Masanori Koide

Evaluation of Pharmaceuticals With a Novel 50-Hour Animal Model of Bone Loss

Osteoporosis remains a major public health problem through its associated fragility fractures. Several animal models for the study of osteoporotic bone loss, such as ovariectomy (OVX) and denervation, require surgical skills and several weeks to establish. Osteoclast differentiation and activation is mediated by RANKL. Here we report the establishment of a novel and rapid bone loss model by the administration of soluble RANKL (sRANKL) to mice. Mice were injected intraperitoneally with sRANKL and used to evaluate existing anti‐osteoporosis drugs. sRANKL decreased BMD within 50 h in a dose‐dependent manner. The marked decrease in femoral trabecular BMD shown by pQCT and the 3D images obtained by μCT were indistinguishable from those observed in the OVX model. Histomorphometry showed that osteoclastic activity was significantly increased in the sRANKL‐injected mice. In addition, serum biochemical markers of bone turnover such as Ca, C‐telopeptide of type 1 collagen (CTX), and TRACP5b were also significantly increased in the sRANKL‐injected mice in a dose‐dependent manner. Bisphosphonates (BPs), selective estrogen receptor modulators (SERMs), and PTH are commonly used for the treatment of osteoporosis. We successfully evaluated the effects of anti–bone‐resorbing agents such as BPs, a SERM, and anti–RANKL‐neutralizing antibody on bone resorption in a couple of weeks. We also evaluated the effects of PTH on bone formation in 2 wk. A combination of sRANKL injections and OVX made it possible to evaluate a SERM. The sRANKL model is the simplest, fastest, and easiest of all osteoporosis models and could be useful in the evaluation of drug candidates for osteoporosis.

Osteoprotegerin-Deficient Male Mice as a Model for Severe Alveolar Bone Loss: Comparison With RANKL-Overexpressing Transgenic Male Mice

Periodontitis, an inflammatory disease of periodontal tissues, is characterized by excessive alveolar bone resorption. An increase in the receptor activator of nuclear factor-κB ligand (RANKL) to osteoprotegerin (OPG) ratio is thought to reflect the severity of periodontitis. Here, we examined alveolar bone loss in OPG-deficient (OPG(-/-)) mice and RANKL-overexpressing transgenic (RANKL-Tg) mice. Alveolar bone loss in OPG(-/-) mice at 12 weeks was significantly higher than that in RANKL-Tg mice. OPG(-/-) but not RANKL-Tg mice exhibited severe bone resorption especially in cortical areas of the alveolar bone. An increased number of osteoclasts was observed in the cortical areas in OPG(-/-) but not in RANKL-Tg mice. Immunohistochemical analyses showed many OPG-positive signals in osteocytes but not osteoblasts. OPG-positive osteocytes in the cortical area of alveolar bones and long bones were abundant in both wild-type and RANKL-Tg mice. This suggests the resorption in cortical bone areas to be prevented by OPG produced locally. To test the usefulness of OPG(-/-) mice as an animal model for screening drugs to prevent alveolar bone loss, we administered an antimouse RANKL antibody or risedronate, a bisphosphonate, to OPG(-/-) mice. They suppressed alveolar bone resorption effectively. OPG(-/-) mice are useful for screening therapeutic agents against alveolar bone loss.

Tetracyclines Convert the Osteoclastic-Differentiation Pathway of Progenitor Cells To Produce Dendritic Cell-like Cells

