Interleukin-4 directly inhibits tumor necrosis factor-alpha-mediated osteoclast formation in mouse bone marrow macrophages

Recently it has been found that osteoclast differentiation is induced by tumor necrosis factor (TNF)-alpha. Interleukin (IL)-4 was reported to suppress osteoclast differentiation and bone resorption. However, no study has investigated the effect of IL-4 on TNF-alpha-induced osteoclast formation. In this study, we investigated whether IL-4 inhibits TNF-alpha-mediated osteoclast formation in mouse bone marrow derived macrophages (BMM). First, IL-4 suppresses RANKL-induced osteoclast formation and bone resorption. Next, when BMM were cultured with TNF-alpha, osteoclast-like cells were formed. When they were cultured with both TNF-alpha and IL-4, osteoclast formation and bone resorption was suppressed by IL-4 in a dose-dependent manner. It has been recently found that TNF-alpha and RANKL synergistically promote osteoclastogenesis. Finally, we investigated whether IL-4 had the ability to inhibit synergistic TNF-alpha and RANKL-induced osteoclastogenesis, with the result that it effectively inhibited the synergistic osteoclast formation in a dose-dependent manner. We conclude that IL-4 can strongly inhibit osteoclast formation that is related to both physiological bone resorption induced by RANKL and pathological bone resorption induced by TNF-alpha.     

Ewing sarcoma cells express RANKL and support osteoclastogenesis

Ewing sarcoma (ES) is a primary malignant round cell tumour of bone characterized by rapid and extensive osteolysis. Cellular mechanisms underlying the rapid bone resorption in ES have not been characterized. Osteoclasts are marrow-derived multinucleated cells that effect tumour osteolysis. The role of ES tumour cells in influencing osteoclast formation and/or directly contributing to the osteolysis in ES has not been determined. Using a tissue culture bioassay, we found that lacunar resorption is not carried out by (CD99(+) ) ES tumour cells, but by (CD68(+) ) macrophage/osteoclast-like cells; this resorption occurred in the absence of the osteoclastogenic factor, receptor activator of nuclear factor κB ligand (RANKL). ES cell lines cultured directly on dentine slices did not resorb the mineral or organic components of the bone matrix. Immunohistochemistry of ES tissue microarrays, western blotting, and RT-PCR studies showed that ES cells strongly expressed both RANKL and macrophage-colony stimulating factor (M-CSF), two major osteoclastogenic factors. When co-cultured with human monocytes, ES cells induced the formation of TRAP(+) osteoclastic cells. Conditioned medium from cultured ES cells did not result in osteoclast formation, indicating that cell-cell contact is required for ES-induced osteoclastogenesis. Our findings indicate that ES cells do not resorb bone directly but that they may support osteoclast formation by a RANKL-dependent mechanism.     

Diminished forkhead box P3/CD25 double-positive T regulatory cells are associated with the increased nuclear factor-kappaB ligand (RANKL+) T cells in bone resorption lesion of periodontal disease

Periodontal disease involves multi-bacterial infections accompanied by inflammatory bone resorption lesions. The abundant T and B lymphocyte infiltrates are the major sources of the osteoclast differentiation factor, receptor activator for nuclear factor-kappaB ligand (RANKL) which, in turn, contributes to the development of bone resorption in periodontal disease. In the present study, we found that the concentrations of RANKL and regulatory T cell (T(reg))-associated cytokine, interleukin (IL)-10, in the periodontal tissue homogenates were correlated negatively, whereas RANKL and proinflammatory cytokine, IL-1beta, showed positive correlation. Also, according to the fluorescent-immunohistochemistry, the frequency of forkhead box P3 (FoxP3)/CD25 double-positive cells was diminished strikingly in the bone resorption lesion of periodontal disease compared to healthy gingival tissue, while CD25 or FoxP3 single positive cells were still observed in lesions where abundant RANKL+ lymphocytes were present. Very importantly, few or no expressions of FoxP3 by the RANKL+ lymphocytes were observed in the diseased periodontal tissues. Finally, IL-10 suppressed both soluble RANKL (sRANKL) and membrane RANKL (mRANKL) expression by peripheral blood mononuclear cells (PBMC) activated in vitro in a bacterial antigen-specific manner. Taken together, these results suggested that FoxP3/CD25 double-positive T(reg) cells may play a role in the down-regulation of RANKL expression by activated lymphocytes in periodontal diseased tissues. This leads to the conclusion that the phenomenon of diminished CD25+FoxP3+ T(reg) cells appears to be associated with the increased RANKL+ T cells in the bone resorption lesion of periodontal disease.     

