Upregulation Of Osteoprotegerin Research Articles

Endogenous osteoprotegerin (OPG) represses ERα and promotes stemness and chemoresistance in breast cancer cells

Breast cancer (BC) is the most prevalent cancer and the leading cause of death among women worldwide. The osteoprotegerin (OPG) cytokine, a decoy receptor for RANKL and a key player in bone homeostasis, has pro-and anti-carcinogenic effects in various types of cancer, including breast neoplasms. In the present study, we have shown that ectopic expression of OPG in breast epithelial/cancer cells promotes the pro-metastatic processes epithelial-to-mesenchymal transition (EMT), stemness, angiogenesis as well as the activation of breast stromal fibroblasts. Furthermore, proteomics analysis, which allows the identification and quantification of a plethora of known and unknown proteins, has shown a strong and significant correlation between OPG upregulation and the expression of proteins with functions in EMT and stemness. On the other hand, OPG knockdown in triple-negative breast cancer (TNBC) cells inhibited the formation of cancer stem cells. Importantly, while OPG upregulation significantly enhanced the resistance of luminal BC cells to cisplatin and docetaxel, OPG downregulation sensitized TNBC cells to these chemotherapeutic drugs. We have also shown that OPG negatively controls estrogen receptor α (ERα), and OPG upregulation correlated well with the expression of genes related to ER-negative claudin low cells. Collectively, these results show that OPG promotes stemness and the consequent chemoresistance of breast cancer cells.

Neuronal nuclear calcium signaling suppression of microglial reactivity is mediated by osteoprotegerin after traumatic brain injury

BackgroundTraumatic brain injury (TBI) is characterized by massive changes in neuronal excitation, from acute excitotoxicity to chronic hyper- or hypoexcitability. Nuclear calcium signaling pathways are involved in translating changes in synaptic inputs and neuronal activity into discrete transcriptional programs which not only affect neuronal survival and synaptic integrity, but also the crosstalk between neurons and glial cells. Here, we report the effects of blunting neuronal nuclear calcium signals in the context of TBI.MethodsWe used AAV vectors to express the genetically encoded and nuclear-targeted calcium buffer parvalbumin (PV.NLS.mCherry) or the calcium/calmodulin buffer CaMBP4.mCherry in neurons only. Upon TBI, the extent of neuroinflammation, neuronal death and synaptic loss were assessed by immunohistochemistry and targeted transcriptome analysis. Modulation of the overall level of neuronal activity was achieved by PSAM/PSEM chemogenetics targeted to parvalbumin interneurons. The functional impact of neuronal nuclear calcium buffering in TBI was assessed by quantification of spontaneous whisking.ResultsBuffering neuronal nuclear calcium unexpectedly resulted in a massive and long-lasting increase in the recruitment of reactive microglia to the injury site, which was characterized by a disease-associated and phagocytic phenotype. This effect was accompanied by a substantial surge in synaptic loss and significantly reduced whisking activity. Transcriptome analysis revealed a complex effect of TBI in the context of neuronal nuclear calcium buffering, with upregulation of complement factors, chemokines and interferon-response genes, as well as the downregulation of synaptic genes and epigenetic regulators compared to control conditions. Notably, nuclear calcium buffering led to a substantial loss in neuronal osteoprotegerin (OPG), whereas stimulation of neuronal firing induced OPG expression. Viral re-expression of OPG resulted in decreased microglial recruitment and synaptic loss. OPG upregulation was also observed in the CSF of human TBI patients, underscoring its translational value.ConclusionNeuronal nuclear calcium signals regulate the degree of microglial recruitment and reactivity upon TBI via, among others, osteoprotegerin signals. Our findings support a model whereby neuronal activity altered after TBI exerts a powerful impact on the neuroinflammatory cascade, which in turn contributes to the overall loss of synapses and functional impairment.

Hypoxia Preconditioned Serum (HPS) Promotes Osteoblast Proliferation, Migration and Matrix Deposition.

