Mechanical Stress Induces Sodium Entry and Osmoprotective Responses in Murine Synovial Fibroblasts.

Osteoarthritis (OA) is a multifactorial disease depending on molecular, genetic, and environmental factors like mechanical strain. Next to the cartilage and the subchondral bone, OA also affects the synovium, which is critically involved in the maintenance of joint homeostasis. As there is a correlation between the extracellular sodium content in the knee joint and OA, this study investigates the impact of sodium on OA-associated processes like inflammation and bone remodeling without and with mechanical loading in synovial fibroblasts. For that purpose, murine synovial fibroblasts from the knee joint were exposed to three different extracellular sodium chloride concentrations (-20 mM, ±0 mM and +50 mM NaCl) in the absence or presence of compressive or intermittent tensile strain. In addition to the intracellular Na+ content and gene expression of the osmoprotective transcription factor nuclear factor of activated T cells 5 (Nfat5), the gene and protein expression of inflammatory mediators (interleukin-6 (IL6), prostaglandin endoperoxide synthase-2 (Ptgs2)/prostaglandin E2 (PGE2)), and factors involved in bone metabolism (receptor activator of NF-κB ligand (RANKL), osteoprotegerin (OPG)) were analyzed by qPCR and ELISA. Mechanical strain already increased intracellular Na+ and Nfat5 gene expression at standard salt conditions to levels obtained by exposure to increased extracellular Na+ content. Both high salt and compressive strain resulted in elevated IL6 and PGE2 release. Intermittent tensile strain did not increase Il6 mRNA expression or IL6 protein secretion but triggered Ptgs2 expression and PGE2 production. Increased extracellular Na+ levels and compressive strain increased RANKL expression. In contrast, intermittent tension suppressed RANKL expression without this response being subject to modification by extracellular sodium availability. OPG expression was only induced by compressive strain. Changes in extracellular Na+ levels modified the inflammatory response and altered the expression of mediators involved in bone metabolism in cells exposed to mechanical strain. These findings indicate that Na+ balance and Nfat5 are important players in synovial fibroblast responses to mechanical stress. The integration of Na+ and Na+-dependent signaling will help to improve the understanding of the pathogenesis of osteoarthritis and could lead to the establishment of new therapeutic targets.

RANK/RANKL axis promotes migration, invasion, and metastasis of osteosarcoma via activating NF-κB pathway.

Osteosarcoma (OS) is one of the most prevalent primary bone tumors with a high degree of metastasis and poor prognosis. Epithelial-to-mesenchymal transition (EMT) is a cellular mechanism that contributes to the invasion and metastasis of cancer cells, and OS cells have been reported to exhibit EMT-like characteristics. Our previous studies have shown that the interaction between tumor necrosis factor superfamily member 11 (TNFRSF11A; also known as RANK) and its ligand TNFSF11 (also known as RANKL) promotes the EMT process in breast cancer cells. However, whether the interaction between RANK and RANKL enhances aggressive behavior by inducing EMT in OS cells has not yet been elucidated. In this study, we showed that the interaction between RANK and RANKL increased the migration, invasion, and metastasis of OS cells by promoting EMT. Importantly, we clarified that the RANK/RANKL axis induces EMT by activating the nuclear factor-kappa B (NF-κB) pathway. Furthermore, the NF-κB inhibitor dimethyl fumarate (DMF) suppressed migration, invasion, and EMT in OS cells. Our results suggest that the RANK/RANKL axis may serve as a potential tumor marker and promising therapeutic target for OS metastasis. Furthermore, DMF may have clinical applications in the treatment of lung metastasis in patients with OS.

Icariin regulates RANKL-induced osteoclast differentiation via the ERα/c-Src/RANK signaling.

