RANKL-RANK Signaling Research Articles

Identification of novel RANKL inhibitors through in silico analysis

Receptor activator of nuclear factor-κB ligand (RANKL) is considered the principal regulator of osteoclast differentiation. Therefore, strategies interfering with the RANKL-RANK signaling pathway may effectively inhibit osteoclast differentiation and mitigate bone resorption. Consequently, RANKL has become a promising target for new drug design strategies. Despite extensive research on specific drugs and antibodies, only a few have shown efficacy in treating osteoporosis. To address this challenge, we aimed to explore new approaches for designing drugs for osteoporosis. In this study, a 3D quantitative structure–activity relationship (QSAR) pharmacophore model was built for RANKL with reference to known inhibitor IC50 values. The optimal pharmacophore model was then employed as a 3D query to screen databases for novel lead compounds. The obtained compounds were subjected to ADMET and TOPKAT analyses to predict drug pharmacokinetics and toxicity. Molecular docking and de novo evolution approaches were applied to verify the docking binding affinities of the compounds. Five candidate compounds were subjected to further in vitro analyses to assess their anti-osteoporotic effects, among which compound 4 demonstrated significant inhibitory activity, achieving an inhibitory rate of 92.6 % on osteoclastogenesis at a concentration of 10 μM. Subsequent molecular dynamics (MD) simulations to assess the stability and behavior of compound 4 and its evolved variant, ZINC00059014397_Evo, within the RANKL binding site revealed that the variant is a potential therapeutic agent for targeting osteoclasts. This study offers valuable insights for developing next generation RANKL inhibitors for osteoporosis treatments.

Toosendanin inhibits osteoclast formation and alleviate postmenopausal osteoporosis by regulating the p38 signaling pathway

Disruption of the balance between osteoclasts and osteoblasts could lead to bone diseases including osteoporosis. It’s well known that RANKL-RANK signaling plays a vital role in activating osteoclasts. Herein, we explored the therapeutic effects of toosendanin (TSN) in osteoporosis, showing that TSN attenuated RANKL-stimulated osteoclastogenesis and osteoclast-specific gene expression in vitro. Bioinformatics predicted that TSN could interfere p38 subunits and regulate the MAPK cascade, and we further verified and demonstrated that TSN significantly inhibited RANKL-induced p38 signaling through western blot. In ovariectomized mouse model, TSN effectively inhibited the formation of TRAP-positive osteoclasts and exhibited protective effect against bone loss. Altogether, these data indicate that TSN targeted p38 activation to inhibit osteoclastogenesis, suggesting the possible therapeutic use of TSN in osteoporosis in the future.

Receptor Activator of Nuclear Factor κB Ligand-Receptor Activator of Nuclear Factor κB Signaling Pathway in Myeloma Bone Disease

Multiple myeloma is an incurable malignant disease characterized by proliferation of clonal plasma cells in the bone marrow.About 90% of the patients with multiple myeloma develop myeloma bone disease(MBD),which seriously affects the quality of life and prognosis of the patients.Traditional therapies for MBD include bisphosphonates,radiotherapy,and surgery.The recent studies have confirmed that the receptor activator of nuclear factor κB ligand (RANKL)-receptor activator of nuclear factor κB(RANK) signaling pathway plays a key role in MBD,providing a new therapeutic target for MBD.This review summarized the role of RANKL-RANK signaling pathway in the pathogenesis of MBD and the advance in the targeted therapy.

Anoctamin 1 controls bone resorption by coupling Cl− channel activation with RANKL-RANK signaling transduction

Osteoclast over-activation leads to bone loss and chloride homeostasis is fundamental importance for osteoclast function. The calcium-activated chloride channel Anoctamin 1 (also known as TMEM16A) is an important chloride channel involved in many physiological processes. However, its role in osteoclast remains unresolved. Here, we identified the existence of Anoctamin 1 in osteoclast and show that its expression positively correlates with osteoclast activity. Osteoclast-specific Anoctamin 1 knockout mice exhibit increased bone mass and decreased bone resorption. Mechanistically, Anoctamin 1 deletion increases intracellular Cl− concentration, decreases H+ secretion and reduces bone resorption. Notably, Anoctamin 1 physically interacts with RANK and this interaction is dependent upon Anoctamin 1 channel activity, jointly promoting RANKL-induced downstream signaling pathways. Anoctamin 1 protein levels are substantially increased in osteoporosis patients and this closely correlates with osteoclast activity. Finally, Anoctamin 1 deletion significantly alleviates ovariectomy induced osteoporosis. These results collectively establish Anoctamin 1 as an essential regulator in osteoclast function and suggest a potential therapeutic target for osteoporosis.