Interest in discovering new methods of employing natural growth factor preparations to promote bone fracture healing is becoming increasingly popular in the field of regenerative medicine. In this study, we were able to demonstrate the osteogenic potential of hypoxia preconditioned serum (HPS) on human osteoblasts in vitro. Human osteoblasts were stimulated with two HPS concentrations (10% and 40%) and subsequently analyzed at time points of days 2 and 4. In comparison to controls, a time- and dose-dependent (up to 14.2× higher) proliferation of osteoblasts was observed after 4 days of HPS-40% stimulation with lower lactate dehydrogenase (LDH)-levels detected than controls, indicating the absence of cytotoxic/stress effects of HPS on human osteoblasts. With regards to cell migration, it was found to be significantly faster with HPS-10% application after 72 h in comparison to controls. Further osteogenic response to HPS treatment was evaluated by employing culture supernatant analysis, which exhibited significant upregulation of OPG (Osteoprotegerin) with higher dosage (HPS-10% vs. HPS-40%) and longer duration (2 d vs. 4 d) of HPS stimulation. There was no detection of anti-osteogenic sRANKL (soluble Receptor Activator of NF-κB Ligand) after 4 days of HPS stimulation. In addition, ALP (alkaline phosphatase)-enzyme activity, was found to be upregulated, dose-dependently, after 4 days of HPS-40% application. When assessing ossification through Alizarin-Red staining, HPS dose-dependently achieved greater (up to 2.8× higher) extracellular deposition of calcium-phosphate with HPS-40% in comparison to controls. These findings indicate that HPS holds the potential to accelerate bone regeneration by osteogenic promotion of human osteoblasts.

Effect of DNA methylation modulators on the production of osteoprotegerin by rheumatoid fibroblast-like synoviocytes in vitro: their migration and invasion

Objective. The purpose of the research was to study the effect of DNA methylation modulators on the production of proinflammatory cytokines by fibroblast-like synovial cells (FLC).Materials and methods. We used the cells derived from the synovial tissue of 6 patients with active rheumatoid arthritis (RA) after 3–7 in vitro culturing passages.Results. There was an IL-1β-induced up-regulation of osteoprotegerin (OPG) synthesis in the RA FLC cultures. The addition of methylating compounds S-Adenosyl methionine (SAMe) and genistein into the cultures resulted in a statistically significant decrease in the production of OPG, while the addition of the demethylating agent hydralazine did not change the synthesis of the cytokine. All three DNA methylation modulators used at different concentrations significantly reduced the percentage of spontaneous migration and invasion of FLC in the Boyden chamber.Conclusion. Enzymes and molecular complexes involved in DNA methylation could be potential therapeutic targets, and in vitro FLC cultures of RA patients can be used as a model for preclinical screening of new drug compounds.

Heamatococcus pluvialis ameliorates bone loss in experimentally-induced osteoporosis in rats via the regulation of OPG/RANKL pathway

BackgroundsOsteoporosis prevailing in elderly involves a marked increase in bone resorption showing an initial fall in bone mineral density leading to a significant reduction in bone formation. AimThe present study aimed to investigate the effect of Heamatococcus pluvialis microalgae on osteoporosis in D-galactose-treated rats. The underlying mechanism was tracked targeting the osteoprotegerin (OPG)/ nuclear factor-κβ ligand (RANKL) pathway using micro-computed tomography scanning. MethodsOsteoporosis was induced in rats by intraperitoneal injection of D-galactose (200 mg/kg/day) for eight consecutive weeks. Osteoporotic rats were orally treated with H. pluvialis biomass (BHP; 450 mg/kg), its polar (PHP; 30 mg/kg) and carotenoid (CHP; 30 mg/kg) fractions for the last 2 weeks of D-Gal injection. Twenty four hours after the last dose of the treatments, tibia bones of the rats were scanned using micro-computed tomography scanning for bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness/separation/number (Tb.Th, Tb.Sp, Tb.N) evaluation, blood samples were withdrawn and sera were used for biochemical assessment. Moreover, femur bones were examined histopathologically using several stains. ResultsInduction of osteoporosis was associated with a marked reduction in BMD, BV/TV, Tb.Th, Tb.Sp, Tb.N and in serum levels of phosphorus and catalase. On the other hand, a significant elevation in serum levels of calcium, bone alkaline phosphatase (BALP) and interleukin-6 was observed. Moreover, up-regulation of OPG was detected in osteoporotic rats. Oral treatment with BHP, and PHP incremented tibia BMD and serum phosphorus level along with the decrease in serum levels of calcium, BALP, interleukin-6, OPG and RANKL. However, treatment with CHP almost restored all the fore mentioned parameters to normal values. Furthermore, the histopathological evaluation emphasized the biochemical outcomes. ConclusionH. pluvialis fractions rich in astaxanthin ameliorated bone loss in experimentally-induced osteoporosis in rats probably through the down-regulation of serum OPG in concurrence with up-regulation of serum RANKL.

