RANK ligand-induced elevation of cytosolic Ca2+ accelerates nuclear translocation of nuclear factor kappa B in osteoclasts

RANK ligand (RANKL) induces activation of NFkappaB, enhancing the formation, resorptive activity, and survival of osteoclasts. Ca(2+) transduces many signaling events, however, it is not known whether the actions of RANKL involve Ca(2+) signaling. We investigated the effects of RANKL on rat osteoclasts using microspectrofluorimetry and patch clamp. RANKL induced transient elevation of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) to maxima 220 nm above basal, resulting in activation of Ca(2+)-dependent K(+) current. RANKL elevated [Ca(2+)](i) in Ca(2+)-containing and Ca(2+)-free media, and responses were prevented by the phospholipase C inhibitor. Suppression of [Ca(2+)](i) elevation using the intracellular Ca(2+) chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) abolished the ability of RANKL to enhance osteoclast survival. Using immunofluorescence, NFkappaB was found predominantly in the cytosol of untreated osteoclasts. RANKL induced transient translocation of NFkappaB to the nuclei, which was maximal at 15 min. or BAPTA delayed nuclear translocation of NFkappaB. Delays were also observed upon inhibition of calcineurin or protein kinase C. We conclude that RANKL acts through phospholipase C to release Ca(2+) from intracellular stores, accelerating nuclear translocation of NFkappaB and promoting osteoclast survival. Such cross-talk between NFkappaB and Ca(2+) signaling provides a novel mechanism for the temporal regulation of gene expression in osteoclasts and other cell types.     

Interaction of Fas ligand and Fas expressed on osteoclast precursors increases osteoclastogenesis

We incidentally found that osteoclast precursors and mature osteoclasts express Fas ligand (FasL) as well as Fas, which was confirmed by flow cytometry, immunofluorescent staining, and RT-PCR. The aim of this study was to determine the role of FasL in differentiation and cell death of osteoclasts. To study the role of FasL in osteoclastogenesis, neutralizing anti-FasL mAb or rFasL was added during receptor activator of NF-kappaB ligand (RANKL)-induced osteoclastogenesis using bone marrow-derived macrophages. Neutralization of endogenous FasL by anti-FasL mAb decreased osteoclastogenesis, whereas rFasL enhanced osteoclast differentiation in a dose-dependent manner. In addition, rFasL up-regulated the secretion of osteoclastogenic cytokines, such as IL-1beta and TNF-alpha, and the activation of NF-kappaB. Functional blocking of IL-1beta and TNF-alpha using IL-1 receptor antagonist and soluble TNFR confirmed that those cytokines mediated the effect of FasL on osteoclastogenesis. The osteoclast precursors were relatively resistant to rFasL-induced apoptosis especially before RANKL treatment, resulting in minimal cell loss by rFasL treatment during osteoclastogenesis. Although rFasL increased the cell death of mature osteoclasts, growth factor withdrawal induced much more cell death. However, anti-FasL mAb did not affect the survival of mature osteoclasts, suggesting that the endogenous FasL does not have a role in the apoptosis of osteoclasts. Finally, in contrast to the effect on apoptosis, rFasL-assisted osteoclastogenesis was not mediated by caspases. In conclusion, FasL has a novel function in bone homeostasis by enhancing the differentiation of osteoclasts, which was not considered previously.     

IL-4 inhibits bone-resorbing activity of mature osteoclasts by affecting NF-kappa B and Ca2+ signaling

IL-4 is an important immune cytokine that regulates bone homeostasis. We investigated the molecular mechanism of IL-4 action on bone-resorbing mature osteoclasts. Using a highly purified population of mature osteoclasts, we show that IL-4 dose-dependently inhibits receptor activator of NF-kappaB ligand (RANKL)-induced bone resorption by mature osteoclasts. We detected the existence of IL-4R mRNA in mature osteoclasts. IL-4 decreases TRAP expression without affecting multinuclearity of osteoclasts, and inhibits actin ring formation and migration of osteoclasts. Interestingly, IL-4 inhibition of bone resorption occurs through prevention of RANKL-induced nuclear translocation of p65 NF-kappaB subunit, and intracellular Ca(2+) changes. Moreover, IL-4 rapidly decreases RANKL-stimulated ionized Ca(2+) levels in the blood, and mature osteoclasts in IL-4 knockout mice are sensitive to RANKL action to induce bone resorption and hypercalcemia. Furthermore, IL-4 inhibits bone resorption and actin ring formation by human mature osteoclasts. Thus, we reveal that IL-4 acts directly on mature osteoclasts and inhibits bone resorption by inhibiting NF-kappaB and Ca(2+) signaling.     