Molecular Mechanisms Leading from Periodontal Disease to Cancer.

Periodontitis is prevalent in half of the adult population and raises critical health concerns as it has been recently associated with an increased risk of cancer. While information about the topic remains somewhat scarce, a deeper understanding of the underlying mechanistic pathways promoting neoplasia in periodontitis patients is of fundamental importance. This manuscript presents the literature as well as a panel of tables and figures on the molecular mechanisms of Porphyromonas gingivalis and Fusobacterium nucleatum, two main oral pathogens in periodontitis pathology, involved in instigating tumorigenesis. We also present evidence for potential links between the RANKL–RANK signaling axis as well as circulating cytokines/leukocytes and carcinogenesis. Due to the nonconclusive data associating periodontitis and cancer reported in the case and cohort studies, we examine clinical trials relevant to the topic and summarize their outcome.

Receptor Activator of Nuclear Factor (Nf)-kb Ligand Promotes T Helper 17 Cell Differentiation through Fas

ABSTRACT T helper 17 (Th17) cells play important role in the defense against pathogens and autoimmune diseases. Many cytokines can induce Th17 cell differentiation. However, the mechanism of Th17 cell differentiation is not well clarified. RankL, a member of the TNF superfamily, binds with Rank and then participates in the proliferation and differentiation of many kinds of cells. Recent studies showed that RankL-Rank signaling is closely related to Th17 differentiation and function. The detail of the Rank-RankL pathway in Th17 cell differentiation is still unclear. To illustrate the role of Rank-RankL in Th17 differentiation, naive CD4 + T cells were differentiated into Th17 cells with or without RankL stimulation. During Th17 differentiation, the expression of Rank obviously increased. The RankL stimulation significantly increased Th17 cell differentiation indicated by increased IL-17-positive cell number, highly expressed IL-17 and IL-22 and elevated IL-17 secretion. These effects were canceled by Rank-Fc addition. In further study, RankL treatment during Th17 differentiation up-regulated Fas expression. Fas knockdown inhibited the Th17 differentiation promoted by RankL. In this study, it was confirmed that Rank-RankL signaling could promote Th17 cell differentiation through Fas induction.

Calcium phosphate-based materials regulate osteoclast-mediated osseointegration

Calcium phosphate-based materials (CaP) have been widely used as bone graft substitutes with a decent osseointegration. However, the mechanism whereby cells function and repair the bone defect in CaP micro-environment is still elusive. The aim of this study is to find the mechanism how osteoclast behaviors mediate bone healing with CaP scaffolds. Recent reports show that behaviors of osteoclast are closely related with osteogenesis, thus we make a hypothesis that active osteoclast behaviors induced by CaP facilitate bone healing. Here, we found a new mechanism that CaP can regulate osteoclast-mediated osseointegration. Calcium phosphate cement (CPC) is selected as a representative CaP. We demonstrate that the osteoclast-mediated osseointegration can be strongly modulated by the stimulation with CaP. An appropriate Ca/P ratio in CaP can effectively promote the RANKL-RANK binding and evoke more activated NF-κB signaling transduction, which results in vigorous osteoclast differentiation. We observe significant improvement of bone healing in vivo, owing to the active coupling effect of osteoclasts. What is more noteworthy is that the phosphate ions released from CaP can be a pivotal role regulating osteoclast activity by changing Ca/P ratio readily in materials. These studies suggest the potential of harnessing osteoclast-mediated osteogenesis in order to develop a materials-manipulated approach for improving osseointegration.

