Soluble Decoy Receptor Osteoprotegerin Research Articles

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.

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.

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.

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.

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.

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.

Muscle RANK is a key regulator of Ca2+ storage, SERCA activity, and function of fast-twitch skeletal muscles.

Receptor-activator of nuclear factor-κB (RANK), its ligand RANKL, and the soluble decoy receptor osteoprotegerin are the key regulators of osteoclast differentiation and bone remodeling. Here we show that RANK is also expressed in fully differentiated myotubes and skeletal muscle. Muscle RANK deletion has inotropic effects in denervated, but not in sham, extensor digitorum longus (EDL) muscles preventing the loss of maximum specific force while promoting muscle atrophy, fatigability, and increased proportion of fast-twitch fibers. In denervated EDL muscles, RANK deletion markedly increased stromal interaction molecule 1 content, a Ca(2+)sensor, and altered activity of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) modulating Ca(2+)storage. Muscle RANK deletion had no significant effects on the sham or denervated slow-twitch soleus muscles. These data identify a novel role for RANK as a key regulator of Ca(2+)storage and SERCA activity, ultimately affecting denervated skeletal muscle function.

Changes in RANKL and osteoprotegerin expression after chronic exposure to indoor air pollution as a result of cooking with biomass fuel.

The impact of indoor air pollution as a result of cooking with unprocessed biomass on membrane-bound and serum receptor activator of nuclear factor-kappa ligand 1 (RANKL), its soluble decoy receptor osteoprotegerin (OPG) and osteoclast precursor CD14(+) CD16(+) monocytes was investigated. Seventy-four pre-menopausal women from eastern India using biomass and 65 control women who cooked with cleaner liquefied petroleum gas were enrolled. PM10 and PM2.5 levels in their indoor air were measured with real-time aerosol monitors. The levels of membrane-bound RANKL on leukocytes and percentage CD14(+) CD16(+) monocytes in the subjects' blood were assayed by flow cytometry. Soluble RANKL and OPG in serum were measured by ELISA. The results showed that PM10 and PM2.5 levels were significantly higher in the indoor air of biomass-using households. Compared with the control women, the levels of CD4(+) and CD19(+) lymphocytes and circulating granulocytes with elevated levels of membrane-bound RANKL were higher in biomass users. The serum levels of RANKL were increased by 41% whereas serum OPG was reduced by 22% among biomass users. The absolute number of CD14(+) CD16(+) monocytes was significantly increased in biomass users than the control women. After controlling for potential confounders, PM10 and PM2.5 levels were found to be positively associated with leukocyte and serum RANKL and CD14(+) CD16(+) monocyte levels, but negatively with serum OPG. From these results, we can conclude that chronic exposure to biomass smoke increased membrane-bound and soluble RANKL and circulating osteoclast precursors but decreased OPG, suggesting an increased risk of bone resorption and consequent osteoporosis in biomass-exposed women of a child-bearing age. Copyright © 2015 John Wiley & Sons, Ltd.

Osteoprotegerin Protects against Muscular Dystrophy

Receptor-activator of NF-κB, its ligand RANKL, and the soluble decoy receptor osteoprotegerin are the key regulators of osteoclast differentiation and bone remodeling. Although there is a strong association between osteoporosis and skeletal muscle atrophy/dysfunction, the functional relevance of a particular biological pathway that synchronously regulates bone and skeletal muscle physiopathology still is elusive. Here, we show that muscle cells can produce and secrete osteoprotegerin and pharmacologic treatment of dystrophic mdx mice with recombinant osteoprotegerin muscles. (Recombinant osteoprotegerin-Fc mitigates the loss of muscle force in a dose-dependent manner and preserves muscle integrity, particularly in fast-twitch extensor digitorum longus.) Our data identify osteoprotegerin as a novel protector of muscle integrity, and it potentially represents a new therapeutic avenue for both muscular diseases and osteoporosis.

