Stimulate Osteoclast Formation Research Articles

Osteoclast progenitors from cats with and without tooth resorption respond differently to 1,25-dihydroxyvitamin D and interleukin-6

Both vitamin D and inflammatory cytokines can stimulate osteoclast formation and activity. We studied the effect of 1,25-dihydroxycholecalciferol (1,25(OH)2D), and interleukin-6 (IL-6), on the formation and activity of feline osteoclasts, using peripheral blood mononuclear cells (PBMCs) from cats with and without tooth resorption (TR+ and TR−) as a source of osteoclast precursors. The formation of osteoclast-like cells (defined as multinucleated, tartrate-resistant acid phosphatase-positive cells) was assessed at 7 and 14days. In the presence of M-CSF and RANKL, with and without IL-6, more osteoclasts were formed from TR− PBMCs than from TR+ PBMCs on plastic. More osteoclasts were formed from TR+ PBMCs on bone slices in the presence of M-CSF/RANKL with 1,25(OH)2D. This opposite effect may be due to a higher expression of the vitamin D receptor (VDR) in TR+ osteoclasts and precursors on bone. Formation of resorption pits was analyzed and confirmed with scanning electron microscopy. In conclusion, we propose that TR+ PBMCs when cultured on bone are sensitive to 1,25(OH)2D, whereas the differentiation of TR− PMBCs on bone seem more sensitive to IL-6, suggesting that osteoclast precursors from cats with and without tooth resorption respond differently to osteoclast stimulating factors.

TWEAK and Fn14 expression in the pathogenesis of joint inflammation and bone erosion in rheumatoid arthritis

IntroductionTNF-like weak inducer of apoptosis (TWEAK) has been proposed as a mediator of inflammation and bone erosion in rheumatoid arthritis (RA). This study aimed to investigate TWEAK and TWEAK receptor (Fn14) expression in synovial tissue from patients with active and inactive rheumatoid arthritis (RA), osteoarthritis (OA) and normal controls and assess soluble (s)TWEAK levels in the synovial fluids from patients with active RA and OA. Effects of sTWEAK on osteoclasts and osteoblasts were investigated in vitro.MethodsTWEAK and Fn14 expression were detected in synovial tissues by immunohistochemistry (IHC). Selected tissues were dual labelled with antibodies specific for TWEAK and lineage-selective cell surface markers CD68, Tryptase G, CD22 and CD38. TWEAK mRNA expression was examined in human peripheral blood mononuclear cells (PBMC) sorted on the basis of their expression of CD22. sTWEAK was detected in synovial fluid from OA and RA patients by ELISA. The effect of sTWEAK on PBMC and RAW 264.7 osteoclastogenesis was examined. The effect of sTWEAK on cell surface receptor activator of NF Kappa B Ligand (RANKL) expression by human osteoblasts was determined by flow cytometry.ResultsTWEAK and Fn14 expression were significantly higher in synovial tissue from all patient groups compared to the synovial tissue from control subjects (P < 0.05). TWEAK was significantly higher in active compared with inactive RA tissues (P < 0.05). TWEAK expression co-localised with a subset of CD38+ plasma cells and with CD22+ B-lymphocytes in RA tissues. Abundant TWEAK mRNA expression was detected in normal human CD22+ B cells. Higher levels of sTWEAK were observed in synovial fluids isolated from active RA compared with OA patients. sTWEAK did not stimulate osteoclast formation directly from PBMC, however, sTWEAK induced the surface expression of RANKL by human immature, STRO-1+ osteoblasts.ConclusionsThe expression of TWEAK by CD22+ B cells and CD38+ plasma cells in RA synovium represents a novel potential pathogenic pathway. High levels of sTWEAK in active RA synovial fluid and of TWEAK and Fn14 in active RA tissue, together with the effect of TWEAK to induce osteoblastic RANKL expression, is consistent with TWEAK/Fn14 signalling being important in the pathogenesis of inflammation and bone erosion in RA.

