Osteoprotegerin Protein Levels Research Articles

Mechanisms underlying reversed TRAIL sensitivity in acquired bortezomib-resistant non-small cell lung cancer cells.

Aim: The therapeutic targeting of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) death receptors in cancer, including non-small cell lung cancer (NSCLC), is a widely studied approach for tumor selective apoptotic cell death therapy. However, apoptosis resistance is often encountered. The main aim of this study was to investigate the apoptotic mechanism underlying TRAIL sensitivity in three bortezomib (BTZ)-resistant NSCLC variants, combining induction of both the intrinsic and extrinsic pathways. Methods: Sensitivity to TRAIL in BTZ-resistant variants was determined using a tetrazolium (MTT) and a clonogenic assay. A RT-qPCR profiling mRNA array was used to determine apoptosis pathway-specific gene expression. The expression of these proteins was determined through ELISA assays and western Blotting, while apoptosis (sub-G1) and cytokine expression were determined using flow cytometry. Apoptotic genes were silenced by specific siRNAs. Lipid rafts were isolated with fractional ultracentrifugation. Results: A549BTZR (BTZ-resistant) cells were sensitive to TRAIL in contrast to parental A549 cells, which are resistant to TRAIL. TRAIL-sensitive H460 cells remained equally sensitive for TRAIL as H460BTZR. In A549BTZR cells, we identified an increased mRNA expression of TNFRSF11B [osteoprotegerin (OPG)] and caspase-1, -4 and -5 mRNAs involved in cytokine activation and immunogenic cell death. Although the OPG, interleukin-6 (IL-6), and interleukin-8 (IL-8) protein levels were markedly enhanced (122-, 103-, and 11-fold, respectively) in the A549BTZR cells, this was not sufficient to trigger TRAIL-induced apoptosis in the parental A549 cells. Regarding the extrinsic apoptotic pathway, the A549BTZR cells showed TRAIL-R1-dependent TRAIL sensitivity. The shift of TRAIL-R1 from non-lipid into lipid rafts enhanced TRAIL-induced apoptosis. In the intrinsic apoptotic pathway, a strong increase in the mRNA and protein levels of the anti-apoptotic myeloid leukemia cell differentiation protein (Mcl-1) and B-cell leukemia/lymphoma 2 (Bcl-2) was found, whereas the B-cell lymphoma-extra large (Bcl-xL) expression was reduced. However, the stable overexpression of Bcl-xL in the A549BTZR cells did not reverse the TRAIL sensitivity in the A549BTZR cells, but silencing of the BH3 Interacting Domain Death Agonist (BID) protein demonstrated the importance of the intrinsic apoptotic pathway, regardless of Bcl-xL. Conclusion: In summary, increased sensitivity to TRAIL-R1 seems predominantly related to the relocalization into lipid rafts and increased extrinsic and intrinsic apoptotic pathways.

Diagnostic potential of salivary interleukin-17, RANKL, and OPG to differentiate between periodontal health and disease and discriminate stable and unstable periodontitis: A case-control study.

Limitations of the conventional diagnostic techniques urged researchers to seek novel methods to predict, diagnose, and monitor periodontal disease. Use of the biomarkers available in oral fluids could be a revolutionary surrogate for the manual probing/diagnostic radiograph. Several salivary biomarkers have the potential to accurately discriminate periodontal health and disease. This study aimed to determine the diagnostic sensitivity and specificity of salivary interleukin (IL)-17, receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), RANKL/OPG for differentiating (1) periodontal health from disease and (2) stable and unstable periodontitis. Participants with periodontitis (n = 50) and gingivitis (n = 25), both diseases represented the cases, and subjects with healthy periodontium (n = 15) as a control were recruited for this study. Periodontitis cases were further equally subdivided into stable and unstable. Whole unstimulated salivary sample were collected from all participants. Periodontal parameters including bleeding on probing, probing pocket depth, clinical attachment loss, and number of missing teeth were recorded. The protein levels of salivary IL-17, RANKL, and OPG were determined by using enzyme-linked immunosorbent assays technique. Salivary IL-17, OPG, RANKL, and RANKL/OPG showed high sensitivity and specificity to differentiate periodontal health from gingivitis and periodontitis. Similar pattern was observed in discriminating stable and unstable periodontitis. Salivary IL-17 and RANKL showed a good accuracy to differentiate gingivitis from periodontitis. However, OPG and RANKL/OPG did not exhibit enough sensitivity and specificity to differentiate the latter conditions. Salivary IL-17, RANKL, OPG, and RANKL/OPG system are potential candidates for differentiating periodontal health and disease and discriminate stable and unstable periodontitis.

