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Lipopolysaccharide (LPS) and Receptor Activator of Nuclear Factor-κB Ligand (RANKL) are the two important factors causing bone loss, which is an important pathogenesis for osteoporosis. However, the relationship between LPS and RANKL is not yet clear. LPS can be involved in the weakened osteoblast formation as an autophagy regulator, and osteoblasts and their precursors are the source cells for RANKL production. Our study aimed to explore the relationship between autophagy changes and RANKL production during LPS-regulated osteoblasts. Our results showed that LPS inhibited autophagy (LC3 conversion and autophagosome formation) and enhanced the protein and mRNA expression of RANKL in MC3T3-E1 osteoblast precursor line. Autophagy upregulation with Rapamycin over BECN1 overexpression rescued LPS-inhibited osteoblast formation and -promoted RANKL protein production in MC3T3-E1 cells. In vivo experiments supported that damaged bone mass, bone microstructure, osteoblastic activity (ALP and P1NP production by ELISA assays) and enhanced RANKL production by LPS administration were partially rescued by Rapamycin application. In conclusion, LPS can inhibit autophagy in osteoblast precursors, thereby inhibiting osteoblast formation and RANKL autophagic degradation.

BACKGROUND: Interleukin-1 is an important pro-inflammatory cytokine that has been documented in the regulation of bone inflammation and bone remodeling. A previous study has demonstrated that interleukin-1α can induce apoptosis while inhibiting osteoblast differentiation in MC3T3-E1 cells. OBJECTIVE: To investigate the role and mechanism of interleukin-1α on osteoclast activation and bone loss in mice. METHODS: (1) Cell test: RAW264.7 cells were either treated with interleukin-1α alone or with receptor activator of nuclear factor-κB ligand (RANKL) for 1 and 4 days. Cell viability was tested by cell counting kit-8 assay. The number of multinuclear osteoclasts was detected by tartrate resistant acid phosphatase assay. The mRNA and protein levels of osteoclast-specific genes and genes related to nuclear factor-κB pathway and Wnt/β-catenin pathway were tested by real-time fluorescence quantitative PCR, immunofluorescence staining or western blot. Bone marrow-derived macrophages were either treated with interleukin-1α alone or with RANKL and macrophage colony-stimulating factor for 7 days. The number of multinuclear osteoclasts was detected by tartrate resistant acid phosphatase assay. The protein levels of osteoclast-specific genes were tested by western blot. (2) Animal test: Twenty-four male C57BL/6J mice (6-8 weeks old) were assigned into two groups at random: control group and test group. Mice were subsequently treated with interleukin-1α solution or PBS by intraperitoneal injection twice a week for 5 weeks. Bone tissues from the femurs were performed with micro-computed tomography analysis and hematoxylin-eosin staining, tartrate resistant acid phosphatase, and immunofluorescence analysis. RESULTS AND CONCLUSION: Cell test: Interleukin-1α alone significantly increased RAW264.7 cell proliferation, but stimulated cell differentiation into osteoclasts in combination with RANKL (P < 0.05). Interleukin-1α significantly increased the expression of osteoclast-related markers and the number of tartrate resistant acid phosphatasepositive multinuclear cells in RAW264.7 cells and bone marrow-derived macrophages in the existence of RANKL or RANKL+macrophage colony-stimulating factor (both P < 0.05). Interleukin-1α was found to significantly enhance the nuclear factor-κB and Wnt/beta-catenin signaling in RAW264.7 cells (P < 0.05). Blocking of nuclear factor-κB or Wnt3 signaling not only reversed the activation of nuclear factor-κB and Wnt3 signaling but also weakened the enhanced expression of osteoclast-specific genes induced by interleukin-1α in RAW264.7 cells (P < 0.05). Animal test: interleukin1α induced bone loss in mice while also upregulating the expression of osteoclast-specific markers, RANK, TRAF6 and p65, and Wnt3 in vivo (P < 0.05). The findings indicate that interleukin-1α can induce osteoclast activation and bone loss by promoting the nuclear factor-κB and Wnt signaling pathways. [ABSTRACT FROM AUTHOR]

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.

