Calcineurin/NFAT signaling in osteoblasts regulates bone mass

Development and repair of the vertebrate skeleton requires the precise coordination of bone-forming osteoblasts and bone-resorbing osteoclasts. In diseases such as osteoporosis, bone resorption dominates over bone formation, suggesting a failure to harmonize osteoclast and osteoblast function. Here, we show that mice expressing a constitutively nuclear NFATc1 variant (NFATc1(nuc)) in osteoblasts develop high bone mass. NFATc1(nuc) mice have massive osteoblast overgrowth, enhanced osteoblast proliferation, and coordinated changes in the expression of Wnt signaling components. In contrast, viable NFATc1-deficient mice have defects in skull bone formation in addition to impaired osteoclast development. NFATc1(nuc) mice have increased osteoclastogenesis despite normal levels of RANKL and OPG, indicating that an additional NFAT-regulated mechanism influences osteoclastogenesis in vivo. Calcineurin/NFATc signaling in osteoblasts controls the expression of chemoattractants that attract monocytic osteoclast precursors, thereby coupling bone formation and bone resorption. Our results indicate that NFATc1 regulates bone mass by functioning in both osteoblasts and osteoclasts.     

N-[2-(4-benzoyl-1-piperazinyl)phenyl]-2-(4-chlorophenoxy) acetamide is a novel inhibitor of resorptive bone loss in mice

The dynamic balance between bone formation and bone resorption is vital for the retention of bone mass. The abnormal activation of osteoclasts, unique cells that degrade the bone matrix, may result in many bone diseases such as osteoporosis. Osteoporosis, a bone metabolism disease, occurs when extreme osteoclast-mediated bone resorption outstrips osteoblast-related bone synthesis. Therefore, it is of great interest to identify agents that can regulate the activity of osteoclasts and prevent bone loss-induced bone diseases. In this study, we found that N-[2-(4-benzoyl-1-piperazinyl)phenyl]-2-(4-chlorophenoxy) acetamide (PPOAC-Bz) exerted a strong inhibitory effect on osteoclastogenesis. PPOAC-Bz altered the mRNA expressions of several osteoclast-specific marker genes and blocked the formation of mature osteoclasts, suppressing F-actin belt formation and bone resorption activity in vitro. In addition, PPOAC-Bz prevented OVX-induced bone loss in vivo. These findings highlighted the potential of PPOAC-Bz as a prospective drug for the treatment of osteolytic disorders.     

The Proteasome Inhibitor Carfilzomib Suppresses Parathyroid Hormone-induced Osteoclastogenesis through a RANKL-mediated Signaling Pathway

Parathyroid hormone (PTH) induces osteoclast formation and activity by increasing the ratio of RANKL/OPG in osteoblasts. The proteasome inhibitor carfilzomib (CFZ) has been used as an effective therapy for multiple myeloma via the inhibition of pathologic bone destruction. However, the effect of combination of PTH and CFZ on osteoclastogenesis is unknown. We now report that CFZ inhibits PTH-induced RANKL expression and secretion without affecting PTH inhibition of OPG expression, and it does so by blocking HDAC4 proteasomal degradation in osteoblasts. Furthermore, we used different types of culture systems, including co-culture, indirect co-culture, and transactivation, to assess the effect of CFZ on PTH action to induce osteoclastogenesis. Our results demonstrated that CFZ blocks PTH-induced osteoclast formation and bone resorption by its additional effect to inhibit RANKL-mediated IκB degradation and NF-κB activation in osteoclasts. This study showed for the first time that CFZ targets both osteoblasts and osteoclasts to suppress PTH-induced osteoclast differentiation and bone resorption. These findings warrant further investigation of this novel combination in animal models of osteoporosis and in patients.     

Cajaninstilbene acid inhibits osteoporosis through suppressing osteoclast formation and RANKL-induced signaling pathways

Osteoporosis is a form of osteolytic disease caused by an imbalance in bone homeostasis, with reductions in osteoblast bone formation, and augmented osteoclast formation and resorption resulting in reduced bone mass. Cajaninstilbene acid (CSA) is a natural compound derived from pigeon pea leaves. CSA possesses beneficial properties as an anti-inflammatory, antibacterial, antihepatitis, and anticancer agent; however, its potential to modulate bone homeostasis and osteoporosis has not been studied. We observed that CSA has the ability to suppress RANKL-mediated osteoclastogenesis, osteoclast marker gene expression, and bone resorption in a dose-dependent manner. Mechanistically, it was revealed that CSA attenuates RANKL-activated NF-κB and nuclear factor of activated T-cell pathways and inhibited phosphorylation of key signaling mediators c-Fos, V-ATPase-d2, and ERK. Moreover, in osteoclasts, CSA blocked RANKL-induced ROS activity as well as calcium oscillations. We further evaluated the therapeutic effect of CSA in a preclinical mouse model and showed that in vivo treatment of ovariectomized C57BL/6 mice with CSA protects the mice from osteoporotic bone loss. Thus, this study demonstrates that osteolytic bone diseases can potentially be treated by CSA.     