Stimulation of osteoclast formation by inflammatory synovial fluid

Peri-articular bone resorption is a feature of arthritis due to crystal deposition and rheumatoid disease. Under these conditions, the synovial fluid contains numerous inflammatory cells that produce cytokines and growth factors which promote osteoclast formation. The aim of this study was to determine whether inflammatory synovial fluid stimulates the formation of osteoclasts. Synovial fluid from rheumatoid arthritis (RA), pyrophosphate arthropathy (PPA) and osteoarthritis (OA) patients was added to cultures (n=8) of human peripheral blood mononuclear cells (PBMCs) in the presence and absence of macrophage colony-stimulating factor (M-CSF) and the receptor activator of NF-κB ligand (RANKL). Osteoclast formation was assessed by the formation of cells positive for tartrate-resistant acid phosphatase (TRAP) and vitronectin receptor (VNR) and the extent of lacunar resorption. The addition of 10% OA, RA and PPA synovial fluid to PBMC cultures resulted in the formation of numerous multinucleated or mononuclear TRAP⁺ and VNR⁺ cells which were capable of lacunar resorption. In contrast to PBMC cultures incubated with OA synovial fluid, there was marked stimulation of osteoclast formation and resorption in cultures containing inflammatory RA and PPA synovial fluid which contained high levels of tumour necrosis factor alpha, a factor which is known to stimulate RANKL-induced osteoclast formation.     

IFN-gamma-producing human T cells directly induce osteoclastogenesis from human monocytes via the expression of RANKL

The current study explored our hypothesis that IFN-gamma-producing human T cells inhibit human osteoclast formation. Activated T cells derived from human PBMC were divided into IFN-gamma-producing T cells (IFN-gamma(+) T cells) and IFN-gamma-non-producing T cells (IFN-gamma(-) T cells). IFN-gamma(+) T cells were cultured with human monocytes in the presence of macrophage-CSF alone. The concentration of soluble receptor activator of NF-kappaB ligand (RANKL) and IFN-gamma, and the amount of membrane type RANKL expressed on T cells, were measured by ELISA. In the patients with early rheumatoid arthritis (RA) treated with non-steroidal anti-inflammatory drugs alone, CD4+ T cells expressing both IFN-gamma and RANKL were detected by flow cytometry. Surprisingly, IFN-gamma(+) T cells, but not IFN-gamma(-) T cells, induced osteoclastogenesis from monocytes, which was completely inhibited by adding osteoprotegerin and increased by adding anti-IFN-gamma antibodies. The levels of both soluble and membrane type RANKL were elevated in IFN-gamma(+) T cells. The ratio of CD4+ T cells expressing both IFN-gamma and RANKL in total CD4+ T cells from PBMC was elevated in RA patients. Contrary to our hypothesis, IFN-gamma(+) human T cells induced osteoclastogenesis through the expression of RANKL, suggesting that Th1 cells play a direct role in bone resorption in Th1 dominant diseases such as RA.     

