Decoy Receptor Osteoprotegerin Research Articles

RANKL biology.

Since the receptor activator of nuclear factor-kappa B ligand (RANKL), its cognate receptor activator of nuclear factor-kappa B (RANK), and the decoy receptor osteoprotegerin (OPG) were discovered, a number of studies have uncovered the crucial role of the RANKL-RANK-OPG pathway in controlling the key aspect of bone homeostasis, the immune system, inflammation, cancer, and other systems under pathophysiological condition. These findings have expanded the understanding of the multifunctional biology of the RANKL-RANK-OPG pathway and led to the development of therapeutic potential targeting this pathway. The successful development and application of anti-RANKL antibody in treating diseases causing bone loss validates the utility of therapeutic approaches based on the modulation of this pathway. Moreover, recent studies have demonstrated the involvement of the RANKL-RANK pathway in osteoblast differentiation and bone formation, shedding light on the RANKL-RANK dual signaling in coupling bone resorption and bone formation. In this review, we will summarize the current understanding of the RANKL-RANK-OPG system in the context of the bone and the immune system as well as the impact of this pathway in disease conditions, including cancer development and metastasis.

Rank-Rankl-Opg Axis in Multiple Sclerosis: The Contribution of Placenta

Women with multiple sclerosis (MS) can safely become pregnant and give birth, with no side effects or impediments. Pregnancy is generally accepted as a period of well-being in which relapses have a softer evolution, particularly in the third trimester. Herein, we hypothesized that the placenta, via its “secretome”, could contribute to the recognized beneficial effects of pregnancy on MS activity. We focused on a well-known receptor/ligand/decoy receptor system, such as the one composed by the receptor activator of nuclear factor-kB (RANK), its ligand (RANKL), and the decoy receptor osteoprotegerin (OPG), which have never been investigated in an integrated way in MS, pregnancy, and placenta. We reported that pregnancy at the term of gestation influences the balance between circulating RANKL and its endogenous inhibitor OPG in MS women. We demonstrated that the placenta at term is an invaluable source of homodimeric OPG. By functional studies on astrocytes, we showed that placental OPG suppresses the mRNA expression of the CCL20, a chemokine responsible for Th17 cell recruitment. We propose placental OPG as a crucial molecule for the recognized beneficial effect of late pregnancy on MS and its potential utility for the development of new and more effective therapeutic approaches.

Exendin‐4 enhances osteogenic differentiation of adipose tissue mesenchymal stem cells through the receptor activator of nuclear factor‐kappa B and osteoprotegerin signaling pathway

The capability of mesenchymal stem cells (MSCs) to repair bone damage and defects has long been investigated. The receptor activator of nuclear factor-kappa B (RANK), its ligand (RANKL) and the decoy receptor osteoprotegerin (OPG) axis is crucial to keep the equilibrium between osteoblastic and osteoclastic activity. Exendin-4 utilization increased bone formation and enhanced bone integrity. This study aimed to investigate the mentioned axis and determine the effect of exendin-4 upon adipose mesenchymal stem cells (Ad-MSCs) osteogenic differentiation. Ad-MSCs were isolated from rat epididymal fat, followed by characterization and then differentiation into osteocytes both in the presence or absence of exendin-4. Osteogenic differentiation was evaluated by alizarin red staining and the expression of osteogenic markers; using reverse transcriptase-quantitative polymerase chain reaction, western blotting and enzyme-linked immunoassay. MSCs derived from rat epididymal fat were isolated and characterized, along with their differentiation into osteocytes. The differentiated cells were alizarin red-stained, showing increased staining intensity upon addition of exendin-4. Moreover, the addition of exendin-4 elevated the messenger RNA expression levels of osteogenic markers; runt-related transcription factor-2 (RUNX-2), osteocalcin, and forkhead box protein O-1while reducing the expression of the adipogenic marker peroxisome-proliferator-activated receptor-gamma. Exendin-4 addition elevated OPG levels in the supernatant of osteogenic differentiated cells. Moreover, exendin-4 elevated the protein levels of glucagon-like peptide-1 receptor and RUNX-2, while decreasing both RANK and RANKL. In conclusion, osteogenic differentiation of Ad-MSCs is associated with increased osteoblastic rather than osteoclastic activity. The findings of this study suggest that exendin-4 can enhance Ad-MSCs osteogenic differentiation partially through the RANK/RANKL/OPG axis.