Osteoclast and Sclerostin Expression in Osteocytes in the Femoral Head with Risedronate Therapy in Patients with Hip Fractures: A Retrospective Comparative Study.

Background and Objectives: The majority of research on the effects of osteoporosis drugs has measured the bone mineral density (BMD) of the spine and femur through dual-energy X-ray absorptiometry (DEXA) and compared and analyzed the effects of the drugs through changes in the BMD values. This study aims to compare osteoclast and sclerostin expression in osteocytes after risedronate therapy by obtaining femoral heads from patients with hip fractures. Materials and Methods: We obtained the femoral heads of 10 female patients (age: ≥65 years) who received risedronate therapy for at least 1 year through hip arthroplasty during 2019-2021 (risedronate group). Meanwhile, 10 patients who had never received osteoporosis treatment were selected as controls using propensity scores with age, body mass index, and bone density as covariates (control group). While the osteoclast count was evaluated using tartrate-resistant acid phosphatase (TRAP) staining, the sclerostin expression in osteocytes was assessed using immunohistochemistry. Moreover, Western blotting and polymerase chain reaction (PCR) were performed for receptor activation of nuclear factor kappa-Β ligand (RANKL), RANK, osteoprotegerin (OPG), sclerostin, and bone morphogenetic protein-2 (BMP2). Results: TRAP staining revealed significantly more TRAP-positive cells in the control group (131.75 ± 27.16/mm2) than in the risedronate group (28.00 ± 8.12/mm2). Moreover, sclerostin-positive osteocytes were expressed more in the control group (364.12 ± 28.12/mm2) than in the risedronate group (106.93 ± 12.85/mm2). Western blotting revealed that the expressions of RANKL, RANK, sclerostin, and BMP2 were higher in the control group than in the risedronate group (p < 0.05). Furthermore, RANK, sclerostin, and OPG protein levels were higher in the control group than in the risedronate group. Conclusions: In this study, the risedronate group demonstrated lower osteoclast activity and sclerostin expression in osteocytes in the femoral head than the control group.

Jianpi Qingchang Bushen decoction improves inflammatory response and metabolic bone disorder in inflammatory bowel disease-induced bone loss.

BACKGROUNDBone loss and osteoporosis are commonly described as extra-intestinal manifestations of inflammatory bowel disease (IBD). Jianpi Qingchang Bushen decoction (JQBD) is a prescription used in clinical practice. However, further studies are needed to determine whether JQBD regulates the receptor activator of nuclear factor kappa B (NF-κB) (RANK)/receptor activator of NF-κB ligand (RANKL)/ osteoprotegerin (OPG) pathways and could play a role in treating IBD-induced bone loss.AIMTo evaluate the therapeutic effect of JQBD in IBD-induced bone loss and explore the underlying mechanisms.METHODSAn IBD-induced bone loss model was constructed by feeding 12 6-to-8-wk-old interleukin-10 (IL-10)-knockout mice with piroxicam for 10 d. The mice were randomly divided into model and JQBD groups. We used wild-type mice as a control. The JQBD group was administered the JQBD suspension for 2 wk by gavage, while the control and model groups were given normal saline at the corresponding time points. All mice were killed after the intervention. The effect of JQBD on body weight, disease activity index (DAI), and colon length was analyzed. Histopathological examination, colon ultrastructure observation, and micro-computed tomographic scanning of the lumbar vertebrae were performed. The gene expression of NF-κB, tumor necrosis factor-α (TNF-α), IL-1β, IL-6, and IL-8 in the colon was evaluated by real-time polymerase chain reaction. Colon samples were assessed by Western blot for the expression of RANKL, OPG, RANK, and NF-κB proteins.RESULTSThe model group lost body weight, had a shorter colon, and showed a dramatic increase in DAI score, whereas JQBD had protective and therapeutic effects. Treatment with JQBD significantly improved inflammatory cell infiltration and reduced crypt abscess and ulcer formation. Three-dimensional imaging of the vertebral centrum in the model group revealed a lower bone mass, loose trabeculae, and “rod-shaped” changes in the structure compared to the control group and JQBD groups. The bone volume/total volume ratio and bone mineral density were significantly lower in the model group than in the control group. JQBD intervention downregulated the NF-κB, TNF-α, IL-1β, IL-6, and IL-8 mRNA expression levels. The RANKL and OPG protein levels were also improved.CONCLUSIONJQBD reduces inflammation of the colonic mucosa and inhibits activation of the RANK/ RANKL/OPG signaling pathway, thereby reducing osteoclast activation and bone resorption and improving bone metabolism.

