Presence Of Receptor Activator Of Nuclear Factor kappaB Ligand Research Articles

Abstract 2481: Human osteoclast assays for studying the effects of bone metastasis treatments with different mechanisms of action

Abstract Metastasis to bone is one of the common complications associated with different types of advanced cancer. Zoledronic acid, a third generation nitrogen containing bisphosphonate, accumulates in bones where it binds to hydroxyapatite crystals. For years, zoledronic acid has been regarded as the standard of care for patients with advanced cancer and bone metastasis to prevent skeletal complications. As an alternative therapeutic option, denosumab has also been found to be effective in delaying skeletal-related events in advanced cancer. Denosumab, a human monoclonal antibody against receptor activator of nuclear factor kappa-B ligand (RANKL), blocks the RANKL/RANK signalling pathway. In addition to the skeletal effects, both zoledronic acid and denosumab have shown direct effect on cancer cells. In this study, effects of zoledronic acid and denosumab on osteoclast differentiation and bone resorption activity were studied using human CD34+ osteoclast precursor cells that were cultured on bovine bone slices in 96-well plates for 7 days in the presence of RANKL and M-CSF, allowing them to differentiate into mature osteoclasts. In the osteoclast differentiation assay, zoledronic acid (0.0001 - 1 000 µM) and denosumab (0.01 - 10 µg/ml) were added into the culture medium at day 0. Tartrate-resistant acid phosphatase 5b (TRACP 5b) activity was measured in the culture medium at day 7 as an index of osteoclast differentiation. In the osteoclast activity assay, the test compounds were added into the culture medium at day 7, and the formed osteoclasts were allowed to resorb bone for 3 days. C-terminal crosslinked telopeptides of type I collagen (CTX-I) was measured in the culture medium at day 10 as an index of bone resorption. Denosumab and zoledronic acid showed strong concentration dependent inhibition of osteoclast differentiation and resorption activity. We conclude that this osteoclast culture system can be used for identifying new potential compounds affecting the disease process of bone metastases with different mechanisms of action. The manifold effects of zoledronic acid and denosumab on cancer cells remain to be studied in more detail. Citation Format: Katja M. Fagerlund, Mervi Ristola, Mari I. Suominen, Jenni H. Mäki-Jouppila, Jukka P. Rissanen. Human osteoclast assays for studying the effects of bone metastasis treatments with different mechanisms of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2481.

Osteoclast differentiation by RANKL and OPG signaling pathways.

In bone tissue, bone resorption by osteoclasts and bone formation by osteoblasts are repeated continuously. Osteoclasts are multinucleated cells that derive from monocyte-/macrophage-lineage cells and resorb bone. In contrast, osteoblasts mediate osteoclastogenesis by expressing receptor activator of nuclear factor-kappa B ligand (RANKL), which is expressed as a membrane-associated cytokine. Osteoprotegerin (OPG) is a soluble RANKL decoy receptor that is predominantly produced by osteoblasts and which prevents osteoclast formation and osteoclastic bone resorption by inhibiting the RANKL-RANKL receptor interaction. In this review, we would like to summarize our experimental results on signal transduction that regulates the expression of RANKL and OPG. Using OPG gene-deficient mice, we have demonstrated that OPG and sclerostin produced by osteocytes play an important role in the maintenance of cortical and alveolar bone. In addition, it was shown that osteoclast-derived leukemia inhibitory factor (LIF) reduces the expression of sclerostin in osteocytes and promotes bone formation. WP9QY (W9) is a peptide that was designed to be structurally similar to one of the cysteine-rich TNF-receptortype-I domains. Addition of the W9 peptide to bone marrow culture simultaneously inhibited osteoclast differentiation and stimulated osteoblastic cell proliferation. An anti-sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) antibody inhibited multinucleated osteoclast formation induced by RANKL and macrophage colony-stimulating factor (M-CSF). Pit-forming activity of osteoclasts was also inhibited by the anti-Siglec-15 antibody. In addition, anti-Siglec-15 antibody treatment stimulated the appearance of osteoblasts in cultures of mouse bone marrow cells in the presence of RANKL and M-CSF. Bone mass loss depends on the RANK-RANKL-OPG system, which is a major regulatory system of osteoclast differentiation induction, activation, and survival.