Blockade of receptor activator of nuclear factor‐κB signaling greatly improves SERCA activity and contractility in atrophic and dystrophic skeletal muscles (731.7)

Receptor‐activator of nuclear factor‐κB (RANK), its ligand RANKL and the soluble decoy receptor osteoprotegerin (OPG) are members of the tumor necrosis factor (TNF) superfamily that control osteoclast differentiation, bone remodelling and osteoporosis. Since bone and skeletal muscle physiopathology occurs synchronously, we tested whether RANK/RANKL pathway is involved in muscle dysfunction following different muscle wasting conditions. Contractile properties of EDL muscles were obtained following 14d of denervation in mice specifically deficient in RANK skeletal muscle or in dystrophic mdx mice treated daily for 10d with OPG [0.3mg/kg], the natural inhibitor of RANKL. The activity and expression of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) were determined in denervated or dystrophic muscles. RANK muscle deletion did not prevent atrophy but greatly reduced the losses of force production and SERCA activity in denervated EDL muscles. Force production of EDL muscles also increased by 140% in OPG‐treated relative to PBS‐treated mdx mice while the treatment with OPG prevented the loss of the fast twitch SERCA1 protein content in dystrophic mice. Together, these data indicate that the deletion or blockage of RANK/RANKL pathway has inotropic effects and increases SERCA activity and expression in fast‐twitch skeletal muscles opening promising new avenues for several muscle diseases.

Skeletal and extraskeletal actions of denosumab

Osteoclasts and osteoblasts define skeletal mass, structure and strength through their respective actions in resorbing and forming bone. This remodeling process is orchestrated by the actions of hormones and growth factors, which regulate a cytokine system comprising the receptor activator of nuclear factor κB ligand (RANKL), its receptor RANK and the soluble decoy receptor osteoprotegerin (OPG). Bone resorption depends on RANKL, which determines osteoclast formation, activity and survival. Importantly, cells of the osteoblastic lineage mainly provide RANKL and therefore, are central in the regulation of osteoclast functions. Catabolic effects of RANKL are inhibited by OPG, a TNF receptor family member that binds RANKL, thereby preventing the activation of its receptor RANK, which is expressed by osteoclast precursors. Because this cytokine network is pivotal for the regulation of bone mass in health and diseases, including osteoporosis, rheumatoid arthritis and malignant bone conditions, it has been successfully used for the generation of a targeted therapy to block osteoclast actions. The clinical approval of denosumab, a fully monoclonal antibody against RANKL, provides a novel option to treat bone diseases with a potent, targeted and reversible inhibitor of bone resorption. Although RANKL is also expressed by endothelial cells, T lymphocytes, synovial fibroblasts and various tumor cells, no meaningful clinical extraskeletal effects have been reported after administration of denosumab. This article summarizes the molecular and cellular basis of the RANKL/RANK/OPG system and presents preclinical and clinical studies on the skeletal actions of denosumab.

Molecular Pathways: Osteoclast-Dependent and Osteoclast-Independent Roles of the RANKL/RANK/OPG Pathway in Tumorigenesis and Metastasis

Receptor activator of nuclear factor-kappa B ligand (RANKL) is a TNF ligand superfamily member that is essential for the formation, activation, and function of osteoclasts. RANKL functions via its cognate receptor RANK, and it is inhibited by the soluble decoy receptor osteoprotegerin (OPG). In skeletal metastases, the ratio of RANKL to OPG is upregulated, which leads to increased osteoclast-mediated bone destruction. These changes in the bone microenvironment not only compromise the structural integrity of bone, leading to severe clinical morbidities, but have also been implicated in establishment of de novo bone metastasis and the progression of existing skeletal tumors. Evaluation of RANKL inhibitors, including the fully human anti-RANKL antibody denosumab, in patients with cancer has shown reductions in tumor-induced bone resorption activity and successful management of skeletal complications of bone metastases. RANKL also functions as a major paracrine effector of the mitogenic action of progesterone in mouse mammary epithelium, and it has a role in ovarian hormone-dependent expansion and regenerative potential of mammary stem cells. RANKL inhibition attenuates mammary tumorigenesis and pulmonary metastases in mouse models. These data suggest that the contribution of progesterone to increased mammary cancer incidence is mediated, at least in part, by RANKL-dependent changes in the mammary epithelium; RANKL also directly promotes distant metastases. In summary, the antitumor and antimetastatic effects of RANKL inhibition can occur by at least 2 distinct mechanisms, one in the bone via osteoclast-dependent effects, and the second via direct effects on the tumor cells of various origins and/or mammary epithelium.