Osteoporosis (OP) is a common metabolic bone disease. Excessive osteoclastic activity significantly contributes to the development of OP. Icariin (ICA) is a flavonol glycoside derived from herbal plants and possesses curative effects on postmenopausal OP and bone fracture. This study aimed to investigate the effects of ICA on osteoclast differentiation induced by receptor activator of nuclear factor kappa B (RANK) ligand (RANKL) and the involvement of estrogen receptorα(ERα) and RANK signaling cascade in this process. RANKL was used to induce the differentiation of RAW264.7 cells to into osteoclasts. Small interfering RNA technique was used to knockdown ERαin cells. Cell counting kit-8 assay was performed to determine the cytotoxicity of ICA. The number of tartrate-resistant acid phosphatase (TRAP)-positive cells was quantified by TRAP staining. RANKL induced the differentiation of RAW264.7 cells into osteoclasts, while ICA abolished the pro-osteoporotic effect of RANKL. Moreover, ERαknockdown abolished the effects of ICA on RANKL-induced osteoclastogenesis. Further exploration revealed that ICA inhibited the phosphorylation ofc-Src in osteoclasts via regulating ERα, while inactivation ofc-Src reversed ERαknockdown-promoted osteoclastogenesis. Lastly, ICA inhibited the activation of the mitogen-activated protein kinase signaling pathway and downregulated the expressions of target osteoclastogenic proteins in RANKL-treated RAW 264.7 cells, while ERαknockdown almost completely diminished the effects of ICA. ICA inhibited RANKL-induced osteoclast differentiation via regulating the ERα/c-Src/RANK signaling. These findings elucidated a novel mechanism by which ICA exerts an anti-osteoporotic effect.

Iguratimod suppresses sclerostin and receptor activator of NF-κB ligand production via the extracellular signal-regulated kinase/early growth response protein 1/tumor necrosis factor alpha pathway in osteocytes and ameliorates disuse osteoporosis in mice.

Disuse osteoporosis is a prevalent complication among patients afflicted with rheumatoid arthritis (RA). Although reports have shown that the antirheumatic drug iguratimod (IGU) ameliorates osteoporosis in RA patients, details regarding its effects on osteocytes remain unclear. The current study examined the effects of IGU on osteocytes using a mouse model of disuse-induced osteoporosis, the pathology of which crucially involves osteocytes. A reduction in distal femur bone mass was achieved after 3 weeks of hindlimb unloading in mice, which was subsequently reversed by intraperitoneal IGU treatment (30 mg/kg; five times per week). Histology revealed that hindlimb-unloaded (HLU) mice had significantly increased osteoclast number and sclerostin-positive osteocyte rates, which were suppressed by IGU treatment. Moreover, HLU mice exhibited a significant decrease in osteocalcin-positive cells, which was attenuated by IGU treatment. In vitro, IGU suppressed the gene expression of receptor activator of NF-κB ligand (RANKL) and sclerostin in MLO-Y4 and Saos-2 cells, which inhibited osteoclast differentiation of mouse bone marrow cells in cocultures. Although IGU did not affect the nuclear translocation or transcriptional activity of NF-κB, RNA sequencing revealed that IGU downregulated the expression of early growth response protein 1 (EGR1) in osteocytes. HLU mice showed significantly increased EGR1- and tumor necrosis factor alpha (TNFα)-positive osteocyte rates, which were decreased by IGU treatment. EGR1 overexpression enhanced the gene expression of TNFα, RANKL, and sclerostin in osteocytes, which was suppressed by IGU. Contrarily, small interfering RNA-mediated suppression of EGR1 downregulated RANKL and sclerostin gene expression. These findings indicate that IGU inhibits the expression of EGR1, which may downregulate TNFα and consequently RANKL and sclerostin in osteocytes. These mechanisms suggest that IGU could potentially be used as a treatment option for disuse osteoporosis by targeting osteocytes.

Immunomodulatory Effects of RANK/RANKL Blockade in Patients with Cancer.

In cancer, multiple factors converge upon receptor activator of nuclear factor κB (RANK) and its ligand (RANKL) signaling to promote the development of bone metastases; agents that inhibit RANKL signaling reduce skeletal-related events (SRE) in patients with cancer. In addition, RANKL signaling is important in augmenting the ability of dendritic cells (DC) to stimulate both naïve T-cell proliferation and the survival of RANK+ T cells. In this issue, Chang and colleagues using high-dimensional cytometry to evaluate immunomodulatory effects of denosumab in patients with advanced solid, observe early on treatment changes in multiple compartments, and greater effects in patients receiving concurrent chemotherapy or steroids. See related article by Chang et al., p. 453 (4).

