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Introduction: We investigated receptor activator of nuclear factor-κB ligand (RANKL) expression by B lymphocytes during early and late aspects of the immune response to Aggregatibacter actinomycetemcomitans, a gram-negative, anaerobic bacterium associated with aggressive periodontal disease. Methods: Expression of messenger RNA transcripts (tumor necrosis factor-α, Toll-like receptors 4 and 9, interleukins 4 and 10, and RANKL) involved in early (1-day) and late (10-day) responses in cultured rat splenocytes was examined by reverse transcription–polymerase chain reaction (RT-PCR). The immune cell distribution (T, B, and natural killer cells and macrophages) in cultured rat splenocytes and RANKL expression in B cells were determined by flow cytometric analyses. B-cell capacity for induction of osteoclast differentiation was evaluated by coculture with RAW 264.7 cells followed by a tartrate-resistant acid phosphatase (TRAP) activity assay. Results: The expression levels of interleukins 4 and 10 in cultured cells were not changed in the presence of A. actinomycetemcomitans until cultured for 3 days, and peaked after 7 days. After culture for 10 days, the percentages of B and T cells, the overall RANKL messenger RNA transcripts, and the percentage of RANKL-expressing immunoglobulin G-positive cells were significantly increased in the presence of A. actinomycetemcomitans. These increases were considerably greater in cells isolated from A. actinomycetemcomitans-immunized animals than from non-immunized animals. RAW 264.7 cells demonstrated significantly increased TRAP activity when cocultured with B cells from A. actinomycetemcomitans-immunized animals. The addition of human osteoprotegerin-Fc to the culture significantly diminished such increases. Conclusion: This study suggests that B-lymphocyte involvement in the immune response to A. actinomycetemcomitans through upregulation of RANKL expression potentially contribute to bone resorption in periodontal disease. [ABSTRACT FROM AUTHOR]

Orthodontic therapy is known to have an aggravating effect on the progression of destructive periodontitis if oral hygiene is not maintained. However, it is largely unknown how active periodontitis affects the velocity of orthodontic tooth movement. In this study, we examined the effect of periodontal inflammation on orthodontic tooth movement using a mouse model. Orthodontic force was applied on the maxillary first molar of mice, with or without ligature wire to induce experimental periodontitis. The distance moved by the first molar was significantly reduced by the ligature-induced experimental periodontitis. Tartrate-resistant acid phosphatase staining revealed that the number of osteoclasts present during orthodontic treatment was lower in the pressure zone of alveolar bone in the presence of periodontal inflammation. Consistently, the expression level of receptor activator of nuclear factor-κB ligand (RANKL) in the pressure zone was decreased in the ligature group. By contrast, experimental periodontitis increased the expression of cyclooxygenase-2 mRNA in the periodontal tissues, while in vitro treatment with prostaglandin E2 decreased extracellular signal-regulated kinase phosphorylation and RANKL expression induced by mechanical stress in osteoblasts. Taken together, these results suggest that the orthodontic force-induced osteoclastogenesis in alveolar bone was inhibited by the accompanying periodontal inflammation, at least partly through prostaglandin E2, resulting in reduced orthodontic tooth movement. [ABSTRACT FROM AUTHOR]

Background and Objective: The cellular response of human gingival fibroblasts to a mechanical force is considered to be primarily anti-osteoclastic because they produce relatively high levels of osteoprotegerin. However, there is little information available on the effects of compression force on the production of osteoprotegerin and osteoclastic differentiation by these cells. In this study, we examined how mechanical force affects the nature of human gingival fibroblasts to produce osteoprotegerin and inhibit osteoclastogenesis. Material and Methods: Human gingival fibroblasts were exposed to mechanical force by centrifugation for 90 min at a magnitude of approximately 50 g/cm2. The levels of osteoprotegerin, receptor activator of nuclear factor-κB ligand (RANKL), interleukin-1β and tumor necrosis factor-α were measured at various time-points after applying the force. The effect of the centrifugal force on the formation of osteoclast-like cells was also determined using a co-culture system of human gingival fibroblasts and bone marrow cells. Results: Centrifugal force stimulated the expression of osteoprotegerin, RANKL, interleukin-1β and tumor necrosis factor-α by the cells, and produced a relatively high osteoprotegerin to RANKL ratio at the protein level. Both interleukin-1β and tumor necrosis factor-α accelerated the force-induced production of osteoprotegerin, which was inhibited significantly by the addition of anti-(interleukin-1β) immunoglobulin Ig isotype; IgG (rabbit polyclonal). However, the addition of anti-(tumor necrosis factor-α) immunoglobulin Ig isotype; IgG1 (mouse monoclonal) had no effect. Centrifugal force also had an inhibitory effect on osteoclast formation. Conclusion: Application of centrifugal force to human gingival fibroblasts accelerates osteoprotegerin production by these cells, which stimulates the potential of human gingival fibroblasts to suppress osteoclastogenesis. Overall, human gingival fibroblasts might have natural defensive mechanisms to inhibit bone resorption induced by a mechanical stress. [ABSTRACT FROM AUTHOR]

Purpose: Osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) have been implicated in osteoclastogenesis. However, the relationship between the OPG-RANKL system and bone status in biliary atresia (BA) has not, as yet, been clarified. Thus, the aim of this study has been to evaluate the relationship between the OPG-RANKL system and bone mineral metabolism in patients with BA.Methods: Fifty patients with BA and 13 healthy controls were investigated. The mean age of BA patients and controls was 7.3 +/- 0.6 and 8.0 +/- 1.1 years, respectively. Serum levels of OPG, RANKL, osteocalcin, and C-terminal telopeptide of type I collagen (CTX) were measured by sandwich enzyme-linked immunosorbent assay. Bone mineral density (BMD) of the lumbar spine was determined by dual energy X-ray absorptiometry.Results: Biliary atresia patients had significantly elevated serum OPG levels compared with controls (4.0 +/- 0.3 vs. 3.0 +/- 0.3 pmol/L, P = 0.02) and serum OPG levels in BA patients with jaundice were higher than in those without jaundice (4.6 +/- 0.4 vs. 3.6 +/- 0.4 pmol/L, P = 0.04). Likewise, serum RANKL levels were significantly higher in BA patients than in controls (2.9 +/- 0.2 vs. 1.2 +/- 0.7 pmol/L, P = 0.001). In addition, serum RANKL levels were increased in BA patients with jaundice compared to those without jaundice, but this difference was not statistically significant (3.2 +/- 0.3 vs. 2.7 +/- 0.2 pmol/L, P = 0.2). The serum osteocalcin levels in BA patients were not significantly different from those in the healthy controls, whereas the serum CTX levels were elevated in BA patients compared with the controls (0.4 +/- 0.1 vs. 0.2 +/- 0.1 ng/mL, P = 0.02). Furthermore, BMD of BA children with jaundice was significantly lower than that of BA children without jaundice (P = 0.0005). BMD of BA patients was inversely correlated with serum levels of OPG (r = -0.452, P < 0.001).Conclusion: Elevated serum OPG levels are associated with reduced BMD and the outcome of BA. The increase of serum OPG in BA patients with severe disease could reflect a compensatory response to bone loss. [ABSTRACT FROM AUTHOR]

