Osteoprotegerin mRNA Research Articles

Tetrahydroxystilbene Glucoside Regulates Proliferation, Differentiation, and OPG/RANKL/M-CSF Expression in MC3T3-E1 Cells via the PI3K/Akt Pathway.

Tetrahydroxystilbene glucoside (TSG) is a unique component of the bone-reinforcing herb Radix Polygoni Multiflori Preparata (RPMP). It has the ability to promote bone formation and protect osteoblasts. However, the underlying mechanism remains unclear. To better understand its biological function, we determined TSG’s effect on murine pre-osteoblastic MC3T3-E1 cells by the MTT assay, flow cytometry, FQ-PCR, Western blot, and ELISA. The results showed that TSG caused an elevation of the MC3T3-E1 cell number, the number of cells in the S phase, and the mRNA levels of the runt-related transcription factor-2 (Runx2), osterix (Osx), and collagen type I α1 (Col1a1). In addition, the osteoprotegerin (OPG) mRNA level was up-regulated, while the nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) mRNA levels were down-regulated by TSG. Furthermore, TSG activated the phosphoinosmde-3-kinase/protein kinase B (also known as PI3K/Akt) pathway, and blocking this pathway by the inhibitor LY-294002 could impair TSG’s functions in relation to the MC3T3-E1 cells. In conclusion, TSG could activate the PI3K/Akt pathway and thus promote MC3T3-E1 cell proliferation and differentiation, and influence OPG/RANKL/M-CSF expression. TSG merits further investigation as a potential therapeutic agent for osteoporosis treatment.

Osteoprotegerin deficiency causes morphological and quantitative damage in epithelial rests of Malassez.

Epithelial rests of Malassez (ERM), the only odontogenic epithelial structures in periodontal tissue, are proposed to correlate with root resorption, but the detailed mechanism remains unclear. Osteoprotegerin (OPG), the main inhibitor of osteoclastogenesis, plays a pivotal role in inhibiting root resorption, and ERM cells express OPG mRNA in vitro. Thus, in this study, we aimed to clarify OPG expression in ERM in vivo and to explore the role of OPG in ERM to determine whether ERM are associated with root resorption via OPG. We established Opg-knockout (Opg-KO) mice and detected the OPG expression in ERM by immunohistochemical staining in 4-, 6-, 10-, 26- and 52-week-old mice. The ERM of wild-type (WT) mice and Opg-KO mice were evaluated histologically at 4, 10 and 26 weeks of age. Orthodontic root resorption models were established, maxillae were collected after 4weeks, and ERM were analysed by histomorphometric analysis. In our study, OPG displayed sustained expression in ERM, and OPG deficiency caused the destruction of ERM, characterized by irregular morphology and reduced numbers. Moreover, after orthodontic treatment, the loss of OPG severely damaged ERM, aggravating root resorption. Together, our results demonstrated that ERM expressed the OPG protein in vivo and that OPG deficiency resulted in morphological and quantitative damage to ERM. Furthermore, ERM may be associated with root resorption via OPG, thus helping to explain the mechanism underlying root resorption.

Inhibition effects of total flavonoids from Sculellaria barbata D. Don on human breast carcinoma bone metastasis via downregulating PTHrP pathway.

It is abundantly clear that tumor-derived parathyroid hormone-related protein (PTHrP), receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) are central contributors in promoting osteolytic process of breast carcinoma bone metastasis. Forcusing on this molecular basis, the study was undertaken to explore the inhibition effects of total flavonoids from Scutellaria barbata D. Don (TF-SB) on human breast carcinoma bone metastasis. MDA-MB-231 cells and nude mouse models of breast cancer bone metastasis were given TF-SB in different concentrations. The proliferation, migration and invasion potentials of MDA-MB-231 cells were respectively tested. The effects of TF-SB on tumor weights and bone destruction were investigated. The mRNA and protein expression of PTHrP, OPG and RANKL were assessed by qPCR and western blot analysis. In vitro, TF-SB inhibited the proliferation, migration and invasion of MDA-MB-231 cells in a dose-dependent manner. In vivo, TF-SB prevented bone metastasis of breast cancer by decreasing the number of osteoclast cells per field in a dose-dependent manner, but not affecting tumor growth or mouse survival. Molecular analysis revealed that TF-SB controled the secretion of osteolysis-related factors PTHrP and its downstream RANKL/OPG. Together, by controlling the expression of PTHrP and its downstream OPG/RANKL, TF-SB has significant inhibition effects on breast cancer bone metastasis, which indicates a new therapeutic method.