Could Bone Biomarkers Predict Bone Turnover after Kidney Transplantation?—A Proof-of-Concept Study

Aim: Bone disease after kidney transplant (KT) results from multiple factors, including previous bone and mineral metabolism disturbances and effects of transplant-related medications. New biomolecules have been recently associated with the development and progression of the chronic kidney disease–associated bone and mineral disorder (CKD-MBD). These include sclerostin and the soluble receptor activator of nuclear factor-kB ligand (sRANKL). Methods: To better understand the role of biomarkers in post-transplant bone disease, this study was designed to prospectively evaluate and correlate results from the histomorphometric analysis of bone biopsies after KT with emerging serum biomarkers of the CKD-MBD: sclerostin, Dickkopf-related protein 1 (Dkk-1), sRANKL and osteo-protegerin (OPG). Results: Our data shows a significant increase in plasma levels of bioactive sclerostin after KT accompanied by a significant reduction in plasma levels of Dkk-1, suggesting a promotion of the inhibition of bone formation by osteoblasts through the activation of these inhibitors of the Wnt signaling pathway. In addition, we found a significant increase in plasma levels of free sRANKL after KT accompanied by a significant reduction in plasma levels of its decoy receptor OPG, suggesting an enhanced bone resorption by osteoclasts mediated by this mechanism. Conclusions: Taken together, these results suggest that the loss of bone volume observed after KT could be explain mainly by the inhibition of bone formation mediated by sclerostin accompanied by an enhanced bone resorption mediated by sRANKL.

Salivary Gene Expression of RANK, RANKL, and OPG in Type 1 Diabetes Mellitus and Periodontal Disease Patients.

The relationship between type 1 diabetes mellitus (T1DM) and periodontal disease may exhibit by the alteration of bone metabolism. However, evidence for this relationship is scarce and inconclusive. Thus, the aims of the present study were to investigate salivary receptor activator of nuclear factor kappa-β (RANK), receptor activator of nuclear factor kappa-β ligand (RANKL), osteoprotegerin (OPG) gene expression and the RANKL:OPG ratio in T1DM and non-T1DM. Secondary objective was to determine the relationships of RANK, RANKL and OPG gene expression to clinical parameters of T1DM and periodontal disease. Twenty patients with T1DM and twenty age-matched non-T1DM were recruited. Clinical periodontal parameters were measured. Total RNA was isolated from non-stimulated saliva, and the relative gene expressions of RANK, RANKL, OPG and RANKL:OPG ratio were determined by quantitative real-time polymerase chain reaction. The T1DM group had significantly higher mean periodontal parameters than the non-T1DM group, while the mean plaque scores of both groups were not significantly different. There was a trend of higher relative gene expression of RANK, RANKL, and the RANKL:OPG ratio and lower expression of OPG in T1DM group but no statistic significant different when compared to non-T1DM. In the T1DM group, RANKL:OPG correlated with the percentage of bleeding sites, whereas RANK, RANKL, and HbA1c levels correlated with pocket depth. Bone metabolisms demonstrating by decreased OPG gene expression and upregulated of RANK, RANKL, RANKL:OPG with higher pocket depth and bleeding in T1DM may play an important role in periodontal destruction in T1DM.

Blockade of RANKL/RANK interaction prevents allergic airway inflammation in a mouse model of asthma

Abstract W are constantly looking for new therapeutic options for asthma treatment. In this study, we investigated whether the signaling pathway of the receptor activator of nuclear factor (NF)-κB (RANK), RANK ligand (RANKL), and its decoy receptor osteoprotegerin (OPG) may be considered as a novel drug target for therapy of allergic asthma. Therefore, we tested the effect of blockade of RANK-L with OPG on the development of ovalbumin (OVA)-induced airway allergic inflammation (AAI) and accompanying immune response. The absolute counts of CD11chighCD11b+CD103− (CD11b+) dendritic cells (DC), ST2+Foxp3−CD4+ (Th2) cells and BrdU+(i.e., proliferating)CD4+ T cells were determined in the mediastinal lymph nodes (MLNs) and lungs of untreated non-immunized mice, untreated OVA-immunized mice and OPG-treated OVA-immunized mice. Immunization with OVA induced a significant increase in the mean scores of lung histologic lesions and administration of OPG completely prevented this effect. The absolute counts of CD11b+ DC, Th2 cells and BrdU+CD4+ T cells were increased in the MLNs and lungs of untreated OVA-immunized mice. Treatment with OPG attenuated, but did not abolish, the OVA-induced increase in the count of CD11b+ DC in the MLNs. In turn, administration of this agent prevented the OVA-induced increase in the count of Th2 cells and BrdU+CD4+ T cells in the MLNs and lungs. In conclusion, blockade of RANK/RANKL interaction prevents OVA-induced AAI and this effect is mediated through inhibition the allergen-induced clonal expansion of CD4+ T cells in the MLNs. Clinical implication: the local inhibition of RANK/RANK-L interaction within the respiratory system can be considered as a novel therapeutic strategy in treatment of allergic asthma.

