Downregulated fat mass and obesity-associated protein inhibits bone resorption and osteoclastogenesis by nuclear factor-kappa B inactivation

During osteoporosis, fat mass and obesity-associated protein (FTO) promotes the shift of bone marrow mesenchymal stem cells to adipocytes and represses osteoblast activity. However, the role and mechanisms of FTO on osteoclast formation and bone resorption remain unknown. In this study, we investigated the effect of FTO on RAW264.7 cells and bone marrow monocytes (BMMs)-derived osteoclasts in vitro and observed the influence of FTO on ovariectomized (OVX) mice model to mimic postmenopausal osteoporosis in vivo. Results found that FTO was up-regulated in BMMs from OVX mice. Double immunofluorescence assay showed co-localization of FTO with tartrate-resistant acid phosphatase (TRAP) in femurs of OVX mice. FTO overexpression enhanced TRAP-positive osteoclasts and F-actin ring formation in RAW264.7 cells upon RANKL stimulation. The expression of osteoclast differentiation-related genes, including nuclear factor of activated T cells c1 (NFATc1) and c-FOS, was upregulated in BMMs and RAW264.7 cells after FTO overexpression. FTO overexpression induced the phosphorylation and nuclear translocation of factor-kappa B (NF-κB) p65 in BMMs and RAW264.7 cells exposed to RANKL. ChIP and dual-luciferase assays revealed that FTO overexpression contributed to RANKL-induced binding of NF-κB to NFATc1 promoter. Rescue experiments suggested that FTO overexpression-mediated osteoclast differentiation was suppressed after intervention with a NF-κB inhibitor pyrrolidine dithiocarbamate. Further in vivo evidence revealed that FTO knockdown increased bone trabecula and bone mineral density, inhibited bone resorption and osteoclastogenesis in osteoporotic mice. Collectively, our research demonstrates that downregulated FTO inhibits bone resorption and osteoclastogenesis through NF-κB inactivation, which provides a novel reference for osteoporosis treatment.     

Caffeic acid phenethyl ester,an active component of honeybee propolis attenuates osteoclastogenesis and bone resorption via the suppression of RANKL‐induced NF‐κB and NFAT activity

Receptor activator NF‐κB ligand (RANKL)‐activated signaling is essential for osteoclast differentiation, activation and survival. Caffeic acid phenethyl ester (CAPE), a natural NF‐κB inhibitor from honeybee propolis has been shown to have anti‐tumor and anti‐inflammatory properties. In this study, we investigated the effect of CAPE on the regulation of RANKL‐induced osteoclastogenesis, bone resorption and signaling pathways. Low concentrations of CAPE (<1 µM) dose dependently inhibited RANKL‐induced osteoclastogenesis in RAW264.7 cell and bone marrow macrophage (BMM) cultures, as well as decreasing the capacity of human osteoclasts to resorb bone. CAPE inhibited both constitutive and RANKL‐induced NF‐κB and NFAT activation, concomitant with delayed IκBα degradation and inhibition of p65 nuclear translocation. At higher concentrations, CAPE induced apoptosis and caspase 3 activities of RAW264.7 and disrupts the microtubule network in osteoclast like (OCL) cells. Taken together, our findings demonstrate that inhibition of NF‐κB and NFAT activation by CAPE results in the attenuation of osteoclastogenesis and bone resorption, implying that CAPE is a potential treatment for osteolytic bone diseases. J. Cell. Physiol. 221: 642–649, 2009. © 2009 Wiley‐Liss, Inc.     

