Effect Of Osteoprotegerin Research Articles

Osteoprotegerin Is Essential for the Development of Endothelial Dysfunction Induced by Angiotensin II in Mice.

Opinions on the effects of osteoprotegerin (OPG) have evolved over the years from a protein protecting the vasculature from calcification to a cardiovascular risk factor contributing to inflammation within the vascular wall. Nowadays, the link between OPG and angiotensin II (Ang II) appears to be particularly important. In this study, the endothelial function was investigated in OPG-knockout mice (B6.129.S4-OPG, OPG-) and wild-type (C57BL/6J, OPG+) mice under basic conditions and after Ang II exposure by assessing the endothelium-dependent diastolic response of aortic rings to acetylcholine in vitro. A further aim of the study was to compare the effect of Ang II on the expression of cytokines in the aortic wall of both groups of mice. Our study shows that rings from OPG- mice had their normal endothelial function preserved after incubation with Ang II, whereas those from OPG+ mice showed significant endothelial dysfunction. We conclude that the absence of OPG, although associated with a pro-inflammatory cytokine profile in the vascular wall, simultaneously protects against Ang II-induced increases in pro-inflammatory cytokines in the murine vascular wall. Our study also demonstrates that the absence of OPG can result in a decrease in the concentration of pro-inflammatory cytokines in the vascular wall after Ang II exposure. The presence of OPG is therefore crucial for the development of Ang II-induced inflammation in the vascular wall and for the development of Ang II-induced endothelial dysfunction.

Bone mineral density in egyptian children with juvenile idiopathic arthritis: possible correlation to serum RANKL / osteoprotegerin (OPG) ratio and OPG gene polymorphisms

BackgroundChildren with juvenile idiopathic arthritis (JIA) are at higher risk of decreased bone mineral density (BMD) compared with healthy children due to genetic, disease and medication-related causes. This study aims to investigate the possible effects of osteoprotegerin (OPG) gene polymorphisms and serum levels of osteoprotegerin (OPG) and receptor activator of nuclear factor κB-ligand (RANKL) and RANKL/OPG ratio on BMD in children with JIA.MethodsOPG gene rs2073617, rs3134069, serum RANKL, OPG and RANKL/OPG ratio were evaluated in 60 JIA children and 100 matched healthy controls. BMD was evaluated by lumbar dual energy X-ray absorptiometry (DEXA) according to which patients were classified in 2 groups (DEXA z-score above and below − 2). Composite disease activity was measured using the Juvenile Arthritis Disease Activity Score (JADAS) 27-joints. Articular damage was scored using the juvenile arthritis damage index (JADI).ResultsPatients aged 12.05 ± 3.2 years, included 38 females and 31% had BMD z-score below-2. Systemic-onset JIA was the most frequent phenotype (38%). Genotypes and alleles frequencies of the 2 studied polymorphisms did not differ between patients and controls (p > 0.05 for all) while serum RANKL and RANKL/OPG ratio were significantly higher in patients compared to controls (p = < 0.001 and 0.03 respectively). Patients with BMD < -2 had significantly greater frequencies of rs2073617 TT genotype and T allele (p < 0.001), higher serum RANKL, RANKL/OPG ratio (p = 0.01, 0.002), female predominance (p = 0.02), higher articular and extra-articular damage index (p = 0.008,0.009) and more frequent steroid usage (p = 0.02) compared to patients with BMD z-score >-2. Multivariate analysis showed rs2073617 TT genotype, RANKL/OPG ratio, long disease duration (above 36 months) and use of steroid to be associated with decreased BMD (p = 0.03,0.04,0.01,0.01 respectively) in JIA children.ConclusionsEgyptian children with JIA have decreased BMD. rs2073617 TT genotype and T allele, RANKL/OPG ratio are possible determinants of reduced BMD in JIA. Our results underline the importance of frequent monitoring of BMD in JIA children and trying to control disease activity to preserve long term bone health.