RANKL induces beige adipocyte differentiation in preadipocytes.

The receptor activator of nuclear factor-κB (NF-κB) (RANK), its ligand (RANKL), and the decoy receptor osteoprotegerin (OPG) are a triad of proteins that regulate bone metabolism, and serum OPG is considered a biomarker for cardiovascular diseases and Type 2 diabetes; however, the implications of OPG in adipose tissue metabolism remains elusive. In this study, we investigate RANK-RANKL-OPG signaling in white adipose tissue browning. Histological analysis of osteoprotegerin knockout (OPG-/-) mice showed subcutaneous white adipose tissue (sWAT) browning, resistance for high-fat diet-induced weight gain, and preserved glucose metabolism compared with wild-type (WT) mice. Stromal vascular fraction (SVF) cells from sWAT of OPG-/- mice showed multilocular morphology and higher expression of brown adipocyte marker genes compared with those from the WT group. Infusion of RANKL induced browning and elevated respiratory rates in sWAT, along with increased whole body oxygen consumption in mice measured by indirect calorimetry. Subcutaneous WAT-derived SVF and 3T3-L1 cells, but not mature white adipocytes, differentiated into beige adipose tissue in the presence of RANKL. Moreover, SVF cells, even under white adipocyte differentiation, showed multilocular lipid droplet, lower lipid content, and increased expression of beige adipocyte markers with RANKL stimulation. In this study, we show for the first time the contribution of RANKL to increase energy expenditure by inducing beige adipocyte differentiation in preadipocytes.

At the Crossroads of the Adipocyte and Osteoclast Differentiation Programs: Future Therapeutic Perspectives.

The coordinated development and function of bone-forming (osteoblasts) and bone-resorbing (osteoclasts) cells is critical for the maintenance of skeletal integrity and calcium homeostasis. An enhanced adipogenic versus osteogenic potential of bone marrow mesenchymal stem cells (MSCs) has been linked to bone loss associated with diseases such as diabetes mellitus, as well as aging and postmenopause. In addition to an inherent decrease in bone formation due to reduced osteoblast numbers, recent experimental evidence indicates that an increase in bone marrow adipocytes contributes to a disproportionate increase in osteoclast formation. Therefore, a potential strategy for therapeutic intervention in chronic bone loss disorders such as osteoporosis is to interfere with the pro-osteoclastogenic influence of marrow adipocytes. However, application of this approach is limited by the extremely complex regulatory processes in the osteoclastogenic program. For example, key regulators of osteoclastogenesis such as the receptor activator of nuclear factor-kappaB ligand (RANKL) and the soluble decoy receptor osteoprotegerin (OPG) are not only secreted by both osteoblasts and adipocytes, but are also regulated through several cytokines produced by these cell types. In this context, biologically active signaling molecules secreted from bone marrow adipocytes, such as chemerin, adiponectin, leptin, visfatin and resistin, can have a profound influence on the osteoclast differentiation program of hematopoietic stem cells (HSCs), and thus, hold therapeutic potential under disease conditions. In addition to these paracrine signals, adipogenic transcription factors including CCAAT/enhancer binding protein alpha (C/EBPα), C/EBP beta (C/EBPβ) and peroxisome proliferator-associated receptor gamma (PPARγ) are also expressed by osteoclastogenic cells. However, in contrast to MSCs, activation of these adipogenic transcription factors in HSCs promotes the differentiation of osteoclast precursors into mature osteoclasts. Herein, we discuss the molecular mechanisms that link adipogenic signaling molecules and transcription factors to the osteoclast differentiation program and highlight therapeutic strategies targeting these mechanisms for promoting bone homeostasis.

Osteoprotective action of low-salt diet requires myeloid cell-derived NFAT5.

Dietary salt consumption leads to cutaneous Na+ storage and is associated with various disorders, including osteopenia. Here, we explore the impact of Na+ and the osmoprotective transcription factor nuclear factor of activated T cell 5 (NFAT5) on bone density and osteoclastogenesis. Compared with treatment of mice with high-salt diet, low-salt diet (LSD) increased bone density, decreased osteoclast numbers, and elevated Na+ content and Nfat5 levels in the BM. This response to LSD was dependent on NFAT5 expressed in myeloid cells. Simulating in vivo findings, we exposed osteoclast precursors and osteoblasts to elevated Na+ content (high-salt conditions; HS¢), resulting in increased NFAT5 binding to the promotor region of RANKL decoy receptor osteoprotegerin (OPG). These data not only demonstrate that NFAT5 in myeloid cells determines the Na+ content in BM, but that NFAT5 is able to govern the expression of the osteoprotective gene OPG. This provides insights into mechanisms of Na+-induced cessation of osteoclastogenesis and offers potentially new targets for treating salt-induced osteopenia.