S100A8 enhances osteoclast-mediated bone resorption in experimental antigen-induced arthritis through activation of toll-like receptor 4

ObjectiveRheumatoid arthritis (RA) is characterised by severe bone erosions caused by enhanced osteoclast formation and activity. Significantly elevated S100A8 and S100A9 levels in serum and synovial fluid of RA patients...

Interaction between the skeletal and immune systems in cancer: mechanisms and clinical implications.

The skeletal and immune systems have a complex relationship. Both systems are intimately coupled, with osteoclastogenesis and hematopoiesis occurring in the bone marrow. Bone and immune cells also share common hematopoietic precursors. Furthermore, the skeletal and immune systems share various cytokines, receptors, and transcription factors that regulate signal transduction pathways involved in osteoclastogenesis and immune system activation, including the receptor activator of nuclear factor-κΒ ligand/receptor activator of nuclear factor-κΒ/osteoprotegerin (RANKL-RANK-OPG) pathway. Cancer cells can disrupt both the skeletal and immune systems. Interaction between cancer and bone cells results in a vicious cycle of bone destruction and cancer growth. Bone remodeling generates a growth-factor-rich environment that attracts cancer cells and promotes their proliferation. In turn, cancer cells stimulate osteoclast formation and activity, resulting in additional bone resorption that further stimulates cancer cell growth. Currently available bone-targeted therapies may also modulate the immune system. Bisphosphonates such as zoledronic acid exert stimulating effects on the immune system, resulting in possible anticancer activity against malignant cells. Denosumab, an anti-RANKL monoclonal antibody with proven antiosteoclast activity, may suppress immune responses. This may result in the reported association with an increased risk of neoplasms, as well as serious skin and other infections as reported in some studies, mainly in the postmenopausal setting. When assessing bone-targeted therapies, it is important to consider the shared signaling pathways between bone and the immune system, as well as the clinical risk:benefit ratio.

Augmented Osteolysis in SPARC-Deficient Mice with Bone-Residing Prostate Cancer

Prostate cancer preferentially metastasizes to bone, which is rich in structural and matricellular proteins capable of altering prostate cancer progression. This study explores the role of the bone stromal matricellular protein SPARC (osteonectin/BM-40) in the progression of bone metastatic prostate cancer. Quantification of bone destruction analyzed by micro–computed tomography showed augmented osteoclastic resorption, characterized by decreases in several morphometric bone parameters in SPARC knock out (KO) tibiae harboring RM1 murine prostate cancer cells compared with wild type (WT) animals. Tumor progression stimulated osteoclast formation, which was augmented in SPARC KO mice. In vitro differentiation of SPARC KO osteoclasts indicated accelerated progenitor expansion and formation of tartrate-resistant acid phosphatase–positive osteoclast-like cells with increased resorptive capacity, a mechanism resulting in enhanced tumor-induced bone loss in vivo. Whereas altered bone structure due to SPARC KO played a role in increased osteolysis, the enhanced osteolysis was primarily the result of increased resorption by SPARC KO osteoclasts. Our findings indicate that bone stromal SPARC suppresses tumor-induced bone lesion expansion by limiting osteoclast maturation and function.

Hypoxia stimulates osteoclast formation from human peripheral blood

Active pathological bone destruction in humans often occurs in locations where oxygen tension (pO(2)) is likely to be low, for example, at the sites of tumours, inflammation, infections and fractures, or the poorly vascularized yellow fatty marrow of the elderly. We examined the effect of pO(2) on formation of osteoclasts, the cells responsible for bone resorption, in 14-day cultures of normal human peripheral blood mononuclear cells (hPBMCs) on ivory discs. Hypoxia (1-2% O(2)) caused threefold increases in the number of osteoclasts formed, compared with 20% O(2). Hypoxia also caused a twofold increase in the number of nuclei per osteoclast, leading to stimulations of resorption pit formation of up to 10-fold. Exposure to hypoxia led to stabilization of the hypoxia-inducible factors, HIF1alpha and HIF2alpha, and upregulation of vascular endothelial growth factor and interleukin-6 expression by hPBMCs. These findings help explain why extravasation of mononuclear precursors into relatively O(2)-deficient bone microenvironments could result in osteoclast formation and suggest a new mechanism for the bone loss associated with the pathophysiological conditions where hypoxia commonly occurs.