Abstract WMP32: Osteoprotegerin Attenuates the Growth of Intracranial Aneurysms Activating Collagen Biosynthesis and Vascular Smooth Muscle Cell Proliferation via Transforming Growth Factor-Beta1 Signaling

Introduction: The pathogenesis of intracranial aneurysms (IAs) are characterized by reduced extracellular matrix and decreased number of vascular smooth muscle cells (VSMCs) in cerebral vascular walls. It is previously reported that osteoprotegerin (OPG) increase collagen content and activate VSMCs proliferation via transforming growth factor-beta1 (TGF-beta1). We investigated whether to attenuate IAs growth through the effect of OPG on collagen expression and VSMCs proliferation in IAs model rats. Materials and Methods: IAs were surgically induced in 7-week-old male Sprague Dawley rats. 1 week after the operation, mouse recombinant OPG at 125 μg/mL or vehicle was continuously infused into the lateral ventricle via an osmotic pump. 5 weeks after the first operation, aneurysmal size, media thickness of IAs were measured in the both groups. The expression of type-I and type-III collagen, TGF-beta1 and phosphorylated Smad2/3 in IA walls were examined by immunohistochemistry and RT-PCR. Primary cell culture of mouse VSMCs were analyzed by RT-PCR and the cell proliferation assay with SB431542 (a selective TGF-β receptor I inhibitor). Results: In the OPG treatment group, size and media thickness ratio of IAs were significantly smaller (control vs. OPG: 42 ± 6.5 μm vs 66 ± 6.1 μm; P &lt; 0.05) and higher (control vs. OPG: 55 ± 7 % vs 34 ± 3 %; P &lt; 0.05) than in the vehicle-control group. In the immunohistochemistry, collagen and phosphorylated Smad2/3 were upregulated in IA walls in the OPG group. In the RT-PCR study, OPG treatment significantly upregulated expression of collagen and TGF-beta1 genes ( P &lt; 0.05). In mouse VSMC cultures, OPG administration enhanced the expression of collagen and TGF-β1 genes. The cell proliferation assay revealed the OPG administration promoted VSMCs proliferation and SB431542 canceled this effect ( P &lt; 0.05). Conclusions: Our results demonstrate that OPG has a suppressive effect on IAs growth through the activation of collagen biosynthesis and VSMC proliferation via TGF-beta1 signaling in IA walls. OPG may represent a novel therapeutic target of the medical treatment for IAs.

Osteoprotegerin promotes intimal hyperplasia and contributes to in-stent restenosis: Role of an αVβ3/FAK dependent YAP pathway

ObjectiveAbnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are related to in-stent-restenosis (ISR) following percutaneous coronary intervention (PCI). Osteoprotegerin (OPG) has been implicated in various vascular diseases. However, the effects of OPG on ISR and the underlying mechanism remained elusive. We here investigated the association between OPG and ISR, and to demonstrate the role and potential mechanisms of OPG in neointimal hyperplasia. Approach and resultsFrom 2962 patients who received coronary angiography and follow-up coronary angiography at approximately one year, 291 patients were diagnosed with ISR, and another 291 gender- and age- matched patients without ISR were selected as controls. Serum OPG levels were significantly increased in patients with ISR. Multivariable logistic regression analysis indicated that OPG level was independently associated with the increased risk of ISR. In a mouse femoral artery wire injury model, upregulated OPG was evidenced in vascular tissue after injury. OPG deletion attenuated the vascular injury-induced neointimal hyperplasia and related gene expression in mice. OPG promoted neointimal hyperplasia and human aortic smooth muscle cell (hASMC) proliferation and migration through activation of yes-associated protein (YAP), a major downstream effector of the Hippo signaling pathway, whereas knockdown or inhibition of YAP in hASMCs blunted OPG-induced above effects. Moreover, we found that OPG, as a ligand for integrin αVβ3, mediated phosphorylation of focal adhesion kinase (FAK) and actin cytoskeleton reorganization, resulting in YAP dephosphorylation in hASMCs. OPG-dependent YAP and VSMC activation was prevented by treatment with αVβ3-blocking antibodies and inhibitors of FAK and actin stress fibers. ConclusionsIncreased serum OPG levels are associated with increased risk of ISR following PCI and OPG could promote neointimal hyperplasia in response to injury through integrin αVβ3 mediated FAK and YAP activation, indicating OPG/YAP inhibition might serve as an attractive novel target for the prevention of ISR after PCI.