Tetracyclines, such as doxycycline and minocycline, are used to suppress the growth of bacteria in patients with inflammatory diseases. Tetracyclines have been shown to prevent bone loss, but the mechanism involved is unknown. Osteoclasts and dendritic cells (DCs) are derived from common progenitors, such as bone marrow-derived macrophages (BMMs). In this article, we show that tetracyclines convert the differentiation pathway, resulting in DC-like cells not osteoclasts. Doxycycline and minocycline inhibited the receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis of BMMs, but they had no effects on cell growth and phagocytic activity. They influenced neither the proliferation nor the differentiation of bone-forming osteoblasts. Surprisingly, doxycycline and minocycline induced the expression of DC markers, CD11c and CD86, in BMMs in the presence of RANKL. STAT5 is involved in DC differentiation induced by GM-CSF. Midostaurin, a STAT5-signaling inhibitor, and an anti–GM-CSF–neutralizing Ab suppressed the differentiation induced by GM-CSF but not by tetracyclines. In vivo, the injection of tetracyclines into RANKL-injected mice and RANKL-transgenic mice suppressed RANKL-induced osteoclastogenesis and promoted the concomitant appearance of CD11c+ cells. These results suggested that tetracyclines prevent bone loss induced by local inflammation, including rheumatoid arthritis and periodontitis, through osteoclast–DC-like cell conversion.

Diphenylhydantoin Inhibits Osteoclast Differentiation and Function Through Suppression of NFATc1 Signaling

Diphenylhydantoin (DPH) is widely used as an anticonvulsant drug. We examined the effects of DPH on osteoclast differentiation and function using in vivo and in vitro assay systems. Transgenic mice overexpressing a soluble form of RANKL (RANKL Tg) exhibited increased osteoclastic bone resorption. Injection of DPH into the subcutaneous tissue overlying calvaria of RANKL Tg mice suppressed the enhanced resorption in the calvaria. In co‐cultures of mouse osteoblasts and bone marrow cells, DPH inhibited lipopolysaccharide (LPS)‐induced osteoclast formation. DPH affected neither the mRNA expression of RANKL and osteoprotegerin nor the growth of mouse osteoblasts in culture. On the other hand, DPH inhibited the RANKL‐induced formation of osteoclasts in cultures of mouse bone marrow–derived macrophages (BMMϕs) and of human peripheral blood‐derived CD14+ cells. DPH concealed LPS‐induced bone resorption in mouse calvarial organ cultures and inhibited the pit‐forming activity of mouse osteoclasts cultured on dentine slices. DPH suppressed the RANKL‐induced calcium oscillation and expression of nuclear factor of activated T cells c1 (NFATc1) and c‐fos in BMMϕs. Moreover, DPH inhibited the RANKL‐induced nuclear localization and auto‐amplification of NFATc1 in mature osteoclasts. Both BMMϕs and osteoclasts expressed mRNA of a T‐type calcium channel, Cav3.2, a target of DPH. Blocking the expression of Cav3.2 by short hairpin RNAs significantly suppressed RANKL‐induced osteoclast differentiation. These results suggest that DPH inhibits osteoclast differentiation and function through suppression of NFATc1 signaling. The topical application of DPH may be a therapeutic treatment to prevent bone loss induced by local inflammation such as periodontitis.

Prostaglandin E2 receptor EP4-selective agonist (ONO-4819) increases bone formation by modulating mesenchymal cell differentiation

Prostaglandin E(2) (PGE(2)) positively regulates bone resorption and formation mainly mediated through the EP(4) receptor, a subtype of PGE(2) receptors. ONO-4819, an EP(4) receptor-selective agonist, has been shown to increase bone volume, density, and strength; however, the mechanism of these effects has yet to be fully elucidated. To explore this matter, ONO-4819 (10μg/kg) was injected into intact rats twice a day for 5weeks, and their bones were then analyzed by morphological techniques. The effects of ONO-4819 on the differentiation of bone cells were also examined in vitro. Bone morphometric analysis showed that osteoblast number, bone volume, mineral apposition rate, and bone formation rate were significantly increased by ONO-4819, whereas osteoclast number was not affected. Immunohistochemical examination demonstrated that ONO-4819 increased the number of Runx2- and Osterix-positive osteoblasts in rats. In vitro studies using the multipotent mesenchymal cell line C3H10T1/2 showed that ONO-4819 induced alkaline phosphatase (ALPase) activity and up-regulated the mRNA expression of ALPase and Osterix. In contrast, ONO-4819 reduced the mRNA expression of peroxisome proliferator-activated receptor γ (PPARγ) and inhibited adipocyte differentiation of C3H10T1/2 cells, which findings are consistent with the observation that the age-dependent increase in adipocyte number in the bone marrow was significantly suppressed in the ONO-4819-treated animals. ONO-4819 also dose-dependently increased osteoclast-like cell formation in vitro, but the required concentrations were much higher than those to induce osteoblast differentiation. These results collectively suggest that ONO-4819 increased bone formation by stimulating osteoblast differentiation and function, possibly through modulating mesenchymal cell differentiation in the bone.