Continuous Compressive Force Induces Differentiation of Osteoclasts with High Levels of Inorganic Dissolution.

BackgroundOsteoclast precursor cells are constitutively differentiated into mature osteoclasts on bone tissues. We previously reported that the continuous stimulation of RAW264.7 precursor cells with compressive force induces the formation of multinucleated giant cells via receptor activator of nuclear factor κB (RANK)-RANK ligand (RANKL) signaling. Here, we examined the bone resorptive function of multinucleated osteoclasts induced by continuous compressive force.Material/MethodsCells were continuously stimulated with 0.3, 0.6, and 1.1 g/cm2 compressive force created by increasing the amount of the culture solution in the presence of RANKL. Actin ring organization was evaluated by fluorescence microscopy. mRNA expression of genes encoding osteoclastic bone resorption-related enzymes was examined by quantitative real-time reverse transcription-polymerase chain reaction. Mineral resorption was evaluated using calcium phosphate-coated plates.ResultsMultinucleated osteoclast-like cells with actin rings were observed for all three magnitudes of compressive force, and the area of actin rings increased as a function of the applied force. Carbonic anhydrase II expression as well as calcium elution from the calcium phosphate plate was markedly higher after stimulation with 0.6 and 1.1 g/cm2 force than 0.3 g/cm2. Matrix metalloproteinase-9 expression decreased and cathepsin K expression increased slightly by the continuous application of compressive force.ConclusionsOur study demonstrated that multinucleated osteoclast-like cells induced by the stimulation of RAW264.7 cells with continuous compressive force exhibit high dissolution of the inorganic phase of bone by upregulating carbonic anhydrase II expression and actin ring formation. These findings improve our understanding of the role of mechanical load in bone remodeling.

8-Nitro-cGMP is a promoter of osteoclast differentiation induced by RANKL

Osteoclasts are multinucleated giant cells differentiated from monocyte-macrophage-lineage cells under stimulation of receptor activator of nuclear factor κ-B (RANK) ligand (RANKL) produced by osteoblasts and osteocytes. Although it has been reported that nitric oxide (NO) and reactive oxygen species (ROS) are involved in this process, the mechanism by which these labile molecules promote osteoclast differentiation are not fully understood. In this study, we investigated the formation and function of 8-nitro-cGMP, a downstream molecule of NO and ROS, in the process of osteoclast differentiation in vitro. 8-Nitro-cGMP was detected in mouse bone marrow macrophages and osteoclasts differentiated from macrophages in the presence of RANKL. Inhibition of NO synthase suppressed the formation of 8-nitro-cGMP as well as RANKL-induced osteoclast differentiation from macrophages. On the other hand, RANKL-induced osteoclast differentiation was promoted by addition of 8-nitro-cGMP to the cultures. In addition, 8-nitro-cGMP enhanced the mRNA expression of RANK, the receptor for RANKL. However, 8-bromo-cGMP, a membrane-permeable derivative of cGMP, did not have an effect on either RANKL-induced osteoclast differentiation or expression of the RANK gene. These results suggest that 8-nitro-cGMP is a novel positive regulator of osteoclast differentiation, which might help to explain the roles of NO and ROS in osteoclast differentiation.