Inverse regulation of serum osteoprotegerin and tumor necrosis factor-related apoptosis-inducing ligand levels in patients with leg lesional vascular calcification: An observational study.

We hypothesized that circulating osteoprotegerin (OPG) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) levels could be associated with vascular calcification, which is predominant in diabetes.The study included 71 Korean participants (36 with diabetes and 35 without diabetes), who were sub-grouped according to the results of the ankle–brachial index (ABI) and/or X-ray computed tomography scan (CT scan). Serum OPG and TRAIL levels were assayed using the respective enzyme-linked immunosorbent assay kits. Statistical significance was analyzed using Student's t test between the 2 groups or analysis of variance (ANOVA) among the 4 groups.Serum OPG was up-regulated in the participants with diabetes, with peripheral arterial disease (PAD), and/or with vascular calcification. TRAIL down-regulation was more strictly controlled than OPG up-regulation; it was significantly downregulated in the participants with PAD and vascular calcification, but not in the participants with diabetes. Serum OPG and TRAIL were regulated in the participants with femoral, popliteal, and peroneal artery calcification but not in the participants with aortic calcification.OPG up-regulation and TRAIL down-regulation were found to be associated with leg lesional vascular calcification; therefore, the average OPG/TRAIL ratio was significantly increased by 3.2-fold in the leg lesional vascular calcification group.

Osteoprotegerin increases parallel to insulin resistance in obese adolescents

ABSTRACTPurpose/Aim of the Study: Osteoprotegerin (OPG) is an α tumor necrosis factor receptor superfamily glucoprotein that acts as a decoy receptor for the receptor activator of nuclear factor kappa B ligand (RANKL), exerting an antiresoptive bone effect. It was recently shown that OPG/RANKL axis is activated during vascular calcification, contributing to atherosclerotic lesions formation. Additionally, OPG levels are charachterized as an independent risk factor for overall vascular mortality in obese adults. We aimed to investigate OPG levels in children/adolescents with obesity and explore possible relations with obesity-related insulin resistance (IR). Material and Methods: A total of 160 participants (85 obese) were enrolled. Participants with obesity underwent an oral glucose tolerance test. IR was evaluated according to the homeostasis model assessment-insulin resistance index. Serum OPG levels were determined. Results: OPG levels did not differ significantly between obese subjects and controls in the total sample (p = 0.133). However, in the adolescents’ subgroup, serum OPG levels were significantly increased in obesity (p = 0.019). After stratifying participants according to their IR status, only subjects with both obesity and IR exhibited increased OPG levels compared to controls (p < 0.001). Factor analysis further associated OPG levels variation to insulin levels variation and to IR. Conclusions: Obese individuals demonstrate increased serum OPG levels during puberty. Obesity per se is not the potent factor for this increase; indeed, IR accompanying obesity seems to exert a fundamental role in OPG upregulation.

RANKL Inhibits the Production of Osteoprotegerin from Smooth Muscle Cells under Basal Conditions and following Exposure to Cyclic Strain

Receptor activator of nuclear factor-κB ligand (RANKL) promotes vascular calcification, while osteoprotegerin (OPG) opposes it by blocking RANKL activity. It remains unclear which vascular cell populations produce and secrete OPG/RANKL. This study characterizes the production of OPG/RANKL from human aortic endothelial and smooth muscle cells (HAECs and HASMCs) under various conditions. HAECs and HASMCs were exposed to inflammatory stimuli (tumor necrosis factor-α or hyperglycemia) or physiological levels of hemodynamic (cyclic) strain. After 72 h, both cells and supernatant media were harvested for assessment of OPG/RANKL production. Based on initial findings, the experiments involving HASMCs were then repeated in the presence of exogenous RANKL. OPG was produced and secreted by HASMCs and (to a considerably lesser degree) HAECs under basal conditions. Inflammatory stimuli upregulated OPG production in both cell populations. Cyclic strain significantly upregulated OPG production in HASMCs. Neither cell population produced RANKL. Exposing HASMCs to exogenous RANKL inhibited basal OPG production and completely abrogated the strain-mediated upregulation of OPG. These data suggest that HASMCs are a significant source of OPG within the vasculature but that RANKL, once present, downregulates this production and appears capable of preventing the “protective” upregulation of OPG seen with HASMCs exposed to physiological levels of cyclic strain.