Introduction: Periodontal disease is a chronic inflammation of the attachment structures of the teeth, triggered by potentially hazardous microorganisms and the consequent immune-inflammatory responses. In humans, the T helper type 17 (Th17) lineage, characterized by interleukin-17 (IL-17) production, develops under transforming growth factor-β (TGF-β), IL-1β, and IL-6 signaling, while its pool is maintained by IL-23. Although this subset of cells has been implicated in various autoimmune, inflammatory, and bone-destructive conditions, the exact role of T lymphocytes in chronic periodontitis is still controversial. Therefore, in this study we investigated the presence of Th17 cells in human periodontal disease. Methods: Gingival and alveolar bone samples from healthy patients and patients with chronic periodontitis were collected and used for the subsequent assays. The messenger RNA expression for the cytokines IL-17, TGF-β, IL-1β, IL-6, and IL-23 in gingiva or IL-17 and receptor activator for nuclear factor-κB ligand in alveolar bone was evaluated by real-time polymerase chain reaction. The production of IL-17, TGF-β, IL-1β, IL-6, and IL-23 proteins was evaluated by immunohistochemistry and the presence of Th17 cells in the inflamed gingiva was confirmed by immunofluorescence confocal microscopy for CD4 and IL-17 colocalization. Results: Our data demonstrated elevated levels of IL-17, TGF-β, IL-1β, IL-6, and IL-23 messenger RNA and protein in diseased tissues as well as the presence of Th17 cells in gingiva from patients with periodontitis. Moreover, IL-17 and the bone resorption factor RANKL were abundantly expressed in the alveolar bone of diseased patients, in contrast to low detection in controls. Conclusion: These results provided strong evidence for the presence of Th17 cells in the sites of chronic inflammation in human periodontal disease. [ABSTRACT FROM AUTHOR]

Receptor activator of nuclear factor-κB ligand (RANKL), which functions as a major determinant of osteoclast differentiation and activation, is a type II transmembrane protein and is expressed in osteoblasts-stromal cells. The aim of this study was to clarify the role of chicken RANKL (chRANKL) in chicken osteoclast differentiation and to determine its effect on mature chicken osteoclasts. In the present study, chRANKL protein was first cloned and expressed in Escherichia coli. We then treated chicken bone marrow cells with chRANKL protein and found that it induced the formation of chicken osteoclast-like multinucleated cells in a dose-dependent manner in the presence of human macrophage colony-stimulating factor. Moreover, the addition of chicken osteoprotegerin could block the effect of chRANKL with regard to osteoclast-like multi-nucleated cell formation and bone resorption. Using primary cultures of chicken osteoclasts on bone slices, we also found that bone resorption pits per cell increased with chRANKL concentration in a dose-dependent manner. The chRANKL-treated hens exhibited increased blood Ca[sup++] levels within 2 h after injection, showing that chRANKL also activates osteoclasts in vivo. These results clearly indicate that the expressed protein is functional and may also be a critical factor for chicken osteoclastogenesis and bone resorption. [ABSTRACT FROM AUTHOR]

Abstract: Multinuclear osteoclasts are derived from CD11b-positive mononuclear cells in bone marrow and in circulation. FACS sorting experiments showed impaired osteoclastogenesis in RAW264.7 cells with low CD11b expression. Neutralizing antibodies and siRNA against CD11b inhibited osteoclastogenesis induced by RANKL. Although primary cultured mouse bone marrow macrophages expressed CD11a and CD11b, osteoclastogenesis induced by M-CSF and RANKL was inhibited in the presence of anti-CD11b or anti-CD18 but not anti-CD11a antibodies. Furthermore, anti-CD11b antibodies inhibited NFATc1 expression induced by M-CSF and RANKL in BMMs. These findings suggest, at least partly, an important role of CD11b in osteoclastogenesis. [Copyright &y& Elsevier]

Osteoclast formation from mononuclear precursors is believed to require accessory cells expressing receptor activator of nuclear factor-κB ligand (RANKL). We recently identified a human cell population originated from circulating CD14+ monocytes, called monocyte-derived multipotential cells (MOMCs), which can differentiate into several distinct mesenchymal cells, neuron and endothelial cells. This study was undertaken to examine whether MOMCs can differentiate into functional osteoclasts. MOMCs prepared from peripheral blood of healthy volunteers cultured on fibronectin for 7 days at high density (8 × 105 cells cm−2), but not at regular density (2 × 104 cells cm−2), resulted in the appearance of tartrate-resistant acid phosphatase-positive giant multi-nucleated cells forming actin ring without exogenous osteoclastogenic factors. A subset of these cells showed bone resorption capacity on dentine slices and expression of genes for cathepsin K and calcitonin receptor, characteristic of functional osteoclasts. Such osteoclast differentiation was not observed in high-density culture of circulating monocytes, macrophages or dendritic cells, or the high-density culture of MOMCs on type I collagen. Among cells of the monocyte lineage, untreated MOMCs exclusively showed gene and protein expression of RANKL. When osteoprotegerin/IgG1 Fc chimera was added to high-density MOMC cultures, osteoclast formation was completely inhibited by neutralizing the endogenous RANKL. These results indicate that human MOMCs derived from circulating monocytes can express RANKL and differentiate into functional osteoclasts without RANKL-expressing accessory cells.Immunology and Cell Biology (2008) 86, 453–459; doi:10.1038/icb.2008.4; published online 26 February 2008 [ABSTRACT FROM AUTHOR]

Background/aims: In the present study, we attempted to develop a simulated model to explore the causal effects of periodontal pathogens on skeletal homeostasis in postmenopausal osteoporosis. Methods: Fifty-three female adult ICR mice were randomly assigned to an experimental group (ovariectomized) or a control group. A single injection of Porphyromonas gingivalis lipopolysaccharide ( P. gingivalis-LPS, ATCC 33277) or Escherichia coli lipopolysaccharide ( E. coli-LPS) was administered intraperitoneally 4 weeks after an ovariectomy. Concentrations of interleukin-6 (IL-6), osteoprotegerin (OPG), and the receptor activator of nuclear factor-κB ligand (RANKL) in serum were subsequently analyzed using an enzyme-linked immunosorbent assay (ELISA). Results: Under stimulation with P. gingivalis-LPS or E. coli-LPS, the concentration of OPG rose in both groups. The serum level of RANKL showed a decreasing trend 24 h after the injection in both groups. After injection of P. gingivalis-LPS in both the experimental and control animals, the OPG : RANKL ratio increased 24 h after the booster (22.26–620.99, P < 0.05). The serum level of IL-6 in the experimental group significantly increased 1–6 h after administration of E. coli-LPS and 1–3 h after administration of P. gingivalis-LPS ( P < 0.05). Conclusions: A single booster injection of P. gingivalis-LPS induced short-term changes in OPG, RANKL, and IL-6 serum levels in this ovariectomized mouse model. [ABSTRACT FROM AUTHOR]