MCP-5 suppresses osteoclast differentiation through Ccr5 upregulation.

Human monocyte chemoattractant protein-1 (MCP-1) in mice has two orthologs, MCP-1 and MCP-5. MCP-1, which is highly expressed in osteoclasts rather than in osteoclast precursor cells, is an important factor in osteoclast differentiation. However, the roles of MCP-5 in osteoclasts are completely unknown. In this study, contrary to MCP-1, MCP-5 was downregulated during receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclast differentiation and was considered an inhibitory factor in osteoclast differentiation. The inhibitory role of MCP-5 in osteoclast differentiation was closely related to the increase in Ccr5 expression and the inhibition of IκB degradation by RANKL. Transgenic mice expressing MCP-5 controlled by Mx-1 promoter exhibited an increased bone mass because of a decrease in osteoclasts. This result strongly supported that MCP-5 negatively regulated osteoclast differentiation. MCP-5 also prevented severe bone loss caused by RANKL.

Assessment of osteoprotegerin and RANKL levels and several cardiovascular risk scoring systems in acromegaly.

PURPOSE: The OPG/RANKL (osteoprotegerin/receptor activator of nuclear factor kappa-B) system, which plays a crucial role in bone metabolism, is also associated with vascular calcification. Acromegaly is characterized by excessive secretion of growth hormone and insulin-like growth factor, and studies have demonstrated an elevated risk of cardiovascular disease in individuals with acromegaly. In this study, our objective was to investigate the relationship between OPG/RANKL and various cardiovascular risk scoring systems. METHODS: We recruited 44 consecutive acromegaly patients and 41 healthy controls with a similar age and gender distribution for this study. RESULTS: While RANKL levels were significantly higher in the acromegaly group compared to the controls, OPG levels were not found to be significantly different between the two groups. Furthermore, within the acromegaly group, RANKL levels were significantly higher in patients with active acromegaly compared to those with controlled acromegaly. Osteoprotegerin levels showed a positive correlation with the Framingham risk score (FRS) in the acromegaly group. Linear regression analysis revealed an association of OPG with FRS (adjusted R2 value of 21.7%). CONCLUSION: OPG and RANKL may serve as potential markers for assessment of cardiovascular calcification and prediction of the cardiovascular risk status in acromegalic patients.

NOTCH2 promotes osteoclast maturation and metabolism and modulates the transcriptome profile during osteoclastogenesis.

Notch signaling plays a key regulatory role in bone remodeling and NOTCH2 enhances osteoclastogenesis, an effect that is mostly mediated by its target gene Hes1. In the present study, we explored mechanisms responsible for the enhanced osteoclastogenesis in bone marrow-derived macrophages (BMM) from Notch2tm1.1Ecan, harboring a NOTCH2 gain-of-function mutation, and control mice. Notch2tm1.1Ecan mice are osteopenic and have enhanced osteoclastogenesis. Bulk RNA-Seq and gene set enrichment analysis of Notch2tm1.1Ecan BMMs cultured in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand revealed enrichment of genes associated with enhanced cell metabolism, aerobic respiration, and mitochondrial function, all associated with osteoclastogenesis. These pathways were not enhanced in the context of a Hes1 inactivation. Analysis of single cell RNA-Seq data of pooled control and Notch2tm1.1Ecan BMMs treated with M-CSF or M-CSF and receptor activator of NF-κB ligand for 3 days identified 11 well-defined cellular clusters. Pseudotime trajectory analysis indicated a trajectory of clusters expressing genes associated with osteoclast progenitors, osteoclast precursors, and mature cells. There were an increased number of cells expressing gene markers associated with the osteoclast and with an unknown, albeit related, cluster in Notch2tm1.1Ecan than in control BMMs as well as enhanced expression of genes associated with osteoclast progenitors and precursors in Notch2tm1.1Ecan cells. In conclusion, BMM cultures display cellular heterogeneity, and NOTCH2 enhances osteoclastogenesis, increases mitochondrial and metabolic activity of osteoclasts, and affects cell cluster allocation in BMMs.