Levels of oxidative stress biomarkers and bone resorption regulators in apical periodontitis lesions infected by Epstein-Barr virus.

To investigate whether apical periodontitis lesions infected by Epstein-Barr virus (EBV) exhibit higher levels of oxidative stress biomarkers [8-hydroxydeoxyguanosine (8-OHdG) and oxidized glutathione (GSSG)] and bone resorption regulators [receptor activator of nuclear factor (NF-κB) ligand (RANKL) and osteoprotegerin (OPG)] compared to EBV-negative periapical lesions and healthy pulp tissues. The experimental group consisted of 30 EBV-positive and 30 EBV-negative periapical lesions collected in conjunction with apicoectomy. The pulp tissues of 20 impacted third molars were used as healthy controls. The qualitative and quantitative analysis of EBV was performed by nested and real-time polymerase chain reaction (PCR), respectively. The levels of RANKL and OPG were analysed by reverse transcriptase real-time PCR. The levels of 8-OHdG and GSSG were determined by enzyme-linked immunosorbent assay (ELISA). Mann-Whitney U-test and Spearman's correlation were used for statistical analysis. The levels of RANKL, OPG, 8-OHdG and GSSG were significantly higher in apical periodontitis lesions compared to healthy pulp controls (P=0.001, P<0.001, P<0.001 and P<0.05, respectively). RANKL and OPG mRNA expression was significantly higher in EBV-positive compared to EBV-negative periapical lesions (P<0.05). There was no significant correlation between EBV copy numbers and levels of RANKL, OPG, 8OH-dG and GSSG in apical periodontitis. Levels of bone resorption regulators and oxidative stress biomarkers were increased in apical periodontitis compared to healthy pulp tissues. EBV-positive periapical lesions exhibited higher levels of RANKL and OPG compared to EBV-negative periapical lesions. EBV may contribute to progression of apical periodontitis via enhanced production of bone resorption regulators.

Anti-osteoporotic activities of the collagen extracted from Carapacis et Plastri Testudinis on a ROS1728 cell line in vitro

Purpose: The decoction of collagen extracted from Carapacis et Plastri Testudinis (CCPT) has been reported to have an effect of anti-osteoporosis. The present study was to investigate the in vitro molecular mechanisms effect of CCPT on ROS1728 differentiation and proliferation. Methods: Rat osteoblast cell line (ROS 1728) was cultured and exposed to CCPT at different concentrations. The effect of CCPT on the proliferation of ROS1728 was evaluated through detecting the activity of Alkaline Phosphatase (ALP) and the type I collagen (Coll-I) content. The gene expression of ALP, Coll-I, Osteocalcin (OC), Runx2, Osterix (Osx), Osteoprotegerin (OPG) and RANKL (receptor activator of NF-MB ligand) was observed using RT-PCR analysis. Results: The results showed that CCPT treatment (2.5, 5.0 and 10.0 mg/ml) affected the proliferation of ROS1728 in a concentration-dependent manner. The highest expression was obtained by treatment with 5 mg/ml of CCPT after a nine-day treatment duration based on ALP activity and Coll-I content (P<0.01) with the control group, whereas, the expression of ALP, Coll-I, OC, Runx2, Osx and OPG mRNA was facilitated (P<0.01). Only the level of RANKL mRNA decreased compared with the control group (P<0.01). Conclusion: Use of CCPT can induce ROS1728 proliferation into osteoblasts and enhance the function of osteogenesis through regulating the gene expression of Runx2 and the osteogenesis-specific transcription factors of Osx, and inhibit bone absorption by OPG/RANKL/RANK signaling pathways.

A study on OPG/RANK/RANKL axis in osteoporotic bile duct-ligated rats and the involvement of nitrergic and opioidergic systems.