Coil poly(ethylene glycol)-brush-like poly(L-lysine) block copolymers as efficient nanocarriers for insulin delivery

Osteoclasts are multinucleated giant cells differentiated from monocyte-macrophage-lineage cells under stimulation of receptor activator of nuclear factor κ-B (RANK) ligand (RANKL) produced by osteoblasts and osteocytes. Although it has been reported that nitric oxide (NO) and reactive oxygen species (ROS) are involved in this process, the mechanism by which these labile molecules promote osteoclast differentiation are not fully understood. In this study, we investigated the formation and function of 8-nitro-cGMP, a downstream molecule of NO and ROS, in the process of osteoclast differentiation in vitro. 8-Nitro-cGMP was detected in mouse bone marrow macrophages and osteoclasts differentiated from macrophages in the presence of RANKL. Inhibition of NO synthase suppressed the formation of 8-nitro-cGMP as well as RANKL-induced osteoclast differentiation from macrophages. On the other hand, RANKL-induced osteoclast differentiation was promoted by addition of 8-nitro-cGMP to the cultures. In addition, 8-nitro-cGMP enhanced the mRNA expression of RANK, the receptor for RANKL. However, 8-bromo-cGMP, a membrane-permeable derivative of cGMP, did not have an effect on either RANKL-induced osteoclast differentiation or expression of the RANK gene. These results suggest that 8-nitro-cGMP is a novel positive regulator of osteoclast differentiation, which might help to explain the roles of NO and ROS in osteoclast differentiation.

Transdifferentiation of Bone Marrow Pro-B Cells into Bone-Resorbing Osteoclasts- an Unexpected Role for Erythropoietin

Bone turnover is regulated by the coupled actions of osteoblasts, the bone-forming cells, and monocyte-derived osteoclasts, which mediate bone resorption. B cells were also shown to regulate bone metabolism, chiefly via paracrine signals. Depending on their state and/or mode of activation, B cells may inhibit or enhance osteoclastogenesis. In mammals, B cell development and maturation occurs in the bone marrow (BM), spleen, and other peripheral lymphoid tissues. In the BM, Pro-B cells sequentially differentiate into Pre-B and immature B cells. Whether BM B cells can transdifferentiate into osteoclasts remains controversial, since osteoclast differentiation from residual monocytic precursors in the cultures was not excluded in the earlier studies. Osteoclasts and B cells arise from distinct myeloid and lymphoid progenitors, respectively, which are downstream of a common multipotent progenitor cell. Monocyte differentiation into osteoclasts relies on monocyte-macrophage colony-stimulating factor (M-CSF) and the receptor activator of nuclear factor kappa B ligand (RANKL). Here, we investigated the possibility that BM B-cells contribute to bone loss by transdifferentiating into bone-resorbing osteoclasts. We found that B220+CD19+ cells can transdifferentiate into multinucleated tartrate-resistant acid phosphatase (TRAP) positive osteoclasts in the presence of RANKL and M-CSF. Our results show that Pro-B cells (B220+CD19+CD43HighIGM-), but not Pre-B cells (B220+CD19+CD43LowIGM-), nor immature B cells (B220+CD19+CD43-IGM+), could transdifferentiate into osteoclasts (16%±3.7 vs. 0.79%±0.28 and 0.48%±0.13 osteoclasts area, respectively). Moreover, among the Pro-B cells, only those expressing M-CSF receptor (CD115) could transdifferentiate into functional osteoclasts (18%±6.55 vs. 0.11%±0.05 osteoclasts area, respectively, Figure 1A and B). To unequivocally establish the generation of osteoclasts from B-cells, we next utilized a mouse model in which all B cell lineage-derived progenies express EYFP. We found that B cells isolated from BM of CD19-Cre:EYFP mice differentiated into TRAP+ multinucleated osteoclasts that were also positive for EYFP (Figure1C).Erythropoietin (EPO) is a crucial kidney-derived hormone responsible for erythropoiesis. Once thought to act solely on the erythroid compartment to potentiate red blood cell production, it became evident that EPO receptors are also found on the monocytic lineage (monocytes, macrophages and dendritic cells). In that respect, we have reported that EPO directly stimulates bone loss via activation of EPO-R signaling in the monocytic lineage (Hiram-Bab et al., 2015). Here we report that B cells express EPO-R, and that EPO enhances Pro-B cell differentiation into osteoclasts by 70% (p<0.05) (Figure 1D).Conceivably, in other scenarios, e.g., sex hormone deficiency, certain hematological cancers, and treatment with anti-CD20, increased B cell lymphocytes contribute to the bone loss phenotype due to transdifferentiation of B-cell precursors into osteoclasts. Taken together, our data suggest a new physio-pathological role for BM B cell precursors in bone metabolism, via their capacity to differentiate into functional osteoclasts, and a possible role for EPO in this process. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Massive elimination of multinucleated osteoclasts by eupatilin is due to dual inhibition of transcription and cytoskeletal rearrangement