Effect of Osteoprotegerin and XRCC3 Genes Polymorphisms with the Occurrence of Left Ventricular Hypertrophy in Hypertensive Patients

Background: High blood pressure is associated with adverse morphological and functional changes in the cardiovascular system, including left ventricular hypertrophy (LVH). Osteoprotegerin (OPG) is a member of the tumor necrosis factor receptor superfamily of cytokines. X-ray repair cross-complementing protein 3 (XRCC3) is involved in the repair pathway for double-strand breaks (DSBs). We assessed the association of osteoprotegerin and XRCC3 gene polymorphisms with the occurrence of left ventricular hypertrophy in hypertensive patients. Patients and methods: The study included 50 hypertensive patients: 25 with LVH (group A) and 25 without LVH (group B). All cases were subjected to complete history taking and clinical examination. ECG and echocardiography were done. LV mass was calculated to detect the presence or absence of LV hypertrophy. DNA was extracted from blood samples, and then, each DNA sample was amplified in PCRs, to detect osteoprotogrin and XRCC3 gene polymorphisms. Results: Mean age in the cases in group A is 63.12 years and in group B was 58.24 years with statistically significant difference between the two groups. The duration of the disease and SBP revealed statistically significant difference between the two groups. The LV mass index and E/A ratio revealed high statistically significant difference between the two groups. OPG sequence revealed no statistically significant difference between the two groups, but XRCC3 sequence revealed statistically significant difference. The age was a risk factor for LVH. Conclusion: Osteoprotogrin and XRCC3 genes polymorphism mutations may be associated with left ventricular hypertrophy in hypertensive patients.

Comparative study of the effects of osteoprotegerin and testosterone on bone quality in male orchidectomised rats

Aim: The aim of this work is to compare the effects of osteoprotegerin (OPG) and testosterone on bone quality in a model of orchidectomised (ORX) rats.Methods: Three-month-old ORX or SHAM operated groups (n = 15 each group) were used. The SHAM and ORX groups received saline. There were two ORX groups, receiving OPG–Fc (10 mg/kg twice weekly) (ORX + OPG–Fc) or testosterone cypionate (1.7 mg/kg/weekly) for 8 weeks. After sacrifice, bone analysis by femoral and lumbar dual-energy X-ray absorptiometry and micro-computed tomography in femora were performed. Histological sections of vertebrae were dyed with hematoxylin–eosin or safranin. Serum osteocalcin (BGP), total alkaline phosphatase (ALP), and C-terminal telopeptide of type I collagen (CTX) were analyzed.Results: ORX resulted in femoral and vertebral bone loss and in microarchitectural deterioration. Treatment with OPG–Fc and testosterone recovered lumbar (L) and femoral (F) bone mineral densitometry bone mineral density (BMD) to SHAM levels. Femoral BMD was significantly higher after treatment with OPG–Fc than after testosterone treatment due to the presence of osteopetrotic changes in the metaphyseal region of long bones. Serum levels of ALP and CTX increased, while OPG levels were unchanged in ORX rats. Treatment with OPG–Fc decreased the levels of BGP, ALP, and CTX. Treatment with testosterone maintained biochemical markers of bone turnover at levels similar to or higher than those of ORX rats.

Adipose-derived stem cell-released osteoprotegerin protects cardiomyocytes from reactive oxygen species-induced cell death

BackgroundThe paracrine effect is likely the major mechanism of the adipose-derived stem cell (ASC)-mediated cardioprotective effect. However, the exact composition and nature of ASC-released paracrine factors remain elusive. In the present study, we examined the effect of osteoprotegerin (OPG), a stem cell-released decoy receptor for death ligand, on the survival of cardiomyocytes exposed to oxidative stress.MethodsThe production of OPG from ASCs under oxidative stress was determined by ELISA and immunohistochemistry. The effects of OPG and the OPG-containing conditioned media of ASCs on the survival of cardiomyocytes were determined using a cell viability assay.ResultsHydrogen peroxide (H2O2) significantly increased OPG production from ASCs in vitro, and OPG production from the ASCs transplanted into the ischemia–reperfusion-injured heart was also observed. OPG significantly attenuated cardiomyocyte death in vitro. OPG-containing conditioned media of ASCs also significantly protected cardiomyocytes. Delivery of siRNA specific to OPG significantly decreased the OPG production of ASCs, and also offset the protective effect of the conditioned media of ASCs.ConclusionsOur study strongly suggests that OPG is one of the prosurvival factors released from ASCs that may contribute to the ASC-mediated cardioprotection and calls for further studies to elucidate detailed underlying mechanisms.