Receptor activator of nuclear factor κB ligand is a biomarker for osteitis of chronic rhinosinusitis.

Evidence of osteitis is frequently observed in patients with chronic rhinosinusitis (CRS), especially in recalcitrant cases. However, studies focusing on biological markers of osteitis are limited and it remains unclear whether osteitis is associated with different phenotypes of CRS. This study aimed to analyze the expression and assess the roles of receptor activator of nuclear factor κB ligand (RANKL) in patients with CRS and osteitis. CRS patients with nasal polyps (CRSwNP, n = 63), CRS patients without nasal polyps (CRSsNP, n = 8), and control subjects (n = 12) were enrolled. Histologic phenotypes, clinical information, and computed tomography (CT) scores were investigated. The Global Osteitis Scoring Scale (GOSS) and RANKL, a molecular marker of bone remodeling, were analyzed in each type of CRS. CRS mouse models were treated with anti-RANKL. GOSS values were significantly higher in all CRS patients than in the control group. The GOSS value in non-eosinophilic CRSwNP was higher than in eosinophilic CRSwNP. RANKL was upregulated whereas decoy receptor osteoprotegerin (OPG) was downregulated in CRS. RANKL messenger RNA (mRNA) and protein levels were positively correlated with GOSS. RANKL/OPG was increased in recurrent cases compared with primary cases. Multiple inflammatory mediators were positively correlated with the protein level of RANKL in CRS tissues. In the mouse CRSwNP model, anti-RANKL treatment abrogated mucosal inflammation and bone remodeling. RANKL expression is associated with clinical osteitis and disease severity in CRSwNP. These findings shed light on the importance of RANKL as a potential biomarker of CRS and a key player in CRS pathogenesis.

TRAIL, OPG, and TWEAK in kidney disease: biomarkers or therapeutic targets?

Ligands and receptors of the tumor necrosis factor (TNF) superfamily regulate immune responses and homeostatic functions with potential diagnostic and therapeutic implications. Kidney disease represents a global public health problem, whose prevalence is rising worldwide, due to the aging of the population and the increasing prevalence of diabetes, hypertension, obesity, and immune disorders. In addition, chronic kidney disease is an independent risk factor for the development of cardiovascular disease, which further increases kidney-related morbidity and mortality. Recently, it has been shown that some TNF superfamily members are actively implicated in renal pathophysiology. These members include TNF-related apoptosis-inducing ligand (TRAIL), its decoy receptor osteoprotegerin (OPG), and TNF-like weaker inducer of apoptosis (TWEAK). All of them have shown the ability to activate crucial pathways involved in kidney disease development and progression (e.g. canonical and non-canonical pathways of the transcription factor nuclear factor-kappa B), as well as the ability to regulate cell proliferation, differentiation, apoptosis, necrosis, inflammation, angiogenesis, and fibrosis with double-edged effects depending on the type and stage of kidney injury. Here we will review the actions of TRAIL, OPG, and TWEAK on diabetic and non-diabetic kidney disease, in order to provide insights into their full clinical potential as biomarkers and/or therapeutic options against kidney disease.

Abstract P6-09-01: In primary, non-metastatic breast cancer patients, increased serum levels of RANKL significantly correlate with tumor cell spread to the bone and the occurrence of bone metastasis whereas high levels of its soluble decoy receptor osteoprotegerin predict poor survival