A hybrid peptide derived from cecropin-A and magainin-2 inhibits osteoclast differentiation

The adult skeleton is in a dynamic state, being continually broken down and reformed by the coordinated actions of osteoclasts and osteoblasts. Increased osteoclast activity may contribute to the development of osteoporosis. Therefore, the intervention of osteoclast-mediated bone resorption is considered as an effective therapeutic approach in the treatment of osteoporosis. In the course of searching for agents that inhibit osteoclast differentiation and activation, we found that a novel hybrid peptide P1 derived from cecropin-A and magainin-2 reduced osteoclast differentiation in various osteoclast culture systems. As this peptide had no cytotoxicity on various cultures of primary cells and established cell lines, its inhibitory effect on osteoclastogenesis was not due to general cytotoxicity. The effects of P1 on osteoclasts appear to be mediated through the inhibition of NF-kappaB and JNK activation induced by the osteoclastogenic cytokine, receptor activator of NF-kappaB ligand (RANKL). These results provide an evidence for the potential usefulness of P1 for the treatment of bone-resorbing diseases.     

Dexamethasone inhibits bone resorption by indirectly inducing apoptosis of the bone-resorbing osteoclasts via the action of osteoblastic cells

Although glucocorticoids (GCs) are physiologically essentialfor bone metabolism, it is generally accepted that high dosesof GCs cause bone loss through a combination of decreased boneformation and increased bone resorption. However, the actionof GCs on mature osteoclasts remains contradictory. In thisstudy, we have examined the effect of GCs on osteoclasticbone-resorbing activity and osteoclast apoptosis, by using twodifferent cell types, rabbit unfractionated bone cells andhighly enriched mature osteoclasts (>95% of purity).Dexamethasone (Dex, 10^-10–10^-7 M) inhibited resorption pit formation on a dentine slice by the unfractionated bone cells in a dose- and time-dependent manner.However, Dex had no effect on the bone-resorbing activity of the isolated mature osteoclasts. When the isolated osteoclastswere co-cultured with rabbit osteoblastic cells, the osteoclastic bone resorption decreased in response to Dex,dependent on the number of osteoblastic cells. Like the effecton the bone resorption, Dex induced osteoclast apoptosis in cultures of the unfractionated bone cells, whereas it did not promote the apoptosis of the isolated osteoclasts. An inhibitorof caspases, Z-Asp-CH2-DCB attenuated both the inhibitory effecton osteoclastic bone resorption and the stimulatory effect onthe osteoclast apoptosis. In addition, the osteoblastic cellswere required for the osteoclast apoptosis induced by Dex. These findings indicate that the main target cells of GCs arenon-osteoclastic cells such as osteoblasts and that GCsindirectly inhibit bone resorption by inducing apoptosis ofthe mature osteoclasts through the action of non-osteoclasticcells. This study expands our knowledge about the multifunctional roles of GCs in bone metabolism.     

TRAF Family Member-Associated NF-��B Activator (TANK) Induced by RANKL Negatively Regulates Osteoclasts Survival and Function

Osteoclasts are the principle bone-resorbing cells. Precise control of balanced osteoclast activity is indispensable for bone homeostasis. Osteoclast activation mediated by RANK-TRAF6 axis has been clearly identified. However, a negative regulation-machinery in osteoclast remains unclear. TRAF family member-associated NF-κB activator (TANK) is induced by about 10 folds during osteoclastogenesis, according to a genome-wide analysis of gene expression before and after osteoclast maturation, and confirmed by western blot and quantitative RT-PCR. Bone marrow macrophages (BMMs) transduced with lentivirus carrying tank-shRNA were induced to form osteoclast in the presence of RANKL and M-CSF. Tank expression was downregulated by 90% by Tank-shRNA, which is confirmed by western blot. Compared with wild-type (WT) cells, osteoclastogenesis of Tank-silenced BMMs was increased, according to tartrate-resistant acid phosphatase (TRAP) stain on day 5 and day 7. Number of bone resorption pits by Tank-silenced osteoclasts was increased by 176% compared with WT cells, as shown by wheat germ agglutinin (WGA) stain and scanning electronic microscope (SEM) analysis. Survival rate of Tank-silenced mature osteoclast is also increased. However, acid production of Tank-knockdown cells was not changed compared with control cells. IκBα phosphorylation is increased in tank-silenced cells, indicating that TANK may negatively regulate NF-κB activity in osteoclast. In conclusion, Tank, whose expression is increased during osteoclastogenesis, inhibits osteoclast formation, activity and survival, by regulating NF-κB activity and c-FLIP expression. Tank enrolls itself in a negative feedback loop in bone resorption. These results may provide means for therapeutic intervention in diseases of excessive bone resorption.     