Nicotine Affects Bone Resorption and Suppresses the Expression of Cathepsin K,MMP-9 and Vacuolar-Type H+-ATPase d2 and Actin Organization in Osteoclasts

Tobacco smoking is an important risk factor for the development of several cancers, osteoporosis, and inflammatory diseases such as periodontitis. Nicotine is one of the major components of tobacco. In previous study, we showed that nicotine inhibits mineralized nodule formation by osteoblasts, and the culture medium from osteoblasts containing nicotine and lipopolysaccharide increases osteoclast differentiation. However, the direct effect of nicotine on the differentiation and function of osteoclasts is poorly understood. Thus, we examined the direct effects of nicotine on the expression of nicotine receptors and bone resorption-related enzymes, mineral resorption, actin organization, and bone resorption using RAW264.7 cells and bone marrow cells as osteoclast precursors. Cells were cultured with 10⁻⁵, 10⁻⁴, or 10⁻³ M nicotine and/or 50 µM α-bungarotoxin (btx), an 7 nicotine receptor antagonist, in differentiation medium containing the soluble RANKL for up 7 days. 1–5, 7, 9, and 10 nicotine receptors were expressed on RAW264.7 cells. The expression of 7 nicotine receptor was increased by the addition of nicotine. Nicotine suppressed the number of tartrate-resistant acid phosphatase positive multinuclear osteoclasts with large nuclei(≥10 nuclei), and decreased the planar area of each cell. Nicotine decreased expression of cathepsin K, MMP-9, and V-ATPase d2. Btx inhibited nicotine effects. Nicotine increased CA II expression although decreased the expression of V-ATPase d2 and the distribution of F-actin. Nicotine suppressed the planar area of resorption pit by osteoclasts, but did not affect mineral resorption. These results suggest that nicotine increased the number of osteoclasts with small nuclei, but suppressed the number of osteoclasts with large nuclei. Moreover, nicotine reduced the planar area of resorption pit by suppressing the number of osteoclasts with large nuclei, V-ATPase d2, cathepsin K and MMP-9 expression and actin organization.     

Inositol Hexakisphosphate Inhibits Osteoclastogenesis on RAW 264.7 Cells and Human Primary Osteoclasts

Background

Inoxitol hexakisphosphate (IP6) has been found to have an important role in biomineralization and a direct effect inhibiting mineralization of osteoblasts in vitro without impairing extracellular matrix production and expression of alkaline phosphatase. IP6 has been proposed to exhibit similar effects to those of bisphosphonates on bone resorption, however, its direct effect on osteoclasts (OCL) is presently unknown.

Methodology/Principal Findings

The aim of the present study was to investigate the effect of IP6 on the RAW 264.7 monocyte/macrophage mouse cell line and on human primary osteoclasts. On one hand, we show that IP6 decreases the osteoclastogenesis in RAW 264.7 cells induced by RANKL, without affecting cell proliferation or cell viability. The number of TRAP positive cells and mRNA levels of osteoclast markers such as TRAP, calcitonin receptor, cathepsin K and MMP-9 was decreased by IP6 on RANKL-treated cells. On the contrary, when giving IP6 to mature osteoclasts after RANKL treatment, a significant increase of bone resorption activity and TRAP mRNA levels was found. On the other hand, we show that 1 µM of IP6 inhibits osteoclastogenesis of human peripheral blood mononuclear cells (PBMNC) and their resorption activity both, when given to undifferentiated and to mature osteoclasts.

Conclusions/Significance

Our results demonstrate that IP6 inhibits osteoclastogenesis on human PBMNC and on the RAW264.7 cell line. Thus, IP6 may represent a novel type of selective inhibitor of osteoclasts and prove useful for the treatment of osteoporosis.     

Bacterial lipopolysaccharide induces osteoclast formation in RAW 264.7 macrophage cells

Lipopolysaccharide (LPS) is a potent bone resorbing factor. The effect of LPS on osteoclast formation was examined by using murine RAW 264.7 macrophage cells. LPS-induced the formation of multinucleated giant cells (MGC) in RAW 264.7 cells 3 days after the exposure. MGCs were positive for tartrate-resistant acid phosphatase (TRAP) activity. Further, MGC formed resorption pits on calcium-phosphate thin film that is a substrate for osteoclasts. Therefore, LPS was suggested to induce osteoclast formation in RAW 264.7 cells. LPS-induced osteoclast formation was abolished by anti-tumor necrosis factor (TNF)-alpha antibody, but not antibodies to macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-kappaB ligand (RANKL). TNF-alpha might play a critical role in LPS-induced osteoclast formation in RAW 264.7 cells. Inhibitors of NF-kappaB and stress activated protein kinase (SAPK/JNK) prevented the LPS-induced osteoclast formation. The detailed mechanism of LPS-induced osteoclast formation is discussed.     