Abstract Background: Receptor activator of nuclear factor kappa-B ligand (RANKL) is an essential protein for osteoclast regulation and its activity is controlled by its soluble decoy receptor osteoprotegerin (OPG). RANKL has been associated with benign as well as malignant bone disease and there is increasing evidence that RANKL may also directly affect breast cancer (BC) progression and metastasis to the bone. Here we assessed serum concentrations of RANKL and OPG in 509 patients with primary, non-metastatic BC and correlated the results with clinical parameters including the presence of disseminated tumor cells (DTCs) in the bone marrow (BM), survival and the risk of developing metastatic disease. Patients and Methods: Patients with first diagnosis of BC between Aug 2006 and Dec 2009 were included in our study. BM sampling was performed before surgery in an adjuvant setting and two BM aspirates were analyzed for DTCs using density centrifugation followed by immunocytochemistry applying the pan-cytokeratin antibody A45-B/B3. Blood was collected from each patient and sRANKL and OPG levels in the serum were measured by ELISA (Biomedica, Vienna, Austria). Results: Mean serum values for RANKL and OPG were 0.23±0.20 pmol/l and 4.24±1.70 pmol/l, respectively. RANKL levels were significantly lower in women above the age of 60 (p<0.0001) and RANKL/OPG ratios were higher in patients with lymph node involvement (p<0.05). High OPG levels were associated with a higher risk of death from BC (HR 1.94 95%CI 1.23-3.07; p=0.005) and multivariate analyses revealed OPG to be an independent prognostic marker for BC specific survival (p=0.035). DTCs were detected in 207/507 (41%) patients and RANKL levels were 33% higher in DTC-positive patients (p<0.0001). Interestingly, in DTC-negative patients, high RANKL levels were associated with a significantly better BC specific survival compared to low levels (HR 0.524; 95%CI 0.30-0.95; p=0.04). RANKL serum levels were significantly enhanced in patients that developed bone metastases (p=0.01) and patients in the highest quartile of RANKL had a significantly increased risk of developing bone metastases compared to those in the lowest RANKL quartile (HR 4.62, 95%CI 1.49-14.34, p=0.03). Conclusion: Increased OPG levels indicated poor survival and high RANKL significantly associated with an increased risk of developing bone metastases while indicating a positive prognostic marker in DTC-negative patients. These findings warrant further investigation as it may provide a rational for novel diagnostic or therapeutic approaches. Citation Format: Bittner A-K, Goebel A, Hoffmann O, Rauner M, Hofbauer LC, Kimmig R, Kasimir-Bauer S, Rachner TD. In primary, non-metastatic breast cancer patients, increased serum levels of RANKL significantly correlate with tumor cell spread to the bone and the occurrence of bone metastasis whereas high levels of its soluble decoy receptor osteoprotegerin predict poor survival [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-09-01.

Effect of Alendronate Treatment on Salivary Levels of Osteoprotegrin and TNF-α in Postmenopausal Woman with Osteoporosis and Periodontal Diseases

Background: All diseases concerning bone destruction such as osteoporosis and periodontal diseases share common pattern in which the osteoclast cells are absolutely responsible for bone resorption that occurred when osteoclast activity exceeds osteoblast activity. Osteoprotegrin (OPG) considered as novel soluble decoy receptor known as “bone protector” since it prevents extreme bone resorption through inhibition of differentiation and activity of osteoclast by competing for binding site. It binds to receptor activator of nuclear factor kappa-B ligand (RANKL) and prevent its interaction with receptor activator of nuclear factor kappa-B (RANK), thus inhibits osteoclast formation. TNF-α is a pro-inflammatory cytokines having a broad range of important roles in regulation of immune system and bone resorption through the stimulation of osteoclastogenesis. Alendronate (ALN) diminishes the expression of osteoclast activating factors and cytokines such as RANKL and enhances the production of decoy receptor osteoprotegerin in osteoblast cells. Moreover, it decreases the production of proinflammatory cytokines such as TNF-α by macrophage, stimulates apoptosis of monocyte-macrophage cell lines derivative and reduces inflammatory response. Aims of the Study: 1. To assess the effect of alendronate treatment on salivary levels of osteoprotegrin and TNF-α in postmenopausal women with osteoporosis and periodontal disease 2. To find any possible correlation between salivary levels of osteoprotegrin and TNF-α in control and study groups. Materials and Methods: Total sample of 90 female subjects (55-65 years) were divided into 3 groups, (30 subjects in each group): first control group involved systemically healthy subjects with healthy periodontium, second group involved postmenopausal women with osteoporosis under alendronate treatment for(3-6)months (alendronate group), third group involved postmenopausal women with osteoporosis without alendronate treatment(osteoporosis group). The last two groups were sub- divided in- to two sub –groups (15 subjects in each sub-group) of gingivitis and periodontitis subjects respectively. Salivary samples were collected from all subjects and salivary levels of osteoprotegrin and TNF- α were determined by enzyme –linked immune sorbent assay (ELISA). Results: Highest median value of salivary (OPG) was found in alendronate group followed by control group while the lowest value was found in osteoporosis group. Highest median value of TNF- α was found in osteoporosis group followed by control group and alendronate group respectively with highly significant differences between them. Spearman correlation between salivary levels of TNF-α and OPG showed non- significant correlation at all subgroups. Conclusion: Subjects with osteoporosis in this study had greater levels of TNF-α and decrease in the level of OPG comparing with patients under alendronate treatment. Alendronate treatment for women with osteoporosis and periodontal disease may have beneficial outcome.