Osteoporosis is an aging-associated disease requiring better therapeutic modality. Eupatilin is a major flavonoid from Artemisia plants such as Artemisia princeps and Artemisia argyi which has been reported to possess various beneficial biological effects including anti-inflammation, anti-tumor, anti-cancer, anti-allergy, and anti-oxidation activity. Complete blockade of RANK-dependent osteoclastogenesis was accomplished upon stimulation prior to the receptor activator of nuclear factor κB (RANK)-ligand (RANKL) treatment or post-stimulation of bone marrow macrophages (BMCs) in the presence of RANKL with eupatilin. This blockade was accompanied by inhibition of rapid phosphorylation of Akt, GSK3β, ERK and IκB as well as downregulation of c-Fos and NFATc1 at protein, suggesting that transcriptional suppression is a key mechanism for anti-osteoclastogenesis. Transient reporter assays or gain of function assays confirmed that eupatilin was a potent transcriptional inhibitor in osteoclasts (OC). Surprisingly, when mature osteoclasts were cultured on bone scaffolds in the presence of eupatilin, bone resorption activity was also completely blocked by dismantling the actin rings, suggesting that another major acting site of eupatilin is cytoskeletal rearrangement. The eupatilin-treated mature osteoclasts revealed a shrunken cytoplasm and accumulation of multi-nuclei, eventually becoming fibroblast-like cells. No apoptosis occurred. Inhibition of phosphorylation of cofilin by eupatilin suggests that actin may play an important role in the morphological change of multinucleated cells (MNCs). Human OC similarly responded to eupatilin. However, eupatilin has no effects on osteoblast differentiation and shows cytotoxicity on osteoblast in the concentration of 50μM. When eupatilin was administered to LPS-induced osteoporotic mice after manifestation of osteoporosis, it prevented bone loss. Ovariectomized (OVX) mice remarkably exhibited bone protection effects. Taken together, eupatilin is an effective versatile therapeutic intervention for osteoporosis via; 1) transcriptional suppression of c-Fos and NFATc1 of differentiating OC and 2) inhibition of actin rearrangement of pathogenic MNCs.

Effect of BioAggregate on Receptor Activator of Nuclear Factor-Kappa B Ligand–induced Osteoclastogenesis from Murine Macrophage Cell Line In Vitro