Background and Objective: Lipopolysaccharide from gram-negative bacteria is one of the microbial-associated molecular patterns that initiate the immune/inflammatory response, leading to the tissue destruction observed in periodontitis. The aim of this study was to evaluate the role of the p38 mitogen-activated protein kinase (MAPK) signaling pathway in lipopolysaccharide-induced receptor activator of nuclear factor-κB ligand (RANKL) expression by murine periodontal ligament cells. Material and Methods: Expression of RANKL and osteoprotegerin mRNA was studied by reverse transcription-polymerase chain reaction upon stimulation with lipopolysaccharide from Escherichia coli and Aggregatibacter actinomycetemcomitans. The biochemical inhibitor SB203580 was used to evaluate the contribution of the p38 MAPK signaling pathway to lipopolysaccharide-induced RANKL and osteoprotegerin expression. Stable cell lines expressing dominant-negative forms of MAPK kinase (MKK)-3 and MKK6 were generated to confirm the role of the p38 MAPK pathway. An osteoclastogenesis assay using a coculture model of the murine monocytic cell line RAW 264.7 was used to determine if osteoclast differentiation induced by lipopolysaccharide-stimulated periodontal ligament was correlated with RANKL expression. Results: Inhibiting p38 MAPK prior to lipopolysaccharide stimulation resulted in a significant decrease of RANKL mRNA expression. Osteoprotegerin mRNA expression was not affected by lipopolysaccharide or p38 MAPK. Lipopolysaccharide-stimulated periodontal ligament cells increased osteoclast differentiation, an effect that was completely blocked by osteoprotegerin and significantly decreased by inhibition of MKK3 and MKK6, upstream activators of p38 MAPK. Conditioned medium from murine periodontal ligament cultures did not increase osteoclast differentiation, indicating that periodontal ligament cells produced membrane-bound RANKL. Conclusion: Lipopolysaccharide resulted in a significant increase of RANKL in periodontal ligament cells. The p38 MAPK pathway is required for lipopolysaccharide-induced membrane-bound RANKL expression in these cells. [ABSTRACT FROM AUTHOR]

Abstract: We investigated here whether adiponectin can exhibit an inhibitory effect on tumor necrosis factor-alpha (TNF-α)- and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis by using RAW264 cell D clone with a high efficiency to form osteoclasts. Globular adiponectin (gAd) strongly inhibited TNF-α/RANKL-induced differentiation of osteoclasts by interfering with TNF receptor-associated factor 6 production and calcium signaling; consequently, the induction of nuclear factor of activated T cells c1 (NFATc1) was strongly inhibited. Moreover, we observed that inhibition of AMP-activated protein kinase abrogated gAd inhibition for TNF-α/RANKL-induced NFATc1 expression. Our data suggest that adiponectin acts as a potent regulator of bone resorption observed in diseases associated with cytokine activation. [Copyright &y& Elsevier]

Bone metastases and skeletal complications are major causes of morbidity in men with prostate cancer. Despite the osteoblastic appearance of bone metastases on imaging studies, patients with bone metastases have elevated markers of bone resorption, indicative of high osteoclast activity. Indeed, increased osteoclast activity is independently associated with higher risk of subsequent skeletal complications. Therapies aimed at reducing osteoclast activity would theoretically diminish disease-related skeletal complications, bone metastases and treatment-related fractures. Phase III clinical trials to address the role of osteoclast-targeted therapy in metastatic prostate cancer are reviewed here. Zoledronic acid, a potent bisphosphonate, significantly decreased the risk of skeletal complications in men with androgen-independent prostate cancer and bone metastases. Ongoing studies will evaluate the use of bisphosphonates or denosumab, a monoclonal antibody that inhibits the receptor activator of nuclear factor-?B ligand signaling in the prevention of bone metastases or skeletal complications. Additional studies are needed to determine the optimal timing, schedule and duration of bisphosphonate treatment in men with bone metastases. The results of these trials in the near future may herald further evolution in the targeting of osteoclasts and optimal supportive care in men with prostate cancer. [ABSTRACT FROM AUTHOR]

Background: Osteoprotegerin (OPG) is a recently identified cytokine that acts as a decoy receptor for the receptor activator of nuclear factor-κB ligand (RANKL). OPG and RANKL have been shown to be important regulators of osteoclastogenesis. The aim of this study was to investigate the relationship between the OPG-RANKL system and bone mineral metabolism in patients with chronic renal failure (CRF). Methods: Serum OPG, RANKL, osteocalcin, cross-linked c-telopeptide of type I collagen (ICTP), intact parathyroid hormone (PTH), bone alkaline phosphatase and cystatin C levels were measured in 40 chronic hemodialysis male patients and 32 age- and sex-matched healthy controls. Their lumbar spine bone mineral density (LS-BMD) was measured by dual energy X-ray absorptiometry. Results: Serum OPG, RANKL, PTH, bone alkaline phosphatase and cystatin C levels were significantly increased in patients with CRF. Serum OPG was positively correlated to serum RANKL and cystatin C. Positive correlations were found between serum RANKL and cystatin C and ICTP. LS-BMD was significantly lower in patients with CRF than in controls. In patients with CRF, LS-BMD was inversely correlated to serum RANKL and cystatin C, whereas it was positively correlated to serum OPG. Conclusions: The OPG-RANKL system is involved in the pathogenesis and regulation of bone turnover in CRF. Circulating levels of OPG and RANKL may be useful markers to assess turnover renal osteopathies. Clin Chem Lab Med 2007;45:1498–503. [ABSTRACT FROM AUTHOR]

