TNF‐α has both stimulatory and inhibitory effects on mouse monocyte‐derived osteoclastogenesis

Phenotypically different osteoclasts may be generated from different subsets of precursors. To what extent the formation of these osteoclasts is influenced or mediated by the inflammatory cytokine TNF‐α, is unknown and was investigated in this study. The osteoclast precursors early blasts (CD31^hiLy‐6C^?), myeloid blasts (CD31⁺Ly‐6C⁺), and monocytes (CD31^?Ly‐6C^hi) were sorted from mouse bone marrow using flow cytometry and cultured with M‐CSF and RANKL, with or without TNF‐α. Surprisingly, TNF‐α prevented the differentiation of TRAcP⁺ osteoclasts generated from monocytes on plastic; an effect not seen with early blasts and myeloid blasts. This inhibitory effect could not be prevented by other cytokines such as IL‐1β or IL‐6. When monocytes were pre‐cultured with M‐CSF and RANKL followed by exposure to TNF‐α, a stimulatory effect was found. TNF‐α also stimulated monocytes’ osteoclastogenesis when the cells were seeded on bone. Gene expression analysis showed that when TNF‐α was added to monocytes cultured on plastic, RANK, NFATc1, and TRAcP were significantly down‐regulated while TNF‐αR1 and TNF‐αR2 were up‐regulated. FACS analysis showed a decreased uptake of fluorescently labeled RANKL in monocyte cultures in the presence of TNF‐α, indicating an altered ratio of bound‐RANK/unbound‐RANK. Our findings suggest a diverse role of TNF‐α on monocytes’ osteoclastogenesis: it affects the RANK‐signaling pathway therefore inhibits osteoclastogenesis when added at the onset of monocyte culturing. This can be prevented when monocytes were pre‐cultured with M‐CSF and RANKL, which ensures the binding of RANKL to RANK. This could be a mechanism to prevent unfavorable monocyte‐derived osteoclast formation away from the bone.

     

RANKL/RANK/OPG: new therapeutic targets in bone tumours and associated osteolysis

The emergence of the molecular triad osteoprotegerin (OPG)/Receptor Activator of NF-kB (RANK)/RANK Ligand (RANKL) has helped elucidate a key signalling pathway between stromal cells and osteoclasts. The interaction between RANK and RANKL plays a critical role in promoting osteoclast differentiation and activation leading to bone resorption. OPG is a soluble decoy receptor for RANKL that blocks osteoclast formation by inhibiting RANKL binding to RANK. The OPG/RANK/RANKL system has been shown to be abnormally regulated in several malignant osteolytic pathologies such as multiple myeloma [MM, where enhanced RANKL expression (directly by tumour cells or indirectly by stromal bone cells or T-lymphocytes)] plays an important role in associated bone destruction. By contrast, production of its endogenous counteracting decoy receptor OPG is either inhibited or too low to compensate for the increase in RANKL production. Therefore, targeting the OPG/RANK/RANKL axis may offer a novel therapeutic approach to malignant osteolytic pathologies. In animal models, OPG or soluble RANK was shown both to control hypercalcaemia of malignancy and the establishment and progression of osteolytic metastases caused by various malignant tumours. To this day, only one phase I study has been performed using a recombinant OPG construct that suppressed bone resorption in patients with multiple myeloma or breast carcinoma with radiologically confirmed bone lesions. RANK-Fc also exhibits promising therapeutic effects, as revealed in animal models of prostate cancer and multiple myeloma. If the animal results translate to similar clinical benefits in humans, using RANK-Fc or OPG may yield novel and potent strategies for treating patients with established or imminent malignant bone diseases and where standard therapeutic regimens have failed.     