Human cord blood CD34+ cells transduced by a lentiviral marking vector can be differentiated into functional GFP expressing osteoclasts at high efficiency

Human recombinant cystatin D also inhibited M-CSF/RANKL stimulated osteoclast formation in BMM cultures, whereas the MMP-inhibitor TIMP-1 had no effect. The amino-terminal segment Arg-Leu-Val-Gly (RLVG) of the cystatin C molecule is an important part of the cysteine proteinase inhibitory center. Similar to cystatin C, Z-RLVG-CHN2 inhibited M-CSF/RANKL stimulated osteoclast formation in BMM cultures. Z-RLVG-CHN2 in which the reactive diazomethane group was substituted for by non-reactive groups (-OH, -NH2, -N(CH3)2) did not affect osteoclastogenesis. The fungal cysteine proteinase inhibitor E-64 also inhibited M-CSF/ RANKL stimulated osteoclast formation. The inhibitory effect by cystatin C was observed when cystatin C was added 1-6 h after RANKL, partially lost when added 12 h after RANKL, and completely lost when added 24 h after RANKL stimulation. Inhibition of osteoclast formation was associated with cystatin C induced inhibition of RANKL stimulated mRNA expression of Calcr, Acp5, Ctsk, Mmp-9, Intb3 and Atp6i, as well as of TRAP protein. Treatment with cystatin C maintained the M-CSF/RANKL stimulated BMM at a macrophage phenotype as assessed by the ability to phagocytose FITC-labelled zymosan particles and increased mRNA expression of the macrophage transcription factor Irf-8. The enhanced expression of osteoclastic transcription factors c-Fos and NFATc1 induced by RANKL was strongly inhibited by cystatin C. These data show that cystatin C can act directly on purified osteoclast progenitors to inhibit differentiation from macrophages to osteoclasts by affecting the differentiation process at an early step. The mechanism is due to inhibition of the osteoclastic transcription factors c-Fos and NFATc1. Disclosure of Interest: None declared

Short-Term Effects of Whole-Body Exposure to56Fe Ions in Combination with Musculoskeletal Disuse on Bone Cells

Space travel and prolonged bed rest cause bone loss due to musculoskeletal disuse. In space, radiation fields may also have detrimental consequences because charged particles traversing the tissues of the body can elicit a wide range of cytotoxic and genotoxic lesions. The effects of heavy-ion radiation exposure in combination with musculoskeletal disuse on bone cells and tissue are not known. To explore this, normally loaded 16-week-old male C57BL/6 mice were exposed to (56)Fe ions (1 GeV/nucleon) at doses of 0 cGy (sham), 10 cGy, 50 cGy or 2 Gy 3 days before tissue harvest. Additional mice were hindlimb unloaded by tail traction continuously for 1 week to simulate weightlessness and exposed to (56)Fe-ion radiation (0 cGy, 50 cGy, 2 Gy) 3 days before tissue harvest. Despite the short duration of this study, low-dose (10, 50 cGy) irradiation of normally loaded mice reduced trabecular volume fraction (BV/TV) in the proximal tibiae by 18% relative to sham-irradiated controls. Hindlimb unloading together with 50 cGy radiation caused a 126% increase in the number of TRAP(+) osteoclasts on cancellous bone surfaces relative to normally loaded, sham-irradiated controls. Together, radiation and hindlimb unloading had a greater effect on suppressing osteoblastogenesis ex vivo than either treatment alone. In sum, low-dose exposure to heavy ions (50 cGy) caused rapid cancellous bone loss in normally loaded mice and increased osteoclast numbers in hindlimb unloaded mice. In vitro irradiation also was more detrimental to osteoblastogenesis in bone marrow cells that were recovered from hindlimb unloaded mice compared to cells from normally loaded mice. Furthermore, irradiation in vitro stimulated osteoclast formation in a macrophage cell line (RAW264.7) in the presence of RANKL (25 ng/ml), showing that heavy-ion radiation can stimulate osteoclast differentiation even in the absence of osteoblasts. Thus heavy-ion radiation can acutely increase osteoclast numbers in cancellous tissue and, under conditions of musculoskeletal disuse, can enhance the sensitivity of bone cells, in particular osteoprogenitors, to the effects of radiation.