Chronic liver disease (CLD) affects millions of people and its impact on bone loss has become a subject of interest. Nitric oxide and endogenous opioids are suggested to increase during cholestasis/cirrhosis and may impact bone resorption by different mechanisms. The receptor activator of nuclear factor-κB (RANK)/RANK-ligand (RANKL)/osteoprotegerin (OPG) signaling pathway regulates bone resorption, but its role in metabolic bone disease subsequent to CLD is unknown. We aimed to investigate the involvement of nitrergic and opioidergic systems in bone loss relative to the RANK/RANKL/OPG pathway, in bile duct-ligated (BDL) rats. Eighty BDL/sham-operated (SO) rats received injections of 3 mg/kg/day Nω-Nitro-L-arginine methyl ester ± naltrexone (10 mg/kg/day) or saline for 28 days. Plasma bone turnover markers, OPG, RANK, and RANKL along with mRNA expression levels of the latter three were assessed. Plasma bone turnover markers and OPG level increased, but RANKL decreased in the BDL group compared with their SO controls (both: P ≤ 0.05). Administration of naltrexone reduced bone turnover markers and OPG level while increased RANKL content in comparison to BDL rats (P ≤ 0.05). As compared to untreated BDL rats, nitric oxide inhibition showed no effect on bone turnover marker i.e. OPG, RANK, and RANKL levels. BDL significantly increased RANK mRNA, but had no significant effect on RANKL and OPG mRNA expression. The lack of association between plasma levels and quantitative gene expression of RANKL and OPG suggests an indirect function of these markers in BDL rats. Considering that opioid receptor blockage by naltrexone in BDL animals caused a significant decrease in OPG and an increase in RANKL plasma contents, it could be postulated that the opioidergic system may have a regulatory effect on these bone markers.

Icariin Restores Bone Structure and Strength in a Rat Model of Chronic High-Dose Alcohol-Induced Osteopenia

Background/Aims: Chronic alcohol abuse is an important risk factor for osteopenia. However, few studies have focused on the efficacy and mechanism of action of icariin on alcohol-induced osteopenia. The aim of this study was to investigate the efficacy and underlying mechanism of action of icariin in the treatment of chronic high-dose alcohol-induced osteopenia in a rat model. Methods: Thirty-six adult male Sprague-Dawley rats were randomly divided into four groups: sham, alcohol, and low-dose and high-dose icariin groups. Bone volume fraction (BV/TV), bone mineral density (BMD), bone biomechanical properties, and bone morphology were assessed after 16 weeks. Reverse-transcription PCR was used to detect mRNA expression levels of alkaline phosphatase (ALP), collagen type I (Col I), osteocalcin (OC), runt-related transcription factor 2 (Runx2), bone morphogenetic protein-2 (BMP-2), and osteoprotegerin (OPG). Results: Bone metabolic markers and biomechanical properties in the alcohol group were decreased significantly compared with the sham group. BV/TV, BMD, mineral apposition rate (MAR), percent trabecular area (%Tb.Ar), and bone biomechanical properties were elevated in the low-dose and high-dose icariin groups relative to the alcohol group. ALP, Col I, OC, Runx2, BMP-2, and OPG mRNA levels in the icariin group were significantly elevated in comparison with the alcohol group. Conclusion: Icariin can prevent overall progression of chronic high-dose alcohol-induced osteopenia in a rat model, in a dose-dependent manner. Icariin promotes bone formation and inhibits bone loss, and effectively restores bone structure and strength in chronic high-dose alcohol-induced osteopenic rats. Bone metabolism reversal is evidenced by increased BV/TV, BMD, MAR, %Tb.Ar, and biomechanical properties and elevated ALP, Col I, OC, Runx2, BMP-2, and OPG mRNA levels.

RANKL/RANK/OPG cytokine receptor system: mRNA expression pattern in BPH, primary and metastatic prostate cancer disease

The cytokine system RANKL (receptor activator of NF-κB ligand), its receptor RANK and the antagonist OPG (osteoprotegerin) play a critical role in bone turnover. Our investigation was conducted to describe the gene expression at primary tumour site in prostate cancer patients and correlate the results with Gleason Score and PSA level. Seventy-one samples were obtained from prostate cancer patients at the time of radical prostatectomy and palliative prostate resection (n=71). Patients with benign prostate hyperplasia served as controls (n=60). We performed real-time RT-PCR after microdissection of the samples. The mRNA expression of RANK was highest in tumour tissue from patients with bone metastases (p<0.001) as compared to BPH or locally confined tumours, also shown in clinical subgroups distinguished by Gleason Score (<7 or≥7, p=0.028) or PSA level (<10 or≥10µg/l, p=0.004). RANKL and OPG mRNA expression was higher in tumour tissue from patients with metastatic compared to local disease. The RANKL/OPG ratio was low in normal prostate tissue and high tumours with bone metastases (p<0.05). Expression of all three cytokines was high in BPH tissue but did not exceed as much as in the tumour tissue. We demonstrated that RANK, RANKL and OPG are directly expressed by prostate cancer cells at the primary tumour site and showed a clear correlation with Gleason Score, serum PSA level and advanced disease. In BPH, mRNA expression is also detectable, but RANK expression does not exceed as much as compared to tumour tissue.

Effects of triethylene glycol dimethacrylate (TEGDMA) on the odontoclastic differentiation ability of human dental pulp cells.

