Receptor activator of nuclear factor-kappa B (RANK) ligand (RANKL) binds RANK on the surface of osteoclast precursors to trigger osteoclastogenesis. Recent studies have indicated that osteocytic RANKL has an important role in osteoclastogenesis during bone remodelling; however, the role of osteoblastic RANKL remains unclear. Here we show that vesicular RANK, which is secreted from the maturing osteoclasts, binds osteoblastic RANKL and promotes bone formation by triggering RANKL reverse signalling, which activates Runt-related transcription factor 2 (Runx2). The proline-rich motif in the RANKL cytoplasmic tail is required for reverse signalling, and a RANKL(Pro29Ala) point mutation reduces activation of the reverse signalling pathway. The coupling of bone resorption and formation is disrupted in RANKL(Pro29Ala) mutant mice, indicating that osteoblastic RANKL functions as a coupling signal acceptor that recognizes vesicular RANK. RANKL reverse signalling is therefore a potential pharmacological target for avoiding the reduced bone formation associated with inhibition of osteoclastogenesis.

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.

Bone degenerative diseases, including osteoporosis, impair the fine balance between osteoclast bone resorption and osteoblast bone formation. Single-agent therapy for anabolic and anticatabolic effects is attractive as a drug target to ameliorate such conditions. Inhibition of nuclear factor (NF)-κB reduces the osteoclast bone resorption. The role of NF-κB inhibitors on osteoblasts and bone formation, however, is minimal and not well investigated. Using an established NF-κB inhibitor named S1627, we demonstrated that inhibition of NF-κB increases osteoblast differentiation and bone formation in vitro by up-regulating the mRNAs of osteoblast-specific genes like type I collagen, alkaline phosphatase, and osteopontin. In addition, S1627 was able to increase bone formation and repair bone defect in a murine calvarial defect model. To determine the effect of NF-κB on a model of osteoporosis, we injected two doses of inhibitor (25 and 50 mg/kg·d) twice a day in sham-operated or ovariectomized 12-wk-old mice and killed them after 4 wk. The anabolic effect of S1627 on trabecular bone was determined by micro focal computed tomography and histomorphometry. Bone mineral density of inhibitor-treated ovariectomized animals was significantly increased compared with sham-operated mice. Osteoblast-related indices like osteoblast surface, mineral apposition rate, and bone formation rate were increased in S1627-treated animals in a dose-dependent manner. NF-κB inhibition by S1627 increased the trabecular bone volume in ovariectomized mice. Furthermore, S1627 could inhibit the osteoclast number, and osteoclast surface to bone surface. In vitro osteoclastogenesis and bone resorbing activity were dose-dependently reduced by NF-κB inhibitor S1627. Taken collectively, our results suggest that NF-κB inhibitors are effective in treating bone-related diseases due to their dual anabolic and antiresorptive activities.

Excessive exposure to glucocorticoids causes osteoporosis in children and adults. Occlusal disharmony is known to induce an increase in serum corticosteroid levels in murine models, but the influence of occlusal disharmony-induced stress on the bone mass during the growth period has not yet been clarified. The purpose of this study was to investigate whether occlusal disharmony-induced stress decreases bone mass. Five-week-old C57BL/6J male mice were used. A 0.5-mm increase in the vertical height of occlusion was used to induce occlusal disharmony for a period of 7 days. Serum corticosterone levels were significantly higher on post-induction day 7, with radiological evidence of osteopenia of the third lumbar vertebra and long bones of the hind limbs. Osteopenia was associated with a reduction of the mechanical properties of the tibia and femur, with significant suppression of bone formation parameters and an increase in bone resorption parameters, as evaluated by bone histomorphometric analysis of the tibial/femur metaphysis. Our findings at the level of bones were supported by our assessment of serum markers of systemic metabolism. Therefore, occlusal disharmony-induced stress may lead to osteopenia and reduce the mechanical strength of bone through an increase in serum glucocorticoid levels in mice.Occlusal disharmony frequently occurs during growth due to mixed dentition, as the primary dentition is changing to permanent dentition. Several lines of evidence indicate that occlusal disharmony increases serum corticosteroid levels in young animals. As the serum corticosteroid level is considered to be a marker of 'stress' in rodents, it is plausible that occlusal disharmony would cause stress 1-4 . The high frequency of bruxism (teeth grinding) during the period of growth further suggests the possible existence of occlusal disharmony-induced stress in childhood [5][6][7] . By contrast, it is well known that excessive exposure to glucocorticoids causes osteopenia 8 . As an example, the use of steroid medications in children for the treatment of immune-related diseases, such as asthma and skin allergies, commonly results in bone-related side effects, including fractures and slowed bone growth 9 . Given the significant effects of stress-induced hormone on bone metabolism 10 , evaluation of the effect of occlusal disharmony-induced stress on skeletal tissue is warranted.Our aim in this study was to use a mouse model of occlusal disharmony to evaluate the effects of occlusal disharmony-induced stress on bone mass and bone mineral density, as well as on the mechanical properties of the long bones and lumbar vertebrae, which are expected to be differentially affected by mechanical loading [11][12][13] . ResultsIncrease in serum corticosterone level with occlusal disharmony. Serum corticosterone levels were measured at baseline and after the induction of occlusal disharmony (Fig. 1). Serum corticosterone levels were significantly higher in the disharmony than in the control group on post-induction days 1, 3 and 7 (Fig. ...

