Osteoclast-mediated Bone Loss Research Articles

The purinergic receptor P2X5 regulates inflammasome activity and hyper-multinucleation of murine osteoclasts

Excessive bone resorption by osteoclasts (OCs) can result in serious clinical outcomes, including bone loss that may weaken skeletal or periodontal strength. Proper bone homeostasis and skeletal strength are maintained by balancing OC function with the bone-forming function of osteoblasts. Unfortunately, current treatments that broadly inhibit OC differentiation or function may also interfere with coupled bone formation. We therefore identified a factor, the purinergic receptor P2X5 that is highly expressed during the OC maturation phase, and which we show here plays no apparent role in early bone development and homeostasis, but which is required for osteoclast-mediated inflammatory bone loss and hyper-multinucleation of OCs. We further demonstrate that P2X5 is required for ATP-mediated inflammasome activation and IL-1β production by OCs, and that P2X5-deficient OC maturation is rescued in vitro by addition of exogenous IL-1β. These findings identify a mechanism by which OCs react to inflammatory stimuli, and may identify purinergic signaling as a therapeutic target for bone loss-related inflammatory conditions.

Synthesis of new indole-based bisphosphonates and evaluation of their chelating ability in PE/CA-PJ15 cells

Bisphosphonates are the most important class of antiresorptive agents used against osteoclast-mediated bone loss, and, more recently, in oncology. These compounds have high affinity for calcium ions (Ca2+) and therefore target bone mineral, where they appear to be internalized selectively by bone-resorbing osteoclasts and inhibit osteoclast function. They are extensively used in healthcare, however they are affected by severe side effects; pharmacological properties of bisphosphonates depend on their molecular structure, which is frequently the cause of poor intestinal adsorption and low distribution. In this work we synthesized six novel bisphosphonate compounds having a variably substituted indole moiety to evaluate their extra- and intracellular calcium chelating ability in PE/CA-PJ15 cells. Preliminary in silico and in vitro ADME studies were also performed and the results suggested that the indole moiety plays an important role in cell permeability and metabolism properties.

Adseverin plays a role in osteoclast differentiation and periodontal disease-mediated bone loss.

Osteoclast differentiation and function are highly dependent on the assembly and turnover of actin filaments, but little is known about the roles of actin binding proteins in these processes. Adseverin (Ads), a member of the gelsolin superfamily of actin capping and severing proteins, regulates actin filament turnover and can regulate the turnover of cortical actin filaments of chromaffin cells during exocytosis. Using a conditional Ads knockout mouse model, we confirmed our previous finding in cultured cells that Ads plays a role in osteoclastogenesis (OCG) and actin cytoskeletal organization in osteoclasts. Here we show that Ads is required for osteoclast formation and that when alveolar bone resorption is experimentally induced in mice, genetic deletion of Ads prevents osteoclast-mediated bone loss. Further, when Ads-null osteoclasts are cultured, they exhibit defective OCG, disorganized podosome-based actin filament superstructures, and decreased bone resorption. Reintroduction of Ads into Ads-null osteoclast precursor cells restored these osteoclast defects. Collectively, these data demonstrate a unique and osteoclast-specific role for Ads in OCG and osteoclast function.

The orally available Btk inhibitor ibrutinib (PCI-32765) protects against osteoclast-mediated bone loss

Bone-resorbing osteoclasts play an essential role in normal bone homeostasis, as well as in various bone disorders such as osteoporosis and rheumatoid arthritis. Previously we showed that the Tec family of tyrosine kinases is essential for the differentiation of osteoclasts and the inhibition of Btk is a promising strategy for the prevention of the bone loss in osteoclast-associated bone disorders. Here we demonstrate that an orally available Btk inhibitor, ibrutinib (PCI-32765), suppresses osteoclastic bone resorption by inhibiting both osteoclast differentiation and function. Ibrutinib downregulated the expression of NFATc1, the key transcription factor for osteoclastogenesis, and disrupted the formation of the actin ring in mature osteoclasts. In addition, genome-wide screening revealed that Btk regulates the expression of the genes involved in osteoclast differentiation and function in both an NFATc1-dependent and -independent manner. Finally, we showed that ibrutinib administration ameliorated the bone loss that developed in a RANKL-induced osteoporosis mouse model. Thus, this study suggests ibrutinib to be a promising therapeutic agent for osteoclast-associated bone diseases.