Objectives:The primary purpose of this study was to examine the effects of triethylene glycol dimethacrylate (TEGDMA) on odontoclastic differentiation in the dental pulp tissue.Material and Methods:The effects of different TEGDMA dosages on the odontoclastic differentiation capability of dental pulp cells were analyzed in vitro using the following methodologies: i) flow cytometry and tartrate-resistant acid phosphatase (TRAP) staining; ii) apoptotic effects using Annexin V staining; iii) mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor (NF)-kB ligand (RANKL) genes by quantitative Real-time PCR (qRT-PCR); and iv) OPG and RANKL protein expression by enzyme-linked immunosorbent assay (ELISA).Results:TEGDMA caused relatively less odontoclastic differentiation in comparison with the control group; however, odontoclastic differentiation augmented with increasing doses of TEGDMA (p<0.05). The mRNA and protein expression of OPG was lower in TEGDMA treated pulp cells than in the control group (p<0.05). While the mRNA expression of RANKL remained unchanged compared to the control group (p>0.05), its protein expression was higher than the control group (p<0.05). In addition, TEGDMA increased the apoptosis of dental pulp cells dose dependently.Conclusions:TEGDMA reduced the odontoclastic differentiation ability of human dental pulp cells. However, odontoclastic differentiation ratios increased proportionally with the increasing dose of TEGDMA.

P38 MAPK and Its Upstream Activators MKK3 and MKK6 Play an Important Role on Lipopolysaccharide-Induced RANKL Expression by Periodontal Ligament Cells

Resident, non-immune cells express various pattern-recognition receptors and produce inflammatory cytokines in response to microbial antigens, during the innate immune response. Alveolar bone resorption is the hallmark of destructive periodontitis and it is caused by the host response to bacteria and their mediators present on the biofilm. The balance between the expression levels of receptor activator of nuclear factorkappa B ligand (RANKL) and osteoprotegerin (OPG) is pivotal for osteoclast differentiation and activity and has been implicated in the progression of bone loss in periodontitis. To assess the contribution of resident cells to the bone resorption mediated by innate immune signaling, we stimulated fibroblasts and osteoblastic cells with LPS from. Escherichia coli (TLR4 agonist), Porphyromonas gingivalis (TLR2 and -4 agonist), and interleukin-1 beta (as a control for cytokine signaling through Toll/IL-1receptor domain) in time-response experiments. Expression of RANKL and OPG mRNA was studied by RT-PCR, whereas the production of RANKL protein and the activation of p38 MAPK and NF-kB signaling pathways were analyzed by western blot. We used biochemical inhibitors to assess the relative contribution of p38 MAPK and NF-kB signaling to the expression of RANKL and OPG induced by TLR2, -4 and IL1β in these cells. Both p38 MAPK and NFkB pathways were activated by these stimuli in fibroblasts and osteoblasts, but the kinetics of this activation varied in each cell type and with the nature of the stimulation. E. coli LPS was a stronger inducer of RANKL mRNA in fibroblasts, whereas LPS from P. gingivalis downregulated RANKL mRNA in periodontal ligament cells but increased its expression in osteoblasts. IL-1β induced RANKL in both cell types and without a marked effect on OPG expression. p38 MAPK was more relevant than NF-kB for the expression of RANKL and OPG in these cell types.

Isopsoralen regulates PPAR‑γ/WNT to inhibit oxidative stress in osteoporosis.

The present study aimed to examine the effects of isopsoralen against postmenopausal osteoporosis in an ovariectomized rat model. The ovariectomized rats were treated with three days 10mg/kg isopsoralen or with three days 20mg/kg isopsoralen. Alkaline phosphatase, the oxidative stress indicators and caspase‑3/9 were measured using ELISA assay kits. Reverse transcription‑quantitative polymerase chain reaction was used to measure collagen type I (Col I), osteocalcin and osteoprotegerin mRNA levels. Wnt, β‑catenin and peroxisome proliferators‑activated receptor γ (PPAR‑γ) were analyzed using western blot analysis. Isopsoralen suppressed mature adipocyte differentiation of C2C12 cells, inhibited serum calcium and urinary calcium levels, and reduced the structural scores of articular cartilage and cancellous bone in the proximal tibia metaphysis of mice with postmenopausal osteoporosis. Isopsoralen also promoted the activity of alkaline phosphatase and the mRNA expression levels of Col 1, osterix and osteopontin in mice with postmenopausal osteoporosis. Oxidative stress and activities of caspase‑3/9 in the mice with postmenopausal osteoporosis were effectively suppressed by isopsoralen treatment, which upregulated the protein expression of Wnt/β‑catenin and downregulated the protein expression of PPAR‑γ. These findings demonstrated that isopsoralen prevented osteoporosis through the regulation of PPAR‑γ/WNT, inhibiting oxidative stress by targeting the PPAR‑γ/WNT pathway. These results provide evidence of the potential targeted therapy for isopsoralen in the clinical treatment of postmenopausal osteoporosis.