Vitamin A differentially regulates RANKL and OPG expression in human osteoblasts

Mouse marrow, which contains osteoblast and osteoclast precursors, was grown in the presence of calcitriol and/or basic fibroblast growth factor (FGF-2). RAW 264.7 cells were differentiated into osteoclast-like cells in the presence of receptor activator of NF-kappaB-Ligand (RANK-L) and/or FGF-2. FGF-2 alone supported osteoclastogenesis in mouse marrow cultures, but not by RAW 264.7 cells alone. Although FGF-2 supported low levels of osteoclastogenesis in mouse marrow cultures, it strongly inhibited the high levels of osteoclastogenesis triggered by calcitriol. Adding excess recombinant-RANK-L to the cultures did not relieve this inhibition. After mouse marrow osteoclasts were differentiated, FGF-2 dose-dependently inhibited bone resorptive activity. FGF-2 increased the tendency of RAW 264.7 osteoclast-like cells to fuse into very large giant cells and induced reorganizations of the actin cytoskeleton in mature, RANK-L-induced RAW 264.7 osteoclast-like cells. These results suggest that FGF-2 has both direct and indirect effects on osteoclast formation and bone resorption.     

Osteoprotegerin induces podosome disassembly in osteoclasts through calcium,ERK, and p38 MAPK signaling pathways

Osteoclasts are critical for bone resorption and use podosomes to attach to bone matrix. Osteoprotegerin (OPG) is a negative regulator of osteoclast function that can affect the formation and function of podosomes. However, the signaling pathways that link OPG to podosome function have not been well characterized. Therefore, this study examined the roles of intracellular calcium and MAPKs in OPG-induced podosome disassembly in osteoclasts. We assessed the effects of the intracellular calcium chelator Bapta-AM, ERK inhibitor U0126, and p38 inhibitor SB202190 on OPG-treated osteoclast differentiation, adhesion structures, intracellular free Ca²⁺ concentration and the phosphorylation state of podosome associated proteins (Pyk2 and Src). Mouse monocytic RAW 264.7 cells were differentiated to osteoclasts using RANKL (30 ng/mL) and M-CSF (25 ng/mL). The cells were pretreated with Bapta-AM (5 μM), U0126 (5 μM), or SB202190 (10 μM) for 30 min, followed by 40 ng/mL OPG for 3 h. Osteoclastogenesis, adhesion structure, viability and morphology, intracellular free Ca²⁺ concentration and the phosphorylation state of Pyk2 and Src were measured by TRAP staining, scanning electron microscopy, real-time cell analyzer, flow cytometry and western blotting, respectively. OPG significantly inhibited osteoclastogenesis, the formation of adhesion structures, and reduced the amount of phosphorylated Pyk2 and Src-pY527, but increased phosphorylation of Src-pY416. Bapta-AM, U0126, and SB202190 partially restored osteoclast differentiation and adhesion structures. Both Bapta-AM and U0126, but not SB202190, restored the levels of intracellular free Ca²⁺ concentration, phosphorylated Pyk2 and Src-pY527. All three inhibitors blocked OPG-induced phosphorylation at Src-pY416. These results suggest OPG disrupts the attachment structures of osteoclasts and activates Src as an adaptor protein that competes for the reduced amount of phosphorylated Pyk2 through calcium- and ERK-dependent signaling pathways. p38 MAPK signaling may have a different role in OPG-induced osteoclast retraction. Our findings potentially offer novel insights into the signaling mechanisms downstream of OPG that affect osteoclast attachment to the extracellular matrix.     