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.

Effects of pigment epithelium-derived factor on osteo-adipogenic transdifferentiation, and osteoprotegerin/receptor activator for nuclear factor-κB ligand ratio of osteoblast after stimulation of high concentrations of dexamethasone

Objective To explore the protective effect of pigment epithelium-derived factor (PEDF) on osteo-adipogenic transdifferentiation, and osteoprotegerin (OPG)/receptor activator of nuclear factor (NF)-κB ligand (RANKL) ratio of osteoblast after stimulation of high concentrations of dexamethasone (DEX). Methods MC3T3-E1 cells were randomly divided into control group, DEX model group and DEX + PEDF group. According to the concentration of PEDF, the DEX + PEDF group was subdivided into 2, 5, 10 nmol/L groups. The inverted phase contrast microscope was used to observe the cell morphology. After 14 d we used cell staining [alkaline phosphatase (ALP) staining, alizarin red staining] to detect the differentiation of osteoblast. Immunofluorescence and Western blotting were used to observe the expression of OPG and RANKL. Results The MC3T3-E1 cells were positive for ALP and expressed OPG and RANKL. After stimulation of high concentrations of DEX, the osteo-adipogenic transdifferentiation of MC3T3-E1 cells was observed. A large number of osteoblasts transfected into adipocytes. High concentrations of DEX significantly raised the expression of RANKL (P=0.008). The osteo-adipogenic transdifferentiation of MC3T3-E1 cells was significantly inhibited by PEDF with an obvious down-regulation of RANKL and up-regulation of OPG showed by Western blotting testing. Compared with DEX group (0.560±0.012), OPG/RANKL expression level of PEDF intervention groups (0.821±0.025, 1.290±0.018 and 1.405±0.120) appeared significantly upregulated (P=0.003, 0.000, 0.019). Conclusion High concentrations of DEX promote osteo-adipogenic transdifferentiation of MC3T3-E1 cells and reduce the expression of OPG/RANKL, whereas PEDF can effectively reverse this process. PEDF play a important role in protecting the osteoblast function. Key words: Dexamethasone; Pigment epithelium-derived factor; Osteoblast; Receptor activator for nuclear factor-κB ligand; Osteoprotegerin; Osteo-adipogenic transdifferentiation

Anabolic Effect of Insulin Therapy on the Bone: Osteoprotegerin and Osteocalcin Up-Regulation in Streptozotocin-Induced Diabetic Rats.

Type 1 diabetes mellitus (T1DM) is associated with several skeletal alterations, particularly in conditions of poor glycaemic control. Insulin therapy is the major conservative treatment for T1DM; however, the effects of this hormone on bone markers of T1DM rats are limited, and the regulatory mechanisms remain elusive. Therefore, the evaluation of molecular and non-molecular parameters in a chronic animal model of T1DM-induced bone loss, treated with and without insulin, may help in elucidating the insulin mechanisms. Male Wistar rats were assigned into three groups: control, T1DM (T1DM rats induced with streptozotocin [STZ] at 40mg/kg intravenously) and T1DM plus insulin therapy (T1DMI). After 8weeks, we evaluated the serum biochemical, tibia histomorphometric and biomechanical parameters, as well as the gene expression of the receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG) and osteocalcin (OC) of femur mRNA. Compared with T1DM, the T1DMI group showed less bone loss, which was revealed by the increased trabecular width (TbWi, p<0.001) and trabecular bone area (BAr, p<0.01), reduced trabecular separation (TbSp, p<0.01) and increased Young's modulus (p<0.05). Moreover, molecular analyses indicated that the expression of OPG and OC was up-regulated (p<0.001 and p<0.05, respectively). In summary, the up-regulation of OPG and OC in the T1DMI group supports an anabolic effect of insulin, which was demonstrated by the maintenance of bone architecture and flexibility. These results suggest that insulin therapy may prevent T1DM-induced bone loss via the effects on the bone formation.