RANKLed by the Complexity of Signaling in Breast Cancer Metastasis to the Brain

BackgroundReceptor activator of nuclear factor κB (RANK) and its ligand, RANKL, are essential for mammary gland development and play a vital role in breast carcinogenesis. RANKL-RANK signaling also drives thermoregulation and modulates inflammatory activation in the brain. The expression of RANKL in primary breast cancer (BC) has been negatively associated with brain metastases, while significantly higher levels of RANK are seen in BC with brain metastases. We examined the expression of RANK and RANKL in BC metastasis to the brain. Patients and MethodsWe examined the expression of RANK and RANKL in 40 cases of BC metastasis to the brain. ResultsRANK was variably expressed in BC cells but minimally expressed in the adjacent brain parenchyma. In contrast, the expression of RANKL was minimal in metastatic BC but highly variable in tumoral stroma. RANKL expression in normal brain stroma obtained during autopsy was negligible. Histologic grade and BC subtypes were not significantly associated with RANK expression in metastatic BC. A significant negative correlation between RANK in metastatic BC and RANKL in tumoral stroma was identified (P < .001). ConclusionRANK expressed by primary BC and RANKL detected in the tumor microenvironment together participate in cancer development, while the same principle may operate at distant sites. Further investigation is necessary to provide additional insight into the role of the RANKL-RANK pathway in BC progression and to investigate the potential efficacy of therapeutic strategies targeting these molecules in BC metastasis to the brain.

Receptor activator of the NFκB ligand system protects renal function during experimental renal ischemia-reperfusion in mice

Renal ischemia-reperfusion (I/R) injury is closely associated with delayed graft function and poor long-term graft survival following transplantation. Various pathophysiologies can cause the deterioration of renal function; however, the immune system plays important roles in promoting and protecting renal tissues. Receptor activator of NFκB ligand (RANKL) is a member of the TNF superfamily and is produced by bone-forming osteoblasts; the receptor for RANKL is called RANK. In bone biology, RANKL-RANK signaling has been extensively studied, but its roles in the immune system are still obscure. We investigated the role of the RANK system in I/R injury of the kidney using an experimental mouse I/R model. The left renal pedicles of 10-week-old male mice were clamped for 60 min, and reperfusion and right nephrectomy were simultaneously performed. Separate groups were administered an anti-RANKL antibody and recombinant RANKL (rRANKL) 24 h prior to I/R. After reperfusion for a set period of time, the serum creatinine level was measured, and the left kidney was removed for histological examination and western blotting to evaluate the expression and localization of RANK-associated molecules and cytokines. The serum creatinine levels were significantly elevated after I/R injury. A time-dependent increase in RANKL was observed up to 24 h, whereas RANK was induced for 12 h after reperfusion. RANK was expressed in infiltrating inflammatory cells, which were positive for CD68, a marker of monocytes/macrophages. The pre-treatment with the anti-RANKL antibody significantly impaired renal function and increased the induction of inflammatory cytokines (TNFα and IL-6), Toll-like receptor (TLR4) and MyD88 (all p < .05) compared to the levels in the I/R group. However, rRANKL significantly improved renal function and decreased the levels of inflammatory cytokines (TNFα and IL-6), TLR4 and MyD88 (p < .05) compared to those in the I/R group. The present study is the first to evaluate the role of the RANK system in renal I/R injury. RANKL-RANK signaling affects macrophage function and results in the downregulation of TLR4 and reduction in TNFα and IL-6, leading to improved renal function following I/R injury.

Airway M Cells Arise in the Lower Airway Due to RANKL Signaling and Reside in the Bronchiolar Epithelium Associated With iBALT in Murine Models of Respiratory Disease.