CCL20 chemokine induces both osteoblast proliferation and osteoclast differentiation: Increased levels of CCL20 are expressed in subchondral bone tissue of rheumatoid arthritis patients

We evaluated the role of CCL20 (MIP-3alpha) chemokine in cells directly involved in the remodeling of bone tissue (osteoblasts and osteoclasts) and we confirmed its expression in the subchondral bone tissue of rheumatoid arthritis (RA) patients. The expression of CCL20 and of its receptor CCR6 was evaluated in osteoblasts isolated from bone tissue of post-traumatic (PT) patients. Functional tests were performed to evaluate osteoblast proliferation and matrix protein modulation. Immunohistochemical analysis for CCR6, CCL20, and RANKL was performed on bone samples from RA patients. The role of CCL20 was then analyzed in osteoclast differentiation. We found that in basal conditions CCR6, but not its ligand CCL20, was highly expressed by osteoblasts. Functional analysis on osteoblasts showed that CCL20 significantly increased cellular proliferation but did not affect matrix protein expression. Pro-inflammatory cytokines significantly induced the release of CCL20 and RANKL by human osteoblasts but did not modulate CCR6 expression. Increased expression of CCR6, CCL20, and RANKL was confirmed in RA subchondral bone tissue biopsies. We demonstrated that CCL20 was also an earlier inducer of osteoclast differentiation by increasing the number of pre-osteoclasts, thus favoring cell fusion and MMP-9 release. Our results add new insight to the important role of the CCL20/CCR6, RANKL system in the bone tissue of RA. The contemporary action of CCL20 on osteoblasts and osteoclasts involved in the maintenance of bone tissue homeostasis demonstrates the important role of this compartment in the evolution of RA, by showing a clear uncoupling between new bone formation and bone resorption.     

IL‐17 induces osteoclastogenesis from human monocytes alone in the absence of osteoblasts,which is potently inhibited by anti‐TNF‐α antibody: A novel mechanism of osteoclastogenesis by IL‐17

IL‐17 is a proinflammatory cytokine crucial for osteoclastic bone resorption in the presence of osteoblasts or synoviocytes in rheumatoid arthritis. However, the role of IL‐17 in osteoclastogenesis from human monocytes alone remains unclear. Here, we investigated the role of IL‐17 in osteoclastogenesis from human monocytes alone and the direct effect of infliximab on the osteoclastogenesis induced by IL‐17. Human peripheral blood mononuclear cells (PBMC) were cultured for 3 days with M‐CSF. After non‐adherent cells were removed, IL‐17 was added with either infliximab or osteoprotegerin (OPG). Seven days later, adherent cells were stained for vitronectin receptor. On the other hand, CD11b‐positive monocytes purified from PBMC were also cultured and stained as described above. CD11b‐positive cells were cultured with TNF‐α and receptor activator of NF‐κB ligand (RANKL). In the cultures of both adherent cells and CD11b‐positive cells, IL‐17 dose‐dependently induced osteoclastogenesis in the absence of soluble‐RANKL. OPG or infliximab inhibited IL‐17‐induced osteoclastogenesis. Interestingly, in the culture of CD11b‐positive cells, the osteoclastogenesis was more potently inhibited by infliximab than by OPG. TNF‐α and RANKL synergistically induced osteoclastogenesis. The present study clearly demonstrated the novel mechanism by which IL‐17 directly induces osteoclastogenesis from human monocytes alone. In addition, infliximab potently inhibits the osteoclastogenesis directly induced by IL‐17. J. Cell. Biochem. 108: 947–955, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

The critical role of IL-34 in osteoclastogenesis

It has been widely believed that the cytokines required for osteoclast formation are M-CSF (also known as CSF-1) and RANKL. Recently, a novel cytokine, designated IL-34, has been identified as another ligand of CSF1R. This study was to explore the biological function, specifically osteoclastogenesis and bone metabolism, of the new cytokine. We produced recombinant mouse IL-34 and found that together with RANKL it induces the formation of osteoclasts both from splenocytes as well as dose-dependently from bone marrow cells in mouse and these cells also revealed bone resorption activity. It also promotes osteoclast differentiation from human peripheral blood mononucleated cells. Finally, we show that systemic administration of IL-34 to mice increases the proportion of CD11b+ cells and reduces trabecular bone mass. Our data indicate that IL-34 is another important player in osteoclastogenesis and thus may have a role in bone diseases. Strategies of targeting CSF1/CSF1R have been developed and some of them are already in preclinical and clinical studies for treatment of inflammatory diseases. Our results strongly suggest the need to revisit these strategies as they may provide a new potential pharmaceutical target for the regulation of bone metabolism in addition to their role in the treatment of inflammatory diseases.     