Estradiol inhibits adhesion and promotes apoptosis in murine osteoclasts in vitro

Osteoporosis caused by estrogen deficiency is characterized by enhanced bone resorption mediated by osteoclasts. Adhesion to bone matrix and survival of differentiated osteoclasts is necessary to resorb bone. The aim of our study was to investigate the in vitro effects of estradiol on murine osteoclasts. RAW 264.7 cells treated with 30 ng/ml RANK-L were used as a model for osteoclastogenesis. Estradiol (10⁻⁸ M) for 5 days induced an inhibition of osteoclast differentiation and β3 expression. Estradiol inhibited significantly the adhesion of mature osteoclasts by 30%. Furthermore estradiol-induced apoptosis shown by with nuclear condensation and Bax/Bcl2 ratio. In addition, estradiol enhanced caspase-3, -8 and -9 activities. This effect completely disappeared using specific caspase-8 inhibitor. However, increased caspase-3 activity by estradiol was observed in the presence of caspase-9 inhibitor, indicating the preferential involvement of caspase-8 pathway. Fas and FasL mRNA expression was not regulated by estradiol. However, estradiol enhanced caspase-3 activity in Fas-induced apoptosis on mature osteoclasts, suggesting that this might interact with the Fas-signaling pathway. These data suggest that estradiol decreases bone resorption by several mechanisms including adhesion and apoptosis of osteoclasts.     

NMDA glutamate receptors are expressed by osteoclast precursors and involved in the regulation of osteoclastogenesis

We previously identified functional N-methyl-D-aspartate (NMDA) glutamate receptors in mature osteoclasts and demonstrated that they are involved in bone resorption in vitro. In the present work, we studied the expression of NMDA receptors (NMDAR) by osteoclast precursors and their role in osteoclastogenesis using two in vitro models, the murine myelomonocytic RAW 264.7 cell line and mouse bone marrow cells, both of which differentiate into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF) and Rank ligand (RankL). Using RT-PCR analysis with specific probes, we showed that RAW 264.7 cells and mouse bone marrow cells express mRNA of NMDAR subunits NMDA receptor 1 (NR1) and NMDA receptor 2 (NR2) A, B, and D. These subunits are expressed all along the differentiation sequence from undifferentiated precursors to mature resorbing osteoclasts. Semi-quantitative PCR analysis showed no regulation of the expression of these subunits during the differentiation process. Two specific non competitive antagonists of NMDAR, MK801 and DEP, dose-dependently inhibited osteoclast formation in both models, indicating that osteoclastogenesis requires the activation of NMDAR expressed by osteoclast precursors. MK801 had no effect when added only during the first 2 days of culture, suggesting that NMDAR are rather involved in the late stages of osteoclast formation. Finally, we demonstrated using Western-blotting and immunofluorescence that activation of NMDAR in RAW 264.7 cells by specific agonists induces nuclear translocation of NF-kappa B, a factor required for osteoclast formation. Altogether, our results indicate that osteoclast precursors express NMDAR that are involved in the osteoclast differentiation process through activation of the NF-kappa B pathway.     