Microfold (M) cells residing in the follicle-associated epithelium of mucosa-associated lymphoid tissues are specialized for sampling luminal antigens to initiate mucosal immune responses. In the past decade, glycoprotein 2 (GP2) and Tnfaip2 were identified as reliable markers for M cells in the Peyer's patches of the intestine. Furthermore, RANKL–RANK signaling, as well as the canonical and non-canonical NFκB pathways downstream, is essential for M-cell differentiation from the intestinal stem cells. However, the molecular characterization and differentiation mechanisms of M cells in the lower respiratory tract, where organized lymphoid tissues exist rarely, remain to be fully elucidated. Therefore, this study aimed to explore M cells in the lower respiratory tract in terms of their specific molecular markers, differentiation mechanism, and functions. Immunofluorescence analysis revealed a small number of M cells expressing GP2, Tnfaip2, and RANK is present in the lower respiratory tract of healthy mice. The intraperitoneal administration of RANKL in mice effectively induced M cells, which have a high capacity to take up luminal substrates, in the lower respiratory epithelium. The airway M cells associated with lymphoid follicles were frequently detected in the pathologically induced bronchus-associated lymphoid tissue (iBALT) in the murine models of autoimmune disease as well as pulmonary emphysema. These findings demonstrate that RANKL is a common inducer of M cells in the airway and digestive tracts and that M cells are associated with the respiratory disease. We also established a two-dimensional culture method for airway M cells from the tracheal epithelium in the presence of RANKL successfully. This model may be useful for functional studies of M cells in the sampling of antigens at airway mucosal surfaces.

The Erk‐Mediated Phosphorylation of Non‐Muscle Caldesmon Alters Osteoclastogenesis in RANKL‐Induced Raw264.7 Cells

The RANKL-RANK signaling pathway is the key event for osteoclastogenesis accompanied by the podosome formation for cell adhesion or extracellular matrix remodeling. Recently, we showed that non-muscle caldesmon (l-CaD) is a key constituent of podosome in RANKL-induced osteoclasts (OCs). Using different decoy peptides mimicking the Pak- or Erk-mediated phosphorylation at the C-terminal end of l-CaD along with full-length mutants of l-CaD also suggested that the l-CaD phosphorylation may involve in the control of osteoclastogenesis. In this study, we used the Erk activator (i.e. t-BHQ) and inhibitors (i.e. PD98059 and PD0325901) to examine the altering effects of the Erk signaling pathway leading to changes of l-CaD phosphorylation on the RANKL-induced TRAP activity and cell-cell fusion in osteoclastogenesis. Our study showed that administrations of t-BHQ concentrations of 1, 10, 25, and 50 microM for 30 min or 24 hr before RANKL induction caused increases of l-CaD phosphorylation by 1.13, 1.14, 1.82, and 1.88 folds, respectively, in concomitant with the increased number of small size of differentiated OCs by 2-, 3-, 4-, and 4-folds, respectively, but the decreased fusion index by 0.66, 0.5, 0.3, and 0.3 in a dose dependent manner. However, complete inhibition of Erk phosphorylation with the concomitant 50~60% decrease of l-CaD phosphorylation by treatment with 10 microM PD0325901 for 30 min or 24 hr before RANKL induction completely abolished the OC differentiation. Results obtained here suggest that the Erk-activated l-CaD phosphorylation is required for RANKL-induced OC differentiation, however the dynamic control of l-CaD unphosphorylation/phosphorylation is important for the control of cell-cell fusion in osteoclastogenesis. Support or Funding Information This study is supported by Ministry of Science and Technology of Taiwan Government ( MOST-106-2320-B-005-003-MY2) RANKL-induced formation of actin ring around the cell periphery in differentiated OCs stained with total l-CaD/F-actin (top panel, A), phosphorylated p-l-CaD/F-actin (bottom, A), and vinculin/F-actin (B), and resolved by confocal immunofluorescence imaging. Actin ring structure is composed of the core (labeled with l-CaD, presumbably unphosphorylated) and the peripheral rim (labeled with vinculin, as well as phosphorylated p-l-CaD). Treatments with t-BHQ concentrations of 1, 10, 25, and 50 microM for 24 hr increased l-CaD phosphorylation. Treatments with t-BHQ concentrations of 1, 10, 25, and 50 microM for 24 hr before RANKL induction increased the number of small size of differentiated OCs, but decreased the fusion index in a dose dependent manner. Erk activator (i.e. t-BHQ) and inhibitors (i.e. PD98059 and PD0325901) were used for 30 min and 24 hr to examine alterations of Erk and l-CaD phosphorylation. Erk activator (i.e. t-BHQ) and inhibitors (i.e. PD98059 and PD0325901) were used for 30 min and 24 hr to examine the altering effects on RANKL-induced TRAP activity and cell-cell fusion in osteoclastogenesis. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RANKL-RANK signaling regulates osteoblast differentiation and bone formation

RANKL-RANK signaling regulates osteoblast differentiation and bone formation

The Intrinsic and Extrinsic Implications of RANKL/RANK Signaling in Osteosarcoma: From Tumor Initiation to Lung Metastases.