Osteolytic diseases are typified by over‐enhanced formation and resorbing function of osteoclasts and have a major impact on human health. Inhibition of osteoclastic differentiation and function is a key strategy for clinical therapy of osteolytic conditions. Maackiain is a natural compound extracted from Sophora flavescens, which has been applied to anti‐allergic and anti‐tumour treatments. The present results showed that Maackiain could restrain receptor activator of nuclear factor‐κB ligand (RANKL)‐stimulated osteoclast formation and hydroxyapatite resorption dose‐dependently, and interrupt the structures of F‐actin belts in the mature osteoclasts. It also repressed the expressions of osteoclast‐specific genes and proteins. Furthermore, Maackiain could inhibit RANKL‐stimulated NF‐κB and calcium signalling pathways, and dampen Nuclear factor of activated T cell cytoplasmic 1 activity, protein expression and translocation into the nucleus. These results revealed that Maackiain may have a potential therapeutic effect on osteoclast‐related disorders. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Postmenopausal osteoporosis (PMOP) is characterized by increased bone loss due to enhanced osteoclastogenesis and bone resorption. A Chinese herbal formula, jiangugranule (JG), exhibited great efficacy in the clinical treatment of PMOP. However, the molecular mechanisms underlying the therapeutic effects remain unclear. The present study aimed to examine the effects of JG-containing serum on receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis. Osteoclast precursor RAW264.7 cells were cultured and treated with JG-containing serum in the presence of RANKL. Following 6 days of culture, the cells were stained with tartrate-resistant acid phosphatase and the rate of differentiation was calculated. In addition, cells were treated with JG-containing serum for 24, 48 and 96 h and total RNA and proteins were extracted for reverse transcription-quantitative polymerase chain reaction and western blot analysis to detect mRNA and protein expression, respectively, of key molecules in the RANK/RANKL signaling pathway, including RANK, tumor necrosis factor receptor-associated factor 6, NF-κB (p50 and p52 subunits), c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1). The results revealed that JG-containing serum inhibited RANKL-induced osteoclastogenesis and reduced mRNA and protein expression of RANK, c-Fos and NFATc1. The results suggested that JG may regulate osteoclast differentiation through the RANK/RANKL signaling pathway, which may be a possible mechanism for the therapeutic effects of JG on PMOP. [ABSTRACT FROM AUTHOR]

Osteoporosis is caused by a functional imbalance between osteoblasts and osteoclasts. The increased activation of osteoclasts that is a hallmark of osteoporosis results in the progressive loss of bone mass and therefore in an increased susceptibility to bone fractures. Diarylheptanoids are a group of phytoestrogens that have been isolated from a number of plant species, including the rhizomes of Curcuma comosa Roxb. In this study, the effect of one of diarylheptanoids, (3 S )-1-(3,4-dihydroxyphenyl)-3-hydroxy-7-phenyl-(6 E )-6-heptene (DHPH), was investigated for anti-inflammatory and anti-osteoclastogenic activity. DHPH significantly inhibited nitric oxide production in RAW264.7 cell line following their activation by lipopolysaccharide and interferon-γ, with no cytotoxicity. In primary mouse bone-marrow-derived macrophage precursors, DHPH suppressed osteoclastogenesis induced by receptor activator of nuclear factor-κB (RANK) ligand at an inhibitory concentration 50 of 325±1.37 nM. DHPH treatment delayed and reduced the expression of master regulators of osteoclast differentiation, NFATc1 and c-Fos. Consistent with this result, the mRNA level of cathepsin K , associated with osteoclast differentiation, was decreased whereas the reduction in the mRNA of irf8 , a negative regulator of osteoclast differentiation, was similar to that measured in the vehicle-treated control cells. DHPH reduced the phosphorylation of p38 MAPK, ERK (p44/42). Furthermore, DHPH suppressed the bone absorption activity of osteoclasts and enhanced osteoblast differentiation. Taken together, DHPH interrupts the immediate downstream signaling cascade of RANK and interferes with osteoclast differentiation and its function while enhances osteoblast differentiation. These results demonstrate the potential of this diarylheptanoid as a new therapeutic agent in osteoporosis. [ABSTRACT FROM AUTHOR]