IntroductionThis study aimed to investigate the effect of BioAggregate, a calcium silicate–based nanoparticulate bioceramic, on the regulation of receptor activator of nuclear factor-kappa B ligand (RANKL)–induced osteoclast differentiation and bone resorption in vitro, as well as to delineate the underlying molecular mechanism. The performance of BioAggregate was compared with that of ProRoot mineral trioxide aggregate (MTA). MethodsCells of a murine macrophage cell line RAW 264.7 were treated with various concentrations of BioAggregate and MTA extracts. Cytotoxicity of material extracts was evaluated with Cell Counting Kit-8 assay. RANKL-induced osteoclast differentiation and function were assessed with tartrate-resistant acid phosphatase staining, F-actin staining, and lacunar resorption pits assay. The mRNA expression associated with osteoclast function was detected with quantitative real-time polymerase chain reaction. Related molecular signaling pathways were investigated with Western blot and immunofluorescence. ResultsBioAggregate extracts dose-dependently inhibited RANKL-induced osteoclast formation and resorption capacity without evident cytotoxicity. RAW 264.7 cells exposed to BioAggregate extracts also presented a decrease in RANKL-stimulated mRNA expression of osteoclast-related genes and transcription factors. Moreover, cells treated with BioAggregate extracts exhibited attenuated TRAF6 expression, suppressed mitogen-activated protein kinase signaling, and decreased nuclear translocation of NFATc1 and c-Fos in the presence of RANKL. Comparable effects were induced by MTA. ConclusionsBioAggregate and MTA exhibit comparable inhibitory effect on osteoclast differentiation and function in vitro, and our findings provide valuable insights into the mechanism of bioceramic-mediated anti-osteoclastogenic activity.

Cell Adhesion Signaling Regulates RANK Expression in Osteoclast Precursors

Cells with monocyte/macrophage lineage expressing receptor activator of NF-κB (RANK) differentiate into osteoclasts following stimulation with the RANK ligand (RANKL). Cell adhesion signaling is also required for osteoclast differentiation from precursors. However, details of the mechanism by which cell adhesion signals induce osteoclast differentiation have not been fully elucidated. To investigate the participation of cell adhesion signaling in osteoclast differentiation, mouse bone marrow-derived macrophages (BMMs) were used as osteoclast precursors, and cultured on either plastic cell culture dishes (adherent condition) or the top surface of semisolid methylcellulose gel loaded in culture tubes (non-adherent condition). BMMs cultured under the adherent condition differentiated into osteoclasts in response to RANKL stimulation. However, under the non-adherent condition, the efficiency of osteoclast differentiation was markedly reduced even in the presence of RANKL. These BMMs retained macrophage characteristics including phagocytic function and gene expression profile. Lipopolysaccharide (LPS) and tumor necrosis factor –αTNF-α activated the NF-κB-mediated signaling pathways under both the adherent and non-adherent conditions, while RANKL activated the pathways only under the adherent condition. BMMs highly expressed RANK mRNA and protein under the adherent condition as compared to the non-adherent condition. Also, BMMs transferred from the adherent to non-adherent condition showed downregulated RANK expression within 24 hours. In contrast, transferring those from the non-adherent to adherent condition significantly increased the level of RANK expression. Moreover, interruption of cell adhesion signaling by echistatin, an RGD-containing disintegrin, decreased RANK expression in BMMs, while forced expression of either RANK or TNFR-associated factor 6 (TRAF6) in BMMs induced their differentiation into osteoclasts even under the non-adherent condition. These results suggest that cell adhesion signaling regulates RANK expression in osteoclast precursors.

RANKL increases the level of Mcl-1 in osteoclasts and reduces bisphosphonate-induced osteoclast apoptosis in vitro.