Mangiferin attenuates osteoclastogenesis,bone resorption,and RANKL‐induced activation of NF‐κB and ERK

Osteolytic bone diseases such as osteoporosis have a common pathological feature in which osteoclastic bone resorption outstrips bone synthesis. Osteoclast formation and activation are regulated by receptor activator of nuclear factor κB ligand (RANKL). The induction of RANKL‐signaling pathways occurs following the interaction of RANKL to its cognate receptor, RANK. This specific binding drives the activation of downstream signaling pathways; which ultimately induce the formation and activation of osteoclasts. In this study, we showed that a natural immunomodulator, mangiferin, inhibits osteoclast formation and bone resorption by attenuating RANKL‐induced signaling. Mangiferin diminished the expression of osteoclast marker genes, including cathepsin K, calcitonin receptor, DC‐STAMP, and V‐ATPase d2. Mechanistic studies revealed that mangiferin inhibits RANKL‐induced activation of NF‐κB, concomitant with the inhibition of IκB‐α degradation, and p65 nuclear translocation. In addition, mangiferin also exhibited an inhibitory effect on RANKL‐induced ERK phosphorylation. Collectively, our data demonstrates that mangiferin exhibits anti‐resorptive properties, suggesting the potential application of mangiferin for the treatment and prevention of bone diseases involving excessive osteoclastic bone resorption. J. Cell. Biochem. 112: 89–97, 2011. © 2010 Wiley‐Liss, Inc.     

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.     

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.     

Osteoprotegerin and inflammation

RANK, RANKL, and OPG have well established regulatory effects on bone metabolism. RANK is expressed at very high levels on osteoclastic precursors and on mature osteoclasts, and is required for differentiation and activation of the osteoclast. The ligand, RANKL binds to its receptor RANK to induce bone resorption. RANKL is a transmembrane protein expressed in various cells type and particularly on osteoblast and activated T cells. RANKL can be cleaved and the soluble form is active. Osteoprotegerin decoy receptor (OPG), a member of the TNF receptor family expressed by osteoblasts, strongly inhibits bone resorption by binding with high affinity to its ligand RANKL, thereby preventing RANKL from engaging its receptor RANK. This system is regulated by the calciotropic hormones. Conversely, the effects of RANKL, RANK, and OPG on inflammatory processes, most notably on the bone resorption associated with inflammation, remain to be defined. The RANK system seems to play a major role in modulating the immune system. Activated T cells express RANKL messenger RNA, and knock-out mice for RANKL acquire severe immunological abnormalities and osteopetrosis. RANKL secretion by activated T cells can induce osteoclastogenesis. These mechanisms are enhanced by cytokines such as TNF-alpha, IL-1, and IL-17, which promote both inflammation and bone resorption. Conversely, this system is blocked by OPG, IL-4, and IL-10, which inhibit both inflammation and osteoclastogenesis. These data may explain part of the abnormal phenomena in diseases such as rheumatoid arthritis characterized by both inflammation and destruction. Activated T cells within the rheumatoid synovium express RANKL. Synovial cells are capable of differentiating to osteoclast-like cells under some conditions, including culturing with M-CSF and RANKL. This suggests that the bone erosion seen in rheumatoid arthritis may result from RANKL/RANK system activation by activated T cells. This opens up the possibility that OPG may have therapeutic effects mediated by blockade of the RANKL/RANK system.     