Thiazolidinediones are traditional anti-diabetic therapeutic agents that have been associated with bone loss and increased fracture risk. However, the underlying mechanisms of this side effect require further elucidation. The present study aimed to investigate the effect of pioglitazone (PIO), a thiazolidinedione, on osteoblastogenesis, osteoclastogenesis and the osteoprotegerin (OPG) / receptor activator of nuclear factor-κB ligand (RANKL) / RANK system. The MC3T3-E1 murine pre-osteoblastic cell line was treated with PIO and processed for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis of OPG, RANKL, peroxisome proliferator-activated receptor γ (PPARγ), Runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN), and western blotting analysis of OPG and RANKL. The culture medium was collected for ELISA analysis of OPG and RANKL. Murine bone marrow monocytes (BMMCs) were treated with PIO in the presence of RANKL and macrophage-colony stimulating factor and subjected to tartrate-resistant acid phosphatase (TRAP) staining and activity measurement, and RT-qPCR analysis of cathepsin K, TRAP and RANK. Co-culture of MC3T3-E1 and BMMCs was performed in the presence of PIO, and TRAP staining was also conducted. PIO inhibited the osteoblastic differentiation of MC3T3-E1 cells, and promoted the osteoclastic differentiation of BMMCs with or without co-culturing with MC3T3-E1 cells. ELISA analysis indicated increased RANKL and decreased OPG expression levels in the medium of MC3T3-E1 cells treated with PIO. PIO upregulated expression of RANKL and PPARγ and downregulated expression of OPG, RUNX2, ALP and OCN in MC3T3-E1 cells, while expression levels of RANK in BMMCs remained unchanged. These results suggest that PIO suppresses osteoblastogenesis and enhances osteoclastogenesis. In addition, PIO may also promote osteoclastogenesis by affecting the OPG-RANKL-RANK system. [ABSTRACT FROM AUTHOR]

A new naphthalene glycoside was isolated from the leaves and stems of Chimaphila umbellata Barton. Its chemical structure was elucidated to be 2,7-dimethyl-1,4-dihydroxynaphthalene-1- O-β- d-glucopyranoside (DMDHNG), based on spectroscopic evidence. DMDHNG significantly inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced tartrate-resistant acid phosphatase (TRAP) activity and the formation of multinucleated osteoclasts in a dose-dependent manner. In addition, the new glycoside inhibited the RANKL-induced mRNA expression of osteoclast-associated genes that encode TRAP, cathepsin K, and another transcription factor-nuclear factor of activated T-cells c1. We believe that the inhibitory effects of DMDHNG on the osteoclast differentiation may be exploited for a therapeutic benefit. [ABSTRACT FROM AUTHOR]

The effect of conophylline (CNP) on the receptor activator of nuclear factor-κB ligand (RANKL) or lipopolysaccharide (LPS)-induced osteoclast formation was studied in vitro using bone marrow-derived macrophages (BMMs) or the mouse macrophage-like cell line RAW 264.7. CNP inhibited RANKL-induced formation of osteoclasts identified as tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in a culture of BMMs. It also inhibited RANKL- or LPS-induced osteoclast formation in RAW 264.7 cells. CNP lowered the osteoclast maturation markers such as calcitonin receptor, MMP9 and cathepsin K in BMMs, suggesting that CNP would inhibit the process of osteoclast differentiation. CNP inhibited the RANKL-induced expressions of c-Fos and nuclear factor of activated T cells (NFATc1), key transcription factors for osteoclastogenesis. On the other hand, CNP did not inhibit the signaling pathway of NF-κB and mitogen-activated protein kinases (MAPKs) in RANKL-stimulated BMMs. Interestingly, CNP inhibited RANKL-induced CREB activation that can mediate c-Fos and NFATc1. CNP also inhibited RANKL- or LPS-induced CREB, c-Fos and NFATc1 activation in RAW 264.7 cells. We have previously found that CNP directly binds to ADP-ribosylation-like factor-6 interacting protein (ARL6ip), although its role in osteoclastogenesis is not clear. Gene knockdown of ARL6ip by siRNA inhibited RANKL-induced c-Fos expression, suggesting that inactivation of ARL6ip may be involved in an inhibitory effect of CNP. Taken together, CNP was shown to inhibit osteoclast formation possibly via CREB inactivation following a decrease in c-Fos and NFATc1 expression. [ABSTRACT FROM AUTHOR]