Background: Osteosarcoma is the most frequent form of malignant pediatric bone tumor. Despite the current therapeutic arsenal, patient life-expectancy remains low if metastases are detected at the time of diagnosis, justifying research into better knowledge at all stages of osteosarcoma ontogenesis and identification of new therapeutic targets. Receptor Activator of Nuclear factor κB (RANK)expression has been reported in osteosarcoma cells, raising the question of Receptor Activator of Nuclear factor κB Ligand (RANKL)/RANK signaling implications in these tumor cells (intrinsic), in addition to previously reported implications through osteoclast activation in the tumor microenvironment (extrinsic). Methods: Based on in vitro and in vivo experimentations using human and mouse osteosarcoma cell lines, the consequences on the main cellular processes of RANK expression in osteosarcoma cells were analyzed. Results: The results revealed that RANK expression had no impact on cell proliferation and tumor growth, but stimulated cellular differentiation and, in an immune-compromised environment, increased the number of lung metastases. The analysis of RANKL, RANK and osteoprotegerin (OPG) expressions in biopsies of a cohort of patients revealed that while RANK expression in osteosarcoma cells was not significantly different between patients with or without metastases at the time of diagnosis, the OPG/RANK ratio decreased significantly. Conclusion: Altogether, these results are in favor of RANKL-RANK signaling inhibition as an adjuvant for the treatment of osteosarcoma.

Anti-IFN-γ Antibody Promotes Osteoclastogenesis in Human Bone Marrow Monocyte-Derived Macrophages Co-Cultured with Tuberculosis-Activated Th1 Cells

Background/Aims: Tuberculosis induces bone loss and activates Th1 cells that play an important role in the host defense of Bacille Calmette-Guérin tuberculosis vaccine. However, the role of tuberculosis-activated Th1 cells in differentiation of osteoclast precursors to osteoclasts is unclear. As secretion of IFN-γ in Th1 cells is induced by tuberculosis, we aimed to investigate the role of anti-IFN-γ antibody on the differentiation and activation of osteoclasts in bone marrow monocyte-derived macrophages (BMMs). Methods: BMMs were isolated and co-cultured with CD4+T helper 1 cells (Th1 cells), pretreated with anti-IFN-γ antibody. Then, cell proliferation, expression and release of cytokines, formation of actin ring, differentiation of osteoclasts and bone resorption function were measured by CCK8 assay, qRT-PCR/Western blot/flow cytometry, ELISA, immunofluorescence, tartrate-resistant acidic phosphatase (TRAP) staining and bone absorbance assay, respectively. Results: Anti-IFN-γ antibody inhibited the cell viability of BMMs, and induced the expressions of RANKL, TNF-α, NF-κB and TRAF6 in BMMs. In addition, it led to increased expression levels of RANK on cell surfaces, and increased production of RANKL, TNF-α, MCP-1 and SDF-1. Anti-IFN-γ antibody also induced the expression of osteoclast differentiation factor and actin ring formation, but inhibited the expression of osteoprotegerin. TRAP staining and bone resorption assays showed that anti-IFN-γ antibody induced an increase in osteoclast formation and bone resorption. Conclusion: The anti-IFN-γ antibody induced osteoclast formation, and is probably mediated by RANKL-induced activation of NF-κB, that induces TRAF6 in the RANKL-RANK signaling pathway. Our data suggest an inhibitory role for IFN-γ in osteoclast formation induced by tuberculosis.

RANK Signaling Pathways and Key Molecules Inducing Osteoclast Differentiation

Mononuclear osteoclast precursors derived from hematopoietic progenitors fuse together and then become multinucleated mature osteoclasts by macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). Especially, the binding of RANKL to its receptor RANK provides key signals for osteoclast differentiation and bone-resorbing function. RANK transduces intracellular signals by recruiting adaptor molecules such as TNFR-associated factors (TRAFs), which then activate mitogen activated protein kinases (MAPKs), Src/PI3K/Akt pathway, nuclear factor-κB (NF-κB) and finally amplify NFATc1 activation for the transcription and activation of osteoclast marker genes. This review will briefly describe RANKL-RANK signaling pathways and key molecules critical for osteoclast differentiation.