Estrogen directly attenuates human osteoclastogenesis, but has no effect on resorption by mature osteoclasts

Estrogen deficiency arising with the menopause promotes marked acceleration of bone resorption, which can be restored by hormone replacement therapy. The inhibitory effects of estrogen seem to involve indirect cytokine- mediated effects via supporting bone marrow cells, but direct estrogen-receptor mediated effects on the bone-resorbing osteoclasts have also been proposed. Little information is available on whether estrogens modulate human osteoclastogenesis or merely inhibit the functional activity of osteoclasts. To clarify whether estrogens directly modulate osteoclastic activities human CD14+ monocytes were cultured in the presence of M-CSF and RANKL to induce osteoclast differentiation. Addition of 0.1-10 nM 17beta-estradiol to differentiating osteoclasts resulted in a dose-dependent reduction in tartrate resistant acid phosphatase (TRACP) activity reaching 60% at 0.1 nM. In addition, 17beta-estradiol inhibited bone resorption, as measured by the release of the C-terminal crosslinked telopeptide (CTX), by 60% at 0.1 nM, but had no effect on the overall cell viability. In contrast to the results obtained with differentiating osteoclasts, addition of 17beta-estradiol (0.001-10 nM) to mature osteoclasts did not affect bone resorption or TRACP activity. We investigated expression of the estrogen receptors, using immunocytochemistry and Western blotting. We found that ER-alpha is expressed in osteoclast precursors, whereas ER- beta is expressed at all stages, indicating that the inhibitory effect of estrogen on osteoclastogenesis is mediated by ER-alpha for the major part. In conclusion, these results suggest that the in vivo effects of estrogen are mediated by reduction of osteoclastogenesis rather than direct inhibition of the resorptive activity of mature osteoclasts.     

RANKL stimulates inducible nitric-oxide synthase expression and nitric oxide production in developing osteoclasts. An autocrine negative feedback mechanism triggered by RANKL-induced interferon-beta via NF-kappaB that restrains osteoclastogenesis and bone resorption

Nitric oxide (NO) is a multifunctional signaling molecule and a key vasculoprotective and potential osteoprotective factor. NO regulates normal bone remodeling and pathological bone loss in part through affecting the recruitment, formation, and activity of bone-resorbing osteoclasts. Using murine RAW 264.7 and primary bone marrow cells or osteoclasts formed from them by receptor activator of NF-kappaB ligand (RANKL) differentiation, we found that inducible nitric-oxide synthase (iNOS) expression and NO generation were stimulated by interferon (IFN)-gamma or lipopolysaccharide, but not by interleukin-1 or tumor necrosis factor-alpha. Surprisingly, iNOS expression and NO release were also triggered by RANKL. This response was time- and dose-dependent, required NF-kappaB activation and new protein synthesis, and was specifically blocked by the RANKL decoy receptor osteoprotegerin. Preventing RANKL-induced NO (via iNOS-selective inhibition or use of marrow cells from iNOS-/- mice) increased osteoclast formation and bone pit resorption, indicating that such NO normally restrains RANKL-mediated osteoclastogenesis. Additional studies suggested that RANKL-induced NO inhibition of osteoclast formation does not occur via NO activation of a cGMP pathway. Because IFN-beta is also a RANKL-induced autocrine negative feedback inhibitor that limits osteoclastogenesis, we investigated whether IFN-beta is involved in this novel RANKL/iNOS/NO autoregulatory pathway. IFN-beta was induced by RANKL and stimulated iNOS expression and NO release, and a neutralizing antibody to IFN-beta inhibited iNOS/NO elevation in response to RANKL, thereby enhancing osteoclast formation. Thus, RANKL-induced IFN-beta triggers iNOS/NO as an important negative feedback signal during osteoclastogenesis. Specifically targeting this novel autoregulatory pathway may provide new therapeutic approaches to combat various osteolytic bone diseases.     