Effect of Alendronate Treatment on Salivary Levels of Osteoprotegrin and TNF-α in Postmenopausal Woman with Osteoporosis and Periodontal Diseases

Background: All diseases concerning bone destruction such as osteoporosis and periodontal diseases share common pattern in which the osteoclast cells are absolutely responsible for bone resorption that occurred when osteoclast activity exceeds osteoblast activity. Osteoprotegrin (OPG) considered as novel soluble decoy receptor known as “bone protector” since it prevents extreme bone resorption through inhibition of differentiation and activity of osteoclast by competing for binding site. It binds to receptor activator of nuclear factor kappa-B ligand (RANKL) and prevent its interaction with receptor activator of nuclear factor kappa-B (RANK), thus inhibits osteoclast formation. TNF-α is a pro-inflammatory cytokines having a broad range of important roles in regulation of immune system and bone resorption through the stimulation of osteoclastogenesis. Alendronate (ALN) diminishes the expression of osteoclast activating factors and cytokines such as RANKL and enhances the production of decoy receptor osteoprotegerin in osteoblast cells. Moreover, it decreases the production of proinflammatory cytokines such as TNF-α by macrophage, stimulates apoptosis of monocyte-macrophage cell lines derivative and reduces inflammatory response. Aims of the Study: 1. To assess the effect of alendronate treatment on salivary levels of osteoprotegrin and TNF-α in postmenopausal women with osteoporosis and periodontal disease 2. To find any possible correlation between salivary levels of osteoprotegrin and TNF-α in control and study groups. Materials and Methods: Total sample of 90 female subjects (55-65 years) were divided into 3 groups, (30 subjects in each group): first control group involved systemically healthy subjects with healthy periodontium, second group involved postmenopausal women with osteoporosis under alendronate treatment for(3-6)months (alendronate group), third group involved postmenopausal women with osteoporosis without alendronate treatment(osteoporosis group). The last two groups were sub- divided in- to two sub –groups (15 subjects in each sub-group) of gingivitis and periodontitis subjects respectively. Salivary samples were collected from all subjects and salivary levels of osteoprotegrin and TNF- α were determined by enzyme –linked immune sorbent assay (ELISA). Results: Highest median value of salivary (OPG) was found in alendronate group followed by control group while the lowest value was found in osteoporosis group. Highest median value of TNF- α was found in osteoporosis group followed by control group and alendronate group respectively with highly significant differences between them. Spearman correlation between salivary levels of TNF-α and OPG showed non- significant correlation at all subgroups. Conclusion: Subjects with osteoporosis in this study had greater levels of TNF-α and decrease in the level of OPG comparing with patients under alendronate treatment. Alendronate treatment for women with osteoporosis and periodontal disease may have beneficial outcome.

Genetic deletion of muscle RANK or selective inhibition of RANKL is not as effective as full-length OPG-fc in mitigating muscular dystrophy

Although there is a strong association between osteoporosis and skeletal muscle atrophy/dysfunction, the functional relevance of a particular biological pathway that regulates synchronously bone and skeletal muscle physiopathology is still elusive. Receptor-activator of nuclear factor κB (RANK), its ligand RANKL and the soluble decoy receptor osteoprotegerin (OPG) are the key regulators of osteoclast differentiation and bone remodelling. We thus hypothesized that RANK/RANKL/OPG, which is a key pathway for bone regulation, is involved in Duchenne muscular dystrophy (DMD) physiopathology. Our results show that muscle-specific RANK deletion (mdx-RANKmko) in dystrophin deficient mdx mice improves significantly specific force [54% gain in force] of EDL muscles with no protective effect against eccentric contraction-induced muscle dysfunction. In contrast, full-length OPG-Fc injections restore the force of dystrophic EDL muscles [162% gain in force], protect against eccentric contraction-induced muscle dysfunction ex vivo and significantly improve functional performance on downhill treadmill and post-exercise physical activity. Since OPG serves a soluble receptor for RANKL and as a decoy receptor for TRAIL, mdx mice were injected with anti-RANKL and anti-TRAIL antibodies to decipher the dual function of OPG. Injections of anti-RANKL and/or anti-TRAIL increase significantly the force of dystrophic EDL muscle [45% and 17% gains in force, respectively]. In agreement, truncated OPG-Fc that contains only RANKL domains produces similar gains, in terms of force production, than anti-RANKL treatments. To corroborate that full-length OPG-Fc also acts independently of RANK/RANKL pathway, dystrophin/RANK double-deficient mice were treated with full-length OPG-Fc for 10 days. Dystrophic EDL muscles exhibited a significant gain in force relative to untreated dystrophin/RANK double-deficient mice, indicating that the effect of full-length OPG-Fc is in part independent of the RANKL/RANK interaction. The sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) activity is significantly depressed in dysfunctional and dystrophic muscles and full-length OPG-Fc treatment increased SERCA activity and SERCA-2a expression. These findings demonstrate the superiority of full-length OPG-Fc treatment relative to truncated OPG-Fc, anti-RANKL, anti-TRAIL or muscle RANK deletion in improving dystrophic muscle function, integrity and protection against eccentric contractions. In conclusion, full-length OPG-Fc represents an efficient alternative in the development of new treatments for muscular dystrophy in which a single therapeutic approach may be foreseeable to maintain both bone and skeletal muscle functions.