IntroductionBisphosphonates are the most widely used class of drug for inhibiting osteoclast-mediated bone loss, but their effectiveness at preventing joint destruction in rheumatoid arthritis has generally been disappointing. We examined whether the ability of bisphosphonates to induce osteoclast apoptosis and inhibit bone resorption in vitro is influenced by the cytokine receptor activator of nuclear factor-kappa B ligand (RANKL), an important mediator of inflammation-induced bone loss.MethodsRabbit osteoclasts were treated with the bisphosphonates clodronate or alendronate for up to 48 hours in the absence or presence of RANKL. Changes in cell morphology and induction of apoptosis were examined by scanning electron microscopy, whilst resorptive activity was determined by measuring the area of resorption cavities. Changes in the level of anti-apoptotic proteins, including Mcl-1, Bcl-2, and Bcl-x>L, were determined in rabbit osteoclasts and in cytokine-starved mouse osteoclasts by Western blotting.ResultsRANKL significantly attenuated the ability of both clodronate and alendronate to induce osteoclast apoptosis and inhibit bone resorption. Treatment of rabbit osteoclasts with RANKL was associated with an increase in the anti-apoptotic protein Mcl-1 but not Bcl-2. A role for Mcl-1 in osteoclast survival was suggested using osteoclasts generated from mouse bone marrow macrophages in the presence of RANKL + macrophage colony-stimulating factor (M-CSF) since cytokine deprivation of mouse osteoclasts caused a rapid loss of Mcl-1 (but not Bcl-2 or Bcl-xL), which preceded the biochemical and morphological changes associated with apoptosis. Loss of Mcl-1 from mouse osteoclasts could be prevented by factors known to promote osteoclast survival (RANKL, M-CSF, tumour necrosis factor-alpha [TNF-α], or lipopolysaccharide [LPS]).ConclusionsRANKL protects osteoclasts from the apoptosis-inducing and anti-resorptive effects of bisphosphonates in vitro. The ability of RANKL (and other pro-inflammatory factors such as TNF-α and LPS) to increase the level of Mcl-1 in osteoclasts may explain the lack of effectiveness of some bisphosphonates in preventing inflammation-induced bone loss.

Characterization of osteoclasts derived from CD14+ monocytes isolated from peripheral blood

Bone resorption is solely mediated by osteoclasts. Therefore, a pure osteoclast population is of high interest for the investigation of biological aspects of the osteoclasts, such as the direct effect of growth factors and hormones, as well as for testing and characterizing inhibitors of bone resorption. We have established a pure, stable, and reproducible system for purification of human osteoclasts from peripheral blood. We isolated CD14-positive (CD14+) monocytes using anti-CD14-coated beads. After isolation, the monocytes are differentiated into mature osteoclasts by stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL). Osteoclast formation was only observed in the CD14+ population, not in the CD14- population, and only in the presence of both M-CSF and RANKL, confirming that the CD14+ system is a pure population of osteoclast precursors. No expression of osteoclast markers was observed in the absence of RANKL, whereas RANKL dose-dependently induced the expression of cathepsin K, tartrate-resistant acid phosphatase (TRACP), and matrix metallo proteinase (MMP)-9. Furthermore, morphological characterization of the cells demonstrated that actin rings were only formed in the presence of RANKL. Moreover, the osteoclasts were capable of forming acidic resorption lacunae, and inhibitors of lysosomal acidification attenuated this process. Finally, we measured the response to known bone resorption inhibitors, and found that the osteoclasts were sensitive to these and thereby provided a robust and valid method for interpretation of the effect of antiresorptive compounds. In conclusion, we have established a robust assay for developing osteoclasts that can be used to study several biological aspects of the osteoclasts and which in combination with the resorption marker CTX-I provides a useful tool for evaluating osteoclast function in vitro.

FLRG, a new ADAM12‐associated protein, modulates osteoclast differentiation

FLRG (follistatin-related gene) is a secreted glycoprotein that is highly homologous with follistatin. These proteins are involved in the regulation of various biological effects mediated by their binding to TGF-beta (transforming growth factor-beta) superfamily members, activin A and bone morphogenetic proteins. To characterize further the function of FLRG, we used a yeast two-hybrid screen to look for other possible functional partners. We report a direct interaction between the cysteine-rich domain of FLRG and ADAM12 (a disintegrin and metalloprotease 12). ADAMs are metalloprotease-disintegrin proteins that have been implicated in cell adhesion, protein ectodomain shedding, matrix protein degradation and cell fusion. Several studies have reported that ADAM12 protein, as well as activin A, are important regulators of osteoclast differentiation. We observed that the expressions of ADAM12 and activin A are modulated during osteoclast formation, whereas the FLRG expression seemed to remain quite constant. We showed that the FLRG protein inhibits osteoclast differentiation from murine primary spleen cells and macrophage RAW264.7 cells cultured in the presence of RANK-L (receptor activator of nuclear factor kappaB ligand) and M-CSF (macrophage colony-stimulating factor). Addition of FLRG protein to precursors significantly reduces the number of osteoclasts, as well as the average number of nuclei in each osteoclast. Our study indicates that the FLRG protein may contribute to bone formation by inhibiting osteoclast differentiation.