The regulation of osteoclast differentiation by Wnt signals.

Wnt ligands activate β-catenin-dependent canonical and -independent noncanonical signaling pathways. Wnt regulates many physiological events such as the development of organs and bone metabolism. In contrast, failed signaling leads to pathological conditions including cancer and osteoporosis. Analyses of loss-of-function mutations in the low-density lipoprotein receptor-related protein (Lrp) 5 gene revealed that Lrp5 acted as a co-receptor of Wnt/β-catenin signals and positively regulated bone mass in humans and mice. Many players in Wnt signals including sclerostin, an osteocyte-derived Wnt antagonist, also have since been found to influence bone mass. Bone mass is regulated by the activities of bone-forming osteoblasts, -resorbing osteoclasts and matrix-embedded osteocytes. The roles of Wnt/β-catenin signals in osteoblastogenesis and osteoclastogenesis have been established by the findings of a large number of in vitro and in vivo studies. In contrast, the roles of noncanonical Wnt signals in bone metabolism are only now being examined. In this review, we introduced and discussed recent information on the roles of Wnt signals in bone resorption.

Roles of cathelicidin‐related antimicrobial peptide in murine osteoclastogenesis

Cathelicidin‐related antimicrobial peptide (CRAMP) not only kills bacteria but also binds to lipopolysaccharide (LPS) to neutralize its activity. CRAMP is highly expressed in bone marrow and its expression is reported to be up‐regulated by inflammatory and infectious stimuli. Here, we examined the role of CRAMP in murine osteoclastogenesis. Osteoclasts were formed in co‐cultures of osteoblasts and bone marrow cells in response to 1α,25‐dihydroxyvitamin D3 [1α,25(OH)2D3], prostaglandin E2 (PGE2), and Toll‐like receptor (TLR) ligands such as LPS and flagellin through the induction of receptor activator of nuclear factor‐κB ligand (RANKL) expression in osteoblasts. CRAMP inhibited the osteoclastogenesis in co‐cultures treated with LPS and flagellin, but not in those treated with 1α,25(OH)2D3 or PGE2. Although bone marrow macrophages (BMMs) highly expressed formyl peptide receptor 2 (a receptor of CRAMP), CRAMP showed no inhibitory effect on osteoclastogenesis in BMM cultures treated with RANKL. CRAMP suppressed both LPS‐ and flagellin‐induced RANKL expression in osteoblasts and tumour necrosis factor‐α (TNF‐α) expression in BMMs, suggesting that CRAMP neutralizes the actions of LPS and flagellin. LPS and flagellin enhanced the expression of CRAMP mRNA in osteoblasts. Extracellularly added CRAMP suppressed LPS‐ and flagellin‐induced CRAMP expression. These results suggest that the production of CRAMP promoted by LPS and flagellin is inhibited by CRAMP released by osteoblasts through a feedback regulation. Even though CRAMP itself has no effect on osteoclastogenesis in mice, we propose that CRAMP is an osteoblast‐derived protector in bacterial infection‐induced osteoclastic bone resorption.