Differential expression of osteopontin, and osteoprotegerin mRNA in epicardial adipose tissue between patients with severe coronary artery disease and aortic valvular stenosis: association with HDL subclasses

BackgroundPrevious studies suggest a relationship of the epicardial adipose tissue (EAT) with progression and calcification of the atherosclerotic plaque; however, it is unknown if this tissue expresses genes that may participate on these processes and if the expression of these genes is regulated by high-density lipoprotein (HDL) subclasses.MethodsTo explore this possibility, we determined the mRNA expression by qPCR of a pro-calcifying gene (osteopontin (OPN)), and two anti-calcifying genes (osteoprotegerin (OPG) and osteonectin (ON)), in biopsies of EAT obtained from 15 patients with coronary artery disease (CAD) determined by angiography, and 15 patients with diagnostic of aortic valve stenosis but without CAD as control group. We determined the distribution and composition of HDL subclasses by electrophoresis and their statistical relationship with the gene expression in EAT.ResultsEAT from CAD patients showed a higher expression level of OPN and OPG than control group, whereas ON expression was similar between groups. Large HDL subclasses were cholesterol-poor in CAD patients as estimated by the cholesterol-to-phospholipid ratio. A linear regression model showed an independent association of OPN expression with HDL3a-cholesterol, and OPG expression with the relative proportion of HDL3b protein. Logistic analysis determined that OPN expression was positively associated with the presence of atherosclerotic plaqueConclusionOPN, ON, and OPG genes are transcribed in EAT; to the exception of ON, the level of expression was different in CAD patients and control group, and correlated with some HDL subclasses, suggesting a new role of these lipoproteins.

Osteoprotegerin Is a New Regulator of Inflammation and Angiogenesis in Proliferative Diabetic Retinopathy.

Osteoprotegerin (OPG) is a novel regulator of endothelial cell function, angiogenesis, and vasculogenesis. We correlated expression levels of OPG with those of the angiogenic and inflammatory factors vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1/CCL2) in proliferative diabetic retinopathy (PDR). We also examined expression of OPG in retinas from diabetic rats and diabetic patients and measured production of OPG by human retinal microvascular endothelial cells (HRMEC) and investigated its angiogenic activity. Vitreous samples from 47 PDR and 28 nondiabetic patients, epiretinal membranes from 14 patients with PDR, human retinas (10 from diabetic patients and 10 from nondiabetic subjects), and rat retinas and HRMEC were studied by using enzyme-linked immunosorbent assay, immunohistochemistry, immunofluorescence, Western blot analysis, and RT-PCR. In vitro and in vivo angiogenesis assays were performed. We showed a significant increase in the expression of OPG, VEGF, and MCP-1/CCL2 in a comparison between vitreous samples from PDR patients and those from nondiabetic controls. Significant positive correlations were found between levels of OPG and levels of VEGF and MCP-1/CCL2. In epiretinal membranes, OPG was expressed in vascular endothelial cells and stromal cells. Significant increases of OPG mRNA and protein were detected in the retinas from diabetic patients. The proinflammatory cytokines TNF-α and IL-1β, but not VEGF, MCP-1/CCL2 or thrombin, induced upregulation of OPG in HRMEC. Osteoprotegerin induced ERK1/2 and Akt phosphorylation in HRMEC and stimulated their migration. Osteoprotegerin potentiated the angiogenic effect of VEGF in the in vivo protein gelatin plug assay. These results suggest that OPG is involved in PDR angiogenesis.

(-)-Epigallocatechin gallate but not chlorogenic acid upregulates osteoprotegerin synthesis through regulation of bone morphogenetic protein-4 in osteoblasts.