Participation of MicroRNA-34a and RANKL on bone repair induced by poly(vinylidene-trifluoroethylene)/barium titanate membrane

The poly(vinylidene-trifluoroethylene)/barium titanate (PVDF) membrane enhances in vitro osteoblast differentiation and in vivo bone repair. Here, we hypothesized that this higher bone repair could be also due to bone resorption inhibition mediated by a microRNA (miR)/RANKL circuit. To test our hypothesis, the large-scale miR expression of bone tissue grown on PVDF and polytetrafluoroethylene (PTFE) membranes was evaluated to identify potential RANKL-targeted miRs modulated by PVDF. The animal model used was rat calvarial defects implanted with either PVDF or PTFE. At 4 and 8 weeks, the bone tissue grown on membranes was submitted to a large-scale analysis of miRs by microarray. The expression of miR-34a and some of its targets, including RANKL, were evaluated by real-time polimerase chain reaction and osteoclast activity was detected by tartrate-resistant acid phosphatase (TRAP) staining. Among more than 250 miRs, twelve, including miR-34a, were simultaneously higher expressed (≥2 fold) at 4 and 8 weeks on PVDF. The higher expression of miR-34a was concomitant with a reduced expression of all its evaluated targets, including RANKL. Additionally, more TRAP-positive cells were observed in bone tissue grown on PTFE compared with PVDF in both time points. In conclusion, our results suggest that the higher bone formation induced by PVDF could be, at least in part, triggered by a miR-34a increase and RANKL decrease, which may inhibit osteoclast differentiation and activity, and bone resorption.     

Lymphocytes and synovial fluid fibroblasts support osteoclastogenesis through RANKL, TNFalpha, and IL-7 in an in vitro model derived from human psoriatic arthritis

Psoriatic arthritis (PsA) is an inflammatory joint disease, characterized by extensive bone resorption, whose mechanisms have not been fully elucidated. Thus, in the present study we investigated the involvement of RANKL, TNFalpha, and IL-7 in the osteoclastogenesis of PsA patients. In vitro osteoclastogenesis models, consisting of unfractionated and T-cell-depleted mononuclear cells from peripheral blood (PBMCs) and synovial fluid (SFMCs) of 20 PsA patients as well as from healthy donors were studied. Freshly isolated T and B cells from PBMCs and T cells and fibroblasts from SFMCs of PsA patients were subjected to RT-PCR to detect the levels of RANKL, TNFalpha, and IL-7. Osteoclastogenesis was studied in the presence of RANK-Fc, anti-TNFalpha, and anti IL-7 functional antibodies. We demonstrate that lymphocytes and fibroblasts support osteoclast (OC) formation in PsA patients through the production of osteoclastogenic cytokines. In particular, OC formation was completely abolished in unstimulated T cell-depleted PBMC cultures, and reduced by approximately 70% in unstimulated T cell-depleted SFMC cultures. Freshly isolated T cells from PBMCs and SFMCs of PsA patients overexpressed RANKL and TNFalpha, while fibroblasts from synovial fluid produced only RANKL. We show that the presence of RANK-Fc and/or anti-TNFalpha functional antibodies reduced OC formation. Moreover, T and B cells from PBMCs as well as T cells and fibroblasts from SFMCs expressed IL-7 mRNA. Finally, the anti-IL-7 functional antibody significantly reduced osteoclastogenesis. Our results suggest that fibroblasts, B and T lymphocytes support OC formation by producing RANKL, TNFalpha, and IL-7, contributing to the aggressive bone resorption in PsA patients.     