TIFA contributes to periodontitis in diabetic mice via activating the NF­κB signaling pathway.

Diabetic periodontitis (DP) refers to destruction of periodontal tissue and absorption of bone tissue in diabetic patients. Tumor necrosis factor receptor­associated factor (TRAF)­interacting protein with forkhead­associated domain (TIFA) as a crucial regulator of inflammation activates the NF­κB signaling pathway to regulate cell biological behavior. However, the function and mechanism of TIFA on DP suffer from a lack of research. In the present study, TIFA was upregulated in the periodontal tissue of a DP mouse model. In addition, the expression of TIFA in RAW264.7 cells was induced by high glucose (HG) culture and increased by lipopolysaccharide (LPS) from Porphyromonas gingivalis treatment in a time­dependent manner. Knockdown of TIFA significantly reduced the levels of inflammatory cytokines, including TNF­α, IL­6, IL­1ß and monocyte chemoattractant protein­1, in HG and LPS­induced RAW264.7 cells. The nuclear translocation of NF­κB p65 was induced by HG and LPS and was clearly suppressed by absence of TIFA. The expression of downstream factors Nod­like receptor family pyrin domain­containing 3 and apoptosis­associated speck­like protein was inhibited by silencing TIFA. Moreover, TIFA was increased by receptor activator of NF­κB (RANK) ligand (RANKL) in a concentration dependent manner. The expression of cathepsin K, MMP9 and nuclear factor of activated T cells cytoplasmic 1 was downregulated by depletion of TIFA. RANKL­induced osteoclast differentiation was inhibited by silencing of TIFA. Meanwhile, the decrease of TIFA blocked activation of the NF­κB pathway in RANKL­treated RAW264.7 cells. In conclusion, TIFA as a promoter regulates the inflammation and osteoclast differentiation via activating the NF­κB signaling pathway.

Expression and functional characterization of bovine receptor activator of NF-κB ligand (RANKL).

Receptor activator of nuclear factor Kappa-B Ligand (RANKL) is a member of the tumor necrosis factor ligand (TNF) family involved in immune responses and immunomodulation. Expressed in various cells types around the body, RANKL plays a crucial role in bone remodeling and development of the thymus, lymph nodes and mammary glands. Research in other species demonstrates that RANKL is required for the development of microfold cells (M cells) in the gut, however limited information specific to cattle is available. Cloning and expression of bovine RANKL (BoRANKL) was carried out and bioactivity of the protein was demonstrated in the induction of osteoclast differentiation from both bovine and ovine bone marrow cells. The effects of BoRANKL on particle uptake in bovine enteroids was also assessed. The production of cross-reactive bovine RANKL protein will enable further investigations into cell differentiation using the available ruminant organoid systems, and their role in investigating host-pathogen interactions in cattle and sheep.

Vascular Pericyte-Derived Exosomes Inhibit Bone Resorption via Traf3.

Purpose: Blood vessels distribute cells, oxygen, and nutrients throughout the body to support tissue growth and balance. Pericytes and endothelial cells form the inner wall of blood vessels, crucial for organ development and tissue homeostasis by producing paracrine signaling molecules. In the skeletal system, pericyte-derived vascular factors along with angiogenic factors released by bone cells regulate angiogenesis and bone formation. Although the involvement of angiogenic factors and skeletal blood vessels in bone homeostasis is relatively clear, the role of pericytes and the underlying mechanisms remain unknown. Here, our objective was to elucidate the significance of pericytes in regulating osteoclast differentiation. Methods: We used tissue staining to detect the coverage of pericytes and osteoclasts in femoral tissues of osteoporotic mice and mice of different ages, analyzing their correlation. We developed mice with conditionally deleted pericytes, observing changes in bone mass and osteoclast activity using micro-computer tomography and tissue staining to detect the regulatory effect of pericytes on osteoclasts. Pericytes-derived exosomes (PC-EVs) were collected and co-cultured with monocytes that induce osteoclast differentiation to detect the effect of the former on the exosomes. Finally, the specific mechanism of PC-EVs regulating osteoclast differentiation was verified using RNA sequencing and Western blotting. Results: Our study indicates a significant correlation between pericytes and age-related bone resorption. Conditional deletion of pericytes activated bone resorption and led to osteopenia in vivo. We discovered that PC-EVs inhibited the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, which is mediated by tumor necrosis factor receptor-associated factor 3 (Traf3), negatively regulating osteoclast development and bone resorption. Silencing Traf3 in PC-EVs canceled their inhibitory effect on osteoclast differentiation. Conclusion: Our study provides a novel perspective into the regulatory role of pericytes on bone resorption and may provide potential strategies for developing novel anti-bone resorption therapies.