Chlorogenic acid (CGA) is a primary phenolic component of coffee and (-)-epigallocatechin gallate (EGCG) is a primary flavonoid component of green tea, both of which have been documented to possess beneficial health properties. A previous study by the present authors demonstrated that p38 mitogen-activated protein kinase (MAPK) may be associated with osteoprotegerin synthesis stimulated by bone morphogenetic protein-4 (BMP-4) in osteoblast-like MC3T3-E1 cells. In the present study, the effects of CGA and EGCG on BMP-4-stimulated osteoprotegerin synthesis in MC3T3-E1 cells were investigated. It was observed that CGA had no effect on osteoprotegerin release stimulated by BMP-4, whereas EGCG significantly enhanced BMP-4-stimulated osteoprotegerin release (P=0.003). Levels of osteoprotegerin mRNA expression induced by BMP-4 were also significantly increased by EGCG (P=0.03). By contrast, EGCG had no significant effect on phosphorylation of Smad1 or p38 MAPK induced by BMP-4. In addition, EGCG had little effect on BMP-induced phosphorylation of p70 S6 kinase; however rapamycin, as an inhibitor of p70 S6 kinase, significantly suppressed osteoprotegerin release (P=0.007). These data suggest that EGCG but not CGA may upregulate the synthesis of osteoprotegerin induced by BMP-4 in osteoblasts.

Amplification by (‑)‑epigallocatechin gallate of prostaglandin F2α‑stimulated synthesis of osteoprotegerin in osteoblasts.

(‑)‑Epigallocatechin gallate (EGCG) and chlorogenic acid (CGA), major flavonoids in green tea, and coffee, respectively, are recognized as possessing potential benefits in a multitude of human health conditions, including bone disorders. We have previously demonstrated that prostaglandin F2α (PGF2α), a potent bone remodeling mediator, stimulates the synthesis of osteoprotegerin (OPG) through the activation of p44/p42 mitogen‑activated protein kinase (MAPK), p38 MAPK and stress activated protein kinase/c‑Jun N‑terminal kinase (SAPK/JNK) in osteoblast‑like MC3T3‑E1 cells. In the present study, the effects of EGCG and CGA on PGF2α‑stimulated OPG synthesis in MC3T3‑E1 cells were investigated. EGCG significantly upregulated PGF2α‑stimulated OPG release, whereas CGA did not affect OPG release. The PGF2α‑induced expression level of OPG mRNA was enhanced by EGCG. Regarding the intracellular signaling underlying the effect of EGCG, EGCG failed to affect PGF2α‑stimulated phosphorylation of p44/p42 MAPK, p38 MAPK or SAPK/JNK. EGCG by itself markedly induced the phosphorylation of p44/p42 MAP kinase for up to 10min and the status decreased subsequently, whereas EGCG did not significantly affect the phosphorylation status of p38 MAPK or SAPK/JNK within 60min. These results indicated that EGCG, but not CGA amplifies the PGF2α‑stimulated OPG synthesis in osteoblasts.

Regulatory effects of bone morphogenetic protein-4 on tumour necrosis factor-α-suppressed Runx2 and osteoprotegerin expression in cementoblasts.

Root resorption is a common phenomenon presented in periodontitis and orthodontic treatment, both of which are accompanied by an elevated TNF-α expression level in the periodontal tissues. Previously, we proved that TNF-α showed an inhibitory effect on cementoblast differentiation, mineralization and proliferation. However, the effect of TNF-α on Runx2 and osteoprotegerin (OPG) expression remains undetermined. This study aimed to identify the influence of TNF-α on Runx2 and OPG expression in cementoblasts and to test whether BMP-2,-4,-6,-7 would affect TNF-α-regulated Runx2 and OPG. An immortalized murine cementoblast cell line OCCM-30 was used in this study. The expression of Runx2 and OPG were examined by qRT-PCR after stimulating cells with TNF-α. The role of signalling pathways, including MAPK, PI3K-Akt and NF-κB, were studied with the use of specific inhibitors. Cells were treated with TNF-α in combination with BMP-2,-4,-6 or -7, then the expression of Runx2 and OPG, the activity of MAPK and NF-κB pathways, and the proliferation ability were evaluated by qRT-PCR, Western blot and MTS assay respectively. TNF-α inhibited Runx2 and OPG mRNAs in OCCM-30 cells, and the inhibitory effects were further aggravated by blocking p38 MAPK or NF-κB pathway. TNF-α-inhibited Runx2 and OPG were up-regulated by BMP-4. The p38 MAPK and Erk1/2 pathways were further activated by the combined treatment of BMP-4 and TNF-α compared with TNF-α alone. Finally, the TNF-α-suppressed proliferation was not obviously affected by BMP-2,-4,-6 or -7. TNF-α inhibited Runx2 and OPG in cementoblasts, and the p38 MAPK and NF-κB pathways acted in a negative-feedback way to attenuate the inhibitory effects. TNF-α-inhibited Runx2 and OPG could be effectively up-regulated by BMP-4; however, further investigations are needed to fully elaborate the underlying mechanisms.

Cabozantinib targets bone microenvironment modulating human osteoclast and osteoblast functions.