Effect of osteoprotegerin in combination with interleukin-6 on inhibition of osteoclast differentiation

ObjectiveTo observe the effect of recombinant interleukin-6 (IL-6) and osteoprotegerin (OPG) on inhibiting bone absorption induced by receptor activator for nuclear factor-κB ligand (RANKL) in murine osteoclast precursor cells (OCPs) model. MethodsRAW 264.7 cells were solely treated with 50 ng/ml RANKL for 1 day, and then they were divided into three groups: RANKL (control group), RANKL+IL-6 (IL-6 group) and RANKL+IL-6+OPG (combination group). These cells were harvested and investigated by means of HE staining under light microscope after consecutive 9 days. Furthermore, staining tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells were detected by inverted phase contrast microscope. The absorption pits of bone slices were observed under scanning electron microscope. ResultsThe number of mature osteoclast cells in control group was more than that in IL-6 alone or IL-6 combined with OPG group (P<0.05). Interestingly, this experiment has also demonstrated that there was a large number of TRAP-positive multinucleated osteoclasts (more than 3 nuclei) and several bone absorption formation in the control group, whereas the outcome was completely different in both IL-6 group and IL-6+OPG group (P<0.05). ConclusionIL-6 can suppress the differentiation of mature osteoclasts as directly adding it into the RAW 264.7 cells induced by 50 ng/ml RANKL, and further the effect of osteolysis is remarkably reduced. When treatment with IL-6 combined with OPG, a more effective strategy for the treatment of osteoporosis is reached.

Abstract MP08: Syndecan-1 is Involved in Osteoprotegerin-induced Vascular Dysfunction

Osteoprotegerin (OPG), an inhibitor of vascular calcification, has pleiotropic vascular effects independently of its actions on calcification. OPG has been associated with vascular inflammation and remodelling and may be important in cardiovascular disease where OPG levels may be elevated. Molecular mechanisms and functional consequences of OPG stimulation in the vasculature are unclear. We propose that syndecan-1, a membrane glycoprotein, may be important and that reactive oxygen species (ROS) play a role in OPG signalling. Vascular reactivity of resistance arteries from WKY rats was studied by wire myography in the presence or absence of OPG (50 ng/mL) and/or synstatin (SSNT - 10-6M - syndecan-1 inhibitor). Rat endothelial cells (EC) and vascular smooth muscle cells (VSMC) were studied. Levels of ROS were measured by chemiluminescence, Amplex Red (H2O2) and ELISA (nitrotyrosine; peroxynitrite - ONOO-). Protein oxidation and levels were measured by immunoblotting. Exposure of resistance arteries to OPG induced endothelial (decreased relaxation to acetylcholine) and VSMC (decreased relaxation to sodium nitroprusside - SNP) dysfunction, as well as, increased contraction to phenylephrine. All responses were blocked by SSNT, N-acetylcisteine (antioxidant) and ML171 (Nox inhibitor). In EC, OPG-induced ROS production (240±46.1% increase vs. veh, p&lt;0.05) was blocked by SSNT. OPG decreased H2O2 production/release (61±5.4% vs. veh) and increased eNOS Thr 495 phosphorylation (inhibitory site) (100±24% vs. veh, p&lt;0.05). In VSMC, OPG increased H2O2 (69±3%) and ONOO- (43±12%) levels, protein oxidation (61±15%), Rho kinase (200±39%) and myosin light chain activation (55±3%) (all vs. veh, p&lt;0.05). Increase in OPG-induced ONOO- levels was exacerbated by SNP (130±16% vs. veh, p&lt;0.05), a nitric oxide donor. In conclusion, vascular dysfunction elicited by OPG is mediated by syndecan-1 and ROS. Whether syndecan-1 also impacts on OPG-sensitive calcification is unclear. Our data identify a novel molecular mechanism through syndecan- 1/ROS that may underlie injurious effects of OPG.

The effect of A163G polymorphism in the osteoprotegerin gene on osteoporosis related traits in Slovak postmenopausal women.