Genistein prevents bone loss in type 2 diabetic rats induced by streptozotocin.

BackgroundDiabetic osteoporosis has become a severe public health problem in the aging societies. Genistein has been reported to play an important role in preventing and treating metabolic diseases via its anti-inflammatory, antioxidant, anti-estrogenic, and estrogen-like functions.ObjectiveWe aimed to investigate whether genistein exerts bone-protective effect on diabetic rats induced by 35 mg/kg streptozotocin (STZ) plus a 4-week high-fat diet.DesignSprague–Dawley rats were randomly divided into four groups: (1) control group, (2) type 2 diabetes mellitus (T2DM) model group, (3) T2DM with 10 mg/kg genistein, and (4) T2DM with 30 mg/kg genistein. After an 8-week treatment with genistein, the femurs, tibias, and blood were collected from all rats for further analysis.ResultsGenistein at 10 mg/kg showed little effect on diabetic osteoporosis, whereas genistein at 30 mg/kg significantly improved glucose and bone metabolisms compared with diabetic rats. Our results showed that 30 mg/kg genistein significantly increased bone mineral density, serum osteocalcin, and bone alkaline phosphatase. Genistein also effectively lowered fasting blood glucose, tartrate-resistant acid phosphatase 5b, tumor necrosis factor-α, interleukin-6, and numbers of adipocytes and osteoclasts. Compared with the T2DM group, protein levels of receptor activator of nuclear factor κB ligand (RANKL) and peroxisome proliferator-activated receptor-γ (PPAR-γ) were decreased, while protein levels of osteoprotegerin (OPG), β-catenin, and runt-related transcription factor 2 (Runx-2) were increased after genistein intervention.ConclusionGenistein could effectively improve abnormal bone metabolism in STZ-induced diabetic rats; the underlying molecular mechanisms might be related to OPG/RANKL, PPAR-γ, and β-catenin/Runx-2 pathways.

Naringin increases osteoprotegerin expression in fibroblasts from periprosthetic membrane by the Wnt/\u03b2-catenin signaling pathway

BackgroundThe osteoclast bone resorption is critical in aseptic loosening after joint replacement. The balance between activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) is considered to play a central role in osteoclast maturation. Fibroblasts from the periprosthetic membrane express RANKL and promote osteoclast formation. Studies have demonstrated that naringin inhibited osteoclastogenesis and wear particle-induced osteolysis. In this study, the naringin-induced OPG/RANKL effects and its underlying mechanism were studied in fibroblasts from periprosthetic membrane.MethodsFibroblasts were isolated from the periprosthetic membrane during hip arthroplasty for revision due to aseptic loosening. Fibroblasts were cultured and treated with or without naringin and DKK-1 (the classical inhibitor of Wnt/β-catenin signaling pathway). OPG and RANKL mRNA and protein levels, gene expression of β-catenin, and cyclin D1, which participate in the Wnt signaling pathway, were examined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay.ResultsThe mRNA and protein levels of OPG were enhanced by naringin in a dose-dependent manner compared to that of the non-treated control. In contrast, naringin did not affect the expression of RANKL. Importantly, DKK-1 attenuated OPG expression in fibroblasts under naringin treatment. Moreover, naringin stimulated the gene expression of β-catenin and cyclin D1 in fibroblasts, and the effect could be inhibited by DKK-1.ConclusionThe results indicated that naringin enhanced OPG expression through Wnt/β-catenin signaling pathway in fibroblasts from periprosthetic membrane, which may be useful to inhibit periprosthetic osteolysis during aseptic loosening after total joint arthroplasty.