Heterologous expression, purification and function of the extracellular domain of human RANK

BackgroundReceptor activator of NF-κB ligand (RANKL)/RANK signaling essentially functions within the skeletal system, particularly participating in osteoclastogenesis and bone resorption. In addition, this signaling pathway has also been shown to influence tumor progression as well as the development and function of the immune system. Therefore, blocking the interaction between RANKL and RANK is a new therapeutic approach to prevent bone-related diseases and cancer.ResultsThe coding sequence encoding the extracellular domain of human RANK (RANK-N) was codon optimized for Pichia pastoris and cloned into the pPIC9K vector, and the recombinant plasmid was then transformed into P. pastoris. The expression of RANK-N protein was confirmed using SDS-PAGE with Coomassie Brilliant Blue stain and western blotting. Recombinant RANK-N protein was purified by a multistep process including ultrafiltration (UF), Sephadex G-50 size-exclusion chromatography and Q-Sepharose Fast Flow ion exchange chromatography, which resulted in a purity >95%. We found that the RANK-N protein can block RANKL-RANK signaling both in vitro and in vivo. Furthermore, using a patient-derived xenograft of human colon cancer, we found that the recombinant RANK-N protein can inhibit the growth of colorectal cancer.ConclusionsThe results show that a simple system to express and purify functional RANK-N protein has been developed. This work has thus laid a foundation for further research and clinical applications of RANK-N protein in treating bone-related diseases or even colorectal cancer.

Moderate-intensity 4 mT static magnetic fields prevent bone architectural deterioration and strength reduction by stimulating bone formation in streptozotocin-treated diabetic rats

Type 1 diabetes mellitus (T1DM) has been associated with deterioration of bone microarchitecture and strength, resulting in increased fracture risk. Substantial studies have revealed the capacity of moderate-intensity static magnetic fields (SMF) on promoting osteoblastogenesis in vitro and stimulating bone growth and bone regeneration in vivo, whereas it is unknown whether SMF can resist T1DM-associated osteopenia/osteoporosis. We herein investigated the potential effects of whole-body SMF exposure with 4mT on bone loss in streptozotocin-induced T1DM rats. We found that SMF exposure for 16weeks inhibited architectural deterioration of trabecular bone and cortical bone and mechanical strength reduction in T1DM rats, as evidenced by the MicroCT and 3-point bending findings. Our serum biochemical, bone histomorphometric and PCR results revealed that SMF induced higher serum osteocalcin, mineral apposition rate and osteoblast number of trabecular bone, and higher skeletal osteocalcin, BMP2 and Runx2 gene expression in T1DM rats, whereas SMF showed no significant alteration in serum CTX, skeletal osteoclast number, or osteoclastogenesis-related RANKL-RANK signaling gene expression. Together, our findings suggest that moderate SMF prevented bone architectural deterioration and strength reduction by inhibiting the reduction of bone formation in T1DM rats, and indicate that SMF might become a promising biophysical countermeasure for T1DM-related osteopenia/osteoporosis.

Effects of low-intensity pulsed electromagnetic fields on bone microarchitecture, mechanical strength and bone turnover in type 2 diabetic db/db mice