Proinflammatory cytokine (TNFalpha/IL-1alpha) induction of human osteoclast formation

TNFalpha and IL-1alpha are potent stimulators of bone resorption in vivo and in vitro. Recently, it has been demonstrated that these two cytokines directly induce osteoclastogenesis in mouse marrow cultures. This study determined whether TNFalpha (+/- IL-1alpha) is also capable of inducing human osteoclastogenesis. The CD14(+) monocyte fraction of human peripheral mononuclear cells was cultured with TNFalpha +/- IL-1alpha in the presence of M-CSF. TNFalpha induced the formation of multinucleated cells (MNCs) which were positive for TRAP, VNR and cathepsin K and showed evidence of resorption pit formation. IL-1alpha stimulated TNFalpha-induced lacunar resorption two- to four-fold. Osteoprotegerin, the decoy receptor for RANKL, did not inhibit this process. Anti-human IL-1alpha neutralizing antibodies significantly inhibited resorption without inhibiting the formation of TRAP(+)/VNR(+) MNCs. These results suggest that, in the presence of M-CSF, TNFalpha is sufficient for inducing human osteoclast differentiation from circulating precursors by a process which is distinct from the RANK/RANKL signalling pathway.     

Inhibition of RANK/RANKL signal transduction pathway: a promising approach for osteoporosis treatment

Osteoporosis is a bone disease causing impaired bone strength. It is characterized by increased osteoclast formation or enhanced bone resorption, leading to an increased risk of fragility fractures. Its prevalence increases with age. The advent of an aging population suggests that progressively more individuals will develop this disease in the aging population. A number of drugs for the prevention and treatment of osteoporosis act by inhibiting bone resorption. However, the effectiveness of osteoporosis treatment in clinical practice is limited. Since the osteoclast is the only cell in the body that is capable of resorbing bone, understanding its biology will be necessary for developing a new therapeutic approach for osteoporosis. Recently, it was discovered that the receptor activator of nuclear factor kappaB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system is an important signal transduction pathway that regulates osteoclast formation. The binding of OPG to RANKL inhibits the binding between RANKL and RANK; this, in turn, prevents osteoclast precursors from differentiating and fusing to form mature osteoclasts. Therefore, the inhibition of the RANK/RANKL pathway inhibits osteoclast formation, differentiation, activation, and bone resorption. A potential clinical antiresorptive therapy can be developed by using an anti-RANKL monoclonal antibody, such as denosumab, that binds to RANKL with high affinity and specificity and blocks RANKL-RANK interactions.     

Osteoclastogenesis is enhanced by activated B cells but suppressed by activated CD8(+) T cells.

Host immune response is known to contribute to the progression of periodontitis, and alveolar bone destruction in periodontitis is associated with enhanced osteoclast activity. Therefore, we evaluated the roles of activated lymphocyte subsets in osteoclastogenesis. Osteoclast precursors were co-cultured with activated lymphocytes (B, CD4(+) T, CD8(+) T) in the presence of either macrophage colony-stimulating factor (M-CSF) alone or M-CSF plus soluble receptor activator of NF-kappaB ligand (sRANKL), and subsequent differentiation into active osteoclasts was evaluated by a resorption assay. The activated B and CD4(+) cells, but not CD8(+) T cells, induced osteoclast differentiation in the presence of M-CSF alone. In the presence of M-CSF and sRANKL, B cells induced the formation of small but highly active osteoclasts and increased resorption, while CD8(+) T cells profoundly suppressed osteoclastogenesis. Co-culture using an insert well or supernatant suggested that both B and CD8(+) T cells acted on osteoclasts mostly via soluble proteins. Activated B cells expressed many osteoclastogenic factors including RANKL, TNF-alpha, IL-6, MIP-1alpha, and MCP-3. CD8(+) T cells expressed a substantial amount of osteoprotegerin (OPG) along with RANKL. However, blocking antibody to OPG did not reverse the suppression by CD8(+) T cells, suggesting that other factor(s) are involved. Taken together, activated B cells promoted osteoclastogenesis, while CD8(+) T cells inhibited the osteoclast formation via direct interaction. The results imply the importance of lymphocyte subpopulations in the development of periodontitis.     