Osteoprotegerin (OPG) plays an important role in the osteoclast differentiation as an effective inhibitor of osteoclast maturation and activation. We examined a potential effect of A163G single nucleotide polymorphism in the promoter region of the OPG gene on femoral neck (FN-BMD) and lumbar spine BMD (LS-BMD), as well as circulating alkaline phosphatase, osteocalcin (ALP, OC; formation markers), beta-CrossLaps (CTx; resorption marker) in Slovak postmenopausal women. In addition, fractures of spine, radius and femur were examined.Altogether 284 women (62.28 ± 8.40 years) were selected according to strict inclusion criteria. The polymorphism was detected by PCR-RFLP method. Genotype frequencies were tested using the chi-square test. The differences of quantitative variables between the genotypes were analyzed by covariance analysis (GLM procedure) after correction of the measurements for age and BMI. Fracture incidence in association with OPG genotype was evaluated by Binary Logistic Regression with the genotype, age, and BMI as covariates. The frequencies of genotypes were 76.8 %, 21.1 %, and 2.1 % for AA, AG, and GG, respectively. Statistically significant associations of OPG genotypes with FN-BMD (p < 0.01) and LS-BMD (p < 0.05) were observed. The GG genotype was associated with higher BMD values likewise decreased CTx concentration (p < 0.05) in compared with the other genotypes, which indicates that the allele G has a protective effect on bone. These associations were not followed by the effect of OPG on fracture incidence. Our results suggest that OPG/A163G polymorphism could contribute to the genetic regulation of BMD or bone turnover markers in Slovak population and thus could increase or decreased osteoporosis risk.

OPG Expression on Endothelial Cells and Modulation by IL-1B, PDGF, Insulin, and Glucose

Introduction: Osteoprotegerin (OPG), an osteoclastogenesis inhibitor implicated in bone remodelling, has emerged as a potential biomarker for cardiovascular diseases. However, OPG circulating sources or the stimuli that increase its release and effects have not been established. Objective: The aim of the present study are to establish whether endothelial cells may be a potential source of OPG, different mediators and OPG effects on endothelial cells. Aim: The aim of the present study were: 1) to establish whether endothelial cells may be a potential source of circulating OPG, 2) to study induced OPG expression and release stimulated by different factors and 3) to explore proatherogenic effects of OPG on endothelial cells. Results: OPG protein and mRNA expression was highly and significantly increased by IL-1β, insulin and glucose. OPG secretion to the supernatant was increased by IL-1β (4758 ± 727 vs. 88 ± 33 pg/mL, p=0.001) and PDGF (451 ± 151 vs. 88 ± 33 pg/mL, p=0.009). Glucose and insulin did not elevate OPG concentration in the supernatant. Endothelial cells stimulated with OPG significantly increased the release of IL-6 to the supernatant (247 ± 60 vs. 148 ± 30, p=0.008) and IL-8 was also increased by OPG (1444 ± 70 vs. 1538 ± 28, p=0.09). Conclusion: Endothelial cells are a source of OPG in circulation and IL-1β, glucose and insulin stimulate its expression.

Inhibition of osteoclast bone resorption activity through osteoprotegerin-induced damage of the sealing zone.

Bone remodeling is dependent on the dynamic equilibrium between osteoclast-mediated bone resorption and osteoblast-mediated osteogenesis. The sealing zone is an osteoclast-specific cytoskeletal structure, the integrity of which is critical for osteoclast-mediated bone resorption. To date, studies have focused mainly on the osteoprotegerin (OPG)‑induced inhibition of osteoclast differentiation through the OPG/receptor activator of the nuclear factor kappa-B ligand (RANKL)/RANK system, which affects the bone resorption of osteoclasts. However, the effects of OPG on the sealing zone have not been reported to date. In this study, the formation of the sealing zone was observed by Hoffman modulation contrast (HMC) microscopy and confocal laser scanning microscopy. The effects of OPG on the existing sealing zone and osteoclast-mediated bone resorption activity, as well as the regulatory role of genes involved in the formation of the sealing zone were examined by immunofluorescence staining, HMC microscopy, quantitative reverse transcription polymerase chain reaction (RT-qPCR), western blot analysis and scanning electron microscopy. The sealing zone was formed on day5, with belt-like protuberances at the cell edge and scattered distribution of cell nuclei, but no filopodia. The sealing zone was intact in the untreated control group. However, defects in the sealing zone were observed in the OPG-treated group (20ng/ml) and the structure was absent in the groups treated with 40 and 80ng/ml OPG. The podosomes showed a scattered or clustered distribution between the basal surface of the osteoclasts and the well surface. Furthermore, resorption lacunae were not detected in the 20ng/ml OPG-treated group, indicating the loss of osteoclast-mediated bone resorption activity. Treatment with OPG resulted in a significant decrease in the expression of Arhgef8/Net1 and DOCK5 Rho guanine nucleotide exchange factors (RhoGEFs), 10 of 18RhoGTPases (RhoA, RhoB, cdc42v1, cdc42v2, RhoU/Wrch1, RhoF/Rif, Rac2, RhoG, Rnd1 and RhoBTB1), ROCK1 and ROCK2. In conclusion, podosome distribution was affected by the OPG-induced inhibition of the expression of genes in the RhoGTPase signaling pathway. This resulted in damage to or destruction of the sealing zone, thus inhibiting osteoclast-mediated bone resorption activity.