Cabozantinib, a c-MET and vascular endothelial growth factor receptor 2 inhibitor, demonstrated to prolong progression free survival and improve skeletal disease-related endpoints in castration-resistant prostate cancer and in metastatic renal carcinoma. Our purpose is to investigate the direct effect of cabozantinib on bone microenvironment using a total human model of primary osteoclasts and osteoblasts.Osteoclasts were differentiated from monocytes isolated from healthy donors; osteoblasts were derived from human mesenchymal stem cells obtained from bone fragments of orthopedic surgery patients. Osteoclast activity was evaluated by tartrate resistant acid phosphatase (TRAP) staining and bone resorption assays and osteoblast differentiation was detected by alkaline phosphatase and alizarin red staining.Our results show that non-cytotoxic doses of cabozantinib significantly inhibit osteoclast differentiation (p=0.0145) and bone resorption activity (p=0.0252). Moreover, cabozantinib down-modulates the expression of osteoclast marker genes, TRAP (p=0.006), CATHEPSIN K (p=0.004) and Receptor Activator of Nuclear Factor k B (RANK) (p=0.001). Cabozantinib treatment has no effect on osteoblast viability or differentiation, but increases osteoprotegerin mRNA (p=0.015) and protein levels (p=0.004) and down-modulates Receptor Activator of Nuclear Factor k B Ligand (RANKL) at both mRNA (p<0.001) and protein levels (p=0.043). Direct cell-to-cell contact between cabozantinib pre-treated osteoblasts and untreated osteoclasts confirmed the indirect anti-resorptive effect of cabozantinib.We demonstrate that cabozantinib inhibits osteoclast functions “directly” and “indirectly” reducing the RANKL/osteoprotegerin ratio in osteoblasts.

Abstract P6-08-10: Osteoprotegerin mediates tumor-promoting effects of Interleukin-1beta in breast cancer cells

Abstract It is widely recognized that inflammation promotes breast cancer invasion and metastasis. Given the complex nature of the breast tumor inflammatory microenvironment, much remains to be understood of the molecular mechanisms that govern these effects. We have previously shown that osteoprotegerin (OPG) knockdown in breast cancer cells resulted in reduced invasion and metastasis. Here we present novel insight into the role of OPG in inflammation-driven tumor progression in breast cancer cells by investigating the link between OPG and the potent pro-inflammatory cytokine IL1B (IL1B). We used human breast cancer cell lines to investigate the effects of IL1B treatment on OPG expression. We analyzed public datasets containing human breast cancer genome-wide mRNA expression data to reveal a significant and positive correlation between OPG mRNA expression and the mRNA expression of IL1B as well as CCL2 (a monocyte chemoattractant protein). We determined the effect of macrophages (which produce IL1B) on OPG expression by co-culturing breast cancer cells and differentiated THP-1 macrophages. In order to demonstrate that OPG mediated functional effects of IL1B we performed cell invasion studies with control and OPG siRNA knockdown breast cancer cells treated with IL1B. We report that OPG expression is induced by IL1B, independent of breast cancer subtype and basal OPG levels. Co-culture of breast cancer cells with IL1B-secreting macrophages resulted in a similar increase in OPG expression in breast cancer cells as IL1B treatment. We show that OPG expression is regulated by IL1B in a p38-dependent manner. We also demonstrate that OPG knockdown represses IL1B expression, IL1B-mediated breast cancer cell invasion and MMP3 expression.It is widely recognized that inflammation promotes breast cancer invasion and metastasis. Given the complex nature of the breast tumor inflammatory microenvironment, much remains to be understood of the molecular mechanisms that govern these effects. We have previously shown that osteoprotegerin (OPG) knockdown in breast cancer cells resulted in reduced invasion and metastasis. Here we present novel insight into the role of OPG in inflammation-driven tumor progression in breast cancer cells by investigating the link between OPG and the potent pro-inflammatory cytokine IL1B (IL1B). We used human breast cancer cell lines to investigate the effects of IL1B treatment on OPG expression. We analyzed public datasets containing human breast cancer genome-wide mRNA expression data to reveal a significant and positive correlation between OPG mRNA expression and the mRNA expression of IL1B as well as CCL2 (a monocyte chemoattractant protein). We determined the effect of macrophages (which produce IL1B) on OPG expression by co-culturing breast cancer cells and differentiated THP-1 macrophages. In order to demonstrate that OPG mediated functional effects of IL1B we performed cell invasion studies with control and OPG siRNA knockdown breast cancer cells treated with IL1B. We report that OPG expression is induced by IL1B, independent of breast cancer subtype and basal OPG levels. Co-culture of breast cancer cells with IL1B-secreting macrophages resulted in a similar increase in OPG expression in breast cancer cells as IL1B treatment. We show that OPG expression is regulated by IL1B in a p38-dependent manner. We also demonstrate that OPG knockdown represses IL1B expression, IL1B-mediated breast cancer cell invasion and MMP3 expression. Citation Format: Tsang Mui Chung S, Geerts D, Roseman K, Renaud A, Connelly L. Osteoprotegerin mediates tumor-promoting effects of Interleukin-1beta in breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-08-10.