Blockade of IL-6 alleviates bone loss induced by modeled microgravity in mice.

This study investigated the effects of blockade of IL-6 on bone loss induced by modeled microgravity (MG). Adult male mice were exposed to hind-limb suspension (HLS) and treated with IL-6-neutralizing antibody (IL-6 nAb) for 4 weeks. HLS in mice led to upregulation of IL-6 expression in both sera and femurs. IL-6 nAb treatment in HLS mice significantly alleviated bone loss, evidenced by increased bone mineral density of whole tibia, trabecular thickness and number, bone volume fraction of proximal tibiae, and ultimate load and stiffness of femoral diaphysis. IL-6 nAb treatment in HLS mice significantly enhanced levels of osteocalcin in sera and reduced levels of deoxypyridinoline. In MC3T3-E1 cells exposed to MG in vitro, IL-6 nAb treatment increased mRNA expression and activity of alkaline phosphatase, mRNA expression of osteopontin and runt-related transcription factor 2, and protein levels of osteoprotegerin and decreased protein levels of receptor activator of the NF-κB ligand. In RAW254.7 cells exposed to MG, IL-6 nAb treatment downregulated mRNA expression of cathepsin K and tartrate-resistant acid phosphatase (TRAP) and reduced numbers of TRAP-positive multinucleated osteoclasts. In conclusion, blockade of IL-6 alleviated the bone loss induced by MG.

Synergistic effects triggered by simultaneous Toll-like receptor-2 and -3 activation in human periodontal ligament stem cells.

BackgroundAlthough periodontitis is associated with disruption of the host‐microbial homeostasis, viruses are currently discussed to influence disease progression. Viral pathogens are recognized by Toll‐like receptor (TLR)‐3, which engages a different signaling pathway than other TLRs. This study aimed to investigate the effect of TLR‐3 agonist polyinosinic:polycytidylic acid (Poly I:C) on the expression of inflammatory markers and bone metabolism proteins by human periodontal ligament stem cells (hPDLSCs) compared with TLR‐2 agonist Pam3CSK4, which mimics the effect of bacterial lipoproteins. To assess potential combined effects of bacterial and viral infections, hPDLSCs response to simultaneous TLR‐2 and TLR‐3 activation was investigated.MethodsHPDLSCs were stimulated with Poly I:C (0.0001‐1 µg/mL), Pam3CSK4 (1 µg/mL), and their combinations for 24 hours. Gene expression and protein levels of interleukin (IL)‐6, IL‐8, monocyte chemoattractant protein (MCP)‐1, and osteoprotegerin (OPG) were measured with qPCR and ELISA.ResultsProduction of IL‐6, IL‐8, MCP‐1, and OPG was significantly increased by Poly I:C or Pam3CSK4 to a similar extent. The levels of all inflammatory mediators induced by simultaneous stimulation with Poly I:C and Pam3CSK4 were significantly higher compared with single stimuli as well as to their summed response. Gene expression and protein levels of OPG were enhanced by Poly I:C, but by lesser extent than by Pam3CSK4. OPG levels upon simultaneous stimulation with Pam3CSK4 and Poly I:C were significantly lower compared with Pam3CSK4 stimulation alone.ConclusionsSimultaneous TLR‐2 and TLR‐3 activation synergistically triggers IL‐6, IL‐8, and MCP‐1 production, which was not observed for OPG. These findings suggest that TLR‐3 activation by viral infections might promote periodontitis progression.

Promoter Methylation Down-regulates Osteoprotegerin Expression in Ovarian Carcinoma.

Previous studies have documented that osteoprotegerin (OPG) is involved in the development and progression of several human malignancies. However, OPG has also been shown to act as a tumor suppressor. The aim of this study was to examine the expression status of OPG in ovarian carcinoma cells and investigate the underlying mechanism responsible for alterations in OPG expression. The expression levels of OPG mRNA and protein were assessed in human ovarian carcinoma cell lines. The methylation status of the OPG promoter region was determined using the bisulfite pyrosequencing technique. The effects of demethylation on OPG expression were also analyzed. The human ovarian carcinoma cell lines, SW 626, OVCAR-3, ES-2, TOV-112D, and TOV-21G, expressed significantly lower levels of OPG mRNA and protein than the normal human ovarian epithelial cell line, HS823.Tc. Moreover, three CpG sites in the OPG promoter region were highly methylated in the SW 626, OVCAR-3, ES-2, and TOV-112D ovarian carcinoma cell lines compared to normal control cells. Furthermore, in all the examined ovarian carcinoma cell lines, treatment with the demethylating agent, 5-aza-2-deoxycytidine, resulted in significantly increased expression levels of OPG mRNA and protein compared to the respective pre-treatment levels. OPG expression was down-regulated in the studied ovarian carcinoma cells compared to the normal control cells, while demethylation significantly restored OPG expression in the OPG-down-regulated cell lines. Our results suggest that OPG down-regulation in ovarian carcinoma occurs, at least partly, through epigenetic repression, suggesting its involvement in ovarian carcinogenesis.