Mitogen- and stress-activated protein kinase 1 activates osteoclastogenesis in vitro and affects bone destruction in vivo

Mitogen- and stress-activated protein kinase (MSK) 1 is an important regulator of immune response and mitogenic signaling. In this study, we report for the first time that MSK1 was activated by the osteoclast differentiation factor receptor activator of nuclear factor kappa B ligand (RANKL) in osteoclast precursor cells. Inhibition of upstream kinases ERK1/2 and p38, but not JNK, suppressed MSK activation upon RANKL stimulation. An MSK1 inhibitor efficiently prevented the induction of c-Fos and NFATc1 and CREB phosphorylation by RANKL. Inhibition of MSK1 also successfully blocked RANKL-induced osteoclastogenesis. MSK knockdown with small interfering RNA significantly inhibited osteoclast differentiation and bone resorption. MSK1 did not affect osteoclast survival. The induction of c-Fos and NFATc1 and the phosphorylation of CREB and ATF2 were also inhibited by MSK1 knockdown. Moreover, knockdown of MSK1 significantly blocked recruitment of c-Fos to the NFATc1 promoter upon RANKL stimulation. Therefore, NFATc1-inducible osteoclast-specific genes were downregulated by MSK1 blockade. NFATc1 retrovirus transduction almost completely rescued the differentiation defect of MSK1-silenced cells. In vivo knockdown of MSK1 reduced RANKL-induced bone resorption as well as osteoclast formation. Thus, our results suggested that MSK1 is an important novel molecule involved in RANKL signaling and osteoclast differentiation.     

B and T lymphocytes are the primary sources of RANKL in the bone resorptive lesion of periodontal disease

Receptor activator of nuclear factor-kappaB (RANKL)-mediated osteoclastogenesis plays a pivotal role in inflammatory bone resorption. The aim of this study was to identify the cellular source of RANKL in the bone resorptive lesions of periodontal disease. The concentrations of soluble RANKL, but not its decoy receptor osteoprotegerin, measured in diseased tissue homogenates were significantly higher in diseased gingival tissues than in healthy tissues. Double-color confocal microscopic analyses demonstrated less than 20% of both B cells and T cells expressing RANKL in healthy gingival tissues. By contrast, in the abundant mononuclear cells composed of 45% T cells, 50% B cells, and 5% monocytes in diseased gingival tissues, more than 50 and 90% of T cells and B cells, respectively, expressed RANKL. RANKL production by nonlymphoid cells was not distinctly identified. Lymphocytes isolated from gingival tissues of patients induced differentiation of mature osteoclast cells in a RANKL-dependent manner in vitro. However, similarly isolated peripheral blood B and T cells did not induce osteoclast differentiation, unless they were activated in vitro to express RANKL; emphasizing the osteoclastogenic potential of activated RANKL-expressing lymphocytes in periodontal disease tissue. These results suggest that activated T and B cells can be the cellular source of RANKL for bone resorption in periodontal diseased gingival tissue.     

Adenoviral transfer of murine oncostatin M elicits periosteal bone apposition in knee joints of mice,despite synovial inflammation and up-regulated expression of interleukin-6 and receptor activator of nuclear factor-kappa B ligand

Oncostatin M (OSM) has been described as a bone-remodeling factor either stimulating osteoblast activity or osteoclast formation in vitro. To elucidate the in vivo effect of OSM on bone remodeling, we injected an adenoviral vector encoding murine OSM in knee joints of mice. OSM strongly induced interleukin (IL)-6 gene expression, a known mediator of osteoclast development. We investigated the OSM effect in wild-type and IL-6-deficient mice and found a similar degree of OSM-induced joint inflammation. Within the first week of inflammation, the periosteum along the femur and tibia increased in cell number and stained positive for the osteoblast marker alkaline phosphatase. At these sites bone apposition occurred in both strains as demonstrated by Goldner and Von Kossa staining. In vitro OSM enhanced the effect of bone morphogenetic protein-2 on osteoblast differentiation. Immunohistochemistry demonstrated expression of receptor activator of nuclear factor-kappa B ligand (RANKL) and its receptor, receptor activator of nuclear factor-kappa B (RANK), in the periosteum but osteoclasts were not detected at sites of bone apposition. Induced mRNA expression for the receptor activator of nuclear factor-kappa B ligand inhibitor osteoprotegerin probably controlled osteoclast development during OSM overexpression. Our results show that OSM favors bone apposition at periosteal sites instead of resorption in vivo. This effect was not dependent on or inhibited by IL-6.     