Further evidence for direct pro-resorptive actions of FSH

We confirm that FSH stimulates osteoclast formation, function and survival to enhance bone resorption. It does so via the activation of a pertussis toxin-sensitive Gi-coupled FSH receptor that we and others have identified on murine and human osteoclast precursors and mature osteoclasts. FSH additionally enhances the production of several osteoclastogenic cytokines, importantly TNFα, likely within the bone marrow microenvironment, to augment its pro-resorptive action. FSH levels in humans rise before estrogen falls, and this hormonal change coincides with the most rapid rates of bone loss. On the basis of accumulating evidence, we reaffirm that FSH contributes to the rapid peri-menopausal and early post-menopausal bone loss, which might thus be amenable to FSH blockade.

The TRPV1 ion channel antagonist capsazepine inhibits osteoclast and osteoblast differentiation in vitro and ovariectomy induced bone loss in vivo

The vanilloid type 1 ion channel (TRPV1) is known to play an important role in the regulation of pain and inflammation. Pharmacological ligands of TRPV1 regulate human osteoclast formation in vitro, but the effects of these agents on osteoblast function have not been studied and their effects on bone loss in vivo are unknown. Here we examined the effects of the TRPV1 ion channel antagonist capsazepine on mouse osteoclast and osteoblast differentiation in vitro and ovariectomy induced bone loss in vivo. Capsazepine inhibited osteoclast formation and bone resorption in a dose dependent manner in bone marrow–osteoblast co-cultures and RANKL generated osteoclast cultures, whereas the TRPV1 agonist capsaicin enhanced RANKL and M-CSF stimulated osteoclast formation. Capsazepine also suppressed RANKL induced IκB and ERK1/2 phosphorylation and caused apoptosis of mature osteoclasts and also inhibited alkaline phosphatase activity and bone nodule formation in calvarial osteoblast cultures. Studies in vivo showed that capsazepine (1 mg/kg/day) inhibited ovariectomy induced bone loss in mice and histomorphometric analysis showed inhibitory effects on indices of bone resorption and bone formation. We conclude that pharmacological blockade of TRPV1 ion channels by capsazepine inhibits osteoclastic bone resorption and protects against ovariectomy induced bone loss in mice, but also inhibits osteoblast activity and bone formation.

Ascorbic acid accelerates osteoclast formation and death

Ascorbic acid (AA) plays a key role in bone formation. However controversy remains about the effect of AA on cells responsible for bone destruction, osteoclasts. We investigated the effect of AA on osteoclastogenesis using primary mouse bone marrow cultures and monocytic RAW 264.7 cells treated with osteoclastogenic factors RANKL and MCSF. Treatment with AA resulted in significant increase in osteoclast number, size and nucleation. To assess osteoclast oxidative stress level, a ratio of reduced (GSH) to oxidized (GSSG) glutathione and the total glutathione content (GSHt) were evaluated. Osteoclast differentiation was associated with a decrease in GSH/GSSG and GSHt. AA induced further decrease in both parameters, and resulted in significant production of H2O2, indicating its pro-oxidant action. At low concentration, H2O2 induced similar effects to AA, although less potently, and catalase partially inhibited AA-induced osteoclastogenesis. To assess the modification in osteoclast metabolism, the mitochondrial activity was evaluated using JC-1 and the ATP levels were assessed. Osteoclast formation was associated with the increase in mitochondrial activity and ATP concentration, which were further increased in the presence of AA. Importantly, the stimulatory effect of AA was only evident at early phase of osteoclastogenesis, whereas at the late stage AA significantly accelerated osteoclast death. Thus, during osteoclastogenesis AA acts as an oxidant, first stimulating osteoclast formation, but later limiting osteoclast lifespan. This duality of AA action allows reconciling the stimulatory action of AA on osteoclastogenesis observed in vitro with an overall attenuation of bone resorption in the presence of AA observed in vivo.