Mechanistic study of the proangiogenic effect of osteoprotegerin

Osteoprotegerin (OPG), a soluble tumour necrosis factor receptor superfamily member, inhibits RANKL-mediated osteoclastogenesis. We have previously reported that OPG enhances the proangiogenic properties of endothelial colony-forming cells (ECFCs) in vitro, and promotes vasculogenesis in vivo. Here we investigated how OPG promotes neovascularisation. Proteomic experiments showed that OPG pretreatment affected ECFCs protein expression in two ways, 23 spots being down-regulated and 6 upregulated. These spots corresponded to proteins involved in cell motility, adhesion, signal transduction and apoptosis. In keeping with these proteomic results, we found that OPG induced ECFCs adhesion to activated endothelium in shear stress conditions, promoting intermediate but not focal adhesion to fibronectin and collagen. Treatment with OPG induced a reorganization of the ECFCs cytoskeleton, with the emergence of cell protrusions characteristic of a migratory phenotype. These effects correlated with decreased FAK phosphorylation and enhanced integrin αVβ3 expression. OPG drastically reduced caspase-3/7 activities and maintained ECFCs viability after 48h of treatment. All these effects were significantly attenuated by ECFCs incubation with the CXCR4 antagonist AMD-3100, and by prior heparan sulphate proteoglycan disruption. The proangiogenic properties of OPG appeared to be mediated by the proteoglycan syndecan-1, although OPG 1-194 lacking its heparin-binding domain still had pro-vasculogenic effects in vitro and in vivo. These results suggest that OPG may interact with ECFCs by binding to HSPGs/syndecan-1, thereby induce an anti-adhesive effect and promoting ECFCs migration through a SDF-1/CXCR4 dependent pathway.

Establishment of OPG Transgenic Mice and the Effect of OPG on Bone Microarchitecture.

Osteoprotegerin (OPG) plays a determinant role in regulating bone metabolism, but the effect of OPG on bone microarchitecture needs to be further elucidated. We attempted to construct pCI-hOPGp-mOPG vector containing human OPG promoter and FLAG tag and to microinject vector into fertilized zygotes from C57BL/6J × CBA mice to prepare transgenic mice. The OPG transgenic positive mice were identified by PCR and western blotting. Twelve-week-old OPG transgenic mice (OPG-Tg mice) and wild-type mice (WT mice) were utilized in the study of bone microarchitecture. Microcomputed tomography (micro-CT) data showed that compared with WT mice, the tibia of OPG-Tg mice showed an increased volumetric BMD (vBMD), tissue BMD (tBMD), trabecular thickness (Tb.Th), and trabecular number (Tb.N), and a decreased trabecular separation (Th.Sp) (P < 0.05) . The cortical bone microarchitecture parameters, such as cortical area (Ct.Ar), cortical thickness (Ct.Th), cortical BMD (Ct.BMD), cortical BMC (Ct.BMC), BMD, and BMC of femur, were increased, and the inner perimeter (In.Pm) was decreased, in OPG-Tg mice, compared to those in WT mice (P < 0.05). The established OPG transgenic mouse model could be valuable for further studying the biological significance and gene regulation of OPG in vivo.

Osteoprotegerin (OPG) protects ovarian cancer cells from TRAIL-induced apoptosis but does not contribute to malignant ascites-mediated attenuation of TRAIL-induced apoptosis