Effect of risperidone on proliferation and apoptosis of MC3T3-E1 cells.

This aim of this study was to assess the molecular mechanism of osteoporosis in schizophrenia patients with risperidone use. Here, we investigated the effects of risperidone on cellular proliferation and apoptosis of a preosteoblast cell line, MC3T3-E1. Cell viability and apoptotic rate of MC3T3-E1 were detected by cell counting kit-8 and flow cytometry at a serial dose of risperidone and at different time points, respectively. Bone transformation relevant gene serum osteocalcin (BGP), collagen 1, tumor necrosis factor-α (TNF-α), osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL) mRNA levels were determined by real-time PCR (qPCR). Their protein expression patterns were evaluated using western blot. The results revealed that risperidone dramatically inhibited MC3T3-E1 cell proliferation in a dose-dependent manner. It also significantly induced MC3T3-E1 cell apoptosis. TNF-α gene and protein levels were greatly enhanced after risperidone treatment. In contrast, BGP, collagen 1, OPG, and RANKL gene and protein levels were markedly downregulated. Our study indicated that risperidone suppressed MC3T3-E1 cell proliferation and induced apoptosis. It also regulated BGP gene and protein expression.

Osteoprotection Through the Deletion of the Transcription Factor Rorβ in Mice.

There is a clinical need to identify new molecular targets for the treatment of osteoporosis, particularly those that simultaneously inhibit bone resorption while stimulating bone formation. We have previously shown in overexpression studies that retinoic acid receptor-related orphan receptor β (Rorβ) suppresses in vitro osteoblast differentiation. In addition, the expression of Rorβ is markedly increased in bone marrow-derived mesenchymal stromal cells with aging in both mice and humans. Here we establish a critical role for Rorβ in regulating bone metabolism using a combination of in vitro and in vivo studies. We used Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing to demonstrate that loss of Rorβ in osteoblasts enhances Wnt signaling, specifically through increased recruitment of β-catenin to T-cell factor/lymphoid enhancer factor (Tcf/Lef) DNA binding sites in the promoters of the Wnt target genes Tcf7 and Opg. This resulted in increased osteogenic gene expression and suppressed osteoclast formation through increased osteoprotegerin (OPG) secretion in Rorβ-deficient cells. Consistent with our in vitro data, genetic deletion of Rorβ in both female and male mice resulted in preserved bone mass and microarchitecture with advancing age due to increased bone formation with a concomitant decrease in resorption. The improved skeletal phenotype in the Rorβ-/- mice was also associated with increased bone protein levels of TCF7 and OPG. These data demonstrate that loss of Rorβ has beneficial skeletal effects by increasing bone formation and decreasing bone resorption, at least in part through β-catenin-dependent activation of the Wnt pathway. Thus, inhibition of Rorβ represents a novel approach to potentially prevent or reverse osteoporosis. © 2017 American Society for Bone and Mineral Research.