Downregulation of osteoprotegerin in colorectal cancer cells promotes liver metastasis via activating tumor-associated macrophage.

Osteoprotegerin (OPG) is a secreted cytokine that functions as a decoy receptor for receptor activator of nuclear factor kappa-B (RANK) ligand (RANKL). Anti-RANKL treatment for bone metastasis has been widely accepted for solid tumors. However, the mechanism of OPG-RANKL-RANK signaling in systemic colorectal cancer (CRC) metastasis remains unclear. In this study, we investigated the relevance and function of OPG expression in CRC liver metastasis. First, we performed in silico analysis using The Cancer Genome Atlas public database and found that lower OPG expression in CRC was associated with poor overall survival. Immunohistochemistry analyses using resected specimen from patients with CRC in our institute confirmed the result. Patient-matched primary CRC and liver metastases showed a significant downregulation of OPG expression in metastatic lesions. In CRC cell lines, OPG expression did not suppress cell proliferation and migration. However, OPG expression inhibited macrophage migration by suppressing the RANKL-RANK pathway. Moreover, in vivo mouse liver metastasis models showed that OPG expression in CRC cells suppressed liver metastases. In addition, treatment with an anti-RANKL neutralizing antibody also suppressed liver metastases. These results showed that downregulation of OPG expression in CRC cells promotes liver metastasis by activating tumor-associated macrophage, which can become a candidate for targeted therapy with anti-RANKL neutralizing antibody for CRC liver metastasis.

RANKL/RANK is required for cytokine-induced beta cell death; osteoprotegerin, a RANKL inhibitor, reverses rodent type 1 diabetes.

Treatment for type 1 diabetes (T1D) requires stimulation of functional ß cell regeneration and survival under stress. Previously, we showed that inhibition of the RANKL/RANK [receptor activator of nuclear factor kappa Β (NF-κB) ligand] pathway, by osteoprotegerin and the anti-osteoporotic drug denosumab, induces rodent and human ß cell proliferation. We demonstrate that the RANK pathway mediates cytokine-induced rodent and human ß cell death through RANK-TRAF6 interaction and induction of NF-κB activation. Osteoprotegerin and denosumab protected ß cells against this cytotoxicity. In human immune cells, osteoprotegerin and denosumab reduce proinflammatory cytokines in activated T-cells by inhibiting RANKL-induced activation of monocytes. In vivo, osteoprotegerin reversed recent-onset T1D in nonobese diabetic/Ltj mice, reduced insulitis, improved glucose homeostasis, and increased plasma insulin, ß cell proliferation, and mass in these mice. Serum from T1D subjects induced human ß cell death and dysfunction, but not α cell death. Osteoprotegerin and denosumab reduced T1D serum-induced ß cell cytotoxicity and dysfunction. Inhibiting RANKL/RANK could have therapeutic potential.

In vivo and in vitro action mechanism of treatment of glucocorticoid-induced osteoporosis by regulation of osteoprotegerin/receptor activator of nuclear factor-κB pathways by denshensu.

The research aimed to discuss the action mechanism of the treatment of glucocorticoid-induced osteoporosis (GIOP) by denshensu. In the research, 60 rats were purchased and divided into a control group, model group, estradiol group, and denshensu treatment group. Except for the control group, GIOP models were established for all other groups, and then the structural changes of osseous tissues as well as osteoprotegerin (OPG), expression of receptor activator of nuclear factor-κB ligands (RANKL) were detected. Besides, the changes in osteoclasts were observed by bone marrow-derived mononuclear phagocytes in vitro. The results showed that the micro-structure of bone trabeculae, bone mineral density (BMD), and bone metabolic markers of rats in the denshensu treatment group were enhanced significantly, while trabecular separation and structural model index were reduced (P<0.05). OPG messenger ribonucleic acid (mRNA) and protein levels in the hypothalamus and femur tissues were increased, while RANKL content was remarkably decreased (P<0.05). In addition, in vitro experiments revealed that denshensu inhibited the differentiation of positive osteoclasts, and osteoclast-related genes were reduced (P<0.05). To conclude, denshensu might inhibit the expressions of OPG and RANKL and further play a role in treating GIOP.