Bone morphogenetic protein-9 activates Smad and ERK pathways and supports human osteoclast function and survival in vitro

BMP-9 is a potent osteogenic factor; however, its effects on osteoclasts, the bone-resorbing cells, remain unknown. To determine the effects of BMP-9 on osteoclast formation, activity and survival, we used human cord blood monocytes as osteoclast precursors that form multinucleated osteoclasts in the presence of RANKL and M-CSF in long-term cultures. BMP-9 did not affect osteoclast formation, but adding BMP-9 at the end of the culture period significantly increased bone resorption compared to untreated cultures, and reduced both the rate of apoptosis and caspase-9 activity. BMP-9 also significantly downregulated the expression of pro-apoptotic Bid, but only after RANKL and M-CSF, which are both osteoclast survival factors, had been eliminated from the culture medium. To investigate the mechanisms involved in the effects of BMP-9, we first showed that osteoclasts expressed some BMP receptors, including BMPR-IA, BMPR-IB, ALK1, and BMPR-II. We also found that BMP-9 was able to induce the phosphorylation of Smad-1/5/8 and ERK 1/2 proteins, but did not induce p38 phosphorylation. Finally, knocking down the BMPR-II receptor abrogated the BMP-9-induced ERK-signaling, as well as the increase in bone resorption. In conclusion, these results show for the first time that BMP-9 directly affects human osteoclasts, enhancing bone resorption and protecting osteoclasts against apoptosis. BMP-9 signaling in human osteoclasts involves the canonical Smad-1/5/8 pathway, and the ERK pathway.     

Reactive oxygen species mediate RANK signaling in osteoclasts

RANKL, a member of tumor necrosis factor (TNF) superfamily, regulates the differentiation, activation, and survival of osteoclasts through binding to its cognate receptor, RANK. RANK can interact with several TNF-receptor-associated factors (TRAFs) and activates signaling molecules including Akt, NF-kappaB, and MAPKs. Although the transient elevation of reactive oxygen species (ROS) by receptor activation has been shown to act as a cellular secondary messenger, the involvement of ROS in RANK signaling pathways has been not characterized. In this study, we found that RANKL stimulated ROS generation in osteoclasts. Pretreatment of osteoclasts with the antioxidants N-acetyl-l-cystein and glutathione reduced RANKL-induced Akt, NF-kappaB, and ERK activation. The reduced NF-kappaB activity by antioxidants was associated with decreased IKK activity and IkappaBalpha phosphorylation. In contrast, antioxidants did not prevent TNF-alpha-induced Akt and NF-kappaB activation. Pretreatment with antioxidants also significantly reduced RANKL-induced actin ring formation, required for bone resorbing activity, and osteoclast survival. Taken together, our results suggest that ROS act as mediators in RANKL-induced signaling pathways and cellular events.     

Phosphatidylinositol 3-kinase coordinately activates the MEK/ERK and AKT/NFkappaB pathways to maintain osteoclast survival

We have examined highly purified osteoclasts that were generated in vitro from murine co-culture of marrow precursors with stromal support cells and have found evidence of activation of the MEK/ERK and AKT/NFkappaB survival pathways. Many mature marrow-derived osteoclasts survived for at least 48 h in culture whether or not they are maintained with stromal cells. Moreover, supplementing purified osteoclasts with RANKL and/or M-CSF had no impact on their survival pattern. In addition, spleen-derived osteoclasts generated with RANKL and M-CSF treatment exhibited a similar survival pattern. Blocking MEK, AKT, or NFkappaB activity resulted in apoptosis of many, but not all, of the osteoclasts in purified marrow-derived osteoclasts, marrow-derived osteoclasts co-cultured with stromal cells, and spleen-derived osteoclasts maintained with RANKL and M-CSF. These data support that both the MEK/ERK and AKT/NFkappaB pathways contribute to osteoclast survival. Since PI3K has been shown to activate either of these pathways, we have examined its role in osteoclast survival. PI3K inhibition caused apoptosis of nearly all osteoclasts in purified and co-cultured marrow-derived osteoclasts and spleen-derived osteoclasts maintained with RANKL and M-CSF. Interestingly, in marrow-derived co-cultures, the apoptotic response was restricted to osteoclasts as there was no evidence of stromal support cell apoptosis. PI3K inhibition also blocked MEK1/2, ERK1/2, and AKT phosphorylation and NFkappaB activation in purified osteoclasts. Simultaneous blockage of both AKT and MEK1/2 caused rapid apoptosis of nearly all osteoclasts, mimicking the response to PI3K inhibition. These data reveal that PI3K coordinately activates two distinct survival pathways that are both important in osteoclast survival.     