Background and Objective: Receptor activator of nuclear factor kappa B ligand (RANKL) is an important factor in osteoclast differentiation, activation and survival; however, its involvement in inflammatory bone resorption, with or without occlusal trauma, is unclear. The purpose of the present study was to investigate the distribution of RANKL-expressing cells in rat periodontium during lipopolysaccharide-induced inflammation with or without occlusal trauma. Material and Methods: Lipopolysaccharide was injected into rat gingiva of the lower left first molar to induce inflammation. In addition, the occlusal surface of the upper left first molar of rat was raised by placing a gold inlay to induce occlusal trauma in the lower left first molars. The distribution of RANKL-expressing cells was immunohistochemically observed. Results: In the inflammatory model, many osteoclasts were observed at the apical inter-radicular septum on day 5 and they were reduced by day 10. On the other hand, in the inflammatory model with occlusal trauma, many osteoclasts were still observed on day 10. RANKL expression was similar to the changes in osteoclast number. The expression of RANKL increased in endothelial cells, inflammatory cells and periodontal ligament cells. Conclusion: These findings clearly demonstrated that RANKL expression on endothelial cells, inflammatory cells and periodontal ligament cells is involved in inflammatory bone resorption and the expression is enhanced by traumatic occlusion. These results suggest that RANKL expression on these cells is closely involved in the increase of osteoclasts induced by occlusal trauma. [ABSTRACT FROM AUTHOR]

Background and Objective: Although induction of heme oxygenase-1 by H2O2 has been reported, the protective role of heme oxygenase-1 against the cytotoxic and osteoclastogenic effects of H2O2 have not been elucidated in human periodontal ligament cells. The aim of this work was to investigate the defense mechanism of heme oxygenase-1 on H2O2-induced cytotoxicity and to analyze the expression of receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin as markers for osteoclast differentiation in periodontal ligament cells. Material and Methods: Using human periodontal ligament cells, cytotoxicity was measured by the 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) assay, and expression of heme oxygenase-1, RANKL, and osteoprotegerin mRNA was determined by reverse transcription–polymerase chain reaction. Results: H2O2 produced a cytotoxic effect by reducing the cell viability and enhancing the expression of heme oxygenase-1 and RANKL mRNAs in a concentration- and time-dependent manner. Additional experiments revealed that heme oxygenase-1 inducer (hemin), a membrane-permeable cGMP analog (8-bromo-cGMP), carbon monoxide, extracellular signal-regulated kinase, p38 mitogen-activated protein kinase inhibitor, protein kinase inhibitor (KT5823), and nuclear factor-κB inhibitor (pyrrolidine dithiocarbamate) also blocked the effects of H2O2 on cell viability and RANKL mRNA expression in periodontal ligament cells. Conclusion: These data suggest that heme oxygenase-1 induction plays a protective role in periodontal ligament cells against the cytotoxic and RANKL-inducing effects of H2O2, through multiple signaling pathways. [ABSTRACT FROM AUTHOR]

Background and Objective: Receptor activator of nuclear factor-κB ligand (RANKL) is responsible for the induction of osteoclastogenesis and bone resorption, whereas its decoy receptor, osteoprotegerin, can directly block this action. Because this dyad of cytokines is crucial for regulating the bone remodelling process, imbalances in their expression may cause a switch from the physiological state to enhanced bone resorption or formation. This study investigated the mRNA expression of RANKL and osteoprotegerin, as well as their relative ratio, in the gingival tissues of patients with various forms of periodontal diseases. Material and Methods: Gingival tissue was obtained from nine healthy subjects and 41 patients, who had gingivitis, chronic periodontitis, generalized aggressive periodontitis, and chronic periodontitis and were receiving immunosuppressant therapy. Quantitative real-time polymerase chain reaction was employed to evaluate the mRNA expression of RANKL and osteoprotegerin in these tissues. Results: Compared with healthy individuals, patients in all periodontitis groups, but not those with gingivitis, exhibited stronger RANKL expression and a higher relative RANKL/osteoprotegerin ratio. In addition, osteoprotegerin expression was weaker in patients with chronic periodontitis. When patients with generalized aggressive periodontitis and chronic periodontitis were compared, the former exhibited stronger RANKL expression, whereas the latter exhibited weaker osteoprotegerin expression, and there was no difference in their relative ratio. When chronic periodontitis patients were compared with chronic periodontitis patients receiving immunosuppressant therapy, osteoprotegerin, but not RANKL, expression was stronger in the latter. Conclusion: This study demonstrates that RANKL and osteoprotegerin expression are differentially regulated in various forms of periodontitis, and the relative RANKL/osteoprotegerin ratio appears to be indicative of disease occurrence. This information may confer diagnostic and therapeutic value in periodontitis. [ABSTRACT FROM AUTHOR]

Background and Objective: Interleukin-1β-stimulated receptor activator of nuclear factor-κB ligand (RANKL) expression in human periodontal ligament cells is partially mediated by endogenous prostaglandin E2, whereas mitogen-activated protein kinases (MAPKs) are implicated in regulating various interleukin-1-responsive genes. We investigated herein the involvement of MAPKs in interleukin-1β-stimulated RANKL expression in human periodontal ligament cells. Material and Methods: Human periodontal ligament cells were pretreated separately with specific inhibitors of MAPKs, including extracellular signal-regulated kinase, p38 MAPK and c-Jun N-terminal kinase, and subsequently treated with interleukin-1β. Following each treatment, the phosphorylation of each MAPK, the expression of RANKL, and the production of prostaglandin E2 were determined. RANKL activity was evaluated using an assay to determine the survival of prefusion osteoclasts. Results: Interleukin-1β induced RANKL expression at the mRNA and protein levels, as well as RANKL activity in human periodontal ligament cells. Interleukin-1β also activated extracellular signal-regulated kinase, p38 MAPK, and c-Jun N-terminal kinase. Pretreatment with each MAPK inhibitor partially, but significantly, suppressed interleukin-1β-induced RANKL expression and its activity, as well as prostaglandin E2 production. Conclusion: In human periodontal ligament cells, three types of MAPK inhibitor may abrogate RANKL expression and activity induced by interleukin-1β, directly or indirectly through partial suppression of prostaglandin E2 synthesis. In addition, extracellular signal-regulated kinase, p38 MAPK, and c-Jun N-terminal kinase signals may co-operatively mediate interleukin-1β-stimulated RANKL expression and its activity in those cells. [ABSTRACT FROM AUTHOR]

There is accumulating evidence that osteoclasts, the primary cells responsible for bone resorption, are involved in bone and joint destruction in rheumatoid arthritis (RA). Preventing joint destruction is one of the most challenging issues in RA therapy. The recent elucidation of the various intracellular signaling pathways in osteoclasts has brought a tremendous understanding of the pathophysiology of inflammatory bone loss, and has heightened expectation of a novel intervention. We here highlight the molecular mechanism of bone and joint destruction in RA and the role of intracellular signaling pathways in osteoclastogenesis and mature osteoclast function. We also describe the recent trials on inhibitor drug and anticytokine therapies of arthritic joint disease-targeting osteoclasts. [ABSTRACT FROM AUTHOR]