Development of 3-acetylindole derivatives that selectively target BRPF1 as new inhibitors of receptor activator of NF-κB ligand (RANKL)-Induced osteoclastogenesis.

Bromodomain and PHD finger-containing (BRPF) proteins function as epigenetic readers that specifically recognize acetylated lysine residues on histone tails. The acetyl-lysine binding pocket of BRPF has emerged as an attractive target for the development of protein interaction inhibitors owing to its potential druggability. In this study, we identified 3-acetylindoles as bone antiresorptive agents with a novel scaffold by performing structure-based virtual screening and hit optimization. Among those derivatives, compound 18 exhibited potent and selective inhibitory activities against BRPF1B (IC50 = 102 nM) as well as outstanding inhibitory activity against osteoclastogenesis (73.8% @ 1 µM) and differentiation (IC50 = 0.19 µM) without cytotoxicity. Besides, cellular mechanism assays demonstrated that compound 18 exhibited a strong bone antiresorptive effect by modulating the RANKL/RANK/NFATc1 pathway. Structural and functional studies on BRPF1 inhibitors aid in making advances to understand the epigenetic mechanisms of bone cell development and create innovative therapeutics for treating bone metastases from solid tumors and other bone erosive diseases.

Expression and ratio of receptor activator of nuclear factor kappa-B ligand and osteoprotegerin following application of Nigella sativa/bovine bone graft combination in post tooth extraction sockets.

Aims: The aim of this study was to analyze the induction effect of a combination of N. sativa and bovine bone graft on the expression and ratio of receptor activator of nuclear factor kappa-B ligand expression (RANKL) and osteoprotegerin (OPG) on alveolar bone socket preservation on days 7 and 14. Settings and Design: The research incorporated a posttest-only control group design. A total of 56 Cavia cobaya were divided into four groups: a control group, an N. sativa group, a bovine bone graft group, and a combined N. sativa and bovine bone graft group. Materials and Methods: The lower incisors of the C. cobaya were extracted with material subsequently being applied to the resulting socket. After the 7th and 14th days, the experimental animals were terminated to enable observation of the socket. Following processing, the tissue was subjected to immunohistochemistry staining consisting of RANKL and OPG antibodies before being observed under a light microscope at × 400. Statistical Analysis Used: Statistical analysis was carried out using the one-way ANOVA and Tukey's honestly significant difference tests. Results: A combination of N. sativa and bovine bone graft reduced both RANKL expression and the RANKL/OPG ratio while increasing OPG expression in comparison to the other groups. In all the results obtained, the N. sativa and bovine bone graft combination was significant (P < 0.05) when compared to the control group on both the 7th and 14th days. Conclusion: A combination of N. sativa and bovine bone graft reduced both RANKL expression and the RANKL/OPG ratio while increasing OPG expression.

The RANK-RANKL-OPG System: A Multifaceted Regulator of Homeostasis, Immunity, and Cancer.

The RANK-RANKL-OPG system is a complex signaling pathway that plays a critical role in bone metabolism, mammary epithelial cell development, immune function, and cancer. RANKL is a ligand that binds to RANK, a receptor expressed on osteoclasts, dendritic cells, T cells, and other cells. RANKL signaling promotes osteoclast differentiation and activation, which leads to bone resorption. OPG is a decoy receptor that binds to RANKL and inhibits its signaling. In cancer cells, RANKL expression is often increased, which can lead to increased bone resorption and the development of bone metastases. RANKL-neutralizing antibodies, such as denosumab, have been shown to be effective in the treatment of skeletal-related events, including osteoporosis or bone metastases, and cancer. This review will provide a comprehensive overview of the functions of the RANK-RANKL-OPG system in bone metabolism, mammary epithelial cells, immune function, and cancer, together with the potential therapeutic implications of the RANK-RANKL pathway for cancer management.