Interleukin 6 and/or Interleukin 17A Modulate the OPG/RANKL System of MC3T3-E1 Murine Osteoblast Cell Line

Cytokines such as interleukin-6 (IL-6) and IL-17 which act as key regulators of the immune response have been identified to have a potential role in the bone remodeling mechanism. Receptor activator of NF-κB ligand (RANKL) has been shown to regulate osteoclast differentiation and function while the osteoprotegerin (OPG) blocks the binding of RANKL and inhibits the differentiation of osteoclasts, thus favoring osteogenesis. Alkaline phosphatase (ALP) on the other hand works as early mineralization indicator in bone regulation. The current study aims to determine the potential role of IL-6 and IL-17A in regulating the OPG/RANKL system of the murine osteoblast cell line (MC3T3-E1). Gene expression analysis showed significant up-regulation of OPG and ALP by all the treated groups (rIL-6, rIL-17A and rIL-6 + rIL-17A). In contrast, treatment of cells with rIL-6 and/or rIL-17A showed down-regulation of RANKL expression. Interestingly, the osteoblast cells treated with combinations of rIL-6 + rIL17A showed marked increased in OPG/RANKL ratio. Similar pattern of protein expression was observed in the osteoblasts treated with rIL-6 and/or rIL-17A as detected by western blotting and ELISA. These findings suggest a new mechanism of regulation by these cytokines on the expression of OPG and RANKL, which could promote osteogenesis and diminish osteoclastogenesis.

Fluoride-induced c-Fos expression in MC3T3-E1 osteoblastic cells

Excessive systemic exposure to fluoride leads to disturbances of bone homeostasis. c-Fos is known to be essential in bone development by affecting osteoblast and osteoclast differentiation. In this study, we examined the effects of fluoride exposure on c-Fos expression and its regulatory signaling pathways in MC3T3-E1 mouse osteoblast cell line. c-fos mRNA level, c-Fos protein level and c-Fos DNA-binding activity were markedly increased, with a peak at 2 or 4 h, in MC3T3-E1 cells exposed to sodium fluoride (NaF). Fra-1 protein, another member of Fos family, was also elevated, whereas FosB and Fra-2 proteins remained unchanged. NaF further induced phosphorylation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase 1/2 (ERK1/2), ERK5, c-Jun NH2-terminal kinase and p38. NaF-induced expression of c-Fos protein was markedly suppressed with U0126, the inhibitor of both activated and non-activated forms of MAPK/ERK kinase 1/2 (MEK1/2) and BIX02189, the MEK5 inhibitor, but partially with SP600125, the JNK inhibitor and SB203580, the p38 inhibitor. Therefore, ERK1/2 and ERK5 signal transduction pathways are important for accumulating c-Fos. siRNA targeting against the mouse c-fos gene further enhanced NaF-induced up-regulation of osteoprotegerin (OPG), an inhibitor of osteoclastogenesis, suggesting that c-Fos might negatively regulate OPG expression induced by fluoride in osteoblastic cells.

Osteoprotegerin Regulates Pancreatic β-Cell Homeostasis upon Microbial Invasion.

Osteoprotegerin (OPG), a decoy receptor for receptor activator of NF-κB ligand (RANKL), antagonizes RANKL’s osteoclastogenic function in bone. We previously demonstrated that systemic administration of lipopolysaccharide (LPS) to mice elevates OPG levels and reduces RANKL levels in peripheral blood. Here, we show that mice infected with Salmonella, Staphylococcus, Mycobacteria or influenza virus also show elevated serum OPG levels. We then asked whether OPG upregulation following microbial invasion had an effect outside of bone. To do so, we treated mice with LPS and observed OPG production in pancreas, especially in β-cells of pancreatic islets. Insulin release following LPS administration was enhanced in mice lacking OPG, suggesting that OPG inhibits insulin secretion under acute inflammatory conditions. Consistently, treatment of MIN6 pancreatic β-cells with OPG decreased their insulin secretion following glucose stimulation in the presence of LPS. Finally, our findings suggest that LPS-induced OPG upregulation is mediated in part by activator protein (AP)-1 family transcription factors, particularly Fos proteins. Overall, we report that acute microbial infection elevates serum OPG, which maintains β-cell homeostasis by restricting glucose-stimulated insulin secretion, possibly preventing microbe-induced exhaustion of β-cell secretory capacity.