The calcium-sensing receptor is involved in strontium ranelate-induced osteoclast apoptosis. New insights into the associated signaling pathways

Strontium ranelate exerts both an anti-catabolic and an anabolic effect on bone cells. To further investigate the molecular mechanism whereby strontium ranelate inhibits bone resorption, we focused our attention on the effects of strontium ranelate on osteoclast apoptosis and on the underlying mechanism(s). Using primary mature rabbit osteoclasts, we demonstrated that strontium (Sro2+) dose-dependently stimulates the apoptosis of mature osteoclasts. As shown previously for calcium (Cao2+), the Sro2+-induced effect on mature osteoclasts is mediated by the Cao2+-sensing receptor, CaR, which in turn stimulates a phospholipase C-dependent signaling pathway and nuclear translocation of NF-kappaB. Unlike Cao2+, however, Sro2+-induced osteoclast apoptosis was shown to depend on PKCbetaII activation and to be independent of inositol 1,4,5-trisphosphate action. As a consequence of these differences in their intracellular signaling pathways, Sro2+ and Cao2+ in combination were shown to exert a greater effect on mature osteoclast apoptosis than did either divalent cation by itself. Altogether, our results show that Sro2+ acts through the CaR and induces osteoclast apoptosis through a signaling pathway similar to but different in certain respects from that of Cao2+. This difference in the respective signaling cascades enables Sro2+ to potentiate Cao2+-induced osteoclast apoptosis and vice versa. In this manner, it is conceivable that Sro2+ and Cao2+ act together to inhibit bone resorption in strontium ranelate-treated patients.     

Effect of high phosphate concentration on osteoclast differentiation as well as bone-resorbing activity

Although high inorganic phosphate (Pi) concentration in culture media directly inhibits generation of new osteoclasts and also inhibits bone resorption by mature osteoclasts, its precise mechanism and the physiological role have not been elucidated. The present study was performed to investigate these issues. Increase in extracellular Pi concentration ([Pi](e)) (2.5-4 mM) concentration dependently inhibited 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] or parathyroid hormone (PTH)-(1-34)-induced osteoclast-like cell formation from unfractionated bone cells in the presence of stromal cells. Increase in [Pi](e) (2.5-4 mM) concentration dependently inhibited 1,25(OH)(2)D(3)-, PTH-(1-34)-, or receptor activator of NF-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF)-induced osteoclast-like cell formation from hemopoietic blast cells in the absence of stromal cells. Increase in [Pi](e) (2.5-4 mM) dose dependently stimulated the expression of osteoprotegerin (OPG) mRNA and increased the expression of OPG mRNA suppressed by PTH-(1-34) or 1,25(OH)(2)D(3) in unfractionated bone cells, while it did not affect RANKL mRNA. Increase in [Pi](e) (2.5-4 mM) concentration dependently inhibited the bone-resorbing activity of isolated rabbit osteoclasts. Increase in [Pi](e) (4 mM) induced the apoptosis of isolated rabbit osteoclasts while it did not affect the apoptosis of osteoclast precursor cells and mouse macrophage-like cell line C7 cells that can differentiate into osteoclasts in the presence of RANKL and M-CSF. These results indicate that increase in [Pi](e) inhibits osteoclast differentiation both by up-regulating OPG expression and by direct action on osteoclast precursor cells. It is also indicated that increase in [Pi](e) inhibits osteoclastic activity at least in part by the direct induction of apoptosis of osteoclasts.     