Parathyroid Hormone, Can Be a New Therapy to Overcome the Bisphosphonate Related Osteonecrosis of Jaw?

For the past 30 years, bisphosphonates (BPs) have been used routinely to control skeletal complications associated with osteoclast mediated bone loss in osteoporosis, osteolytic pathology of bone and complications of metastatic diseases. Because it is a major class of anti-bone resorptive drug, administration of BPs in these patients effectively restores bone mineral density and bone strength, reduces the incidence of bone fracture, and dramatically improves the quality of life

THU0037 Immune complex-induced inhibition of osteoclastogenesis is mediated via activating, but not inhibitory FCGAMMA receptors on myeloid precursor cells

BackgroundRheumatoid arthritis is characterized by osteoclast-mediated bone loss. Co-stimulatory signalling via ITAM- and ITIM-coupled receptors is essential for osteoclast formation and function. The ITAM- and ITIM-coupled Fcγ receptors (FcγR) are...

Activating FCγ RECEPTORS mediate immune complex-induced inhibition of osteoclastogenesis

BackgroundRheumatoid arthritis is characterised by osteoclast-mediated bone loss. Co-stimulatory signalling via ITAM- and ITIM-coupled receptors is essential for osteoclast formation and function. The ITAM- and ITIM-coupled Fcγ receptors (FcγR) play...

Inhibitory effect of chloroquine on bone resorption reveals the key role of lysosomes in osteoclast differentiation and function

The lysosome is an acidic compartment containing certain hydrolytic enzymes necessary for the intracellular digestion of macromolecules. In terms of the activity of bone-resorbing osteoclasts, the secretion of lysosomal vesicles containing protons and matrix-degrading proteinases into the resorption lacunae is essential. Chloroquine (CQ), one of the lysosomotropic agents, has an immunosuppressive effect and is used for the treatment for rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). However, the direct effect of CQ on osteoclasts has not been reported. Here, we show that CQ suppresses the bone resorbing activity of osteoclasts by inhibition of the acidification in the lysosomes, as well as osteoclast differentiation in vitro. CQ treatment ameliorates the bone loss induced by RANKL injection in mice. These results suggest that CQ has a bone-increasing effect by inhibiting osteoclast differentiation and function. In addition, a lysosomal proton pump inhibitor bafilomycin A1 also inhibits osteoclast differentiation. Thus, this study revealed the importance of the lysosomes in osteoclast differentiation and function in vivo as well as in vitro, suggesting the therapeutic efficacy of immunosuppressive CQ in osteoclast-mediated bone loss or destruction.

Fisetin Inhibits Osteoclast Differentiation via Downregulation of p38 and c-Fos-NFATc1 Signaling Pathways.

The prevention or therapeutic treatment of loss of bone mass is an important means of improving the quality of life for patients with disorders related to osteoclast-mediated bone loss. Fisetin, a flavonoid dietary ingredient found in the smoke tree (Continus coggygria), exhibits various biological activities, but its effect on osteoclast differentiation is unknown. In this study, fisetin dose-dependently inhibited the RANKL-induced osteoclast differentiation with downregulation of the activity or expression of p38, c-Fos, and NFATc1 signaling molecules. The p38/c-Fos/NFATc1-regulated expression of genes required for cell fusion and bone resorption, such as DC-STAMP and cathepsin K, was also inhibited by fisetin. Considering the rescue of fisetin's inhibitory action by NFATc1 over-expression, the cascade of p38-c-Fos-NFATc1 could be strongly involved in the inhibitory effect of fisetin on osteoclast differentiation. Furthermore, fisetin inhibited the bone-resorbing activity of mature osteoclasts. In conclusion, fisetin may be of use in the treatment of osteoclast-related disorders, including osteoporosis.