BackgroundResistance to apoptosis is a major problem in ovarian cancer and correlates with poor prognosis. Osteoprotegerin (OPG) is a secreted factor in malignant ascites and acts as a decoy receptor for receptor activator of NF-κB ligand (RANKL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL promotes apoptosis in ovarian cancer cells. Ovarian cancer ascites attenuate TRAIL-induced apoptosis raising the possibility that OPG contained in ascites may abrogate the anti-tumor activity of TRAIL.MethodsDetermination of OPG levels in ascites was measured by ELISA. Effect of OPG on TRAIL-induced cell death was determined by XTT and colony forming assays in ovarian cancer cell lines and primary tumor cells. Apoptosis was assessed by ELISA.ResultsWe found that recombinant OPG and malignant ascites attenuates TRAIL-induced cell death and apoptosis in a dose-dependent manner in ovarian cancer cell lines and primary ovarian tumor cells. OPG is present at high levels in the ascites of patients with ovarian cancer. We found a positive correlation between the levels of OPG in ascites and the ability of the ascites to attenuate TRAIL-induced cell death. The anti-apoptotic effect of ascites was not reversed by co-incubation with an OPG blocking antibody.ConclusionsOPG and malignant ascites protect ovarian cancer cells from TRAIL-induced apoptosis. Although malignant ascites contain high levels of OPG, OPG is not a critical component that contributes to ascites-mediated attenuation of TRAIL-induced apoptosis.

Comparative experimental study of the effect of osteoprotegerin and testosterone in bone remodeling and mass after surgical castration in male rats

Comparative experimental study of the effect of osteoprotegerin and testosterone in bone remodeling and mass after surgical castration in male rats

Abstract 1364: Osteoprotegerin production by breast cancer cells: Regulation by NF-kappaB and role in metastasis

Abstract Human breast cancer cells and tissues express the protein osteoprotegerin (OPG). While many studies have investigated the role of OPG in cancer as a mediator of bone remodeling, few studies consider its impact outside the bone microenvironment. Further investigation is therefore required into endogenous OPG expression by breast cancer cells and its effects on tumor progression. OPG can be regulated by the transcription factor NF-kappaB in several cell types. We investigated whether OPG expression in MDA-MB-231 and MDA-MB-435 breast cancer cells was regulated by NF-kappaB. Cells were treated for 24 hours with 10ng/ml TNF-alpha or 10µM thymoquinone (TQ), to stimulate or inhibit NF-kappaB activity respectively. OPG expression was measured by real time RT-PCR (mRNA) and ELISA (protein). OPG levels were increased after treatment with TNF-alpha while treatment with TQ decreased expression. We used the chick embryo metastasis model to determine the effect of OPG on metastasis. Human breast cancer cells were inoculated onto the chick chorioallantoic membrane (CAM) where they form a tumor that metastasizes to the chick tissue. Metastasis is quantified by real time PCR for Human Alu repeat DNA sequences in the chick tissue. We transfected MDA-MB-231 cells with OPG-specific siRNA which reduced OPG mRNA expression up to 72 hours post-transfection. OPG knockdown or control (non-targeting siRNA) treated cells were inoculated onto the CAM at embryonic day 9; chick tissue was harvested at day 16 and analyzed for breast cancer cell metastasis. We found a significant reduction in metastasis of the OPG knockdown cells compared to the control. We performed genome-wide mRNA microarray data analysis to determine whether OPG expression correlates with breast cancer progression in human tumor samples. OPG expression in 1809 breast cancer tissues was not correlated with prognosis. However, analyses by breast cancer subtypes showed that high OPG mRNA expression correlated with poorer prognosis in basal (p=0.0028, n=383) and also HER2 positive tumor subtypes (p=0.021, n=408). In summary, our data suggest that OPG expression in breast cancer cells is regulated by NF-kappaB, a transcription factor associated with pro-tumor effects. Our in vivo studies suggest that OPG promotes metastasis. We are currently determining the mechanism whereby OPG exerts its effects. Since OPG levels correlate with poorer prognosis in basal and HER2 positive tumors, treatment strategies targeting OPG in the primary tumor may represent a potential therapeutic strategy for these disease subytpes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1364. doi:1538-7445.AM2012-1364

The inhibition of RANKL/RANK signaling by osteoprotegerin suppresses bone invasion by oral squamous cell carcinoma cells