Objective: The metabolism of hard tissue is influenced by the immune system. Research into the bone destruction associated with inflammatory diseases such as periodontal disease and rheumatoid arthritis has highlighted the importance of the interplay of the immune and skeletal systems. This interdisciplinary research field, called ‘osteoimmunology’, has become increasingly important for each system by itself as well as the biology linking them. The history and recent progress of this field are reviewed. ‘Osteoimmunology’ was coined to describe the pioneering work on the T-cell regulation of osteoclastogenesis by the receptor activator of nuclear factor-κB ligand (RANKL) and interferon (IFN)-γ. Accumulating evidence suggests that the immune and skeletal systems share not only cytokines but also various signaling molecules, transcription factors and membrane receptors. The contribution of T cells to the pathogenesis of inflammatory bone destruction is discussed, and our recent findings are summarized to illustrate how the osteoimmunological network functions. RANKL is an osteoclastogenic cytokine that links bone and the immune system. Immunomodulatory cytokines such as IFNs also participate in the regulation of RANKL signaling and inflammatory bone loss. The transcription factor nuclear factor of activated T cells c1 (NFATc1) has been identified as a master switch regulator of osteoclastogenesis. In addition, immunoglobulin-like receptors are critically involved in bone homeostasis. Bone turns out to be a dynamic tissue that is constantly renewed, where the immune system participates to a hitherto unexpected extent. This emerging field will be of great importance to a better understanding and treatment of diseases of the skeletal and immune systems, as well as to the fundamental biology underpinning both. [ABSTRACT FROM AUTHOR]

Ling L-J, Ho F-C, Chen Y-T, Holborow DW, Liu T-Y, Hung S-L. Areca nut extracts modulated expression of alkaline phosphatase and receptor activator of nuclear factorκ B ligand in osteoblasts.J Clin Periodontol 2005; 32: 353–359. doi: 10.1111/j.1600-051X.2005.00687.x.© Blackwell Munksgaard, 2005.Areca chewers have a higher prevalence of periodontal diseases than non-chewers. This study was to determine the possible effects of ripe areca nut extracts (rANE) on viability and gene expression of alkaline phosphatase (ALP), receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) in human osteoblasts.The effects of rANE on cell viability of osteoblast-like MG63 cells were determined using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) that measures metabolic activity. Gene expression of ALP, RANKL and OPG was examined using reverse transcription-polymerase chain reaction. ALP activity and RANKL protein were further examined using substrate assay and confocal laser scanning microscopy, respectively.Relative viability was reduced to approximately 50% when 25 μg/ml of rANE was used. The expression of OPG mRNA in MG63 cells was not altered by rANE. However, decreased levels of mRNA and enzyme activity of ALP were observed. Moreover, the expressions of mRNA and protein of RANKL were stimulated by rANE in a dose-dependent manner.The rANE affected morphology and viability of osteoblasts. We also present novel evidence demonstrating that areca nut may compromise the periodontal health of areca chewers by suppression of ALP gene expression and elevation of RANKL gene expression in osteoblasts. [ABSTRACT FROM AUTHOR]

Abstract: Development of bone depends on a continuous supply of bone-degrading osteoclasts. Although several factors such as cytokines and integrins have been shown to be important for osteoclast recruitment, their mechanism of action is poorly understood. In this study, we demonstrated the enhancement of osteoclast formation by hypoxia and investigated the molecular mechanisms involved. Primary mouse bone marrow cells were cultured in normoxic and hypoxic conditions, and RNA was prepared from each group of cells. Total RNAs were applied to a DNA microarray analysis and then RT-PCR was performed to confirm the microarray data. The most interesting finding of our microarray analysis was upregulation of insulin-like growth factor 2 (IGF2) and stromal cell-derived factor 1 (SDF1) under hypoxic conditions. RT-PCR analysis revealed that IGF2 expression was markedly upregulated in the non-osteoclastic cells. The addition of exogenous IGF2 increased the number of osteoclastic TRAP-positive multinuclear cells formed under normoxic conditions, whereas the addition of exogenous SDF1 did not change osteoclast formation. These results suggest that the upregulation of IGF2 derived from non-osteoclastic cells might be a crucial factor for osteoclast differentiation. [Copyright &y& Elsevier]

Receptor activator of nuclear factor-κB ligand (RANKL) induces osteoclastogenesis by binding with the receptor, receptor activator of nuclear factor-κB in the presence of macrophage colony-stimulating factor. Three human RANKL isoforms, hRANKL1, hRANKL2, and hRANKL3, were identified. hRANKL1 was identical to previously reported RANKL and possessed intracellular, transmembrane, and extracellular domains, hRANKL2 did not have the intracellular domain, and hRANKL3 did not have the intracellular and transmembrane domains. When bone marrow macrophages were cultured with NIH3T3 cells expressing hRANKL1, osteoclasts were formed, but when cultured with NIH3T3 cells expressing hRANKL2 or hRANKL3, no tartrate resistant acid phosphatase-positive cell was observed. In the coculture system, coexpression of hRANKL3 with hRANKL1 significantly inhibited the formation of osteoclasts by hRANKL1, but coexpression of hRANKL2 with hRANKL1 did not affect the osteoclastogenesis by hRANKL1 significantly. These results suggest that the activity of osteoclastogenesis by hRANKL1 is regulated by the attenuator, hRANKL3. [Copyright &y& Elsevier]

Ogasawara T, Yoshimine Y, Kiyoshima T, Kobayashi I, Matsuo K, Akamine A, Sakai H. In situ expression of RANKL, RANK, osteoprotegerin and cytokines in osteoclasts of rat periodontal tissue. J Periodont Res 2004; 39; 42–49. © Blackwell Munksgaard, 2004 This study examined the in situ expression of receptor activator of nuclear factor-κB ligand (RANKL), receptor activator of nuclear factor-κB (RANK), osteoprotegerin, interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) in the osteoclasts of rat periodontal tissue. In periodontal disease, osteoclasts cause resorption of the alveolar bone. The function of osteoclasts is regulated by interaction with periodontal ligament cells (PDLs). Furthermore, various kinds of molecules such as RANKL, RANK, osteoprotegerin, IL-1β and TNFα are known to be related to the osteoclasts differentiation and function. It is therefore important to observe the expression of RANKL, RANK, osteoprotegerin and cytokines in osteoclasts and PDLs. Four-week-old Wistar rats were used. Tooth movement was performed by the Waldo method, and the pathological bone resorption was induced. The demineralized maxillae and mandiblae were embedded with paraffin. In situ hybridization was performed to detect RANKL, RANK, osteoprotegerin, IL-1β, and TNFα mRNAs in osteoclasts and other cells using the specific RNA probes, respectively. Both RANKL and RANK were concomitantly expressed in some osteoclasts. RANKL was also positive in osteoblasts and PDLs. No IL-1β- and TNFα-positive osteoclast was noted. The positive signals of osteoprotegerin were detected in almost all osteoblasts, PDLs and odontoblasts. No osteoprotegerin-positive osteoclasts were observed. The number and the distribution pattern of RANKL- and RANK-expressing osteoclasts changed when orthodontic excessive force was applied to periodontal tissue. In addition, IL-1β and TNFα were shown to be expressed in osteoclasts under pathological status. These findings suggest that an autocrine mechanism of RANKL–RANK exists in osteoclast, which is heightened in the pathological conditions. Furthermore, the autocrine mechanism of IL-1β and TNFα is also provided in osteoclast under pathological condition. These autocrine mechanisms therefore seem to regulate the osteoclast function in both physiological and pathological conditions. [ABSTRACT FROM AUTHOR]