Histone deacetylase inhibitors and periodontal bone loss

Bone loss caused by enhanced osteoclast activity is a significant feature of periodontitis. Histone deacetylase inhibitors (HDACi) can suppress osteoclast-mediated bone loss in vitro and in vivo. This study investigated whether HDACi can suppress bone loss in experimental periodontitis. Experimental periodontitis was induced in mice by oral inoculation with Porphyromonas gingivalis bacteria. Mice were treated orally with olive oil alone, with olive oil and a novel compound - 1179.4b - which targets both Class I and Class II histone deacetylases (HDACs) or with olive oil and MS-275, which targets Class I HDACs. Micro-computed tomography scans of live mice, stereo imaging and histological analyses were used to detect changes in bone. In the absence of treatment there was a 13.2% increase in bone volume in controls compared with a 7.4% decrease in P. gingivalis-inoculated mice. 1179.4b significantly reduced bone loss, with a 3.4% increase in bone volume (p < 0.01). MS-275 did not have a significant effect on P. gingivalis-induced bone loss. Histological analysis revealed that 1179.4b reduced bone loss despite having no effect on inflammation. HDACi were found to effectively suppress bone loss in the mouse model of periodontitis. 1179.4b - the inhibitor of Class I and Class II HDACs - was more effective at suppressing bone loss than MS-275, which targets Class I HDACs only. These compounds may therefore have the potential to be used for the management of periodontitis.

Ionizing Radiation and Bone Loss: Space Exploration and Clinical Therapy Applications

Damage to normal, nontumor bone tissue following therapeutic irradiation increases the risk of fracture among cancer patients. For example, women treated for various pelvic tumors have been shown to have a greater than 65% increased incidence of hip fracture by 5 years postradiotherapy. Another practical situation in which exposure to ionizing radiation may negatively impact skeletal integrity is during extended spaceflight missions. There is a limited understanding of how spaceflight-relevant doses and types of radiation can influence astronaut bone health, particularly when combined with the significant effects of mechanical unloading experienced in microgravity. Historically, negative effects on osteoblasts have been studied. Radiation exposure has been shown to damage osteoblast precursors. Damage to local vasculature has been observed, ranging from decreased lumen diameter to complete ablation within the irradiated volume, causing a state of hypoxia. These effects result in suppression of bone formation and a general state of low bone turnover. More recently, however, we have demonstrated in pre-clinical mouse models, a very rapid but transient increase in osteoclast activity after exposure to spaceflight and clinically relevant radiation doses. Combined with long-term suppression of bone formation, this skeletal damage may cause long-term deficits. This review will present a broad set of literature outlining our current set knowledge of both clinical therapy and space exploration exposure to ionizing radiation. Additionally, we will discuss prevention of the initial osteoclast-mediated bone loss, the need to promote normal bone turnover and long-term quality of bone tissue, and our hypothesized molecular mechanisms.

Denosumab prevents metacarpal shaft cortical bone loss in patients with erosive rheumatoid arthritis

Osteoclast-mediated bone loss in the hand predicts future bone erosions in patients with rheumatoid arthritis (RA). Osteoclast activity depends on RANKL, which is inhibited by denosumab, an investigational fully human monoclonal antibody against RANKL. We measured metacarpal shaft cortical bone thickness using a novel computer-based technique, digital x-ray radiogrammetry (DXR), to evaluate the effects of denosumab on cortical bone in RA. Patients (n = 227) with active, erosive RA were randomized to receive subcutaneous denosumab 60 mg or 180 mg or placebo every 6 months. All patients received stable doses of methotrexate and daily calcium and vitamin D. For this blinded post hoc analysis (n = 218), cortical bone loss was determined by DXR using computer-assisted measurement of cortical thickness and shaft width at 21 midshaft levels of the second through fourth metacarpal bones of both hands. At 12 months, patients receiving denosumab had significantly less metacarpal bone loss versus placebo (denosumab 60 mg: -0.0034, denosumab 180 mg: 0.0001 gain, placebo: -0.0108; P < or = 0.01 for both denosumab doses). Twelve-month decreases from baseline greater than the smallest detectable change occurred in 2 patients in the denosumab 180 mg group, 9 patients in the denosumab 60 mg group, and 12 patients in the placebo group. Negative correlation was significant between static cortical thickness ratios and static erosion scores (6 and 12 months), and for placebo, between changes in erosion scores and changes in cortical thickness ratio. Twice-yearly injections of denosumab with ongoing methotrexate treatment significantly reduced cortical bone loss in RA patients for up to 12 months. These results add to the growing evidence supporting the clinical utility of DXR.

Immune complex-mediated inhibition of osteoclastogenesis: role for the inhibitory Fc γ receptor IIB in bone erosion in antigen-induced arthritis?