Abstract: The effect of pyrroloquinoline quinine (PQQ) on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation was examined using RAW 264.7 macrophage-like cells. RANKL led to the formation of osteoclasts identified as tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in the culture of RAW 264.7 cells. However, PQQ inhibited the appearance of osteoclasts and prevented the decrease of F4/80 macrophage maturation marker on RANKL-stimulated cells, suggesting a preventive action of PQQ on RANKL-induced osteoclast differentiation. PQQ inhibited the activation of nuclear factor of activated T cells (NFATc1), a key transcription factor of osteoclastogenesis, in RANKL-stimulated cells. On the other hand, PQQ did not inhibit the signaling pathway from RANK/RANKL binding to NFATc1 activation, including NF-κB and mitogen-activated protein kinases (MAPKs). PQQ augmented the expression of type I interferon receptor (IFNAR) and enhanced the IFN-β-mediated janus kinase (JAK1) and signal transducer and activator of transcription (STAT1) expression. Moreover, PQQ reduced the expression level of c-Fos leading to the activation of NFATc1. Taken together, PQQ was suggested to prevent RANKL-induced osteoclast formation via the inactivation of NFATc1 by reduced c-Fos expression. The reduced c-Fos expression might be mediated by the enhanced IFN-β signaling due to augmented IFNAR expression. [Copyright &y& Elsevier]

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]

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]

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]

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]

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]

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]

Abstract: Background: Osteoporosis is accompanied by an increase in bone marrow adipose tissue. Bone marrow adipogenesis has emerged as a therapeutic target for prevention of bone loss. Amino-bisphosphonates have been widely used for treatment of osteoporosis, but the mechanism through which amino-bisphosphonates inhibit osteoporosis remains unclear. The purpose of this study is to investigate the effects of bisphosphonates on bone marrow adipogenesis and the pro-osteoclastic factors produced by adipocytes in bone marrow microenvironment. Materials and methods: Human mesenchymal stem cells were obtained and purified from six volunteer donors. Each sample of cells was treated by increasing concentrations of risedronate with or without adipogenic induction for 14 d, and then droplets of the differentiated adipocytes were analyzed. The level of receptor activator of nuclear factor-κB ligand and osteoprotegerin, as well as pro-osteoclastic inflammatory factors interleukin-1, interleukin-6, and tumor necrosis factor α produced by adipocytes were evaluated by Western blot and ELISA assay. Moreover, the effect of risedronate on the activity of mammalian target of rapamycin complex 1, a key Ser/Thr kinase for initiation of adipocyte differentiation, was investigated. Results: Risedronate not only dose-dependently inhibited the bone marrow adipogenesis from human mesenchymal stem cells but also suppressed receptor activator of nuclear factor-κB ligand, not osteoprotegerin, expression in differentiated adipocytes, as well as pro-osteoclastic inflammatory factors. Furthermore, the activity of mammalian target of rapamycin complex 1 was suppressed by risedronate. Conclusion: Our findings that risedronate influences the crosstalk between bone marrow adipocyte–osteoclast represent a novel mechanism for the anti-osteoporotic effects of risedronate. [Copyright &y& Elsevier]