Initial osteoblast functions on a type of near β-type titanium alloys surfaces modified by the double glow plasma nitriding technology

Objective: To evaluate the adhesion, proliferation, alkaline phosphatase (ALP) activity and the expression of osteogenesis-related genes and osteoprotegerin (OPG)/receptor activator of NF-κB ligand (RANKL) of osteoblast-like cells on a type of near β-type titanium alloys (Ti-5Zr-3Sn-5Mo-15Nb, TLM) surfaces modified by the double glow plasma nitriding technology, and to investigate the effect of the modified surfaces on the initial functions of osteoblast-like cells. Methods: The surfaces of TLM were modified by the double glow plasma nitriding technology. TLM surfaces without modification were used as control. Cell morphology was observed with scanning electron microscopy (SEM). Methyl thiazolyl tetrazolium (MTT) method was used to measure cell proliferation. Cell ALP activity was evaluated by using reagent kits. The mRNA expression of Runt-related transcription factor-2 (RUNX2), typeⅠcollagen alpha 1 chain (COLⅠ α1) and OPG/RANKL were examined by quantitative real-time PCR(qRT-PCR). Results: Four hour following cell alture, cells on modified surfaces extend filopodia and intercellular junction was tight. Three days later, cell proliferation (0.277±0.007) was significantly higher than that in control group (0.249±0.004) (P<0.01). After two weeks, ALP activity on TLM modified layer (173.6±1.89) was significantly higher than that on unmodified TLM (162.6±2.4) (P<0.01). The mRNA expression of osteoblast marker RUNX2, COLⅠα1 were stronger than that in control group (P<0.05). The expression of OPG mRNA was higher than that in control group (P<0.01), and RANKL mRNA expression was significantly lower than that in control group (P<0.05). Conclusions: The TLM surface modified by the double glow plasma nitriding technology has a positive effect on osteoblasts initial adhesion, proliferation and differentiation, and it can also improve expression of OPG mRNA and has an inhibitory effect on RANKL mRNA expression of osteoblasts.

Attenuation of prostaglandin E1‑induced osteoprotegerin synthesis in osteoblasts by normoxic HIF inducers.

Mimosine, which is a natural plant amino acid present in the Leucaena genus, is able to induce hypoxia‑inducible factors (HIFs). Previous evidence has indicated that HIF regulates angiogenesis‑osteogenesis coupling in bone metabolism, and it has previously been reported that mimosine inhibits prostaglandin (PG)F2α‑induced osteoprotegerin (OPG) synthesis without affecting interleukin‑6 (IL‑6) production in osteoblast‑like MC3T3‑E1 cells. In addition, PGE1 has been demonstrated to induce OPG synthesis via activation of p38 mitogen‑activated protein (MAP) kinase and stress‑activated protein kinase/c‑Jun N‑terminal kinase (SAPK/JNK) in these cells, and PGE1 stimulates IL‑6 production via the activation of protein kinase A. In the present study, the effects of mimosine on the PGE1‑stimulated synthesis of OPG and IL‑6 were investigated in osteoblast‑like MC3T3‑E1 cells. The concentrations of OPG and IL‑6 were measured using relevant ELISA kits. OPG mRNA was measured by semi‑quantitative reverse transcription polymerase chain reaction. The phosphorylation of p38 MAP kinase and SAPK/JNK was analyzed by western blotting. Mimosine significantly reduced PGE1‑induced release of OPG and OPG mRNA expression levels without affecting the release of IL‑6. In addition, deferoxamine, which is also a normoxic HIF inducer, significantly inhibited PGE1‑induced OPG release and OPG mRNA expression levels; however, it had little effect on IL‑6 release. Furthermore, mimosine and deferoxamine failed to affect PGE1‑stimulated phosphorylation of p38 MAP kinase or SAPK/JNK. These results strongly suggest that normoxic HIF inducers attenuate PGE1‑stimulated OPG synthesis without affecting IL‑6 production in osteoblasts.