We examined the responses of human osteoblastic cell line SaOS-2 to bacterial lipid A, a bioactive center of lipopolysaccharide, during osteoclast differentiation of human peripheral blood mononuclear cells (PBMC). SaOS-2 cells expressed mRNA for Toll-like receptor (TLR) 4, MD-2, CD14, and myeloid differentiation factor 88, whereas they failed to express mRNA for TLR2. Escherichia coli-type synthetic lipid A (compound 506) induced cytokine mRNA expression and nuclear factor (NF)-κB activation in SaOS-2 cells. Compound 506 also increased the expression of receptor activator of NF-κB ligand. Further, cells primed with compound 506 augmented the differentiation of PBMC into osteoclastic cells, and the effect was inhibited by anti-TLR4 monoclonal antibody. These findings suggest that the TLR signaling cascade in osteoblastic cells is involved in regulating the function of osteoclastogenesis. [Copyright &y& Elsevier]

Objectives: Pathological bone resorption is common in chronic periodontitis. However, the effect of exosomes (Exo) secreted by periodontal ligament stem cells (PDLSCs) on bone resorption is unclear. This study explored the Exo of inflammatory PDLSCs, their protein components, and their effects on osteoclast differentiation., Methods: PDLSCs were isolated from the periodontal ligament tissues of orthodontic patients and those with chronic periodontitis. The surface markers of PDLSCs were detected by flow cytometry. Exo were characterized by Western blot, transmission electron microscope (TEM), bicinchoninic acid assay (BCA), nanosight tracking analysis (NTA). The protein components of Exo were detected by protein profiling. The expression levels of differentially expressed proteins tumor necrosis factor-α (TNF-α), receptor activator of nuclear factor-κB ligand (RANKL), interleukin (IL)-1α, transforming growth factor β (TGF-β), and bone morphogenetic protein 2 (BMP-2) were verified by enzyme linked immunosorbent assay (ELISA). Then, 10, 100, and 1 000 μg·mL -1 of Exo-CP or Exo-WT were added to RAW264.7 medium, and the expression levels of osteoclast-related indicators were detected by real time quantitative polymerase chain reaction (RT-qPCR), Western blot, and tartrate resistant acid phosphatase (TRAP) staining at 5 days. Experimental data were statistically analyzed using SPSS 24.0 software., Results: The differentially expressed proteins enriched in Exo-CP were mainly related to the tumor necrosis factor (TNF) signaling, osteoclast differentiation, and nuclear transcription factor κB (NF-κB) signaling pathways. ELISA experiments confirmed Exo-CP had high expression of TNF-α, RANKL, and IL-1α and low expression of TGF-β1 and BMP-2 ( P <0.05). Adding Exo-CP to RAW264.7 significantly increased the expression of mRNA and proteins related to osteoclast differentiation of cells. In a concentration-dependent manner, the effect of Exo-CP on osteoclast differentiation at concentrations of 100 and 1 000 μg·mL -1 was significantly higher than that on the 10 μg·mL -1 concentration group ( P <0.05)., Conclusions: Pathological bone resorption of chronic periodontitis may be caused by the activation of Exo-CP to promote osteoclast differentiation. The main protein in Exo may be RANKL and TNF-α. This research provides a new perspective on pathological bone resorption in chronic periodontitis.

The article discusses a study which is conducted to demonstrate that haemophilics show high sclerostin serum levels and the reduced bone mineral density (BMD) and are at risk for developing osteoporosis and fractures. It mentions that pathogenesis of low BMD in haemophilics is multifactorial and includes immobilization as well as haemophilic arthropathy. It also mentions that correlation with bone remodelling markers and dual X-ray absorptiometry parameters.

Osteoporosis is a bone related disease that is characterised by bone loss that further increases the susceptibility to bone fractures and bone frailty due to disturbances in the micro-architecture of bone tissue. Fisetin (flavonoids) exhibited anti-inflammatory and antioxidative stress effects against various diseases. In this protocol, we make an effort to comfort the anti-osteoporosis effect of fisetin against ovariectomy (OVX) induced osteoporosis. A docking study of fisetin and alendronate on the estrogen (α and β) and vitamin D receptors was carried out. SaOS-2 (osteoblast like human) cells were used for the estimation of cell proliferation. The OVX induced OVX model was used and three doses of fisetin and alendronate was given to rats till 16 weeks. The hormone levels, bone turnover markers and biochemical parameters were estimated. Fisetin was docked into estrogen (α and β) and vitamin D receptors, resulting in stable complexes with lower binding scores. Fisetin significantly (p < 0.001) exhibited the induction of cell proliferation against the SaOS-2 cells. OVX induced osteoporosis rats exhibited a suppression of body weight and uterus index, after the Fisetin treatment. Fisetin treatment significantly (p < 0.001) improved the level of bone mineral content (BMC), bone mineral density (BMD) and biochemical parameters such as energy, maximum load, stiffness, young modules, maximum stress and reduced the level of 1,25(OH) 2 D 3 and E 2 . Fisetin treatment significantly (p < 0.001) declined the level of phosphorus (P), calcium (Ca) and boosted the level of VitD. Fisetin treatment significantly (p < 0.001) reduced the malonaldehyde (MDA) level and enhanced the glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) level in the bone, intestine and hepatic tissue. Fisetin treatment suppressed the cytokines, RANKL/OPG ratio, receptor activator of nuclear factor-κB ligand (RANKL) and improved the level of osteoprotegerin (OPG). The findings suggest that fisetin could be a beneficial phytoconstituent for the treatment and prevention of postmenopausal osteoporotic complications.