Infliximab acts directly on human osteoclast precursors and enhances osteoclast formation induced by receptor activator of nuclear factor κB ligand in vitro

Infliximab is known to protect against the development of joint destruction. In the present study, we sought to determine whether Infliximab acts directly on human osteoclast precursors and influences monocyte-osteoclast differentiation induced by receptor activator of nuclear factor kappaB ligand (RANKL) in vitro. Peripheral blood mononuclear cells (PBMCs) isolated from rheumatoid arthritis (RA) patients and normal controls were cultured in the presence of RANKL and macrophage colony stimulating factor. Infliximab, antihuman tumor necrosis factor alpha (TNFalpha), antihuman TNF soluble receptor p55 (TNFR p55), and antihuman TNF soluble receptor p75 (TNFR p75) antibodies were added. Osteoclast formation was determined by assessing the number of tartrate-resistant acid phosphatase (TRAP) staining cells and the extent of lacunar resorption. Addition of Infliximab resulted in a marked increase in the number of TRAP-positive multinucleated cells (TRAP(+) MNCs) and in the extent of lacunar resorption compared with the control cultures. Antihuman TNFalpha antibody showed the same effect; however, the addition of neither TNFR p55 nor TNFR p75 antibody affected the extent of TRAP(+) MNCs and lacunar resorption. Our results suggest that infliximab acts directly on early osteoclast precursors, and stimulates osteoclast formation and lacunar resorption induced by RANKL in vitro.

TNF-related apoptosis-inducing ligand (TRAIL) blocks osteoclastic differentiation induced by RANKL plus M-CSF

AbstractThe role of the tumor necrosis factor (TNF) superfamily member receptor activator of nuclear factor kappa B ligand (RANKL) in promoting the differentiation of osteoclasts has been extensively characterized. In this study, we have investigated the effect of TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily of cytokines, in osteoclastogenesis, by using human peripheral blood mononuclear cells and the RAW264.7 murine monocytic cell line. Both cell models differentiate into osteoclast-like cells in presence of RANKL plus macrophage-colony-stimulating factor (M-CSF), as evaluated in terms of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and bone resorption activity. Unexpectedly, when added in culture in combination with RANKL plus M-CSF, TRAIL inhibited osteoclastic differentiation in both cell models. To investigate the molecular mechanism underlining such inhibitory activity, we analyzed the effect of TRAIL on the mitogen-activated protein kinases (MAPKs) pathways, which play a key role in osteoclastogenesis. Treatment with RANKL plus M-CSF activated both the ERK1/2 and p38/MAPK pathways, which are essential for proliferation and differentiation of preosteoclasts, respectively. Of note, the addition of TRAIL to RANKL plus M-CSF did not affect ERK1/2 but it profoundly inhibited p38/MAPK phosphorylation. Thus, our data demonstrate that TRAIL blocks osteoclastic differentiation and suggest that inhibition of the p38/MAPK pathway by TRAIL likely plays an important role in this process. (Blood. 2004;104:2044-2050)

The immunosuppressant rapamycin, alone or with transforming growth factor-beta, enhances osteoclast differentiation of RAW264.7 monocyte-macrophage cells in the presence of RANK-ligand.