Type 2 diabetic patients have impaired bone quality, leading to increased fracture risk. Substantial evidence demonstrates that pulsed electromagnetic fields (PEMF) could resist osteopenia/osteoporosis induced by estrogen deficiency and disuse. However, the effects of PEMF on osteopenia/osteoporosis associated with diabetes, especially for more prevalent type 2 diabetes, remain poorly understood. We herein investigated the skeletal effects and mechanisms of PEMF (15 Hz, 20 Gs) on leptin receptor-deficient db/db mice with typical type 2 diabetic symptoms. Our µCT results showed that 12-week PEMF exposure significantly improved both cancellous and cortical bone microarchitecture in db/db mice. Three-point bending and biomechanical indentation testing demonstrated that PEMF improved whole-bone structural properties and tissue-level material properties in db/db mice. PEMF significantly promoted bone formation in db/db mice evidenced by increased serum osteocalcin and bone mineral apposition rate, whereas PEMF exerted no observable alteration in bone resorption. Real-time PCR showed that PEMF upregulated tibial gene expression of osteoblastogenesis-related of canonical Wnt/β-catenin signaling but not osteoclastogenesis-related RANKL-RANK signaling in db/db mice. Our findings demonstrate that PEMF improved bone quantity and quality with obvious anabolic activities in db/db mice, and imply that PEMF might become a clinically applicable treatment modality for improving bone quality in type 2 diabetic patients.

Melittin inhibits osteoclast formation through the downregulation of the RANKL-RANK signaling pathway and the inhibition of interleukin-1β in murine macrophages.

Melittin is a major toxic component of bee venom (Apis mellifera). It is not known whether melittin is involved in bone metabolism and osteoclastogenesis. The aim of this study was to determine the role of melittin in the regulation of osteoclastogenesis. In vitro osteoclastogenesis assays were performed using mouse RAW 264.7 cells and bone marrow-derived macrophages (BMMs) treated with receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Morphologic and functional analyses for osteoclast-like multinucleated cells (MNCs) were performed by tartrate-resistant acid phosphatase (TRAP) staining, F-actin staining and pit formation methods. The gene expression of TRAP, cathepsin K, matrix metalloproteinase-9 (MMP-9) and carbonic anhydrase II was measured by reverse transcription-quantitative PCR. The protein expression levels of mitogen-activated protein kinases (MAPKs), the p65 subunit of nuclear factor-κB (NF-κB), c-Fos, c-Jun, nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), TNF receptor-associated factor-6 (TRAF6), and interleukin-1β (IL-1β) were assessed by western blot analysis. Melittin inhibited the mRNA expression of TRAP, cathepsin K, MMP-9 and carbonic anhydrase II in RANKL-stimulated RAW 264.7 cells. The increased protein expression of TRAF6, p-extracellular signal-regulated kinase (ERK), p-JNK, p-p65, p-c-Fos and NFATc1 induced by RANKL was significantly suppressed in the RAW 264.7 cells treated with melittin. A synergistic effect of IL-1β on the formation of RANKL-induced osteoclast-like MNCs was found in two experimental cells. The increased expression of IL-1β following the stimulation of RAW 264.7 cells with RANKL activated TRAF6, p-ERK, p-JNK, p-p65, p-c-Fos and NFATc1. These effects were attenuated by the downregulation of IL-1β using siRNA against IL-1β, and also by treatment with melittin. On the whole, the findings of this study demonstrate that melittin inhibits the formation of osteoclast-like MNCs by interfering with the RANKL-RANK signaling pathway.

Current Understanding of RANK Signaling in Osteoclast Differentiation and Maturation.

Osteoclasts are bone-resorbing cells that are derived from hematopoietic precursor cells and require macrophage-colony stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL) for their survival, proliferation, differentiation, and activation. The binding of RANKL to its receptor RANK triggers osteoclast precursors to differentiate into osteoclasts. This process depends on RANKL-RANK signaling, which is temporally regulated by various adaptor proteins and kinases. Here we summarize the current understanding of the mechanisms that regulate RANK signaling during osteoclastogenesis. In the early stage, RANK signaling is mediated by recruiting adaptor molecules such as tumor necrosis factor receptor-associated factor 6 (TRAF6), which leads to the activation of mitogen-activated protein kinases (MAPKs), and the transcription factors nuclear factor-κB (NF-κB) and activator protein-1 (AP-1). Activated NF-κB induces the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), which is the key osteoclastogenesis regulator. In the intermediate stage of signaling, the co-stimulatory signal induces Ca2+ oscillation via activated phospholipase Cγ2 (PLCγ2) together with c-Fos/AP-1, wherein Ca2+ signaling facilitates the robust production of NFATc1. In the late stage of osteoclastogenesis, NFATc1 translocates into the nucleus where it induces numerous osteoclast-specific target genes that are responsible for cell fusion and function.