors in purified osteoclasts. OPG induced the phosphorylation of p38 and ERK1/2 in RAW264.7 cells. Only p38 activation was totally abolished by a blocking anti-RANKL antibody or an excess of RANKL. Surface plasmon resonance experiments revealed that RANK, RANKL and OPG are able to form a tertiary complex. These results suggested a potential formation of a tertiary complex RANK-RANKL-OPG on osteoclasts. Thus, OPG is not only a soluble decoy receptor for RANKL but must be also considered as a direct effector of osteoclast functions.     

Aminothiazoles inhibit osteoclastogenesis and PGE2 production in LPS‐stimulated co‐cultures of periodontal ligament and RAW 264.7 cells,and RANKL‐mediated osteoclastogenesis and bone resorption in PBMCs

Inflammatory mediator prostaglandin E₂ (PGE₂) contributes to bone resorption in several inflammatory conditions including periodontitis. The terminal enzyme, microsomal prostaglandin E synthase‐1 (mPGES‐1) regulating PGE₂ synthesis is a promising therapeutic target to reduce inflammatory bone loss. The aim of this study was to investigate effects of mPGES‐1 inhibitors, aminothiazoles TH‐848 and TH‐644, on PGE₂ production and osteoclastogenesis in co‐cultures of periodontal ligament (PDL) and osteoclast progenitor cells RAW 264.7, stimulated by lipopolysaccharide (LPS), and bone resorption in RANKL‐mediated peripheral blood mononuclear cells (PBMCs). PDL and RAW 264.7 cells were cultured separately or co‐cultured and treated with LPS alone or in combination with aminothiazoles. Multinucleated cells stained positively for tartrate‐resistant acid phosphatase (TRAP) were scored as osteoclast‐like cells. Levels of PGE₂, osteoprotegerin (OPG) and interleukin‐6, as well as mRNA expression of mPGES‐1, OPG and RANKL were analysed in PDL cells. PBMCs were treated with RANKL alone or in combination with aminothiazoles. TRAP‐positive multinucleated cells were analysed and bone resorption was measured by the CTX‐I assay. Aminothiazoles reduced LPS‐stimulated osteoclast‐like cell formation both in co‐cultures and in RAW 264.7 cells. Additionally, aminothiazoles inhibited PGE₂ production in LPS‐stimulated cultures, but did not affect LPS‐induced mPGES‐1, OPG or RANKL mRNA expression in PDL cells. In PBMCs, inhibitors decreased both osteoclast differentiation and bone resorption. In conclusion, aminothiazoles reduced the formation of osteoclast‐like cells and decreased the production of PGE₂ in co‐cultures as well as single‐cell cultures. Furthermore, these compounds inhibited RANKL‐induced bone resorption and differentiation of PBMCs, suggesting these inhibitors for future treatment of inflammatory bone loss such as periodontitis.     

Receptor activator of nuclear factor‐kappa B ligand induces osteoclast formation in RAW 264.7 macrophage cells via augmented production of macrophage–colony‐stimulating factor

RAW 264.7 macrophage cells differentiate into osteoclast‐like cells in the presence of RANKL. Participation of M‐CSF in RANKL‐induced osteoclast formation of RAW 264.7 cells was examined. TRAP‐positive osteoclast‐like cells appeared in RAW 264.7 cells cultured in the presence of RANKL. RANKL‐induced osteoclast formation was markedly inhibited by anti‐M‐CSF antibody. RANKL augmented M‐CSF mRNA expression and M‐CSF production in RAW 264.7 cells. Further, anti‐M‐CSF antibody inhibited the expression of RANK, c‐fms, c‐fos and TRAP mRNA in RANKL‐stimulated RAW 264.7 cells. However, anti‐M‐CSF antibody did not affect the expression of DC‐STAMP in the stimulated cells. Therefore, RANKL was suggested to induce osteoclast formation in RAW 264.7 cells via augmented production of M‐CSF. The putative role of M‐CSF in RANKL‐induced osteoclast formation of RAW 264.7 cells is discussed.     

Mechanisms involved in regulation of osteoclastic differentiation by mechanical stress-loaded osteoblasts

Mechanical stress is known to be important for regulation of bone turnover, though the detailed mechanisms are not fully understood. In the present study, we examined the effect of mechanical stress on osteoblasts using a novel compression model. Mouse osteoblastic MC3T3-E1 cells were embedded in three-dimensional (3D) gels and cultured with continuous compressive force (0–10.0 g/cm²) for 48 h, and the conditioned medium were collected. RAW264.7 cells were then incubated with the conditioned medium for various times in the presence of receptor activator of nuclear factor-κB ligand (RANKL). Conditioned medium was found to inhibit the differentiation of RAW264.7 cells into osteoclasts induced by RANKL via down-regulation of the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6), phosphorylation of IκBα, and nuclear translocation of p50 and p65. Interestingly, the conditioned medium also had a high level of binding activity to RANKL and blocked the binding of RANK to RANKL. Furthermore, the binding activity of conditioned medium to RANKL was reduced when the 3D gel was supplemented with KN-93, an inhibitor of non-canonical Wnt/Ca²⁺ pathway. In addition, expression level of osteoprotegerin (OPG) mRNA was increased in time- and force-dependent manners, and remarkably suppressed by KN-93. These results indicate that osteoblastic cells subjected to mechanical stress produce OPG, which binds to RANKL. Furthermore, this binding activity strongly inhibited osteoclastogenesis through suppression of TRAF6 and the nuclear factor-kappa B (NF-κB) signaling pathway, suggesting that enhancement of OPG expression induced by mechanical stress is dependent on non-canonical Wnt/Ca²⁺ pathway.     