Nitric oxide enhances osteoclastogenesis possibly by mediating cell fusion

Osteoclasts are multinucleated bone resorbing cells which form by fusion of pre-osteoclasts. Here, we investigate how nitric oxide (NO) affects osteoclastogenesis. Time lapse photomicrography, using the fluorescent NO indicator dye, 4,5-diaminofluorescein diacetate, revealed an intense NO signal in pre-osteoclasts preceding cell fusion. Osteoclastogenesis in RAW264.7 cells increased when exposed to the NO synthase inhibitor, l-NMMA (0.25 μM), for the initial 48 h. In contrast, pre-osteoclast fusion decreased when RAW264.7 cells were exposed to l-NMMA from 48 to 96 h. Both NO synthase inhibitors, l-NMMA and l-NAME, decreased osteoclast formation during this time period. The inhibitory effect of l-NMMA on osteoclast formation was abolished with increasing concentrations (25–200 ng/ml) of sRANKL suggesting signaling cross talk. NO donors increased osteoclast formation in a dose-dependent manner, with greatest stimulation at 15 μM NOC-12 (2.3 fold) and 5 μM NOC-18 (2.4 fold). Measuring nitrite (NO end product) daily from culture media of RAW264.7 cells undergoing osteoclastogenesis revealed that an increase in NO production coincided with the fusion of pre-osteoclasts (day 4). Inhibiting fusion by plating cells on polystyrene dishes pre-coated with poly-(l-lysine) decreased both osteoclast formation and NO production. To address if NO mediates fusion through the actin cytoskeleton, actin free barbed ends were measured. 0.25 μM l-NMMA decreased, while 15 μM NOC-12 and 5 μM NOC-18 increased actin free barbed ends. We hypothesize that while NO initially negatively regulates pre-osteoclast differentiation; it later facilitates the fusion of mononuclear pre-osteoclasts, possibly by up regulating actin remodeling.     

Fisetin antagonizes cell fusion,cytoskeletal organization and bone resorption in RANKL-differentiated murine macrophages

Osteoclastogenesis is comprised of several stage s including progenitor survival, differentiation to mononuclear preosteoclasts, cell fusion to multinuclear mature osteoclasts, and activation to osteoclasts with bone resorbing activity. Botanical antioxidants are now being increasingly investigated for their health-promoting effects on bone. This study investigated that fisetin, a flavonol found naturally in many fruits and vegetables, suppressed osteoclastogenesis by disturbing receptor activator of nuclear factor (NF)-κB ligand (RANKL)-mediated signaling pathway and demoting osteoclastogenic protein induction. Nontoxic fisetin at ≤10μM inhibited the induction of RANK, tumor necrosis factor receptor associated factor 6 (TRAF6) and the activation of NF-κB in RANKL-stimulated RAW 264.7 macrophages. In RANKL-differentiated osteoclasts cell fusion protein of E-cadherin was induced, which was dampened by fisetin. The formation of tartrate-resistance acid phosphatase-positive multinucleated osteoclasts was suppressed by adding fisetin to RANKL-exposed macrophages. It was also found that fisetin reduced actin ring formation and gelsolin induction of osteclasts enhanced by RANKL through disturbing c-Src-proline-rich tyrosine kinase 2 signaling. Fisetin deterred preosteoclasts from the cell-cell fusion and the organization of the cytoskeleton to seal the resorbing area and to secret protons for bone resorption. Consistently, the 5 day-treatment of fisetin diminished RANKL-induced cellular expression of carbonic anhydrase II and integrin β3 concurrently with a reduction of osteoclast bone-resorbing activity. Therefore, fisetin was a natural therapeutic agent retarding osteoclast fusion and cytoskeletal organization such as actin rings and ruffled boarder, which is a property of mature osteoclasts and is required for osteoclasts to resorb bone.     

RANKL induces components of the extrinsic coagulation pathway in osteoclasts

Prothrombin is converted to thrombin by factor Xa in the cell-associated prothrombinase complex. Prothrombin is present in calcified bone matrix and thrombin exerts effects on osteoblasts as well as on bone resorption by osteoclasts.We investigated whether (1) osteoclasts display factor Xa-dependent prothrombinase activity and (2) osteoclasts express critical regulatory components upstream of the prothrombinase complex.The osteoclast differentiation factor RANKL induced formation of multinucleated TRAP positive cells concomitant with induction of prothrombinase activity in cultures of RAW 264.7 cells and bone marrow osteoclast progenitors.Expression analysis of extrinsic coagulation factors revealed that RANKL enhanced protein levels of factor Xa as well as of coagulation factor III (tissue factor). Inhibition assays indicated that factor Xa and tissue factor were involved in the control of prothrombinase activity in RANKL-differentiated osteoclasts, presumably at two stages (1) conversion of prothrombin to thrombin and (2) conversion of factor X to factor Xa, respectively.Activation of the extrinsic coagulation pathway during osteoclast differentiation through induction of tissue factor and factor Xa by a RANKL-dependent pathway indicates a novel role for osteoclasts in converting prothrombin to thrombin.     