Bone destruction in chronic otitis media is not mediated by the RANKL pathway or estrogen receptor-alpha.

BACKGROUND: Chronic otitis media with or without cholesteatoma progresses with various degrees of bone resorption and remodeling. Estrogen mediates osteoprotective effects through the receptor activator of NF-κB ligand (RANKL) pathway, which is mainly mediated by estrogen receptor-alpha (ER-α). OBJECTIVES: The present study investigated the expression patterns of receptor activator of NF-κB (RANK), osteoprotegerin (OPG), RANKL, and ER-α in pathological tissue from patients with chronic otitis media to determine the roles of those factors in osteolytic mechanisms underlying the pathogenesis of chronic otitis media. METHODS: Normal and pathological specimens from 18 patients with chronic otitis media were examined. RESULTS: There were no significant differences in RANK, OPG, RANKL, or ER-α mRNA expression between normal and pathological specimens of epithelial tissue. CONCLUSIONS: Our findings suggested that RANK, OPG, RANKL, and ER-α are not associated with the bone destruction in chronic otitis media; other cytokines may directly activate the osteoclasts in chronic otitis media.

Xanthoxyletin blocks the RANK/RANKL signaling pathway to suppress the growth of human pancreatic cancer cells.

Xanthoxyletin is a vital plant-derived bioactive coumarin. It has been shown to exhibit anticancer effects against different human cancers. Nonetheless, the anticancer effects of xanthoxyletin against human pancreatic cancer cells have not been evaluated. Against this backdrop, the present study was designed to evaluate the anticancer effects of xanthoxyletin in human pancreatic cancer cells and to decipher the underlying molecular mechanisms. The results revealed a significant (p < 0.05) upregulation of receptor activator of NF-kappaB (RANK), receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) in human pancreatic tissues and cell lines at both transcriptional and translational levels. The administration of pancreatic cancer cells with xanthoxyletin diminished the viability of Capan-2 cells in a concentration-dependent manner and led to a significant decline in RANK, RANKL, and OPG expression. Silencing of RANK and xanthoxyletin treatment declined the viability of Capan-2 pancreatic cancer cells via induction of apoptosis. However, pancreatic cancer cells overexpressing RANK could rescue the growth inhibitory effects. Collectively, xanthoxyletin targets the RANK/RANKL signaling pathway in pancreatic cancer cells to induce cell apoptosis and may prove to be an important lead molecule.

The effects of exenatide and insulin glargine treatments on bone turnover markers and bone mineral density in postmenopausal patients with type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) related bone fracture. The effects of glucagon-like peptide-1 receptor analogs for the treatment of T2DM on bone are controversial in human studies. This study aimed to compare the effects of GLP-1 receptor analogs exenatide and insulin glargine treatment on bone turnover marker levels and bone mineral density (BMD) in postmenopausal female patients with T2DM. Thirty female patients with T2DM who were naive to insulin and incretin-based treatments, with spontaneous postmenopause, were randomized to exenatide or insulin glargine arms and were followed up for 24 weeks. BMD was evaluated using dual-energy X-ray absorptiometry and bone turnover markers by serum enzyme-linked immunosorbent assay. The body mass index significantly decreased in the exenatide group compared to the glargine group (P < .001). Receptor activator of nuclear factor kappa-B (RANK) and RANK ligand (RANKL) levels were significantly decreased with exenatide treatment (P = .009 and P = .015, respectively). Osteoprotegerin (OPG) level significantly increased with exenatide treatment (P = .02). OPG, RANK, RANKL levels did not change with insulin glargine treatment. No statistically significant difference was found between the pre- and posttreatment BMD, alkaline phosphatase, bone-specific alkaline phosphatase, and type 1 crosslinked N-telopeptide levels in both treatment arms. Despite significant weight loss with exenatide treatment, BMD did not decrease, OPG increased, and the resorption markers of RANK and RANKL decreased, which may reflect early antiresorptive effects of exenatide via the OPG/RANK/RANKL pathway.