Bone metabolism and RANKL/RANK/OPG trail in periodontal disease

Abstract Periodontal disease is an inflammatory disease of multifactorial etiology. In order for it to appear there must come to an imbalance between the effects of pathogens and host defense mechanisms. As a result of its course the destruction of structures supporting the teeth appears (periodontium, cement, bone), and consequently leads to teeth loosening and loss. In recent years, the participation of RANKL/RANK/OPG in bone remodeling process was highligted. At the molecular level the bone resorption is regulated through the interaction of the ligand receptor activator of nuclear NF-kappa B (RANKL) and osteoprotegerin (OPG), which is a system of two proteins belonging to the protein tumor necrosis factor (TNF). Recent findings about the RANKL protein and OPG have shed new light on the previously unexplained phenomenon of the basis of bone resorption. Research has shown that both protein OPG and RANKL can be detected in gingival crevicular fluid, which has become a window of opportunity in the analysis of non-invasive markers of periodontal tissues, confirming elevated levels of RANKL protein in periodontal disease, and decreased levels of OPG protein. Bone resorption is initiated by the binding of the RANKL protein to receptors RANK present on the surface of mature osteoclasts, and their precursors, which leads to the differentiation and activation of osteoclasts. OPG, being RANKL’s inhibitor, has, in turn, opposite characteristics to RANKL, resulting in the reduction of osteoclastogenesis process. Despite all this, the exact mechanism of bone resorption has not yet been elucidated.

Fibroblast growth factor 23 inhibits osteoblastic gene expression and induces osteoprotegerin in vascular smooth muscle cells

Background and aimsElevated fibroblast growth factor 23 (FGF23) levels are associated with cardiovascular mortality in patients with chronic kidney disease. However, both clinical and basic research have demonstrated conflicting evidence regarding the pathophysiological role of FGF23 in vascular calcification. The aim of this study was to determine the role of FGF23 in the osteoblastic gene expression in vascular smooth muscle cells (SMCs). Methods and resultsWe transduce human aortic SMCs (HASMCs) expressing klotho and FGF receptors with the adenovirus expressing human FGF23 (Ad-FGF23).We observed significant decreases in the expression of osteoblast-marker genes including BMP2, BMP4, MSX2, RUNX2 and ALP, as well as reduced calcification. Notably, Ad-FGF23 increased mRNA and protein levels of osteoprotegerin (OPG), and human OPG promoter was activated by FGF23. Moreover, in HASMCs overexpressing klotho, FGF23 upregulated OPG expression, whereas depletion of klotho by siRNA attenuated FGF23-induced OPG expression. Furthermore, in 73 consecutive patients with type 2 diabetes mellitus undergoing cardiac computed tomography to determine coronary calcium scores (CCSs), serum FGF23 levels were positively correlated with OPG independent of phosphate and estimated glomerular filtration rate (eGFR, r = 0.65, p < 0.01). Serum FGF23 levels were significantly elevated in patients with high CCSs (≧100) compared to those with low CCSs (<100). ConclusionsOur in vitro results indicate that FGF23 suppresses osteoblastic gene expression and induces OPG expression in HASMCs. Together with our cross-sectional clinical assessment, the present study lends support to our hypothesis that FGF23 counteracts osteogenic conversion of vascular SMCs as a part of a compensatory mechanism to mitigate vascular calcification.

Assessment of OPG, RANKL, bone turnover markers serum levels and BMD after treatment with strontium ranelate and ibandronate in patients with postmenopausal osteoporosis.

The aim of this study was to evaluate quantitative changes in OPG and RANKL proteins after treatment with strontium ranelate (SR) and ibandronate in patients with postmenopausal osteoporosis. A total of 89 women with postmenopausal osteoporosis (PO), aged 51-85 years, patients of the Outpatient Clinic of Osteoporosis of the Military Teaching Hospital in Lodz, were enrolled in the study. The patients were randomly assigned to different therapies: ibandronate and (SR). Patients of the control group received only calcium and vitamin D3 supplements. The patients' visits were repeated after three and six months. Measurements of beta-CTX (C-terminal Telopeptide of type 1 collagen), osteocalcin, RANKL, osteoprotegerin (OPG), alkaline phosphatase concentrations in serum, as well as of total 24-hour calcium and phosphate levels in serum and urine, were carried out in material collected at baseline and after three and six months of therapy. Left hip and lumbar spine densitometry was done twice (at baseline visit and after six months). In all three groups there were no significant differences noted in the concentrations of OPG and RANKL serum protein levels during the study period. Both negative and positive correlations or tendencies of correlations were found between OPG serum concentrations and BMD changes in the SR group. Both ibandronate and SR do not seem to cause any significant changes in OPG and RANKL protein serum levels during the first six months of treatment. OPG may play a role in osteoclast activity suppression in the course of treatment with ibandronate in patients with PO. OPG may play an important role in the mechanism of SR therapy and may be viewed as a potentially valuable parameter for monitoring and predicting the course of treatment with SR in PO.