A novel T cell cytokine, secreted osteoclastogenic factor of activated T cells, induces osteoclast formation in a RANKL-independent manner.

Chronic T cell activation is central to the etiology of rheumatoid arthritis (RA), an inflammatory autoimmune disease that leads to severe focal bone erosions and generalized systemic osteoporosis. Previous studies have shown novel cytokine-like activities in medium containing activated T cells, characterized by potent induction of the osteoblastic production of interleukin-6 (IL-6), an inflammatory cytokine and stimulator of osteoclastogenesis, as well as induction of an activity that directly stimulates osteoclast formation in a manner independent of the key osteoclastogenic cytokine RANKL. This study was undertaken to identify the factors secreted by T cells that are responsible for these activities. Human T cells were activated using anti-human CD3 and anti-human CD28 antibodies for 72 hours in AIM V serum-free medium to obtain T cell-conditioned medium, followed by concentration and fractionation of the medium by fast-protein liquid chromatography. Biologically active fractions were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major bands were analyzed by mass spectrometry, and a major candidate protein was identified. This novel cytokine was cloned, and its expression was analyzed using recombinant DNA technologies. A single novel cytokine that could induce both osteoblastic IL-6 production and functional osteoclast formation in the absence of osteoblasts or RANKL and that was insensitive to the effects of the RANKL inhibitor osteoprotegerin was identified in the activated T cell-conditioned medium; this cytokine was designated secreted osteoclastogenic factor of activated T cells (SOFAT). Further analysis of SOFAT revealed that it was derived from an unusual messenger RNA splice variant coded by the threonine synthase-like 2 gene homolog, which is a conserved gene remnant coding for threonine synthase, an enzyme that functions only in microorganisms and plants. SOFAT may act to exacerbate inflammation and/or bone turnover under inflammatory conditions such as RA or periodontitis and in conditions of estrogen deficiency.

Effects of mechanical strain magnitude on formation and differentiation of osteoclasts

Objective To study the effects of early mechanical strain magnitude on formation and differentiation of osteoclasts. Methods RAW 264.7 cells induced by macrophage colony-stimulating factors and osteoclast differentiation factors were subjected to 0, 1 000, 1 500, 2 000, 2 500 and 5 000 μe mechanical straining for three days. The morphological changes, number of osteoclasts and proliferation of precursor cells were determined at day 7. The activity of the tartrate-reaistant acid phosphatase (TRAP) in the culture medium was detected at days 4 and 7. Results The number of osteoclasts was decreased in 2 500 μe group, while it was increased in 5 000 μe group. The proliferation of precursor cells was increased in 2 000 and 2 500 μe group, while it was decreased significantly in 5 000 μe group. There was no significant difference in the number of osteoclasts and proliferation of precursor cells among 1 000 μe group, 1 500 μe group and 0 μe group. The activity of TRAP was decreased in 1 000, 1 500, 2 000, 2500 and 5 000 μe groups at days 4 and 7 when compared with μe group. Conclusions Early mechanical straining plays a direct role in formation and differentiation of osteoclasts. The high strain magnitude within physiological load inhibits osteoclast formation, while high strain magnitude beyond physiological load stimulates osteoclast formation. Low strain magnitude has nearly no impact on formation of osteoclasts. Early mechanical straining may inhibit differentiation of osteoclasts. Key words: Osteoporosis; Osteoclasts; Mechanical strain