Inhibitory effect of luteolin on osteoclast differentiation and function

Osteoclasts are multinucleated cells that play a crucial role in bone resorption, and are formed by the fusion of mononuclear osteoclasts derived from osteoclast precursors of the macrophage lineage. Compounds that specifically target functional osteoclasts would be ideal candidates for anti-resorptive agents for clinical applications. In the present study, we investigated the effects of luteolin, a flavonoid, on the regulation of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, functions and signaling pathway. Addition of luteolin to a coculture system of mouse bone marrow cells and ST2 cells in the presence of 10⁻⁸ M 1α,25(OH)₂D₃ caused significant inhibition of osteoclastogenesis. Luteolin had no effects on the 1α,25(OH)₂D₃-induced expressions of RANKL, osteoprotegerin and macrophage colony-stimulating factor mRNAs. Next, we examined the direct effects of luteolin on osteoclast precursors using bone marrow macrophages and RAW264.7 cells. Luteolin completely inhibited RANKL-induced osteoclast formation. Moreover, luteolin inhibited the bone resorption by mature osteoclasts accompanied by the disruption of their actin rings, and these effects were reversely induced by the disruption of the actin rings in mature osteoclasts. Finally, we found that luteolin inhibited RANKL-induced osteoclastogenesis through the suppression of ATF2, downstream of p38 MAPK and nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) expression, respectively. Taken together, the present results indicate that naturally occurring luteolin has inhibitory activities toward both osteoclast differentiation and functions through inhibition of RANKL-induced signaling pathway as well as actin ring disruption, respectively.     

Mechanisms involved in enhancement of osteoclast formation and function by low molecular weight hyaluronic acid

Hyaluronic acid (HA) is a component of the extracellular matrix that has been shown to play an important role in bone formation, resorption, and mineralization both in vivo and in vitro. We examined the effects of HA at several molecular weights on osteoclast formation and function induced by RANKL (receptor activator of NF-kappa B ligand) in a mouse monocyte cell line (RAW 264.7). HA at M(r) < 8,000 (low molecular weight HA (LMW-HA)) enhanced tartrate-resistant acid phosphatase-positive multinucleated cell formation and tartrate-resistant acid phosphatase activity induced by RANKL in a dose-dependent manner, whereas HA at M(r) > 900,000 (high molecular weight HA (HMW-HA)) showed no effect on osteoclast differentiation. LMW-HA enhanced pit formation induced by RAW 264.7 cells, whereas HMW-HA did not, and LMW-HA stimulated the expression of RANK (receptor activator of NF-kappa B) protein in RAW 264.7 cells. In addition, we found that LMW-HA enhanced the levels of c-Src protein and phosphorylation of ERKs and p38 MAPK in RAW 264.7 cells stimulated with RANKL, whereas the p38 MAPK inhibitor SB203580 inhibited RANKL-induced osteoclast differentiation. This enhancement of c-Src and RANK proteins induced by LMW-HA was inhibited by CD44 function-blocking monoclonal antibody. These results indicate that LMW-HA plays an important role in osteoclast differentiation and function through the interaction of RANKL and RANK.     