Osteoporosis is a systemic skeletal disease characterized by reduced bone mineral density (BMD), which results in an increased risk of fracture. Melandrium firmum (Siebold & Zucc.) Rohrbach (MFR), 'Wangbulryuhaeng' in Korean, is the dried aerial portion of Melandrii Herba Rohrbach, which is a member of the Caryophyllaceae family and has been used to treat several gynecological conditions as a traditional medicine. However, to the best of our knowledge, the effect of MFR on osteoclast differentiation and osteoporosis has not been assessed. To evaluate the effects of MFR on osteoclast differentiation, tartrate-resistant acid phosphatase staining, actin ring formation and bone resorption assays were used. Additionally, receptor activator of nuclear factor-κB ligand-induced expression of nuclear factor of activated T cell, cytoplasmic 1 (NFATc1) and c-Fos were measured using western blotting and reverse transcription-PCR. The expression levels of osteoclast-related genes were also examined. To further investigate the anti-osteoporotic effects of MFR in vivo, an ovariectomized (OVX) rat model of menopausal osteoporosis was established. Subsequently, the femoral head was scanned using micro-computed tomography. The results revealed that MFR suppressed osteoclast differentiation, formation and function. Specifically, MFR reduced the expression levels of osteoclast-related genes by downregulating transcription factors, such as NFATc1 and c-Fos. Consistent with the in vitro results, administration of MFR water extract to OVX rats reduced BMD loss, and reduced the expression levels of NFATc1 and cathepsin K in the femoral head. In conclusion, MFR may contribute to alleviate osteoporosis-like symptoms. These results suggested that MFR may exhibit potential for the prevention and treatment of postmenopausal osteoporosis. [ABSTRACT FROM AUTHOR]

Osteoblast differentiation is important for the development of bone and the maintenance of bone density. Propionibacterium freudenreichii is a probiotic with an anti-inflammatory property. The aim of this study was to investigate the enhancement effect of P. freudenreichii MJ2 (MJ2) isolated from raw milk on osteoblast differentiation, mineralization, and its signaling pathway. For in vitro and in vivo experiments, human fetal osteoblastic cell line hFOB 1.19 and an ovariectomized rat model were used, respectively. Expression levels of genes and proteins related to osteoblast differentiation and mineralization were measured by real-time polymerase chain reaction (qPCR) and Western blotting, respectively. Alizarin red S staining was performed to measure osteoblast mineralization. Heat-killed MJ2 (hkMJ2)-treated cells showed significantly increased osteoblast differentiation via an increase in the osteoprotegerin (OPG)/receptor activator of nuclear factor-κB ligand (RANKL) ratio and significantly increased osteoblast mineralization by stimulating the expression of bone morphogenetic protein 2 and runt-related transcription factor 2. Additionally, oral administration of live or heat-killed MJ2 to ovariectomized rats inhibited osteoporosis-induced bone loss. Specifically, surface proteins isolated from MJ2 promoted osteoblast differentiation and mineralization. In conclusion, MJ2 enhanced osteoblast differentiation and mineralization through the OPG/RANKL signaling pathway and the effective component of MJ2 might be its surface proteins. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the changes in miRNA-142 and bone morphogenetic protein-2 (BMP-2) expression before and after hip replacement surgery, and to determine their association with receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG). For this purpose, 142 cases of hip arthroplasty in patients with femoral neck fracture were selected as the research group, and 50 cases of healthy individuals who underwent a physical examination during the same time period were selected the control group. Serum miR-142 and BMP-2 levels were measured by RT-qPCR before and after surgery in the research group and the control group. Serum RANKL and OPG levels were detected before and after surgery by enzyme-linked immunosorbent assay (ELISA). The levels of serum miR-142 and BMP-2 in the research group were significantly lower than those in the control group. At 1 month after surgery, the levels of serum miR-142 and BMP-2 in the research group were significantly higher than those before surgery, and at 6 months after surgery, and they were higher than those in the control group. Pearson's correlation analysis revealed that the serum levels of miR-142 positively correlated with the BMP-2 levels before surgery, at 1 month after surgery, and at 6 months after surgery in the research group. The results of ROC curve analysis revealed that the AUC values of serum miR-142 and BMP-2 were 0.911 and 0.861, respectively. At 1 month after surgery, the levels of serum miR-142 and BMP-2 in group A (patients with a good or excellent recovery) were significantly higher than those in group B (patients with a fair or poor recovery). The levels of serum RANKL and OPG in the research group significantly increased at 1 month after surgery. The serum levels of miR-142 and BMP-2 positively correlated with those of RANKL and OPG before surgery and at 1 month after surgery. On the whole, the findings of the present study indicate that pre-operative serum miR-142 and BMP-2 levels are valuable for evaluating the post-operative recovery of patients with femoral neck fracture undergoing hip replacement surgery. In addition, in the present study, at 1 month after surgery, the levels of both miR-142 and BMP-2 were related to the recovery of the patients, and positively correlated with the RANKL and OPG levels. [ABSTRACT FROM AUTHOR]

The effect of triptolide (TP) on the expression of receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) was explored in rat adjuvant induced arthritis (AA). AA was induced in Wistar rats. Arthritis rats were treated with TP and methotrexate (MTX) at the onset (day 9) of arthritis. On the peak of arthritis (day 24), the expression of RANKL and OPG protein in the joints and RANKL mRNA in peripheral blood mononuclear cells (PBMC) was detected. TNF-α and IL-1β levels in peripheral blood were determined. Bone erosion scores were also evaluated. The results showed that bone erosion scores in TP and MTX groups were lower than in AA group ( P<0.01); The expression levels of RANKL in the synovium ( P<0.01) and bone ( P<0.05), and OPG level in synovium ( P<0.05) were lower in TP group than in AA group ( P<0.05). In TP group, the expression levels of RANKL mRNA and TNF-α, IL-1β in PBMC were lower than in AA group (all P<0.01). It was concluded that TP could inhibit rat adjuvant arthritis bone erosion by suppressing the expression of RANKL. [ABSTRACT FROM AUTHOR]

Breast cancer and osteoporosis are common diseases that affect the survival and quality of life in postmenopausal women. Women with breast cancer are more likely to develop osteoporosis than women without breast cancer due to certain factors that can affect both diseases simultaneously. For instance, estrogen and the receptor activator of nuclear factor-κB ligand (RANKL) play important roles in the occurrence and development of these two diseases. Moreover, chemotherapy and hormone therapy administered to breast cancer patients also increase the incidence of osteoporosis, and in recent years, neuropeptide Y (NPY) has also been found to impact breast cancer and osteoporosis.Y1 and Y5 receptors are highly expressed in breast cancer, and Y1 and Y2 receptors affect osteogenic response, thus potentially highlighting a potential new direction for treatment strategies. In this paper, the relationship between breast cancer and osteoporosis, the influence of NPY on both diseases, and the recent progress in the research and treatment of these diseases are reviewed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lin, Li, Sun, Chen, Huang, Lin and Cai.)