In Vivo siRNA Delivery Using JC Virus-like Particles Decreases the Expression of RANKL in Rats.

Bone remodeling requires a precise balance between formation and resorption. This complex process involves numerous factors that orchestrate a multitude of biochemical events. Among these factors are hormones, growth factors, vitamins, cytokines, and, most notably, osteoprotegerin (OPG) and the receptor activator for nuclear factor-kappaB ligand (RANKL). Inflammatory cytokines play a major role in shifting the RANKL/OPG balance toward excessive RANKL, resulting in osteoclastogenesis, which in turn initiates bone resorption, which is frequently associated with osteoporosis. Rebalancing RANKL/OPG levels may be achieved through either upregulation of OPG or through transient silencing of RANKL by means of RNA interference. Here, we describe the utilization of a viral capsid-based delivery system for in vivo and in vitro RNAi using synthetic small interfering RNA (siRNA) molecules in rat osteoblasts. Polyoma JC virus-derived virus-like particles are capable of delivering siRNAs to target RANKL in osteoblast cells both in vitro and in a rat in vivo system. Expression levels were monitored using quantitative real-time polymerase reaction and enzyme-linked immunosorbent assay after single and repeated injections over a 14-day period. Our data indicate that this is an efficient and safe route for in vivo delivery of gene modulatory tools to study important molecular factors in a rat osteoporosis model.

Osteostatin-Coated Porous Titanium Can Improve Early Bone Regeneration of Cortical Bone Defects in Rats

A promising bone graft substitute is porous titanium. Porous titanium, produced by selective laser melting (SLM), can be made as a completely open porous and load-bearing scaffold that facilitates bone regeneration through osteoconduction. In this study, the bone regenerative capacity of porous titanium is improved with a coating of osteostatin, an osteoinductive peptide that consists of the 107-111 domain of the parathyroid hormone (PTH)-related protein (PTHrP), and the effects of this osteostatin coating on bone regeneration were evaluated in vitro and in vivo. SLM-produced porous titanium received an alkali-acid-heat treatment and was coated with osteostatin through soaking in a 100 nM solution for 24 h or left uncoated. Osteostatin-coated scaffolds contained ∼0.1 μg peptide/g titanium, and in vitro 81% was released within 24 h. Human periosteum-derived osteoprogenitor cells cultured on osteostatin-coated scaffolds did not induce significant changes in osteogenic (alkaline phosphatase [ALP], collagen type 1 [Col1], osteocalcin [OCN], runt-related transcription factor 2 [Runx2]), or angiogenic (vascular endothelial growth factor [VEGF]) gene expression; however, it resulted in an upregulation of osteoprotegerin (OPG) gene expression after 24 h and a lower receptor activator of nuclear factor kappa-B ligand (RankL):OPG mRNA ratio. In vivo, osteostatin-coated, porous titanium implants increased bone regeneration in critical-sized cortical bone defects (p=0.005). Bone regeneration proceeded until 12 weeks, and femurs grafted with osteostatin-coated implants and uncoated implants recovered, respectively, 66% and 53% of the original femur torque strength (97±31 and 77±53 N·mm, not significant). In conclusion, the osteostatin coating improved bone regeneration of porous titanium. This effect was initiated after a short burst release and might be related to the observed in vitro upregulation of OPG gene expression by osteostatin in osteoprogenitor cells. Long-term beneficial effects of osteostatin-coated, porous titanium implants on bone regeneration or mechanical strength were not established here and may require optimization of the pace and dose of osteostatin release.