New insights: Elevated follicle-stimulating hormone and bone loss during the menopausal transition

We hypothesize that a rising follicle-stimulating hormone (FSH) level during the menopausal transition, even in the face of a normal estrogen level, contributes to increased bone resorption and profound bone loss that is accompanied by trabecular perforation and diminished bone strength. FSH has been shown to directly stimulate osteoclast formation and bone resorption, and our murine genetic studies indicate that the absence of FSH can, in part, protect against hypogonadal hyperresorption that causes bone loss. Furthermore, carefully conducted human studies, such as the Study of Women Across Nations (SWAN), indicate strong correlations between serum FSH levels and bone loss. Potential therapeutic implications include the development of antagonists to circulating FSH and its osteoclastic receptor.

Undifferentiated osteoblasts stimulate osteoclast formation in the presence of osteoprotegerin

Undifferentiated osteoblasts stimulate osteoclast formation in the presence of osteoprotegerin

Molecular Mechanism of the Bifunctional Role of Lipopolysaccharide in Osteoclastogenesis

Lipopolysaccharide (LPS), a common bacteria-derived product, has long been recognized as a key factor implicated in periodontal bone loss. However, the precise cellular and molecular mechanisms by which LPS induces bone loss still remains controversial. Here, we show that LPS inhibited osteoclastogenesis from freshly isolated osteoclast precursors but stimulated osteoclast formation from those pretreated with RANKL in vitro in tissue culture dishes, bone slices, and a co-culture system containing osteoblasts, indicating that RANKL-mediated lineage commitment is a prerequisite for LPS-induced osteoclastogenesis. Moreover, the RANKL-mediated lineage commitment is long term, irreversible, and TLR4-dependent. LPS exerts the dual function primarily by modulating the expression of NFATc1, a master regulator of osteoclastogenesis, in that it abolished RANKL-induced NFATc1 expression in freshly isolated osteoclast precursors but stimulated its expression in RANKL-pretreated cells. In addition, LPS prolonged osteoclast survival by activating the Akt, NF-kappaB, and ERK pathways. Our current work has not only unambiguously defined the role of LPS in osteoclastogenesis but also has elucidated the molecular mechanism underlying its complex functions in osteoclast formation and survival, thus laying a foundation for future delineation of the precise mechanism of periodontal bone loss.

Oxytocin is an anabolic bone hormone

We report that oxytocin (OT), a primitive neurohypophyseal hormone, hitherto thought solely to modulate lactation and social bonding, is a direct regulator of bone mass. Deletion of OT or the OT receptor (Oxtr) in male or female mice causes osteoporosis resulting from reduced bone formation. Consistent with low bone formation, OT stimulates the differentiation of osteoblasts to a mineralizing phenotype by causing the up-regulation of BMP-2, which in turn controls Schnurri-2 and 3, Osterix, and ATF-4 expression. In contrast, OT has dual effects on the osteoclast. It stimulates osteoclast formation both directly, by activating NF-kappaB and MAP kinase signaling, and indirectly through the up-regulation of RANK-L. On the other hand, OT inhibits bone resorption by mature osteoclasts by triggering cytosolic Ca(2+) release and NO synthesis. Together, the complementary genetic and pharmacologic approaches reveal OT as a novel anabolic regulator of bone mass, with potential implications for osteoporosis therapy.

Increased circulating heat shock protein 60 induced by menopause, stimulates apoptosis of osteoblast-lineage cells via up-regulation of toll-like receptors