Targeted overexpression of osteoactivin in cells of osteoclastic lineage promotes osteoclastic resorption and bone loss in mice

This study sought to test whether targeted overexpression of osteoactivin (OA) in cells of osteoclastic lineage, using the tartrate-resistant acid phosphase (TRAP) exon 1B/C promoter to drive OA expression, would increase bone resorption and bone loss in vivo. OA transgenic osteoclasts showed ～2-fold increases in OA mRNA and proteins compared wild-type (WT) osteoclasts. However, the OA expression in transgenic osteoblasts was not different. At 4, 8, and 15.3 week-old, transgenic mice showed significant bone loss determined by pQCT and confirmed by μ-CT. In vitro, transgenic osteoclasts were twice as large, had twice as much TRAP activity, resorbed twice as much bone matrix, and expressed twice as much osteoclastic genes (MMP9, calciton receptor, and ADAM12), as WT osteoclasts. The siRNA-mediated suppression of OA expression in RAW264.7-derived osteoclasts reduced cell size and osteoclastic gene expression. Bone histomorphometry revealed that transgenic mice had more osteoclasts and osteoclast surface. Plasma c-telopeptide (a resorption biomarker) measurements confirmed an increase in bone resorption in transgenic mice in vivo. In contrast, histomorphometric bone formation parameters and plasma levels of bone formation biomarkers (osteocalcin and pro-collagen type I N-terminal peptide) were not different between transgenic mice and WT littermates, indicating the lack of bone formation effects. In conclusion, this study provides compelling in vivo evidence that osteoclast-derived OA is a novel stimulator of osteoclast activity and bone resorption.     

Ajulemic acid, a nonpsychoactive cannabinoid acid, suppresses osteoclastogenesis in mononuclear precursor cells and induces apoptosis in mature osteoclast-like cells

Oral administration of ajulemic acid (AjA), a cannabinoid acid devoid of psychoactivity, prevents joint tissue injury in rats with adjuvant induced arthritis. Because activation of osteoclasts is central to the pathogenesis of bone erosion in patients with rheumatoid arthritis (RA), we investigated the influence of AjA on osteoclast differentiation and survival. Osteoclast cultures were established by stimulation of RAW264.7 cells and primary mouse bone marrow cultures with receptor activator of NF-kappaB ligand (RANKL). Simultaneous addition of AjA (15 and 30 microM) and RANKL to both culture systems significantly suppressed development of multinucleated osteoclasts (osteoclastogenesis) in a dose dependent manner, as determined by quantification of multinuclear, tartrate-resistant acid phosphatase (TRAP)-positive cells. AjA impaired growth of RAW264.7 monocytes and prevented further osteoclast formation in cultures in which osteoclastogenesis had already begun. Reduction by AjA of both monocyte growth and osteoclast formation was associated with apoptosis, assayed by annexin V and propidium iodide staining, and caspase activity. The anti-osteoclastogenic effects of AjA did not require the continuous presence of AjA in the cell cultures. Based on these findings, we propose that AjA or other nonpsychoactive synthetic analogs of Cannabis constituents may be useful therapy for diseases such as RA and osteoporosis in which bone resorption is a central feature.     

A role for alphaV integrin subunit in TGF-beta-stimulated osteoclastogenesis

TGF-beta increases bone resorption in vivo and greatly increases osteoclast formation stimulated by receptor activator of NF-kappaB ligand (RANKL) in vitro. TGF-beta does not independently affect the differentiation state of RAW264.7 preosteoclasts, but increases cell attachment to vitronectin. This effect is mediated by increased expression of alphaV integrin subunit mRNA and protein. Concomitant with induction of osteoclast differentiation, RANKL causes relocation of alphaV to focal sites in the cell. This effect is potentiated by TGF-beta. Integrin blockade disrupts both attachment to vitronectin and RANKL-induced osteoclast formation, but culture on vitronectin has little effect. Ectopic expression of alphaV stimulates multinucleation of RAW264.7 cells and increases the number of osteoclasts formed in the presence of RANKL. These data suggest that TGF-beta potentiates RANKL-induced osteoclast formation, in part by increased expression of the alphaV integrin subunit, which may contribute to cell fusion.