RANKing intracellular signaling in osteoclasts

RANKL plays a pivotal role in the differentiation, function and survival of osteoclasts, the principal bone-resorbing cells. RANKL exerts the effects by binding RANK, the receptor activator of NF-kappaB, in osteoclasts and its precursors. Upon binding RANKL, RANK activates six major signaling pathways: NFATc1, NF-kappaB, Akt/PKB, JNK, ERK and p38, which play distinct roles in osteoclast differentiation, function and survival. Recent studies have not only provided more insights into RANK signaling but have also revealed that several factors, including INF-gamma, IFN-beta, and ITAM-activated costimulatory signals, regulate osteoclastogenesis via direct crosstalk with RANK signaling. It was recently shown that RANK contains three functional motifs capable of mediating osteoclastogenesis. Moreover, although both IFN-gamma and IFN-beta inhibit osteoclastogenesis, they exert the inhibitory effects by distinct mechanisms. Whereas IFN-gamma has been shown to block osteoclastogenesis by promoting degradation of TRAF6, IFN-beta inhibits osteoclastogenesis by down-regulating c-fos expression. In contrast, the ITAM-activated costimulatory signals positively regulate osteoclastogenesis by mediating the activation of NFATc1 through two ITAM-harboring adaptors: FcRgamma and DAP12. This review is focused on discussing the current understanding of RANK signaling and signaling crosstalk between RANK and the various factors in osteoclasts.     

Osteoprotegerin decreases human osteoclast apoptosis by inhibiting the TRAIL pathway

Osteoprotegerin (OPG) is a secreted decoy receptor that recognizes RANKL, and blocks the interaction between RANK and RANKL, leading to the inhibition of osteoclast differentiation and activation. As OPG is a major inhibitor of bone resorption, we wondered whether OPG could modulate osteoclast survival/apoptosis. Osteoclast apoptosis was evaluated by adding various doses of OPG to human osteoclast cultures obtained from cord blood monocytes. Surprisingly, apoptosis decreased after adding the OPG. We hypothesized that OPG may block its second ligand, TRAIL, which is involved in osteoclast apoptosis. We showed that osteoclasts expressed TRAIL, and that TRAIL levels in the culture medium dose-dependently decreased in presence of OPG, as did the level of activated caspase-8 in osteoclasts. In addition, the expression of TRAIL by osteoclasts was not affected in the presence of OPG. Our findings suggest that OPG inhibits osteoclast apoptosis, at least in part, by binding and thus inhibiting endogenously produced TRAIL in human osteoclast cultures. TRAIL could be an autocrine factor for the regulation of osteoclast survival/apoptosis.     

Cell adhesion is a prerequisite for osteoclast survival

The present study demonstrates that loss of cell adhesion potently promotes apoptosis in osteoclasts, a process termed "anoikis." When purified mature rabbit osteoclasts were cultured on plastic for 18 h, about 25% of them were spontaneously committed to apoptosis. The death rate increased more than twofold, after osteoclasts were subjected to suspension culture in inverted Terasaki plates. The osteoclast anoikis was significantly prevented by bongkrekic acid, an inhibitor of mitochondrial permeability transition (PT), and z-VAD-FMK, a caspase inhibitor, suggesting involvement of mitochondrial PT and caspase activation in the death process. Colony-stimulating factor-1 (CSF-1), receptor activator of NF-kappaB ligand (RANKL), and calcitonin protected adherent osteoclasts, but not floating osteoclasts from anoikis. These data show that adhesion is a primary requirement for osteoclast survival.     

G protein-coupled receptor 119 is involved in RANKL-induced osteoclast differentiation and fusion

G protein-coupled receptor 119 (GPR119) is known to be a promising therapeutic target for type 2 diabetes. Recently, it has been reported that the GPR119 agonist increases bone mineral density in an animal model of diabetes, suggesting that GPR119 may play a key role in bone metabolism. In this study, we investigated the functional role of GPR119 in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. We found that the GPR119 expression was markedly increased in preosteoclasts and then downregulated in mature osteoclasts. Activation of GPR119 with AS1269574, a potent selective agonist for GPR119, inhibited the generation of multinuclear osteoclasts from bone marrow-derived macrophages. Confirming this observation, targeted silencing of GPR119 using short hairpin RNA abrogated the AS1269574-mediated suppressive effect on osteoclast formation. GPR119 activation attenuated the expression of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) and blocked RANKL-stimulated phosphorylation of IκBα, c-Jun N-terminal protein kinase (JNK), and extracellular signal-regulated kinase (ERK) but not p38. In addition, GPR119 activation suppressed preosteoclast fusion by downregulating the expression of the dendritic cell-specific transmembrane (DC-STAMP), a molecule that is essential for cell–cell fusion in osteoclast formation. Furthermore, ectopic expression of DC-STAMP restored AS1269574-mediated inhibition of osteoclast fusion. Taken together, our findings demonstrate that GPR119 plays a negative role in osteoclast differentiation and fusion induced by RANKL, and therefore may represent a potential target for bone resorption-associated diseases.     