Postmenopausal osteoporosis is a heterogeneous disorder characterized by accelerated bone loss after natural or surgical menopause and an increased risk of fractures. The bone loss in estrogen deficiency results from the increased bone resorption and impaired ability of osteoblastic bone formation. Previous studies have reported that the HSP60 stimulates osteoclast formation and bone resorption. Here we found that plasma HSP60 levels were significantly higher in postmenopausal (median 1152.4 ng/ml; range 724.7–2123.4 ng/ml) than in premenopausal (median 316.3 ng/ml; range 164.6–638.4 ng/ml) women. In primary human bone marrow stromal cells (hBMSC) and the HS-5 hBMSC cell line, HSP60 significantly reduced cell viability and increased caspase-dependent apoptosis. Consistent with these observations, HSP60 activated caspase-3 and -9, but not caspase-8 in HS-5 cells, and increased the release of mitochondrial cytochrome c into the cytosol. In addition, HSP60 activated p38 and NFκB, but not ERK or JNK; importantly, inhibitors of p38 (SB203580) and NFκB (PDTC) abolished HSP60-induced apoptosis. Furthermore, Western blotting showed that HSP60 up-regulated TLR-2 and TLR-4 expression, and pretreatment with blocking antibodies for TLR-2 and TLR-4 almost completely eliminated the effects of HSP60 on apoptosis, caspase-3 and -9 activation, and activation of NFκB and p38 MAPK. Most notably, ovariectomy-induced bone loss was attenuated in TLR-2 KO mice. In conclusion, up-regulation of TLR-2 by HSP60 may play a critical role in promoting bone loss in the estrogen-deficient state.

Hypoxia stimulates vesicular ATP release from rat osteoblasts

Many neuronal and non-neuronal cell types release ATP in a controlled manner. After release, extracellular ATP (or, following hydrolysis, ADP) acts on cells in a paracrine manner via P2 receptors. Extracellular nucleotides are now thought to play an important role in the regulation of bone cell function. ATP (and ADP), acting via the P2Y(1) receptor, stimulate osteoclast formation and activity, whilst P2Y(2) receptor stimulation by ATP (or UTP) inhibits bone mineralization by osteoblasts. We found that rat calvarial osteoblasts released ATP constitutively, in a differentiation-dependent manner, with mature, bone-forming osteoblasts releasing up to sevenfold more ATP than undifferentiated, proliferating cells. The inhibitors of vesicular exocytosis, monensin, and N-ethylmaleimide (1-1,000 microM) inhibited basal ATP release by up to 99%. The presence of granular ATP-filled vesicles within the osteoblast cytoplasm was demonstrated by quinacrine staining. Exposure to hypoxia (2% O(2)) for up to 3 min increased ATP release from osteoblasts up to 2.5-fold without affecting cell viability. Peak concentrations of ATP released into culture medium were >1 microM, which equates with concentrations known to exert significant effects on osteoblast and osteoclast function. Monensin and N-ethylmaleimide (100 microM) attenuated the hypoxia-induced ATP release by up to 80%. Depletion of quinacrine-stained vesicles was also apparent after hypoxic stimulation, indicating that ATP release had taken place. These data suggest that vesicular exocytosis is a key mediator of ATP release from osteoblasts, in biologically significant amounts. Moreover, increased extracellular ATP levels following acute exposure to low O(2) could influence local purinergic signaling and affect the balance between bone formation and bone resorption.

Disruption of Rankl/Rank Signaling Reduces TNF-Induced Joint Inflammation In Vivo

TNF is a dominant inflammatory cytokine that is over-produced in the joints of patients with rheumatoid arthritis, as well as animal models of this disease. It promotes bone destruction and inflammation through stimulation of osteoclast formation and activation. While it has been established that the RANKL/RANK system is essential for TNF-induced osteoclast formation and bone erosion in vivo, its role in TNF-induced inflammation remains poorly understood and controversial. Here, we report that genetic and biologic disruption of the RANKL/RANK signaling eliminates knee joint inflammation in TNF-Tg/RANKL-/-, TNF-Tg/RANK-/- and RANK:Fc treated-TNF-Tg mice as determined by functional assessments and histomorphometry. Among several potential mechanisms that could be responsible for this decrease in inflammation, we found that RANKL synergizes with TNF to stimulate TNF production by osteoclast precursors. Thus, blockade of RANKL/RANK signaling may be a viable therapeutic strategy to prevent inflammation and bone loss in diseases where elevated TNF is a key pathogenic factor.