IL-17 is critical in lung lymphoid neogenesis in COPD, but the cellular and molecular mechanisms remain to be elucidated. Receptor activator of nuclear factor-κB ligand (RANKL) functions in lymphoid follicle formation in other organs, whether it is involved in IL-17A-dependent lymphoid neogenesis in COPD is unknown. To elucidate the expression and functional role of IL-17A/RANKL pathway in COPD. We first quantified and localized RANKL, its receptor RANK and IL-17A in lungs of patients with COPD, smokers and non-smokers. Next, IL-17A -/- and wild-type (WT) mice were exposed to air or cigarette smoke (CS) for 24 weeks, and lung lymphoid follicles and RANKL-RANK expression were measured. Lastly, we studied the in vitro biological function of RANKL pertaining to lymphoid neogenesis. We found that the expressions of RANKL-RANK and IL-17A, together with lymphoid follicles, were increased in lung tissues from patients with COPD. In WT mice exposed to CS, RANKL-RANK expressions were prominent in lung lymphoid follicles, which were absent in IL-17A -/- mice exposed to CS. In the lymphoid follicles, RANKL + cells were identified mostly as B cells and RANK was localized in dendritic cells (DCs). In vitro IL-17A increased the expressions of RANKL in B cells and RANK in DCs, which in turn responded to RANKL stimulation by upregulation of CXCL13. Altogether, these results suggest that B lymphocyte RANKL pathway is involved in IL-17A-dependent lymphoid neogenesis in COPD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Xiong, Zhou, Tian, Yang, Li, Jin, Le, Rao and Sun.)

In ultra-rare bone diseases, information on growth during childhood is sparse. Juvenile Paget disease (JPD) is an ultra-rare disease, characterized by loss of function of osteoprotegerin (OPG). OPG inhibits osteoclast activation via the receptor activator of nuclear factor-κB (RANK) pathway. In JPD, overactive osteoclasts result in inflammatory-like bone disease due to grossly elevated bone resorption. Knowledge on the natural history of JPD, including final height and growth, is limited. Most affected children receive long-term antiresorptive treatment, mostly with bisphosphonates, to contain bone resorption, which may affect growth. In this study, we report the follow-up of height, growth velocity, and skeletal maturation in a 16-year-old female patient with JPD. The patient was treated with cyclic doses of pamidronate starting at 2.5 years of age and with 2 doses of denosumab at the age of 8 years, when pamidronate was paused. In the following years, a sustainable decline in a height z-score and a stunted pubertal growth spurt; despite appropriate maturation of the epiphyseal plates of the left hand, the proximal right humerus and both femora were observed. Whether this reflects the growth pattern in JPD or might be associated to the antiresorptive treatments is unclear, since there is very limited information available on the effect of bisphosphonates and denosumab on growth and the growth plate in pediatric patients. Studies are needed to understand the natural history of an ultra-rare bone disease and to assess the effects of antiresorptive treatment on the growing skeleton., (© 2021 S. Karger AG, Basel.)

Osteoclasts with elevated bone resorption are commonly present in postmenopausal osteoporosis, and other osteolytic pathologies. Therefore, suppressing osteoclast generation and function has been the main focus of osteoporosis treatment. Betulinic acid (BA) represents a triterpenoid mainly purified from the bark of Betulaceae. BA shows multiple biological activities, including antitumor and anti-HIV properties, but its effect on osteolytic conditions is unknown. Here, BA suppressed receptor activator of nuclear factor-κB ligand (RANKL)-associated osteoclastogenesis and bone resorptive function, as assessed by tartrate-resistant acid phosphatase (TRAP) staining, fibrous actin ring generation, and hydroxyapatite resorption assays. Mechanistically, BA downregulated the expression of osteoclastic-specific genes. Western blot analysis revealed that BA significantly interrupted ERK, JNK and p38 MAPK activation as well as intracellular reactive oxygen species (ROS) production, thus altering c-Fos and NFATc1 activation. Corroborating the above findings in cell-based assays, BA prevented ovariectomy-associated bone loss in an animal model. In conclusion, these findings suggest that BA can inhibit osteoclast generation and function as well as the RANKL signaling pathway, and might be used for treating osteoclast-related osteoporosis., (Copyright © 2020 Wei, Li, Liu, Lan, Wei, Tian, Wu, Lin, Xu, Zhao and Yang.)

There is still a lack of evidence that minodronate or denosumab prevents bone loss due to androgen deprivation therapy (ADT) in non-Western patients. This study showed that both drugs significantly improved lumbar spine and total hip bone mineral density in Asian men with prostate cancer who received ADT., Introduction: To evaluate whether monthly oral minodronate or semiannual subcutaneous injection of denosumab improves bone mineral density (BMD) in Asian men with prostate cancer (PCa) receiving ADT., Methods: A multicenter, open-label, randomized, controlled study including patients with hormone-sensitive PCa without bone metastasis receiving ADT was performed. Patients were randomized (1:1:1) to minodronate, denosumab, or no agent control groups. The primary end point was the mean percentage change in BMD at the lumbar spine at 12 months. Secondary end points were the mean percentage change in BMD at the femoral neck and total hip and changes in bone turnover markers. Statistical comparison was performed using analysis of covariance., Results: Of the 147 subjects enrolled in this study, 102 were randomly assigned into the minodronate (n = 36), denosumab (n = 36), and control (n = 30) groups. The percentage change in BMD at the lumbar spine was significantly improved in the minodronate (2.5%, p < 0.05) and denosumab groups (4.0%, p < 0.01) compared with that in the control group (- 0.1%). Denosumab increased BMD at the femoral neck and total hip at 12 months, whereas minodronate only increased BMD at the total hip compared with controls (all p < 0.05). The percentage change in bone turnover markers at 12 months was significantly lower in the minodronate and denosumab groups compared with that in the control group (both p < 0.01)., Conclusion: Minodronate or denosumab can be used for preventing bone loss related to ADT in Asian patients with PCa.

Although the role of cathepsin C (Cat C) in inflammation is gradually being elucidated, its function in periapical periodontitis, which is one of the most common infectious diseases worldwide, has not been studied. This study evaluated a surgically-induced model of periapical periodontitis in cathepsin C (Cat C) knock-down (KD) mice, which was constructed with a tetracycline operator, to evaluate the role of Cat C in the pathogenesis and progression of periapical periodontitis. Our results showed, for the first time, that there was a statistically significant increase in the expression of Cat C as periapical periodontitis progressed; this increase started from 1 week after surgery and reached a peak at 3 weeks after surgery, before gradually decreasing. The volume of periapical bone resorption in Cat C KD mice was significantly smaller than that in wild-type mice at 3 and 4 weeks after surgery (P<0.05). Inflammatory cell infiltration into the apical tissues of wild-type mice was also significantly higher than that of Cat C KD mice. The expression of receptor activator of nuclear factor-κB ligand (RANKL) in wild-type mice was also higher than that in Cat C KD mice. The difference in the number of osteoclasts in the apical area between the two groups was statistically significant after 2 weeks. Correlation analysis showed that there was a significant correlation between Cat C and RANKL expression (r= 0.835). Therefore, our data indicated that Cat C promoted the apical inflammation and bone destruction in mice., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2019 Science China Press. Published by Elsevier B.V. All rights reserved.)