Nuclear factor-jB (NF-jB) is constitutively activated in many cancers, including oral squamous cell carcinoma (OSCC), and is involved in the invasive characteristics of OSCC, such as growth, antiapoptotic activity and protease production. However, the cellular mechanism underlying NF-jB's promotion of bone invasion by OSCC is unclear. Therefore, we investigated the role of NF-jB in bone invasion by OSCC in vivo. Immunohistochemical staining of OSCC invading bone in 10 patients indicated that the expression and nuclear translocation of p65, a main subunit of NF-jB, was increased in OSCC compared with normal squamous epithelial cells. An active form of p65 phosphorylated at serine 536 was present mainly in the nucleus in not only differentiated tumor cells but also tumor-associated stromal cells and bone-resorbing osteoclasts. We next injected mouse OSCC SCCVII cells into the masseter region of C 3 H/HeN mice. Mice were treated for 3 weeks with a selective NF-jB inhibitor, NBD peptide, which disrupts the association of NF-jB essential modulator (NEMO) with IjB kinases. NBD peptide treatment inhibited TNFa-induced and constitutive NF-jB activation in SCCVII cells in vitro and in vivo, respectively. Treatment with NBD peptide decreased zygoma and mandible destruction by SCCVII cells, reduced number of osteoclasts by inhibiting RANKL expression in osteoblastic cells and SCCVII cells, increased apoptosis and suppressed the proliferation of SCCVII cells. Taken together, our data clearly indicate that inhibition of NF-jB is useful for inhibiting bone invasion by OSCC.Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the oral cavity, head and neck. 1-3 Despite advances in our understanding of the molecular mechanisms of tumor development, prevention and treatment, the longterm survival for patients with OSCC, a cancer that, worldwide, accounts for more than 500,000 cases each year, 1 remains low. The poor prognosis for OSCC reflects a limited understanding of the mechanisms of local and regional invasion and metastasis present in a significant portion of patients, together with an unsatisfactory responsiveness to conventional systemic therapy in recurrent and advanced disease. 2,3 The ability of OSCC to invade the maxilla or mandibular bone is a critical factor, which, because it leads to metastasis, affects the prognosis of patients. 4 Although controversial, bone destruction that occurs with OSCC invasion is thought to be mediated by osteoclasts rather than by the carcinoma itself. 4 Recent studies have established that bone resorption by osteoclasts is an important step in the process of bone invasion and metastasis in several types of malignancy, 5 indicating that a full understanding of the regulation of osteoclastogenesis by OSCC cells is necessary to prevent bone invasion by OSCC cells. Several in vitro and animal experiments using human OSCC cells have shown that tumor cells

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Receptor activator of NF-κB ligand (RANKL)-binding peptides inhibit bone resorption and were recently shown to activate bone formation. The stimulatory mechanism underlying bone formation associated with these peptides was explained as RANKL-reverse signaling, wherein RANKL molecules on osteoblasts work as receptors to stimulate osteoblast differentiation. However, why RANKL-binding peptides stimulate osteoblast differentiation while osteoprotegerin (OPG), which is well known to bind to RANKL, cannot activate osteoblast differentiation has remained unclear. In this mini-review, we introduce three main issues: (1) The inhibitory effects of two RANKL-binding peptides (W9 and OP3-4) on bone resorption; (2) The stimulatory effects of the RANKL-binding peptides on osteoblast differentiation; and (3) The accumulation and membrane clustering of RANKL molecules at the cell surface of osteoblasts as a potential molecular switch stimulating osteoblast differentiation by RANKL-binding peptides.