Immunosuppressant therapy is known to cause bone loss. Since this may partly result from direct effects on osteoclast development, we investigated whether cyclosporin A (CsA), rapamycin, or FK506 affect osteoclastic differentiation of RAW264.7 monocytic cells induced by RANK-ligand (RANKL). Furthermore, since the rapamycin receptor protein binds transforming growth factor beta (TGF-beta) receptors, and TGF-beta enhances osteoclastogenesis induced by RANKL, we also examined potential synergistic effects of rapamycin and TGF-beta1. Rapamycin inhibited cell proliferation and stimulated tartrate-resistant acid phosphatase (TRAP) activity of RAW cells in a dose-dependent manner. At the optimal concentration of 10 ng/ml, it increased the number of TRAP+ multinucleated cells (MNC) more than 20-fold and enhanced the expression of TRAP and calcitonin receptor (CTR) mRNAs 2.1- and 10-fold, respectively. CsA, at 125-2000 ng/ml, similarly inhibited proliferation, but at high doses (1000-2000 ng/ml) it decreased TRAP activity, TRAP+MNC formation, and the expression of TRAP and CTR mRNAs. FK506 had no effect on cell proliferation or TRAP activity at concentrations up to 2000 ng/ml; however, like CsA, 1000 ng/ml FK506 inhibited TRAP+MNC formation and the expression of TRAP and CTR mRNAs. The combination of rapamycin (10 ng/ml) and TGF-beta1 (1 ng/ml) increased TRAP+MNC 3.1- and 6.9-fold as compared with rapamycin or TGF-beta1 alone, respectively, and enhanced CTR mRNA expression induced by TGF-beta1 by 1.9-fold. Rapamycin also increased osteoclastic resorption activity by 6.5-fold compared with control, and this was enhanced further by the addition of TGF-beta by 3-fold, compared with rapamycin alone. These data thus indicate that rapamycin, alone or in synergy with TGF-beta, directly enhances osteoclastogenesis and may affect bone metabolism in vivo after long-term use.

Osteoclast differentiation from circulating mononuclear precursors in Paget’s disease is hypersensitive to 1,25-dihydroxyvitamin D 3 and RANKL

A characteristic feature of Paget’s disease is an increase in the number of osteoclasts in bone. Osteoclasts are formed from mononuclear phagocyte precursors that circulate in the monocyte fraction of peripheral blood. These cells require the presence of RANK ligand (RANKL)-expressing osteoblastic cells and human macrophage colony-stimulating factor (M-CSF) to form osteoclasts in vitro. To determine the role of osteoclast differentiation from circulating precursors in Paget’s disease, we cultured monocytes from Paget’s patients and gender- and age-matched normal controls with no evidence of bone disease for up to 21 days in the presence of UMR 106 cells and various concentrations of M-CSF (1–25 ng/mL) and 1,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3] (10 −10 to 10 −7 mol/L). Relative to controls, there was a significant increase in the extent of osteoclast differentiation from pagetic monocytes as assessed by expression of tartrate-resistant acid phosphatase (TRAP), vitronectin receptor (VNR), and lacunar bone resorption. Serial dilution experiments (2 × 10 5 to 2 × 10 2 cells/well) showed no difference in the concentration of osteoclast precursors in the peripheral blood. In Paget’s patients with high serum alkaline phosphatase (sAP) levels, increased sensitivity to the osteoclastogenic effect of 1,25(OH) 2D 3 was noted. Osteoclast differentiation did not occur when M-CSF was substituted by interleukin-6 (IL-6) and soluble IL-6 receptor (sIL-6R), and these factors did not stimulate osteoclast differentiation in the presence of M-CSF. In this in vitro coculture system, osteoclast formation was inhibited by osteoprotegerin in a dose-dependent manner. In the presence of RANKL (5–30 ng/mL) and M-CSF (25 ng/mL), osteoclast formation and bone resorption were significantly increased in cultures of monocytes from patients with high and low sAP levels as compared with normal controls. Our findings suggest that the increase in osteoclast numbers seen in Paget’s disease results not from an increase in the number of circulating precursors in peripheral blood but rather from an increased sensitivity of osteoclast precursors to the humoral factors, 1,25(OH) 2D 3 and RANKL, which regulate osteoclast formation.