Recent advances in osteoclast biology and pathological bone resorption

The osteoclast is a bone-degrading polykaryon. Recent studies have clarified the differentiation of this cell and the biochemical mechanisms it uses to resorb bone. The osteoclast derives from a monocyte/macrophage precursor. Osteoclast formation requires permissive concentrations of M-CSF and is driven by contact with mesenchymal cells in bone that bear the TNF-family ligand RANKL. Osteoclast precursors express RANK, and the interaction between RANKL and RANK (which is inhibited by OPG) is the major determinant of osteoclast formation. Hormones, such as PTH/PTHrP, glucocorticoids and 1,25(OH)2D3, and humoral factors, including TNFalpha, interleukin-1, TGFss and prostaglandins, influence osteoclast formation by altering expression of these molecular factors. TNFalpha, IL-6 and IL-11 have also been shown to promote osteoclast formation by RANKL-independent processes. RANKL-dependent/independent osteoclast formation is likely to play an important role in conditions where there is pathological bone resorption such as inflammatory arthritis and malignant bone resorption. Osteoclast functional defects cause sclerotic bone disorders, many of which have recently been identified as specific genetic defects. Osteoclasts express specialized proteins including a vacuolar-type H+-ATPase that drives HCl secretion for dissolution of bone mineral. One v-ATPase component, the 116 kD V0 subunit, has several isoforms. Only one isoform, TCIRG1, is up-regulated in osteoclasts. Defects in TCIRG1 are common causes of osteopetrosis. HCl secretion is dependent on chloride channels; a chloride channel homologue, CLCN7, is another common defect in osteopetrosis. Humans who are deficient in carbonic anhydrase II or who have defects in phagocytosis also have variable defects in bone remodelling. Organic bone matrix is degraded by thiol proteinases, principally cathepsin K, and abnormalities in cathepsin K cause another sclerotic bone disorder, pycnodysostosis. Thus, bone turnover in normal subjects depends on relative expression of key cytokines, and defects in osteoclastic turnover usually reflect defects in specific ion transporters or enzymes that play essential roles in bone degradation.     

RANKL与骨重建

骨是一种动态更新的组织,它不断进行骨吸收（bone resorption）与骨形成（bone formation）的平衡,这个过程称之为骨重建（bone remodeling）．核因子κB受体活化因子配体（receptor activator of nuclear factor κB ligand,RANKL）是骨吸收和骨形成耦联的关键,具有诱导破骨细胞（osteoclast, OC）生成、活化,抑制破骨细胞凋亡的作用．RANKL最初发现于活化的T细胞,但骨重建过程中RANKL主要来源于骨细胞、成骨细胞和骨髓基质细胞．RANKL/核因子κB受体活化因子（receptor activator of nuclear factor κB,RANK）/骨保护素（osteoprotegerin, OPG）信号通路在成骨细胞调控破骨细胞生成的过程中起着重要的调节作用,是维持骨重建平衡的关键．本文就RANKL及其在骨中的分子作用机制作一综述.     

Osteoprotegerin inhibit osteoclast differentiation and bone resorption by enhancing autophagy via AMPK/mTOR/p70S6K signaling pathway in vitro

Osteoclasts are highly differentiated terminal cells formed by fusion of hematopoietic stem cells. Previously, osteoprotegerin (OPG) inhibit osteoclast differentiation and bone resorption by blocking receptor activator of nuclear factor-κB ligand (RANKL) binding to RANK indirect mechanism. Furthermore, autophagy plays an important role during osteoclast differentiation and function. However, whether autophagy is involved in OPG-inhibited osteoclast formation and bone resorption is not known. To elucidate the role of autophagy in OPG-inhibited osteoclast differentiation and bone resorption, we used primary osteoclast derived from mice bone marrow monocytes/macrophages (BMM) by induced M-CSF and RANKL. The results showed that autophagy-related proteins expression were upregulated; tartrate-resistant acid phosphatase-positive osteoclast number and bone resorption activity were decreased; LC3 puncta and autophagosomes number were increased and activated AMPK/mTOR/p70S6K signaling pathway. In addition, chloroquine (as the autophagy/lysosome inhibitor, CQ) or rapamycin (as the autophagy/lysosome inhibitor, Rap) attenuated osteoclast differentiation and bone resorption activity by OPG treatment via AMPK/mTOR/p70S6K signaling pathway. Our data demonstrated that autophagy plays a critical role in OPG inhibiting osteoclast differentiation and bone resorption via AMPK/mTOR/p70S6K signaling pathway in vitro.     

The inhibitors of cyclin-dependent kinases and GSK-3β enhance osteoclastogenesis

Osteoclasts are multinucleated cells with bone resorption activity that is crucial for bone remodeling. RANK‐RANKL (receptor activator of nuclear factor κB ligand) signaling has been shown as a main signal pathway for osteoclast differentiation. However, the molecular mechanism and the factors regulating osteoclastogenesis remain to be fully understood. In this study, we performed a chemical genetic screen, and identified a Cdks/GSK-3β (cyclin-dependent kinases/glycogen synthase kinase 3β) inhibitor, kenpaullone, and two Cdks inhibitors, olomoucine and roscovitine, all of which significantly enhance osteoclastogenesis of RAW264.7 cells by upregulating NFATc1 (nuclear factor of activated T cells, cytoplasmic 1) levels. We also determined that the all three compounds increase the number of osteoclast differentiated from murine bone marrow cells. Furthermore, the three inhibitors, especially kenpaullone, promoted maturation of cathepsin K, suggesting that the resorption activity of the resultant osteoclasts is also activated. Our findings indicate that inhibition of GSK-3β and/or Cdks enhance osteoclastogenesis by modulating the RANK–RANKL signaling pathway.     