Effects of IL-10 and glucose on expression of OPG and RANKL in human periodontal ligament fibroblasts.

The effects of interleukin-10 (IL-10) and glucose on mRNA and protein expression of osteoprotegerin (OPG), and its ligand, receptor activator of nuclear factor-κB ligand (RANKL), were investigated in human periodontal ligament fibroblasts (HPDLFs). Primary HPDLFs were treated with different concentrations of IL-10 (0, 1, 10, 25, 50, and 100 ng/mL) or glucose (0, 5.5, 10, 20, 30, and 40 mmol/L). Changes in mRNA and protein expression were examined using the reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. After IL-10 treatment, mRNA and protein levels of OPG were increased, while mRNA and protein levels of RANKL were decreased (P<0.05), both in a concentration-dependent manner. Glucose stimulation had the opposite concentration-dependent effect to that of IL-10 on OPG and RANKL expression. IL-10 upregulated OPG expression and downregulated RANKL expression, whereas high glucose upregulated RANKL and downregulated OPG in HDPLFs. Abnormal levels of IL-10 and glucose may contribute to the pathogenesis of periodontal disease.

Lentivirus-mediated TNF-α gene silencing and overexpression of osteoprotegerin inhibit titanium particle-induced inflammatory response and osteoclastogenesis in vitro

Macrophages and osteoclasts release proinflammatory factors and promote osteoclastogenesis following the phagocytosis of wear particles. During this pathological process, receptor of nuclear factor κB ligand (RANKL) and tumor necrosis factor (TNF)-α are critical factors contributing to resorption and the inflammatory response. The present study aimed to construct recombination lentivirus vectors carrying TNF-α small interfering (si)RNA and osteoprotegerin (OPG) cDNA, and to examine the effects of Lenti‑siTNFα‑OPG on the wear particle‑induced inflammatory response and osteoclastogenesis in a titanium (Ti) particle‑induced‑inflammatory response cell model. Lenti‑siTNFα‑OPG vectors were constructed and transnfected into RAW264.7 and MC3T3‑E1 cells, respectively, prior to particle stimulation. The protein levels of TNF‑α, OPG and RANKL were evaluated using western blot analysis and enzyme‑linked immunosorbent assays, and the mRNA expression levels of the inflammatory factors, TNF‑α, interleukin (IL)‑1β and IL‑6, as well as OPG and RANKL, were measured using reverse transcription‑quantitative polymerase chain reaction analysis. The activity of alkaline phosphatase (ALP) was examined using an ALP kit. In the presence of the Lenti‑siTNFα‑OPG vector, the mRNA expression levels of the inflammatory factors and RANKL were downregulated, as were the protein levels of TNF‑α. The mRNA expression and protein levels of OPG were upregulated, and ALP activity was increased. These findings suggested that Lenti‑siTNFα‑OPG transfection inhibited the wear particle‑induced inflammatory response and osteoclastogenesis, which warrants further investigation for the prevention and/or treatment of wear particle-induced osteolysis.

Pretreatment levels of serum osteoprotegerin and p53 protein and urine telomerase as prognostic factors affecting survival in Egyptian bladder cancer patients.

A non-invasive marker is required for the diagnosis and follow-up of patients with bladder cancer. The aim of the current study was to evaluate the potential prognostic significance of serum osteoprotegerin (OPG), p53 protein and urine telomerase in patients with bladder cancer. For all patients, serum levels of OPG and p53 protein were determined using enzyme-linked immunosorbent assay (ELISA), and urine telomerase was assessed using a polymerase chain reaction ELISA technique. Patients were assigned into group 1 (cystectomy and adjuvant radiotherapy) or group 2 (transurethral resection and chemoradiotherapy). The results revealed that serum OPG and p53, and urine telomerase levels were significantly higher in bladder cancer patients compared with in healthy individuals (P<0.0001). High serum OPG was associated with significantly lower overall survival and disease-free survival rates (both P=0.001), and was correlated with advanced tumor stages (P<0.0001), high tumor grades (P<0.0001) and the occurrence of disease relapse (P=0.001). Serum p53 and urine telomerase did not demonstrate prognostic significance. These findings indicate that serum OPG level may be used as a diagnostic tool and a prognostic variable for patients with muscle invasive bladder cancer. Future trials are required to elucidate its therapeutic role in such patients.