Caffeic acid phenethyl ester,an active component of honeybee propolis attenuates osteoclastogenesis and bone resorption via the suppression of RANKL‐induced NF‐κB and NFAT activity

Receptor activator NF‐κB ligand (RANKL)‐activated signaling is essential for osteoclast differentiation, activation and survival. Caffeic acid phenethyl ester (CAPE), a natural NF‐κB inhibitor from honeybee propolis has been shown to have anti‐tumor and anti‐inflammatory properties. In this study, we investigated the effect of CAPE on the regulation of RANKL‐induced osteoclastogenesis, bone resorption and signaling pathways. Low concentrations of CAPE (<1 µM) dose dependently inhibited RANKL‐induced osteoclastogenesis in RAW264.7 cell and bone marrow macrophage (BMM) cultures, as well as decreasing the capacity of human osteoclasts to resorb bone. CAPE inhibited both constitutive and RANKL‐induced NF‐κB and NFAT activation, concomitant with delayed IκBα degradation and inhibition of p65 nuclear translocation. At higher concentrations, CAPE induced apoptosis and caspase 3 activities of RAW264.7 and disrupts the microtubule network in osteoclast like (OCL) cells. Taken together, our findings demonstrate that inhibition of NF‐κB and NFAT activation by CAPE results in the attenuation of osteoclastogenesis and bone resorption, implying that CAPE is a potential treatment for osteolytic bone diseases. J. Cell. Physiol. 221: 642–649, 2009. © 2009 Wiley‐Liss, Inc.     

Silibinin inhibits osteoclast differentiation mediated by TNF family members

Silibinin is a polyphenolic flavonoid compound isolated from milk thistle (Silybum marianum), with known hepatoprotective, anticarcinogenic, and antioxidant effects. Herein, we show that silibinin inhibits receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis from RAW264.7 cells as well as from bone marrow-derived monocyte/macrophage cells in a dose-dependent manner. Silibinin has no effect on the expression of RANKL or the soluble RANKL decoy receptor osteoprotegerin (OPG) in osteoblasts. However, we demonstrate that silibinin can block the activation of NF-κB, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein (MAP) kinase, and extracellular signal-regulated kinase (ERK) in osteoclast precursors in response to RANKL. Furthermore, silibinin attenuates the induction of nuclear factor of activated T cells (NFAT) c1 and osteoclast-associated receptor (OSCAR) expression during RANKL-induced osteoclastogenesis. We demonstrate that silibinin can inhibit TNF-α-induced osteoclastogenesis as well as the expression of NFATc1 and OSCAR. Taken together, our results indicate that silibinin has the potential to inhibit osteoclast formation by attenuating the downstream signaling cascades associated with RANKL and TNF-α.     

Direct inhibitory and indirect stimulatory effects of RAGE ligand S100 on sRANKL‐induced osteoclastogenesis

Diabetes results in increased fracture risk, and advance glycation endproducts (AGEs) have been implicated in this pathophysiology. S100 proteins are ligands for the receptor of AGEs (RAGE). An intracellular role of the S100 family member S100A4 (Mts1) to suppress mineralization has been described in pre‐osteoblastic MC3T3‐E1 cells. However, S100 proteins could have additional effects on bone. The goal of the current study was to determine effects of increased extracellular S100 on osteoclastogenesis. We first determined the direct effects of S100 on pre‐osteoclast proliferation and osteoclastic differentiation. RANKL‐treated RAW 264.7 cell proliferation and TRAP activity were significantly inhibited by S100, and the number and size of TRAP‐positive multinucleated cells were decreased. We then determined whether S100 could affect osteoclastogenesis by an indirect process by examining effects of conditioned media from S100‐treated MC3T3‐E1 cells on osteoclastogenesis. In contrast to the direct inhibitory effect of S100, the conditioned media promoted RAW 264.7 cell proliferation and TRAP activity, with a trend toward increased TRAP‐positive multinucleated cells. S100 treatment of the MC3T3‐E1 cells for 14 days did not significantly affect alkaline phosphatase, M‐CSF, or OPG gene expression. RANKL was undetectable in both untreated and treated cells. The treatment slightly decreased MC3T3‐E1 cell proliferation. Interestingly, S100 treatment increased expression of RAGE by the MC3T3‐E1 cells. This suggested the possibility that S100 could increase soluble RAGE, which acts as a decoy receptor for S100. This decrease in availability of S100, an inhibitor of pre‐osteoclast proliferation, could contribute to osteoclastogenesis, ultimately resulting in increased bone resorption. J. Cell. Biochem. 107: 917–925, 2009. © 2009 Wiley‐Liss, Inc.     