Unraveling Binding Interactions between Human RANKL and Its Decoy Receptor Osteoprotegerin.

Recent studies have revealed the importance and the active contribution of the RANKL/OPG/RANK pathway in many bone diseases including different forms of common osteoporosis. In this study, we present an extensive atomistic molecular dynamic study of the OPG/RANKL system. Within the molecular models, we varied the number of OPG molecules bound to the RANKL trimer and carried out a study to determine how the binding affinity of the OPG/RANKL system changes as a function of OPG concentration. The molecular mechanics Poisson-Boltzmann surface area method was used to analyze binding free energies. It is shown that the binding affinity decreases with increasing numbers of OPG molecules. Additionally, conformational changes of RANKL, interactions between the N-terminus outlier module of OPG with RANKL, and residues that play an important role in the binding of OPG to RANKL trimer were investigated. A probable cause for unfavorable binding for a third OPG molecule was found. Along with the currently available experimental studies, this computational study will be valuable for the comprehensive understanding of OPG/RANKL at the atomistic level.

Abstract P6-07-11: Serum levels of RANKL are increased in primary breast cancer patients in the presence of disseminated tumor cells in the bone marrow

Abstract Background: Receptor activator of nuclear factor kappa-B ligand (RANKL) is an essential protein for osteoclast regulation that is associated with benign and malignant bone disease. The activity of RANKL is controlled by its soluble decoy receptor osteoprotegerin (OPG). There is increasing evidence that RANKL may also directly affect breast cancer progression and metastasis to the bone. This study was aimed to assess the levels of RANKL and OPG in 509 newly diagnosed breast cancer patients with regard to the presence of disseminated tumor cells (DTCs) in the bone marrow (BM), circulating tumor cells (CTCs) in blood and clinical parameters. Patients and Methods: 509 patients with first diagnosis of breast cancer between Aug 2006 and Dec 2009 were included in our study. Blood and BM sampling was performed before surgery in an adjuvant setting. Blood was collected from each patient and sRANKL as well as OPG in the serum were measured by ELISA (Immunodiagnostic, Vienna, Austria). Two BM aspirates were analyzed for DTCs by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. In a subgroup of 364 patients, 2 x 5 ml blood was studied for CTCs using the AdnaTest BreastCancer (QIAGEN, Hannover GmbH, Germany) for the detection of EpCAM, MUC-1, HER-2, and beta-Actin transcripts. Results: Mean serum values for RANKL and OPG were 0.23 ± 0.20 pmol/l and 4.24 ± 1.70 pmol/l, respectively. RANKL levels were significantly lower in women above 60 years of age (0.19 pmol/l vs 0.26 pmol/l; p < 0.0001). This finding was reflected by higher RANKL serum levels and RANKL/OPG ratios in premenopausal patients compared to peri- (p<0.05) and postmenopausal patients (p<0.001), respectively. RANKL/OPG ratios were also higher in patients with lymph node involvement (N1-N3, p=0.03). All other clinical parameters did not influence RANKL or OPG levels. DTCs were detected in 213/509 (42%) patients and CTCs in 81/364 (22%) patients, respectively. However, while RANKL levels were unchanged in patients with detectable CTCs, they significantly increased by 33% (p<0.0001) in patients with DTCs. There was no difference in OPG levels, resulting in an increased RANKL to OPG ratio in patients with DTCs in the BM (0.087 vs. 0.060; p < 0.0001). Conclusion: In conclusion, we show that RANKL serum levels and RANKL/OPG ratios are increased in patients with detectable DTCs in the BM, prior to the establishment of detectable bone metastases. This finding warrants further investigation as it may provide a rational for novel diagnostic or therapeutic approaches. Citation Format: Kasimir-Bauer S, Bittner A-K, Goebel A, Hoffmann O, Browne AJ, Rauner M, Hofbauer LC, Wimberger P, Kimmig R, Rachner TD. Serum levels of RANKL are increased in primary breast cancer patients in the presence of disseminated tumor cells in the bone marrow [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 P6-07-11.

Quantitation of the Rank-Rankl Axis in Primary Biliary Cholangitis.