Transactivation of human osteoprotegerin promoter by GATA-3.

Osteoprotegerin (OPG) is a key regulator of bone remodeling. Mutations in OPG are involved in a variety of human diseases. We have shown that cochlear spiral ganglion cells secrete OPG at high levels and lack of OPG causes sensorineural hearing loss in addition to the previously described conductive hearing loss. In order to study the regulation of OPG expression, we conducted a database search on regulatory elements in the promoter region of the OPG gene, and identified two potential GATA-3 binding sites. Using luciferase assays and site directed mutagenesis, we demonstrate that these two elements are GATA-3 responsive and support GATA-3 transactivation in human HEK and HeLa cells. The expression of wild type GATA-3 activated OPG mRNA and protein expression, while the expression of a dominant negative mutant of GATA-3 or a GATA-3 shRNA construct reduced OPG mRNA and protein levels. GATA-3 deficient cells generated by expressing a GATA-3 shRNA construct were sensitive to apoptosis induced by etoposide and TNF-α. This apoptotic effect could be partly prevented by the co-treatment with exogenous OPG. Our results suggest new approaches to rescue diseases due to GATA-3 deficiency – such as in hypoparathyroidism, sensorineural deafness, and renal (HDR) syndrome – by OPG therapy.

Effects of high glucose on expression of OPG and RANKL in rat aortic vascular smooth muscle cells.

To explore effect of high glucose on expression of osteoprotegerin (OPG) and receptor activator of NF- κ B ligand (RANKE) in rat aortic vascular smooth muscle cells. SD rats were intraperitoneally injected with streptozotocin, OPG and RANKL expression in rat thoracic aortas were detected by immunohistochemical staining. In cultured vascular smooth muscle cells (VSMCs) (A7r5), qRT-PCR and Western blot analysis were used to examine the mRNA and protein levels of OPG and RANKL. Our results demonstrated that OPG expression was increased in hyperglycemic rat aortic VSMCs, while RANKL expression was decreased. Besides, in vitro experiments high glucose induced OPG expression, but depressed RANKL expression by dose- and time-dependent manner in cultured A7r5. Our findings suggested that high glucose could promote the expression of OPG, and inhibit the expression of RANKL in VSMCs, which may be partly be the molecular mechanism of diabetic vascular calcification.

Mycobacterium tuberculosis 10-kDa co-chaperonin regulates the expression levels of receptor activator of nuclear factor-κB ligand and osteoprotegerin in human osteoblasts

The aim of the present study was to investigate the effect of recombinant Mycobacterium tuberculosis (r-Mt) 10-kDa co-chaperonin (cpn10) on the expression of osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) in third-generation cultured osteoblasts. The osteoblast-like cultures were isolated from bone fragments taken from patients undergoing surgery. Prior to stimulation with r-Mt cpn10, cells were incubated in serum-free medium for 24 h. r-Mt cpn10 was added into fresh serum-free medium, reaching final concentrations of 0.01–10 μg/ml. The levels of OPG were determined using enzyme-linked immunosorbent assay. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis was performed to determine the levels of RANKL and OPG mRNA. For measurement of the protein levels of OPG and RANKL, a western blotting assay was performed. r-Mt cpn10 downregulated the protein levels of OPG in the third generation cultured osteoblasts at a dose of 10 μg/ml. RT-qPCR revealed that the OPG mRNA level was decreased by 73% after 4 h and by 85.5% after 8 h following incubation with r-Mt cpn10 (10 μg/ml). Western blot analysis demonstrated similar results for the OPG protein level. In the third-generation cultured osteoblasts, the levels of RANKL mRNA and protein were increased by 2.6- and 1-fold, respectively, following incubation with r-Mt cpn10 (10 μg/ml). Furthermore, the RANKL/OPG ratio was markedly increased by r-Mt cpn10 (10 μg/ml) treatment. In conclusion, the results of the current study demonstrated that r-Mt cpn10 decreased the levels of OPG and increased the levels of RANKL in a dose- and time-dependent manner. Notably, the present study indicated that r-Mt cpn10 exerts its effect on osteoblastic cells by increasing the RANKL/OPG ratio.