SLAT negatively regulates RANKL-induced osteoclast differentiation

RANKL induces the formation of osteoclasts, which are responsible for bone resorption. Herein, we investigated the role of SWAP-70-like adapter of T cells (SLAT) in RANKL-induced osteoclastogenesis. Expression levels of SLAT were reduced during RANKL-induced osteoclastogenesis. Overexpression of SLAT in BMMs inhibited TRAP-positive multinuclear osteoclast formation and attenuated the expression of NFATc1, which is an important modulator in osteoclastogenesis. Furthermore, silencing of SLAT by RNA interference enhanced osteoclast formation as well as NFATc1 expression. In addition, SLAT was involved in RANKL-induced JNK activation in osteoclasts. Taken together, our data suggest that SLAT acts as a negative modulator of RANKL-induced osteoclastogenesis.     

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.     

Icariin inhibits RANKL-induced osteoclastogenesis via modulation of the NF-κB and MAPK signaling pathways

The receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-RANK regulatory axis is a major regulator of osteoclast differentiation and activation. Icariin, a flavonol glycoside isolated from the Epimedium herb, has been reported to prevents bone loss in ovariectomized mice and inhibits wear particle-induced osteolysis. However, the molecular mechanism through which icariin inhibits RANKL-induced osteoclastogenesis has not been fully understood. Therefore, we aimed to investigate the effects of icariin on RANKL-induced osteoclastogenesis and to elucidate the mechanism underlying this effect. Our results showed that RANKL-induced osteoclastogenesis was inhibited by icariin in bone marrow macrophages (BMMs) and RAW264.7?cells, and that this effect was due to suppression of NF-κB and mitogen-activated protein kinase (MAPK) activation. In addition, icariin inhibited F-actin ring formation and attenuated the bone resorption ability of mature osteoclasts. Collectively, our results indicate that icariin may be a promising potential candidate for the treatment of osteolytic diseases such as osteoporosis. Moreover, our findings lay the foundation for understanding and intervening in osteoclast-related diseases at the molecular level.     

Linarin and its aglycone acacetin abrogate actin ring formation and focal contact to bone matrix of bone-resorbing osteoclasts through inhibition of αvβ3 integrin and core-linked CD44

BackgroundSince enhanced bone resorption due to osteoclast differentiation and activation cause skeletal diseases, there is a growing need in therapeutics for combating bone-resorbing osteoclasts. Botanical antioxidants are being increasingly investigated for their health-promoting effects on bone. Edible Cirsium setidens contains various polyphenols of linarin, pectolinarin, and apigenin with antioxidant and hepatoprotective effects.PurposeThis study aimed to determine whether linarin present in Cirsium setidens water extracts (CSE) and its aglycone acacetin inhibited osteoclastogenesis of RANKL-exposed RAW 264.7 murine macrophages for 5 days.MethodsThis study assessed the osteoprotective effects of CSE, linarin and acacetin on RANKL-induced differentiation and activation of osteoclasts by using MTT assay, TRAP staining, Western blot analysis, bone resorption assay actin ring staining, adhesion assay and immunocytochemical assay. This study explored the underlying mechanisms of their osteoprotection, and identified major components present in CSE by HPLC analysis.ResultsLinarin and pectolinarin were identified as major components of CSE. Nontoxic linarin and acacetin as well as CSE, but not pectolinarin attenuated the RANKL-induced macrophage differentiation into multinucleated osteoclasts, and curtailed osteoclastic bone resorption through reducing lacunar acidification and bone matrix degradation in the osteoclast-bone interface. Linarin and acacetin in CSE reduced the transmigration and focal contact of osteoclasts to bone matrix-mimicking RGD peptide. Such reduction was accomplished by inhibiting the induction of integrins, integrin-associated proteins of paxillin and gelsolin, cdc42 and CD44 involved in the formation of actin rings. The inhibition of integrin-mediated actin ring formation by linarin and acacetin entailed the disruption of TRAF6-c-Src-PI3K signaling of bone-resorbing osteoclasts. The functional inhibition of c-Src was involved in the loss of F-actin-enriched podosome core protein cortactin-mediated actin assembly due to linarin and acacetin.ConclusionThese observations demonstrate that CSE, linarin and acacetin were effective in retarding osteoclast function of focal adhesion to bone matrix and active bone resorption via inhibition of diffuse cloud-associated αvβ3 integrin and core-linked CD44.     