Rheumatoid arthritis is characterised by osteoclast-mediated bone loss. In several studies it was shown that SHIP-/- mice are severely osteoporotic due to increased numbers of hyperreactive osteoclasts. Because SHIP is...

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.

Bisphosphonates: Mechanism of Action and Role in Clinical Practice

Bisphosphonates are primary agents in the current pharmacological arsenal against osteoclast-mediated bone loss due to osteoporosis, Paget disease of bone, malignancies metastatic to bone, multiple myeloma, and hypercalcemia of malignancy. In addition to currently approved uses, bisphosphonates are commonly prescribed for prevention and treatment of a variety of other skeletal conditions, such as low bone density and osteogenesis imperfecta. However, the recent recognition that bisphosphonate use is associated with pathologic conditions including osteonecrosis of the jaw has sharpened the level of scrutiny of the current widespread use of bisphosphonate therapy. Using the key words bisphosphonate and clinical practice in a PubMed literature search from January 1, 1998, to May 1, 2008, we review current understanding of the mechanisms by which bisphosphonates exert their effects on osteoclasts, discuss the role of bisphosphonates in clinical practice, and highlight some areas of concern associated with bisphosphonate use.

Subgroup analysis of a randomized, phase III study of the effect of denosumab in women with nonmetastatic breast cancer receiving aromatase inhibitor (AI) therapy

546 Background: In postmenopausal women with breast cancer, adjuvant AI therapy contributes to accelerated bone loss and risk for fracture. Denosumab, a fully human monoclonal antibody to RANKL, inhibits osteoclast-mediated bone loss. Methods: In this 2-year, double-blind, placebo-controlled study, we evaluated factors that may influence BMD in patients (pts) ≥ 18 years old with hormone receptor-positive breast cancer who were receiving adjuvant AI therapy and had evidence of low bone mass. Pts were stratified by duration of prior AI therapy (≤ 6 vs >6 months) and randomized to receive, together with calcium and vitamin D, placebo (n = 125) or 60 mg denosumab (n = 127) subcutaneously every 6 months for 4 doses. A sample size of 104 pts/group was planned to provide 95% power to detect a 2% difference between groups (α = 0.05). An analysis of covariance model was used for subgroup analyses, adjusting for treatment, stratification factor, baseline BMD value, densitometer type, and baseline BMD value-by-densi...

The effect of zoledronic acid incorporated in a poly(D,L‐lactide) implant coating on osteoblasts in vitro

Bisphosphonates such as zoledronic acid (ZOL) are used in diseases associated with osteoclast-mediated bone loss. However, their antiresorptive activity is partly due to their effect on osteoblasts. Local application might increase the therapeutical fence and their local efficiency and reduce systemic side effects. Aim of the study was to investigate the effect of ZOL on human osteoblasts like cells in vitro with special focus on the synthesis of factors mediating osteoclast differentiation (RANKL, OPG). ZOL was incorporated in an implant coating based on poly(D,L-lactide) (PDLLA) in different concentrations (10-150 microM). Control groups were treated with uncoated implants, PDLLA-coated implants, and ZOL pure substance in corresponding concentrations. After an experimental period of 144 h, primary human osteoblasts were stained with alamar blue and cell viability was measured. Procollagen I synthesis, osteoprotegerin (OPG) secretion, and soluble receptor activator of nuclear factor-kappaB ligand (sRANKL) were analyzed. Results showed that cell viability was not affected when treated with doses equivalent up to 100 microM ZOL-coated implants (ZOL-CI). Procollagen I synthesis was highest when treated with 50 microM ZOL-CI. OPG increased significantly in the 10 microM ZOL-CI group, whereas sRANKL decreased significantly with different concentrations of ZOL-CI. Higher concentrations or exposure to the pure substance showed a decrease in cell viability, collagen I, OPG, and sRANKL synthesis. In conclusion, exposure to specific concentrations of ZOL-CI showed a beneficial effect on osteoblast differentiation and protein synthesis without influencing their proliferation. Changes in sRANKL and OPG production may contribute to the inhibition of osteoclastic bone resorption. This local antiresorptive effect might be clinically useful in osseous implant integration and fracture healing.