A RANK/TRAF6-dependent signal transduction pathway is essential for osteoclast cytoskeletal organization and resorptive function

Signaling through receptor activator of nuclear factor-kappaB (RANK) is essential for the differentiation and activation of osteoclasts, the cell principally responsible for bone resorption. Animals genetically deficient in RANK or the cognate RANK ligand are profoundly osteopetrotic because of the lack of bone resorption and remodeling. RANK provokes biochemical signaling via the recruitment of intracellular tumor necrosis factor receptor-associated factors (TRAFs) after ligand binding and receptor oligomerization. To understand the RANK-mediated signal transduction mechanism in osteoclastogenesis, we have designed a system to recapitulate osteoclast differentiation and activation in vitro by transfer of the RANK cDNA into hematopoietic precursors genetically deficient in RANK. Gene transfer of RANK constructs that are selectively incapable of binding different TRAF proteins revealed that TRAF pathways downstream of RANK that affect osteoclast differentiation are functionally redundant. In contrast, the interaction of RANK with TRAF6 is absolutely required for the proper formation of cytoskeletal structures and functional resorptive activity of osteoclasts. Moreover, signaling via the interleukin-1 receptor, which also utilizes TRAF6, rescues the osteoclast activation defects observed in the absence of RANK/TRAF6 interactions. These studies are the first to define the functional domains of the RANK cytoplasmic tail that control specific differentiation and activation pathways in osteoclasts.     

Src‐like adaptor protein regulates osteoclast generation and survival

Src‐like adaptor protein (SLAP) is a hematopoietic adaptor containing Src homology (SH)3 and SH2 motifs and a unique carboxy terminus. Unlike c‐Src, SLAP lacks a tyrosine kinase domain. We investigated the role of SLAP in osteoclast development and resorptive function. Employing SLAP‐deficient mice, we find lack of the adaptor enhances in vitro proliferation of osteoclast precursors in the form of bone marrow macrophages (BMMs), without altering their survival. Furthermore, osteoclastogenic markers appear more rapidly in SLAP?/? BMMs exposed to RANK ligand (RANKL). The accelerated proliferation of M‐CSF‐treated, SLAP‐deficient precursors is associated with enhanced ERK activation. SLAP's role as a mediator of M‐CSF signaling, in osteoclastic cells, is buttressed by complexing of the adaptor protein and c‐Fms in lipid rafts. Unlike c‐Src, SLAP does not impact resorptive function of mature osteoclasts but induces their early apoptosis. Thus, SLAP negatively regulates differentiation of osteoclasts and proliferation of their precursors. Conversely, SLAP decreases osteoclast death by inhibiting activation of caspase 3. These counterbalancing events yield indistinguishable bones of WT and SLAP?/? mice which contain equal numbers of osteoclasts in basal and stimulated conditions. J. Cell. Biochem. 110: 201–209, 2010. © 2010 Wiley‐Liss, Inc.     

RANK ligand

RANK ligand (RANKL), a key mediator of bone resorption in normal and pathological states, is expressed as membrane-bound or soluble forms by tissues as diverse as lymph nodes, spleen, thymus and bone-forming cells. In normal bone turnover and in bone metastasis, RANKL stimulates the formation and activity of bone-removing cells, osteoclasts, by binding to its cognate receptor, RANK, on osteoclasts and their progenitors; these processes are disrupted by binding of RANKL to osteoprotegerin (OPG), a soluble decoy receptor. Whilst no mutations in the RANKL gene have yet been identified in human disease, mutations that result in enhanced RANK signalling through inactivation of OPG or activation of RANK are associated with Juvenile Paget's disease and familial expansile osteolysis, respectively. This review focuses on the central role of RANKL in bone resorption and on the therapeutic targeting of RANKL in osteoporosis, humoral hypercalcaemia of malignancy and bone metastasis.