The relationship between calcium accumulation in osteoclast mitochondrial granules and bone resorption

In the process of bone resorption, calcium is considered to be transported within vesicles in osteoclasts and eventually released. We studied the ultramicromorphology of calcium (Ca) transport in osteoclasts by preparing samples of osteoclasts collected from rat femurs in which calcium was maximally preserved and subjected them to high-pressure quick-freezing and freeze-substitution. We then examined the localization of calcium by Electron Energy Loss Spectroscopy (EELS). The structures of cell membranes were preserved, suggesting the suitability of this high-pressure quick-freezing and freeze-substitution technique. Osteoclast mitochondria adjacent to the ruffled border were rich in mitochondrial granules and contained a large amount of Ca. In contrast, mitochondria in the basolateral region contained few granules. Moreover, by an osteoclast-culturing experiment, differences in the morphology of mitochondrial granules were noted between culturing on a dentin slice and that on a gold plate, i.e., few mitochondrial granules were noted in osteoclasts cultured on a non-dentin plate. These findings suggest an association between the morphology of mitochondrial granules in osteoclasts and bone resorption as well as a new transport route for Ca resorbed by osteoclasts. We propose that Ca accumulates in mitochondria granules to prevent increased Ca concentration in cytoplasm of osteoclasts during bone resorption.

Bone Formation Is Coupled to Resorption Via Suppression of Sclerostin Expression by Osteoclasts

Bone formation is coupled to bone resorption throughout life. However, the coupling mechanisms are not fully elucidated. Using Tnfrsf11b‐deficient (OPG–/–) mice, in which bone formation is clearly coupled to bone resorption, we found here that osteoclasts suppress the expression of sclerostin, a Wnt antagonist, thereby promoting bone formation. Wnt/β‐catenin signals were higher in OPG–/– and RANKL‐transgenic mice with a low level of sclerostin. Conditioned medium from osteoclast cultures (Ocl‐CM) suppressed sclerostin expression in UMR106 cells and osteocyte cultures. In vitro experiments revealed that osteoclasts secreted leukemia inhibitory factor (LIF) and inhibited sclerostin expression. Anti‐RANKL antibodies, antiresorptive agents, suppressed LIF expression and increased sclerostin expression, thereby reducing bone formation in OPG–/– mice. Taken together, osteoclast‐derived LIF regulates bone turnover through sclerostin expression. Thus, LIF represents a target for improving the prolonged suppression of bone turnover by antiresorptive agents.

Wnt16 regulates osteoclast differentiation in conjunction with Wnt5a.

The canonical Wnt/β-catenin signaling pathway in osteoblast-lineage cells inhibits osteoclastogenesis through the expression of osteoprotegerin (Opg), a decoy receptor of receptor activator of Nf-κb (Rank) ligands. Wnt5a, a typical non-canonical Wnt ligand, enhances the expression of Rank in osteoclast precursors, which, in turn, promotes the Rank ligand (Rankl)-induced formation of osteoclasts. In contrast, Wnt16 and Wnt4 have been shown to inhibit the Rankl-induced formation of osteoclasts through non-canonical Wnt signals. However, the relationships among these Wnt ligands in osteoclastogenesis remained to be elucidated. We herein showed that Wnt16, but not Wnt4, inhibited the Rankl-induced osteoclastogenesis in bone marrow-derived macrophage (BMM) cultures. Wnt3a and Wnt4 inhibited the 1α,25-dihydroxy vitamin D3 (1,25D3)-induced osteoclastogenesis in co-cultures prepared from wild-type mice, but not in those from Opg(-/-) nice. Wnt16 inhibited the 1,25D3-induced formation of osteoclasts in both wild-type and Opg(-/-) co-cultures. Wnt16, Wnt4, and Wnt3a failed to inhibit the pit-forming activity of osteoclasts. Wnt16 failed to inhibit the Wnt5a-induced expression of Rank in osteoclast precursors. In contrast, Wnt5a abrogated the inhibitory effects of Wnt16 on Rankl-induced osteoclastogenesis. These results suggested that Wnt16 inhibited osteoclastogenesis, but not the function of osteoclasts and that Wnt16, an inhibitory Wnt ligand for osteoclastogenesis, regulates bone resorption in conjunction with Wnt5a.