Effect of rapamycin on hepatic osteodystrophy in rats with portasystemic shunting

To study if T-cell activation related to portasystemic shunting causes osteoclast-mediated bone loss through RANKL-dependent pathways. We also investigated if T-cell inhibition using rapamycin would protect against bone loss in rats. Portasystemic shunting was performed in male Sprague-Dawley rats and rapamycin 0.1 mg/kg was administered for 15 wk by gavage. Rats received powderized chow and supplemental feeds to prevent the effects of malnutrition on bone composition. Weight gain and growth was restored after surgery in shunted animals. At termination, biochemical parameters of bone turnover and quantitative bone histology were assessed. Markers of T-cell activation, inflammatory cytokine production, and RANKL-dependent pathways were measured. In addition, the roles of IGF-1 and hypogonadism were investigated. Portasystemic shunting caused low turnover osteoporosis that was RANKL independent. Bone resorbing cytokine levels, including IL-1, IL-6 and TNFalpha, were not increased in serum and TNFalpha and RANKL expression were not upregulated in PBMC. Portasystemic shunting increased the circulating CD8+ T-cell population. Rapamycin decreased the circulating CD8+ T-cell population, increased CD8+ CD25+ T-regulatory cell population and improved all parameters of bone turnover. Osteoporosis caused by portasystemic shunting may be partially ameliorated by rapamycin in the rat model of hepatic osteodystrophy.

Fos/AP‐1 proteins in bone and the immune system

The skeleton and the immune system share a variety of different cytokines and transcription factors, thereby mutually influencing each other. These interactions are not confined to the bone marrow cavity where bone cells and hematopoietic cells exist in proximity but also occur at locations that are target sites for inflammatory bone diseases. The newly established research area termed 'osteoimmunology' attempts to unravel these skeletal/immunological relationships. Studies towards a molecular understanding of inflammatory bone diseases from an immunological as well as a bone-centered perspective have been very successful and led to the identification of several signaling pathways that are causally involved in inflammatory bone loss. Induction of receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) signals by activated T cells and subsequent activation of the key transcription factors Fos/activator protein-1 (AP-1), NF-kappaB, and NF for activation of T cells c1 (NFATc1) are in the center of the signaling networks leading to osteoclast-mediated bone loss. Conversely, nature has employed the interferon system to antagonize excessive osteoclast differentiation, although this counteracting activity appears to be overruled under pathological conditions. Here, we focus on Fos/AP-1 functions in osteoimmunology, because this osteoclastogenic transcription factor plays a central role in inflammatory bone loss by regulating genes like NFATc1 as well as the interferon system. We also attempt to put potential therapeutic strategies for inflammatory bone diseases in perspective.

The nitrogen-containing bisphosphonate, zoledronic acid, influences RANKL expression in human osteoblast-like cells by activating TNF-alpha converting enzyme (TACE).

Bisphosphonates are used to prevent osteoclast-mediated bone loss. Zoledronic acid inhibits osteoclast maturation indirectly by increasing OPG protein secretion and decreasing transmembrane RANKL expression in human osteoblasts. The decreased transmembrane RANKL expression seems to be related to the upregulation of the RANKL sheddase, TACE. Bisphosphonates (BPs) exhibit high affinity for hydroxyapatite mineral in bone and are used extensively to treat malignancy-associated bone disease and postmenopausal bone loss by inhibiting osteoclast (OC)-mediated bone resorption. We examined the effect of the most potent nitrogen-containing BP available, zoledronic acid (ZOL), on the expression of RANKL and osteoprotegerin (OPG), critical factors in the regulation of OC formation and activation, in primary osteoblast (OB)-like cells derived from human bone, using flow cytometry, ELISA, semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), in situ immunofluorescence staining, and Western blotting. Our studies show that ZOL, while not significantly affecting RANKL or OPG gene expression, markedly increased OPG protein secretion and reduced transmembrane RANKL protein expression in OB-like cells. The reduction in transmembrane RANKL expression was preceded by a marked increase in the expression of the metalloprotease-disintegrin, TNF-alpha converting enzyme (TACE). In addition, the decreased transmembrane expression of RANKL could be partially reversed by a TACE inhibitor, TAPI-2. Our studies indicate that ZOL, in addition to its direct effects on mature OCs, may inhibit the recruitment and differentiation of OCs by cleavage of transmembrane RANKL in OB-like cells by upregulating the sheddase, TACE.