Multiple roles of M-CSF in human osteoclastogenesis

Although the critical role of M-CSF in osteoclastogenesis is well documented, there has been no detailed analysis of how it regulates human osteoclast formation and function in vitro. We used a human osteoclastogenesis model employing CFU-GM osteoclast precursors cultured for 14 days on dentine with RANKL, with varying exposure to exogenous human M-CSF. Short-term treatment of precursors with M-CSF (10-100 ng/mL) resulted in increased proliferation with or without RANKL. Treatment with M-CSF (1-100 ng/mL) for 14 days caused a biphasic concentration-dependent stimulation of formation, fusion, and resorption peaking at 10-50 ng/mL and almost complete abolition of resorption at 100 ng/mL. Time-course studies using M-CSF (25 ng/mL) showed that osteoclast size, nuclei/cell, and resorption increased with longer duration of M-CSF treatment. When treatment was restricted to the first 4 days, M-CSF (25-100 ng/mL) stimulated formation of normal numbers of osteoclasts that resorbed less. Blockade of endogenous M-CSF signaling with neutralizing M-CSF antibody during the first week of culture extensively inhibited osteoclastogenesis, whereas blockade during the second week produced only a small reduction in resorption. Treatment with M-CSF during the second week of culture caused a small increase in osteoclast number and a concentration-dependent increase in cytoplasmic spreading with inhibition of resorption. We have shown that M-CSF modulates multiple steps of human osteoclastogenesis, including proliferation, differentiation and fusion of precursors. In the later stages of osteoclastogenesis, M-CSF modulates osteoclast-resorbing activity, but is not required for survival. Modulation of M-CSF signaling is a potential therapeutic target for conditions associated with excess bone resorption.     

Brain-type creatine kinase has a crucial role in osteoclast-mediated bone resorption

Osteoclasts differentiate from precursor cells of the monocyte-macrophage lineage and subsequently become activated to be competent for bone resorption through programs primarily governed by receptor activator of nuclear factor-kappaB ligand in cooperation with macrophage colony-stimulating factor. Proteins prominently expressed at late phases of osteoclastogenesis and with a supportive role in osteoclast function are potential therapeutic targets for bone-remodeling disorders. In this study, we used a proteomics approach to show that abundance of the brain-type cytoplasmic creatine kinase (Ckb) is greatly increased during osteoclastogenesis. Decreasing Ckb abundance by RNA interference or blocking its enzymatic activity with a pharmacological inhibitor, cyclocreatine, suppressed the bone-resorbing activity of osteoclasts grown in vitro via combined effects on actin ring formation, RhoA GTPase activity and vacuolar ATPase function. Activities of osteoclasts derived from Ckb-/- mice were similarly affected. In vivo studies showed that Ckb-/- mice were better protected against bone loss induced by ovariectomy, lipopolysaccharide challenge or interleukin-1 treatment than wild-type controls. Furthermore, administration of cyclocreatine or adenoviruses harboring Ckb small hairpin RNA attenuated bone loss in rat and mouse models. Our findings establish an important role for Ckb in the bone-resorbing function of osteoclasts and underscore its potential as a new molecular target for antiresorptive drug development.     

MCP-1-induced human osteoclast-like cells are tartrate-resistant acid phosphatase, NFATc1, and calcitonin receptor-positive but require receptor activator of NFkappaB ligand for bone resorption

MCP-1 (monocyte chemotactic protein-1) is a CC chemokine that is induced by receptor activator of NFkappaB ligand (RANKL) in human osteoclasts. In the absence of RANKL, treatment of human peripheral blood mononuclear cells with macrophage colony-stimulating factor and MCP-1 resulted in tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells that are positive for calcitonin receptor (CTR) and a number of other osteoclast markers, including nuclear factor of activated t cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). Although NFATc1 was strongly induced by MCP-1 and was observed in the nucleus, MCP-1 did not permit the formation of bone-resorbing osteoclasts, although these cells had the typical TRAP(+)/CTR(+) multinuclear phenotype of osteoclasts. Despite a similar appearance to osteoclasts, RANKL treatment was required in order for TRAP(+)/CTR(+) multinuclear cells to develop bone resorption activity. The lack of bone resorption was correlated with a deficiency in expression of certain genes related to bone resorption, such as cathepsin K and MMP9. Furthermore, calcitonin blocked the MCP-1-induced formation of TRAP(+)/CTR(+) multinuclear cells as well as blocking osteoclast bone resorption activity, indicating that calcitonin acts at two stages of osteoclast differentiation. Ablation of NFATc1 in mature osteoclasts did not prevent bone resorption activity, suggesting NFATc1 is involved in cell fusion events and not bone resorption. We propose that the MCP-1-induced TRAP(+)/CTR(+) multinuclear cells represent an arrested stage in osteoclast differentiation, after NFATc1 induction and cellular fusion but prior to the development of bone resorption activity.