Biomechanical Loading Modulates Proinflammatory and Bone Resorptive Mediators in Bacterial-Stimulated PDL Cells

The present study aimed to evaluate in vitro whether biomechanical loading modulates proinflammatory and bone remodeling mediators production by periodontal ligament (PDL) cells in the presence of bacterial challenge. Cells were seeded on BioFlex culture plates and exposed to Fusobacterium nucleatum ATCC 25586 and/or cyclic tensile strain (CTS) of low (CTSL) and high (CTSH) magnitudes for 1 and 3 days. Synthesis of cyclooxygenase-2 (COX2) and prostaglandin E2 (PGE2) was evaluated by ELISA. Gene expression and protein secretion of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were evaluated by quantitative RT-PCR and ELISA, respectively. F. nucleatum increased the production of COX2 and PGE2, which was further increased by CTS. F. nucleatum-induced increase of PGE2 synthesis was significantly (P < 0.05) increased when CTSH was applied at 1 and 3 days. In addition, CTSH inhibited the F. nucleatum-induced upregulation of OPG at 1 and 3 days, thereby increasing the RANKL/OPG ratio. OPG and RANKL mRNA results correlated with the protein results. In summary, our findings provide original evidence that CTS can enhance bacterial-induced syntheses of molecules associated with inflammation and bone resorption by PDL cells. Therefore, biomechanical, such as orthodontic or occlusal, loading may enhance the bacterial-induced inflammation and destruction in periodontitis.

Inhibition Of Mir-21 In HS-5 Bone Marrow Stromal Cells In The Presence Of Multiple Myeloma cells Restores RANKL/OPG Ratio: A Potential Therapeutic Approach For Myeloma-Related Bone Disease

Multiple Myeloma (MM)-related osteolytic lesions of the skeleton result from asynchronous bone turnover wherein increased bone resorption by osteoclasts (OCLs) is associated to suppression of bone formation due to inhibition of osteoblasts (OBLs). In this context, the balance between osteoprotegerin (OPG) and the receptor activator for nuclear factor-κB ligand (RANKL) plays a relevant role, because RANKL signaling induces differentiation, activation and survival of OCLs, whereas OPG acts as a decoy receptor of RANKL, thus blocking bone resorption. In the MM milieu, the interaction of malignant plasma cells (PCs) and bone marrow stromal cells (BMSCs) favors the production of RANKL and reduces OPG secretion by BMSCs. The resulting severe imbalance in RANKL/OPG ratio is the main trigger of MM-related bone disease (BD). Emerging evidence indicates that microRNAs (miRNAs) play a crucial role in bone remodelling as they can act on bone effectors (OCLs, OBLs, BMSCs) and PCs. Indeed, several findings suggest that miR-29 family support OBL differentiation, while we have previously demonstrated that miR-29b is down-regulated during osteoclastogenesis and its enforced expression functionally inhibits terminally differentiated OCLs. Moreover, also miR-21 plays an important role in OCL differentiation and is up-regulated by IL-6 via STAT3 that, in turn, promotes survival and proliferation of PCs, favoring bone damage. On these bases, we investigated whether modulation of miR-21 expression in BMSCs may have an impact on MM microenviroment, focusing our attention on OPG production, taking into account that different bioinformatics resources indicate OPG mRNA as a predicted target of miR-21. To this end, we established a co-culture system where primary BMSCs and HS-5, a human BMSC cell line, were exposed to either RPMI 8226 MM cells or primary CD138+ PCs isolated from MM patients. Then, we isolated BMSCs and evaluated miR-21 and OPG expression. We observed strong up-regulation of miR-21 in BMSCs and HS-5 together with a significant reduction of OPG levels in the presence of MM cells. These preliminary data suggested that antagonizing miR-21 in BMSCs exposed to MM cells could restore RANKL/OPG ratio through the up-regulation of OPG. To address this hypothesis, we transduced HS-5 cell line with a lentiviral vector carrying anti-miR-21 sequence (a21 HS-5) or with an empty lentiviral vector carrying only the GFP gene (GFP CNT HS-5). OPG expression was determined by RT-PCR, Western Blotting and ELISA assays after 48h and 72h of exposure to RPMI 8226 cells or primary PCs. We observed a significant increase in OPG production in a21 HS-5 cells compared to controls (Fig. 1, panel A and panel B). We also evaluated RANKL expression in the same samples. Interestingly, constitutive inhibition of miR-21 in HS-5 cell line upon exposure to MM cells induced a significant RANKL down-regulation, thus restoring a physiological RANKL/OPG ratio.We hypothesized that this surprising result may be due to the activity of the protein inhibitor of activated STAT3 (PIAS3). Indeed, PIAS3 binds to STAT3 and blocks its DNA binding ability, thereby inhibiting STAT3-mediated gene activation. It is already known that PIAS3 acts as a negative regulator of the RANKL expression and it is a direct and validated target of miR-21. Therefore, the inhibition of miR-21 induces PIAS3 up-regulation and a decreased RANKL transcription. To confirm the involvement of PIAS3 in RANKL suppression in our system, we performed a WB assay, showing that in a21 HS-5 cells, PIAS3 is indeed over-expressed compared to controls. Taken together, these findings provide the first evidence that inhibition of miR-21 in MM microenviroment restores RANKL/OPG balance, which might result in prevention of skeletal- related events. These findings support the design of innovative miR-21 inhibition-based approaches against MM-related BD.Supported by the Italian Association for Cancer Research (AIRC) “Special Program Molecular Clinical Oncology - 5 per mille”, PI:P.T., n. 9980, 2010/15.” [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Targeting syndecan-1 in breast cancer inhibits osteoclast functions through up-regulation of osteoprotegerin