Human bone marrow adipocytes support dexamethasone-induced osteoclast differentiation and function through RANKL expression

The TNF-family molecule, Receptor Activator of Nuclear factor κ B Ligand (RANKL) is known as a key regulator for bone remodeling, and is essential for the development and activation of osteoclasts. In this study, we examined the regulation of RANKL in primary human bone marrow adipocytes and the relationship between bone marrow adipocytes and bone metabolism. RANKL expression and the RANKL/osteoprotegerin (OPG) mRNA ratio in marrow adipocytes increased following dexamethasone treatment. In co-cultures of human osteoclast precursors and bone marrow adipocytes with dexamethasone, osteoclast precursors differentiated to TRAP-positive multinuclear cells. Moreover, the ability of bone resorption was confirmed in co-culture in flasks coated with calcium phosphate film. Osteoclast precursor differentiation and bone resorption were blocked by RANKL antibody pretreatment. TRAP-positive multinuclear cells did not form in coculture without cell-to-cell contact conditions. We conclude that primary human bone marrow adipocytes have the ability to promote osteoclast differentiation and activities, similar to osteoblasts and other RANKL-expressing cells.     

CD14- mononuclear stromal cells support (CD14+) monocyte-osteoclast differentiation in aneurysmal bone cyst

Aneurysmal bone cyst (ABC) is a benign osteolytic bone lesion in which there are blood-filled spaces separated by fibrous septa containing giant cells. The nature of the giant cells in this lesion and the mechanism of bone destruction in ABC is not certain. In this study, we have analysed several characteristics of mononuclear and multinucleated cells in the ABC and examined the cellular and molecular mechanisms of ABC osteolysis. The antigenic and functional phenotype of giant cells in ABC was determined by histochemistry/immunohistochemistry using antibodies to macrophage and osteoclast markers. Giant cells and CD14+ and CD14- mononuclear cells were isolated from ABC specimens and cultured on dentine slices and coverslips with receptor activator of nuclear factor κB ligand (RANKL)+/- macrophage-colony stimulating factor (M-CSF) and functional and cytochemical evidence of osteoclast differentiation sought. Giant cells in ABC expressed an osteoclast-like phenotype (CD51+, CD14-, cathepsin K+, TRAP+) and were capable of lacunar resorption, which was inhibited by zoledronate, calcitonin and osteoprotegerin (OPG). When cultured with RANKL±M-CSF, CD14+, but not CD14-, mononuclear cells differentiated into TRAP+ multinucleated cells that were capable of lacunar resorption. M-CSF was not necessary for osteoclast formation from CD14+ cell cultures. CD14- cells variably expressed RANKL, OPG and M-CSF but supported osteoclast differentiation. Our findings show that the giant cells in ABC express an osteoclast-like phenotype and are formed from CD14+ macrophage precursors. CD14- mononuclear stromal cells express osteoclastogenic factors and most likely interact with CD14+ cells to form osteoclast-like giant cells by a RANKL-dependent mechanism.     

