Midori Nakamura

MyD88 But Not TRIF Is Essential for Osteoclastogenesis Induced by Lipopolysaccharide, Diacyl Lipopeptide, and IL-1α

Myeloid differentiation factor 88 (MyD88) plays essential roles in the signaling of the Toll/interleukin (IL)-1 receptor family. Toll–IL-1 receptor domain-containing adaptor inducing interferon-β (TRIF)-mediated signals are involved in lipopolysaccharide (LPS)-induced MyD88-independent pathways. Using MyD88-deficient (MyD88−/−) mice and TRIF-deficient (TRIF−/−) mice, we examined roles of MyD88 and TRIF in osteoclast differentiation and function. LPS, diacyl lipopeptide, and IL-1α stimulated osteoclastogenesis in cocultures of osteoblasts and hemopoietic cells obtained from TRIF−/− mice, but not MyD88−/− mice. These factors stimulated receptor activator of nuclear factor-κB ligand mRNA expression in TRIF−/− osteoblasts, but not MyD88−/− osteoblasts. LPS stimulated IL-6 production in TRIF−/− osteoblasts, but not TRIF−/− macrophages. LPS and IL-1α enhanced the survival of TRIF−/− osteoclasts, but not MyD88−/− osteoclasts. Diacyl lipopeptide did not support the survival of osteoclasts because of the lack of Toll-like receptor (TLR)6 in osteoclasts. Macrophages expressed both TRIF and TRIF-related adaptor molecule (TRAM) mRNA, whereas osteoblasts and osteoclasts expressed only TRIF mRNA. Bone histomorphometry showed that MyD88−/− mice exhibited osteopenia with reduced bone resorption and formation. These results suggest that the MyD88-mediated signal is essential for the osteoclastogenesis and function induced by IL-1 and TLR ligands, and that MyD88 is physiologically involved in bone turnover.

Intermittent PTH Administration Stimulates Pre‐Osteoblastic Proliferation Without Leading to Enhanced Bone Formation in Osteoclast‐Less c‐fos−/− Mice

This study aimed to investigate the behavior and ultrastructure of osteoblastic cells after intermittent PTH treatment and attempted to elucidate the role of osteoclasts on the mediation of PTH‐driven bone anabolism. After administering PTH intermittently to wildtype and c‐fos−/− mice, immunohistochemical, histomorphometrical, ultrastructural, and statistical examinations were performed. Structural and kinetic parameters related to bone formation were increased in PTH‐treated wildtype mice, whereas in the osteoclast‐deficient c‐fos−/− mice, there were no significant differences between groups. In wildtype and knockout mice, PTH administration led to significant increases in the number of cells double‐positive for alkaline phosphatase and BrdU, suggesting active pre‐osteoblastic proliferation. Ultrastructural examinations showed two major pre‐osteoblastic subtypes: one rich in endoplasmic reticulum (ER), the hypER cell, and other with fewer and dispersed ER, the misER cell. The latter constituted the most abundant preosteoblastic phenotype after PTH administration in the wildtype mice. In c‐fos−/− mice, misER cells were present on the bone surfaces but did not seem to be actively producing bone matrix. Several misER cells were shown to be positive for EphB4 and were eventually seen rather close to osteoclasts in the PTH‐administered wildtype mice. We concluded that the absence of osteoclasts in c‐fos−/− mice might hinder PTH‐driven bone anabolism and that osteoclastic presence may be necessary for full osteoblastic differentiation and enhanced bone formation seen after intermittent PTH administration.

Dental Pulp and Periodontal Ligament Cells Support Osteoclastic Differentiation

Odontoclasts and cementoclasts are considered to play major roles in the internal resorption of dentin and the external resorption of tooth roots. In this study, we evaluated the osteoclast-inducing ability of human dental pulp and periodontal ligament cells, which are mesenchymal cells in dental tissues. These cells expressed RANKL and OPG mRNA constitutively. As osteoclast precursors, CD14+ monocytes derived from human peripheral blood were isolated, and incubated together with human dental pulp or periodontal ligament cells. Both cell types spontaneously induced the differentiation of CD14+ monocytes into osteoclasts without osteotropic factors. These results suggest that dental pulp and periodontal ligament cells are involved in regulating the differentiation and function of osteoclasts.