There is substantial data that suggests an abnormality of innate immunity in patients with primary biliary cholangitis (PBC) which includes the transcription factor nuclear factor-kB (NF-kB) and well as downstream inflammatory signaling pathways. In addition, ImmunoChip analysis has identified a novel PBC-associated locus near the receptor activator of NF-kB ligand (RANKL) gene. Based on these observations, we investigated the role of the RANKL axis in the liver of patients with PBC compared to controls. We used immunohistochemistry to quantitate liver expression of RANKL, its receptor (RANK), and importantly the decoy receptor osteoprotegerin (OPG), including a total of 122 liver samples (PBC = 37, primary sclerosing cholangitis = 20, autoimmune hepatitis = 26, chronic hepatitis B = 32 and unaffected controls = 7). In addition, we studied RANKL-RANK-OPG co-localization in CD4 and CD8 T cells, B cells, dendritic cells, macrophages, NK, NKT cells, hepatocytes, and cholangiocytes. We report herein that RANK is constitutively expressed by cholangiocytes in both unaffected and diseased liver. However, cholangiocytes from PBC express significantly higher levers of RANK than either the unaffected controls or liver diseased controls. CD4, CD8 and CD19 cells with in the portal areas around bile ducts in PBC express significantly higher levels of RANKL compared to controls. Importantly, the overall hepatic RANKL level and the ratio of hepatic RANKL/OPG correlated with disease severity in PBC. In conclusion, our data indicate a role of RANK-RANKL axis in the innate immune activation in PBC and we hypothesize that the damaged cholangiocytes, which express high levels of RANK, lead to the recruitment of RANKL positive cells and ultimately the classic portal tract infiltrates.

Gene Polymorphisms in the RANKL/RANK/OPG Pathway Are Associated with Type 2 Diabetes Mellitus in Southern Han Chinese Women.

Receptor activator of nuclear factor-kappa B ligand (RANKL), its receptor activator of nuclear factor-kappa B (RANK), and decoy receptor osteoprotegerin (OPG) are three major proteins of the RANKL/RANK/OPG signaling pathway encoded by TNFSF11, TNFRSF11A, and TNFRSF11B, respectively. This pathway plays a critical role in bone remodeling and may have a role in the pathogenesis of type 2 diabetes mellitus (T2DM). The aim of this study was to investigate the relationship between gene polymorphisms in the RANKL/RANK/OPG pathway and T2DM in Southern Han Chinese women. A total of 1233 participants, including 514 T2DM patients and 719 healthy control subjects, were enrolled in this case-control study. Twenty-one single-nucleotide polymorphisms (SNPs) of TNFSF11, TNFRSF11A, and TNFRSF11B were genotyped using an improved multiplex ligation detection reaction technique. Two SNPs of TNFRSF11B (rs11573819 and rs2073618) were significantly associated with T2DM (p = 0.04 and p = 0.009, respectively). Subjects with the GA genotype of rs11573819 had a lower risk of T2DM (odds ratio [OR] = 0.67, 95% confidence interval [CI] = 0.51-0.88, p = 0.005) compared with subjects with the GG genotype. The GG genotype of rs2073618 was associated with increased risk for T2DM (OR = 1.94, 95% CI = 1.14-3.30, p = 0.01) compared with the CC genotype. This study suggests that TNFRSF11B but not TNFSF11 and TNFRSF11A genetic polymorphisms are associated with T2DM in Southern Han Chinese women. These findings provide preliminary support for the potential role of the RANKL/RANK/OPG pathway in T2DM.

Physiological role of receptor activator nuclear factor-kB (RANK) in denervation-induced muscle atrophy and dysfunction.

The bone remodeling and homeostasis are mainly controlled by the receptor-activator of nuclear factor kB (RANK), its ligand RANKL, and the soluble decoy receptor osteoprotegerin (OPG) pathway. While there is a strong association between osteoporosis and skeletal muscle dysfunction, the functional relevance of a particular biological pathway that synchronously regulates bone and skeletal muscle physiopathology remains elusive. Our recent article published in the American Journal of Physiology (Cell Physiology) showed that RANK is also expressed in fully differentiated C2C12 myotubes and skeletal muscles. We used the Cre-Lox approach to inactivate muscle RANK (RANKmko) and showed that RANK deletion preserves the force of denervated fast-twitch EDL muscles. However, RANK deletion had no positive impact on slow-twitch Sol muscles. In addition, denervating RANKmko EDL muscles induced an increase in the total calcium concentration ([CaT]), which was associated with a surprising decrease in SERCA activity. Interestingly, the levels of STIM-1, which mediates Ca2+ influx following the depletion of SR Ca2+ stores, were markedly higher in denervated RANKmko EDL muscles. We speculated that extracellular Ca2+ influx mediated by STIM-1 may be important for the increase in [CaT] and the gain of force in denervated RANKmko EDL muscles. Overall, these findings showed for the first time that the RANKL/RANK interaction plays a role in denervation-induced muscle atrophy and dysfunction.