The cytolethal distending toxin induces receptor activator of NF-kappaB ligand expression in human gingival fibroblasts and periodontal ligament cells

Actinobacillus actinomycetemcomitans is associated with localized aggressive periodontitis, a disease characterized by rapid loss of the alveolar bone surrounding the teeth. Receptor activator of NF-kappaB Ligand (RANKL) and osteoprotegerin (OPG) are two molecules that regulate osteoclast formation and bone resorption. RANKL induces osteoclast differentiation and activation, whereas OPG blocks this process by acting as a decoy receptor for RANKL. The purpose of this study was to investigate the effect of A. actinomycetemcomitans on the expression of RANKL and OPG in human gingival fibroblasts and periodontal ligament cells. RANKL mRNA expression was induced in both cell types challenged by A. actinomycetemcomitans extract, whereas OPG mRNA expression remained unaffected. Cell surface RANKL protein was also induced by A. actinomycetemcomitans, whereas there was no change in OPG protein secretion. A cytolethal distending toxin (Cdt) gene-knockout strain of A. actinomycetemcomitans did not induce RANKL expression, in contrast to its wild-type strain. Purified Cdt from Haemophilus ducreyi alone, or in combination with extract from the A. actinomycetemcomitans cdt mutant strain, induced RANKL expression. Pretreatment of A. actinomycetemcomitans wild-type extract with Cdt antiserum abolished RANKL expression. In conclusion, A. actinomycetemcomitans induces RANKL expression in periodontal connective tissue cells. Cdt is crucial for this induction and may therefore be involved in the pathological bone resorption during the process of localized aggressive periodontitis.     

BMP-2 and titanium particles synergistically activate osteoclast formation

A previous study showed that BMP-2 (bone morphogenetic protein-2) and wear debris can separately support osteoclast formation induced by the receptor activator of NF-κB ligand (RANKL). However, the effect of BMP-2 on wear debris-induced osteoclast formation is unclear. In this study, we show that neither titanium particles nor BMP-2 can induce osteoclast formation in RAW 264.7 mouse leukemic monocyte macrophage cells but that BMP-2 synergizes with titanium particles to enhance osteoclast formation in the presence of RANKL, and that at a low concentration, BMP-2 has an optimal effect to stimulate the size and number of multinuclear osteoclasts, expression of osteoclast genes, and resorption area. Our data also clarify that the effects caused by the increase in BMP-2 on phosphorylated SMAD levels such as c-Fos expression increased throughout the early stages of osteoclastogenesis. BMP-2 and titanium particles stimulate the expression of p-JNK, p-P38, p-IkB, and P50 compared with the titanium group. These data suggested that BMP-2 may be a crucial factor in titanium particle-mediated osteoclast formation.     

Vitamin K2 stimulates osteoblastogenesis and suppresses osteoclastogenesis by suppressing NF-κB activation

Several bone protective factors are reported to exhibit stimulatory activities on bone formation coupled with inhibitory effects on bone resorption; one such factor is vitamin K2. Vitamin K species [K1 (phylloquinone) and K2 (menaquinone)] have long been associated with bone protective activities and are receiving intense interest as nutritional supplements for the prevention or amelioration of bone disease in humans. However, the mechanisms of vitamin K action on the skeleton are poorly defined. Activation of the nuclear factor κB (NF-κB) signal transduction pathway is essential for osteoclast formation and resorption. By contrast, NF-κB signaling potently antagonizes osteoblast differentiation and function, prompting us to speculate that NF-κB antagonists may represent a novel class of dual anti-catabolic and pro-anabolic agents. We now show that vitamin K2 action on osteoblast and osteoclast formation and activity is accomplished by down-regulating basal and cytokine-induced NF-κB activation, by increasing IκB mRNA, in a γ-carboxylation-independent manner. Furthermore, vitamin K2 prevented repression by tumor necrosis factor α (TNFα) of SMAD signaling induced by either transforming growth factor ? (TGF?) or bone morphogenetic protein-2 (BMP-2). Vitamin K2 further antagonized receptor activator of NF-κB (RANK) ligand (RANKL)-induced NF-κB activation in osteoclast precursors. Our data provide a novel mechanism to explain the dual pro-anabolic and anti-catabolic activities of vitamin K2, and may further support the concept that pharmacological modulation of NF-κB signal transduction may constitute an effective mechanism for ameliorating pathological bone loss and for promoting bone health.