Stimulation of Bone Formation in Cortical Bone of Mice Treated with A Receptor Activator of Nuclear Factor-κB Ligand (RANKL) Binding Peptide That Possesses Osteoclastogenesis Inhibitory Activity

Background: A RANKL-binding peptide WP9QY (W9) is known to inhibit osteoclastogenesis. Results: W9 showed an anabolic effect on cortical bone in mice. W9 bound RANKL and differentiated osteoblasts with production of autocrine factors like BMP-4. Conclusion: Signaling through RANKL is involved in part in the W9-induced osteoblast differentiation. Significance: The RANKL pathway could be a novel mechanism in osteoblast differentiation. To date, parathyroid hormone is the only clinically available bone anabolic drug. The major difficulty in the development of such drugs is the lack of clarification of the mechanisms regulating osteoblast differentiation and bone formation. Here, we report a peptide (W9) known to abrogate osteoclast differentiation in vivo via blocking receptor activator of nuclear factor-κB ligand (RANKL)-RANK signaling that we surprisingly found exhibits a bone anabolic effect in vivo. Subcutaneous administration of W9 three times/day for 5 days significantly augmented bone mineral density in mouse cortical bone. Histomorphometric analysis showed a decrease in osteoclastogenesis in the distal femoral metaphysis and a significant increase in bone formation in the femoral diaphysis. Our findings suggest that W9 exerts bone anabolic activity. To clarify the mechanisms involved in this activity, we investigated the effects of W9 on osteoblast differentiation/mineralization in MC3T3-E1 (E1) cells. W9 markedly increased alkaline phosphatase (a marker enzyme of osteoblasts) activity and mineralization as shown by alizarin red staining. Gene expression of several osteogenesis-related factors was increased in W9-treated E1 cells. Addition of W9 activated p38 MAPK and Smad1/5/8 in E1 cells, and W9 showed osteogenesis stimulatory activity synergistically with BMP-2 in vitro and ectopic bone formation. Knockdown of RANKL expression in E1 cells reduced the effect of W9. Furthermore, W9 showed a weak effect on RANKL-deficient osteoblasts in alkaline phosphatase assay. Taken together, our findings suggest that this peptide may be useful for the treatment of bone diseases, and W9 achieves its bone anabolic activity through RANKL on osteoblasts accompanied by production of several autocrine factors.

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.

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.

Mineral trioxide aggregate solution inhibits osteoclast differentiation through the maintenance of osteoprotegerin expression in osteoblasts.

Mineral trioxide aggregate (MTA) is a therapeutic, endodontic repair material that is reported to exhibit calcified tissue-conductive activity. The aim of this study was to investigate whether MTA may prevent osteoclast differentiation in vitro. MTA solution, but not other commonly used retrofilling materials, such as Dycal, Super-EBA, or intermediate restorative material (IRM) solution, dose-dependently inhibited osteoclastogenesis in cocultures of mouse bone marrow cells (BMCs) with primary osteoblast cells (POBs) induced by 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2) D(3) ]. Exogenous CaCl(2) medium supplementation did not inhibit osteoclastogenesis in cocultures. Furthermore, MTA solution did not affect receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis, suggesting that POBs are targets of MTA. MTA solution suppressed the 1α,25(OH)(2) D(3) -induced reduction of osteoprotegerin (OPG) mRNA and protein production without changing RANKL expression in POBs. Consistent with this result, MTA solution did not inhibit osteoclastogenesis in cocultures of BMCs and POBs from OPG-deficient mice. Therefore, the maintenance of OPG expression in POBs appears to be critical for the inhibitory effect of MTA solution on osteoclast differentiation.

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.

Cardiac hypertrophy is exacerbated in aged mice lacking the osteoprotegerin gene

AIMS Osteoprotegerin (OPG) may play a role in the progression of cardiac hypertrophy and heart failure. However, its pathophysiological role in changes in cardiac structure and function with ageing remains to be elucidated. METHODS AND RESULTS We conducted experiments using 2.5- and 12-month-old OPG(-/-) mice and age-matched wild-type (WT) mice and compared the morphology and function of the left ventricle (LV). Both 2.5- and 12-month-old OPG(-/-) mice showed a higher systolic blood pressure and a greater heart weight/body weight ratio than age-matched WT mice. Twelve-month-old OPG(-/-) mice had a significantly larger LV chamber and reduced wall thickness compared with age-matched WT mice, and contractile function was decreased. The morphological differences were accompanied by an increase in the number of apoptotic cells and activation of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) in the LV of 12-month-old OPG(-/-) mice. Correspondingly, OPG small interfering RNA induced the expressions of TRAIL and cleaved caspase-3 in cultured cardiac myocytes. In addition, these mice revealed a decrease in interstitial fibrosis, activation of matrix metalloproteinase (MMP)-2 and tissue inhibitors of MMP-1 and -2, and inactivation of procollagen α1 synthesis. Moreover, intraperitoneal administration of recombinant OPG to either 2.5- or 12-month-old OPG(-/-) mice for 28 days led to partial improvement of LV structure and function without affecting systolic blood pressure. CONCLUSION These results suggest that OPG plays a role in preserving myocardial structure and function with ageing through a reduction in apoptosis and preservation of the matrix structure. In addition, this appears to be independent of effects on the vasculature.