Oral squamous cell carcinomas (OSCCs) are malignant tumors that frequently invade the maxilla and mandibular bone. However, the molecular mechanisms underlying bone invasion by OSCC are unclear. Recent studies showed that receptor activator of nuclear factor κB (RANK) was expressed not only in osteoclast precursors but also in tumor cells. Therefore, we examined whether RANK ligand (RANKL)/RANK signaling regulates bone invasion by OSCC cells in vivo and in vitro. We first injected human OSCC B88 cells into the masseter region of nude mice. Mice were treated for 3 weeks with osteoprotegerin (OPG), the decoy receptor for RANKL. Treatment with OPG decreased bone invasion by B88 cells, reduced the number of osteoclasts and increased B88 cell apoptosis. However, OPG did not affect apoptosis and proliferation in B88 cells in vitro, suggesting that the effects of OPG on apoptosis in B88 cells are restricted in a bone environment. RANK was expressed in the B88 cells and in OSCC cells from patients. RANKL induced NF-κB activation and extracellular signal-regulated kinase phosphorylation in B88 cells and enhanced B88 cell migration in a modified chemotaxis chamber equipped with a gelatin-coated filter. OPG inhibited RANKL-induced NF-κB activation, extracellular signal-regulated kinase phosphorylation and cell migration. Our data clearly indicate that RANKL/RANK inhibition suppresses bone invasion by inhibiting osteoclastogenesis and cancer cell migration and by inducing apoptosis of cancer cells via indirect anticancer action in vivo.

The expression of osteoprotegerin is required for maintaining the intervertebral disc endplate of aged mice.

Human chondrocytes and annulus fibrosus cells of intervertebral disc (IVD) express osteoprotegerin (OPG), but the effect of OPG on the pathogenesis of IVD degeneration remains unknown. Here we assessed the phenotype change of IVD in OPG(-/-) mice. The IVDs from 12-, 20-, and 28-week-old OPG(-/-) mice and WT controls were subjected to histologic analyses including TRAP staining for osteoclasts, immunostaining for OPG and type I collagen protein expression, and TUNEL staining for apoptosis. The IVD tissues were also subjected to real time RT-PCR for mRNA expression of genes for osteoblast-osterix, ALP, and osteocalcin; for osteoclasts-trap, rank, mmp9 and cathepsin K, and for chondrocytes-aggrecan, mmp13 and Col10. OPG protein expresses at the cells of endplate cartilage and annulus fibrosis in IVDs of WT mice. Compared to WT mice, OPG(-/-) mice developed aging related cartilage loss and bony tissue appearance at the endplate. Stating from 20 weeks of age, IVDs from OPG(-/-) mice expressed significantly increased mmp13 and Col10 levels, which is associated with increased osteoblast number and elevated expression of osteoblast marker genes. Furthermore, TRAP+ osteoclasts were presented in the endplate cartilage of OPG(-/-) mice. These osteoclasts localized adjacently to and erosion into the cartilage. Increased expression of RANK, mmp9 and cathepsin k was detected in OPG(-/-) IVDs. OPG at IVD plays an important role for maintaining the integrity of endplate cartilage during aging by preventing endplate cartilage from osteoclast-mediated resorption.

Osteoprotegerin, a new actor in vasculogenesis, stimulates endothelial colony‐forming cells properties

Osteoprotegerin (OPG), a soluble receptor of the tumour necrosis factor family, and its ligand, the receptor activator of nuclear factor-κB ligand (RANKL), are emerging as important regulators of vascular pathophysiology. We evaluated their effects on vasculogenesis induced by endothelial colony-forming cells (ECFC) and on neovessel formation in vivo. Effects of OPG and RANKL on in vitro angiogenesis were evaluated after ECFC incubation with OPG or RANKL (0-50 ng mL(-1)). Effects on microvessel formation were evaluated with an in vivo murin Matrigel plug assay. Vascularization was evaluated by measuring plug hemoglobin and vascular endothelial growth factor (VEGF)-R2 content 14 days after implantation. We found that ECFC expressed OPG and RANK but not RANKL mRNA. Treatment of ECFC with VEGF or stromal cell-derived factor-1 (SDF-1) upregulated OPG mRNA expression. OPG stimulated ECFC migration (P < 0.05), chemotaxis (P < 0.05) and vascular cord formation on Matrigel(®) (P < 0.01). These effects were correlated with SDF-1 mRNA overexpression, which was 30-fold higher after 4 h of OPG stimulation (P < 0.01). OPG-mediated angiogenesis involved the MAPK signaling pathway as well as Akt or mTOR cascades. RANKL also showed pro-vasculogenic effects in vitro. OPG combined with FGF-2 promoted neovessel formation in vivo, whereas RANKL had no effect. OPG induces ECFC activation and is a positive regulator of microvessel formation in vivo. Our results suggest that the OPG/RANK/RANKL axis may be involved in vasculogenesis and strongly support a modulatory role in tissue revascularization.