BackgroundBreast cancer often metastasizes into bone and leads to osteolytic lesions. The underlying mechanisms, however, are complex and not fully understood. Syndecan-1 is a proteoglycan that has various functions relevant for tumor progression including cell–cell communication and cell–matrix interactions. Moreover, its two glycosaminoglycan-binding sites suggest that it may interfere with glycoproteins such as osteoprotegerin, a potent inhibitor of osteoclastogenesis. Thus, we hypothesize that tumor-derived syndecan-1 alters osteoclast biology by modulating osteoprotegerin. MethodsSyndecan-1 expression was down-regulated via siRNA and the cell fate of the breast cancer cell lines MCF-7, T-47D, and MDA-MB-231 was investigated. Furthermore, we determined the regulation of syndecan-1 by dexamethasone, a commonly used antiemetic in breast cancer therapy. Additionally, we analyzed the genesis and activity of osteoclasts in indirect co-culture experiments using supernatants from MCF-7 cells with deficient and sufficient levels of syndecan-1. ResultsDexamethasone time- and dose-dependently increased syndecan-1 expression up to 4-fold but did not alter cell behavior. Syndecan-1 up-regulation did not affect the survival or migration of breast cancer cells. Depletion of syndecan-1 using siRNA led to decreased vitality of progesterone receptor-positive cell lines. In MCF-7 cells osteoprotegerin production was up-regulated 2.5-fold after syndecan-1 knock-down. The culture of osteoclast precursors with the supernatant of MCF-7 cells with reduced syndecan-1 levels suppressed osteoclast formation and activity by 21% and 23%, respectively. Adding neutralizing antibodies to osteoprotegerin to the breast cancer supernatants reversed osteoclastogenesis. ConclusionThus, we identified tumor-derived syndecan-1 as a novel positive regulator of osteoclastogenesis and new player in the tumor-bone dialog.

Osteoprotegerin deficiency attenuates strontium-mediated inhibition of osteoclastogenesis and bone resorption

Strontium (Sr) exerts an anabolic and antiresorptive effect on bone, but the mechanism remains unknown. Osteoprotegerin (OPG) expressed by osteoblasts plays an important role in regulating bone homeostasis by inhibiting osteoclastogenesis and bone resorption. This study aims at evaluating the role of OPG in Sr-mediated inhibition of osteoclastogenesis and bone resorption. Six-week-old Opg knockout (KO) male mice and their wild-type (WT) littermates were treated orally with vehicle (Veh) or Sr compound (4 mmol/kg) daily for 8 weeks. Bone mass and microstructure in the lumbar spine (L(4)) and proximal tibia were analyzed with micro-computed tomography (µCT). Bone remodeling was evaluated with serum biochemical analysis and static and dynamic bone histomorphometry. Osteoclast differentiation potential and gene expression were analyzed in bone marrow cells. The findings demonstrate that Sr compound treatment results in greater bone volume and trabecular number than Veh treatment in WT mice. The anabolic response of trabecular bone to Sr treatment is attenuated in KO mice. Although Sr treatment significantly decreases in vitro osteoclastogenesis and bone resorption in WT mice, these effects are attenuated in KO mice. Furthermore, Sr treatment profoundly increases Opg gene expression in the tibias and OPG protein levels in the sera of WT mice. This study concludes that the inhibition of osteoclastogenesis and bone resorption is possibly associated with OPG upregulation by Sr treatment.