The role of Mac-1 (CD11b/CD18) in osteoclast differentiation induced by receptor activator of nuclear factor-kappaB ligand

Multinuclear osteoclasts are derived from CD11b-positive mononuclear cells in bone marrow and in circulation. FACS sorting experiments showed impaired osteoclastogenesis in RAW264.7 cells with low CD11b expression. Neutralizing antibodies and siRNA against CD11b inhibited osteoclastogenesis induced by RANKL. Although primary cultured mouse bone marrow macrophages expressed CD11a and CD11b, osteoclastogenesis induced by M-CSF and RANKL was inhibited in the presence of anti-CD11b or anti-CD18 but not anti-CD11a antibodies. Furthermore, anti-CD11b antibodies inhibited NFATc1 expression induced by M-CSF and RANKL in BMMs. These findings suggest, at least partly, an important role of CD11b in osteoclastogenesis.     

Irradiation-induced osteocyte damage promotes HMGB1-mediated osteoclastogenesis in vitro

Irradiation-induced bone loss is widely reported, especially in radiotherapy-induced osteoporosis. In addition to the mechanism of osteogenesis inhibition and osteoclastogenesis promotion, the regulation effect of osteocytes, which also send signals to modulate osteoclastogenesis, should be elucidated. In this study, the effect of irradiation on osteocyte and its accommodation to osteoclastogenesis via the release of high mobility group box 1 (HMGB1) was explored. Furthermore, the control response of HMGB1 inhibitor on receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) expression in osteocyte and osteocyte-induced osteoclastogenesis was assessed. It was observed that irradiated osteocyte-like MLO-Y4 cells exhibited polygonal-shaped morphological changes and shortened dendrites, inhibited cell viability and induced cellular apoptosis, along with the reduction in dendritic E11 protein/messenger RNA expression at a doses of 4 Gy. Additionally, the secretion of HMGB1 in supernatants was promoted, accompanied by the decreased OPG and elevated RANKL expression. When the RAW264.7 cells were cocultured with irradiated MLO-Y4 cells or its conditioned medium, enhanced migration and differentiation of osteoclast precursor was observed, and this difference was alleviated with anti-HMGB1 neutralizing antibody. In conclusion, this study demonstrated that irradiation deteriorated osteocytes’ potential to promote recruitment and differentiation of osteoclast precursor via stimulating HMGB1 release and subsequent elevation of RANKL/OPG level. This study will assist in designing the intervention programs for irradiation-induced bone loss.     

Evaluation of efficacy on RANKL induced osteoclast from RAW264.7 cells

Established RAW264.7 cell lines for osteoclastic differentiation has been widely engaged in bone homeostasis research, however, the efficacy of RANKL independently stimulating has rarely been defined, because protocols were usually developed and modified by various laboratories. Otherwise, problematic issues are also lie in the cell's seeding density, RANKL stimulating time point, and distinguishing osteoclastogenesis ability of RANKL-treated RAW264.7 cells. Therefore, in the current study, we examined the efficacy of various concentrations of RANKL-treated RAW264.7 for its osteoclastic differentiation with or without pretreated other costimulators such as: LPS and/or M-CSF. The oteoclastogenesis ability of RANKL-treated RAW264.7 cells was demonstrated by bone resorption pit, F-actin, and osteoclastogenesis specific marker studies. Besides that, through tartrate-resistant acid phosphatase (TRAP) staining, we clarified to start the treatment with 30 ng/ml RANKL at 12 hr after seeded RAW264.7 with the density of 6.25 × 10 ³ cells/cm ² manifested an significantly increased number of multinucleated osteoclastic cells. Overall, our results establishing an optimal method for RANKL independently inducing RAW 264.7 cell osteoclastic differentiation, which could efficiently generate osteoclasts in vitro for significant advances in our understanding of bone biology.     

Impact of the microgravity environment in a 3-dimensional clinostat on osteoblast- and osteoclast-like cells

Mechanical unloading conditions result in decreases in bone mineral density and quantity, which may be partly attributed to an imbalance in bone formation and resorption. To investigate the effect of mechanical unloading on osteoblast and osteoclast differentiation, and the expression of RANKL and OPG genes in osteoblasts, we used a three-dimensional (3D) clinostat system simulating microgravity to culture MC3T3-E1 and RAW264.7 cells. Long-term exposure (7 days) of MC3T3-E1 cells to microgravity in the 3D clinostat inhibited the expression of Runx2, Osterix, type I collagen alphaI chain, RANKL and OPG genes. Similarly, 3D clinostat exposure inhibited the enhancement of beta3-integrin gene expression, which normally induced by sRANKL stimulation in RAW264.7 cells. These results, taken together, demonstrate that long-term 3D clinostat exposure inhibits the differentiation of MC3T3-E1 cells together with suppression of RANKL and OPG gene expression, as well as the RANKL-dependent cellular fusion of RAW264.7 cells, suggesting that long-term mechanical unloading suppresses bone formation and resorption.