The molecular scaffold Gab2 is a crucial component of RANK signaling and osteoclastogenesis

Morphogenesis and remodeling of bone involve synthesis of bone matrix by osteoblasts and coordinate resorption of bone by osteoclasts. Defective bone remodeling caused by altered osteoclast activity underlies a multitude of osteopenic disorders. Receptor activator of NF-kappaB (RANK) and its ligand RANKL have been identified as essential factors involved in osteoclast development and bone remodeling, but their mechanism and interacting factors have not been fully characterized. Here we report that the molecular adapter Grb-2-associated binder-2 (Gab2) associates with RANK and mediates RANK-induced activation of NF-kappaB, Akt and Jnk. Inactivation of the gene encoding Gab2 in mice results in osteopetrosis and decreased bone resorption as a result of defective osteoclast differentiation. We also show that Gab2 has a crucial role in the differentiation of human progenitor cells into osteoclasts. We have thus identified a new, key regulatory scaffold molecule, Gab2, that controls select RANK signaling pathways and is essential for osteoclastogenesis and bone homeostasis.     

Knockdown of TRPV4 suppresses osteoclast differentiation and osteoporosis by inhibiting autophagy through Ca2+–calcineurin–NFATc1 pathway

The aim of this study is to evaluate the effect of transient receptor potential vanilloid 4 (TRPV4) on osteoclast differentiation and osteoporosis, and to investigate the underlying mechanism. The results showed that TRPV4 expression and intracellular Ca²⁺ concentration were significantly upregulated in macrophage colony-stimulating factor (M-CSF)-stimulated and receptor activator of nuclear factor κΒ ligand (RANKL)-stimulated RAW264.7 cells. Furthermore, TRPV4 overexpression further increased the M-CSF- and RANKL-induced number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and expression of osteoclastogenesis-related genes (TRAP, c-Fos, and nuclear factor of activated T cells [NFATc1]), activated the Ca ²⁺–calcineurin–NFATc1 signaling and increased autophagy-related proteins (light chain [LC] 3II and Beclin-1) during osteoclast differentiation. In contrast, TRPV4 knockdown exerted the opposite effects. Mechanically, inhibition of Ca ²⁺–calcineurin–NFATc1 signaling by FK506 or 11R-VIVIT abrogated the TRPV4 overexpression-induced osteoclast differentiation and autophagy induction. Moreover, suppression of autophagy by 3-methyladenine attenuated the TRPV4-induced osteoclast differentiation. In addition, short hairpin RNA TRPV4-lentivirus administration significantly diminished the increased levels of several osteoclastogenesis-related genes (RANKL, TRAP, and tumor necrosis factor-α), alleviated the disturbed microarchitecture of lumbar vertebrae, restored the decreased bone mineral density, ratio of bone volume to total tissue volume, trabecular thickness, and trabecular number, and diminished the increased trabecular separation, in ovariectomy (OVX)-induced osteoporosis mice. Consistent with the in vitro data, TRPV4 knockdown significantly decreased the induced number of TRAP-positive osteoclasts, the increased LC3 and NFATc1 expression in the lumbar vertebrae of OVX mice. In conclusion, TRPV4 knockdown suppresses osteoclast differentiation and osteoporosis by inhibiting autophagy through Ca ²⁺–calcineurin–NFATc1 pathway.     

p44/42 MAPK activation is necessary for receptor activator of nuclear factor-kappaB ligand induction by high extracellular calcium

Although extracellular calcium (Ca(2+)(o)) has been suggested to modulate bone remodeling, the exact mechanism is unclear. This study was performed to explore the signaling pathways of high Ca(2+)(o) that are responsible for controlling the expression of receptor activator of NF-kappaB ligand (RANKL) in mouse osteoblastic cells. As previously reported, high Ca(2+)(o) increased RANKL expression. However, the G protein-coupled Ca(2+)(o)-sensing receptor (CaSR) was not detected in the primary cultured mouse osteoblastic cell. The inhibition of the pertussis-sensitive G protein, phospholipase C, protein kinase C, intracellular calcium mobilization, p38 MAPK, or phosphoinositide 3-kinase did not block RANKL induction caused by high Ca(2+)(o). In contrast, the inhibition of p44/42 MAPK pathway reduced the RANKL expression induced by high Ca(2+)(o). Moreover, high Ca(2+)(o) activated p44/42 MAPK and MEK1/2. These results suggest that RANKL induction by high Ca(2+)(o) might not be mediated by CaSR and its putative downstream signaling pathways, but the pathway employing p44/42 MAPK is involved in the high Ca(2+)(o)-induced RANKL expression in mouse osteoblastic cells.