Role of DNA methylation in the regulation of the RANKL-OPG system in human bone

Osteoblasts are specialized cells that form new bone and also indirectly influence bone resorption by producing factors that modulate osteoclast differentiation. Although the methylation of CpG islands plays an important role in the regulation of gene expression, there is still scanty information about its role in human bone. The aim of this study was to investigate the influence of CpG methylation on the transcriptional levels of two osteoblast-derived critical factors in the regulation of osteoclastogenesis: the receptor activator of nuclear factor NF-κB ligand (RANKL) and its soluble decoy receptor osteoprotegerin (OPG). Quantitative methylation specific PCR (qMSP) and pyrosequencing analysis in various cell types showed that the methylation of regulatory regions of these genes, in the vicinity of the transcription start sites, repressed gene transcription, whereas an active transcription was associated with low levels of methylation. In addition, treatment with the DNA demethylating agent 5-azadeoxycitidine promoted a 170-fold induction of RANKL and a 20-fold induction of OPG mRNA expression in HEK-293 cells, which showed hypermethylation of the CpG islands and barely expressed RANKL and OPG transcripts at baseline. Transcriptional levels of both genes were also explored in bone tissue samples from patients with hip fractures and hip osteoarthritis. Although RANKL transcript abundance and the RANKL:OPG transcript ratio were significantly higher in patients with fractures than in those with osteoarthritis (RANKL: 0.76 ± 0.23 vs. 0.24 ± 0.08, p = 0.012; RANKL/OPG: 7.66 ± 2.49 vs. 0.92 ± 0.21, p = 0.002), there was no evidence for differential methylation across patient groups. In conclusion, the association between DNA methylation and the repression of RANKL and OPG expression strongly suggests that methylation-dependent mechanisms influence the transcription of these genes, which play a critical role in osteoclastogenesis. However, other mechanisms appear to be involved in the increased RANKL/OPG ratio of patients with osteoporotic fractures.

The RANKL–OPG system is differentially regulated by supragingival and subgingival biofilm supernatants

Periodontitis is an inflammatory condition that destroys the tooth supporting tissues, including the alveolar bone. It is triggered by polymicrobial biofilms attaching on tooth surfaces, which can be supragingival or subgingival. Bone resorption is triggered by receptor activator of NF-κB ligand (RANKL) and blocked by its soluble decoy receptor osteoprotegerin (OPG), which are cytokines of the tumor necrosis factor ligand and receptor families, respectively. The present study aimed to comparatively investigate the effects of the Zürich in vitro supragingival and subgingival biofilm models, on RANKL and OPG gene expression in primary human gingival fibroblasts (GF) cultures. The cells were challenged with biofilm culture supernatants for up-to 24 h. RANKL and OPG gene expression in the cells was analyzed by quantitative real-time polymerase chain reaction (qPCR) and their relative RANKL/OPG ratio was calculated. Both biofilm supernatants induced RANKL expression, but the subgingival caused a more pronounced up-regulation compared to the supragingival (10-fold at 6 h and 100-fold at 24 h). Changes in OPG expression in response to either biofilm were more limited. Accordingly, the subgingival biofilm caused a greater enhancement of the relative RANKL/OPG ratio (4-fold at 6 h and 110-fold 24 h). In conclusion, subgingival biofilms exhibit a stronger potency for inducing molecular mechanisms of bone resorption than supragingival biofilms, in line with their higher virulence nature for the development of periodontitis.

Mechanisms and clinical relevance of TRAIL-triggered responses in the synovial fibroblasts of patients with rheumatoid arthritis

Results of studies in mice suggest a protective role for TRAIL in arthritis. The aim of this study was to investigate the role of TRAIL in patients with rheumatoid arthritis (RA). In the present study, we compared RA fibroblast-like synoviocytes (FLS) that were resistant or sensitive to TRAIL-induced apoptosis and the expression of TRAIL receptors in these cells, and also investigated the clinical features of the patients from whom the FLS were derived. Furthermore, we evaluated the levels of TRAIL and its soluble decoy receptor osteoprotegerin (OPG) in patients with RA, patients with osteoarthritis (OA), and patients with spondylarthritis (SpA). Sensitivity to TRAIL-induced apoptosis varied in FLS from different patients, and the severity of disease in patients with RA was inversely correlated with the susceptibility of their FLS to TRAIL-induced apoptosis. TRAIL-sensitive cells expressed significantly lower levels of TRAILR-1, and silencing of TRAILR-1 increased TRAIL-induced apoptosis in RA FLS. TRAIL levels were elevated in the arthritic joints of patients with established RA, and TRAIL levels in the synovial fluid of these patients were elevated compared with levels in the synovial fluid of patients with OA or SpA. At baseline, a low OPG-to-TRAIL ratio in the sera of patients with early RA was associated with a better evolution of disease activity, but high serum levels of TRAIL at followup were associated with joint damage. These findings suggest that TRAIL has a dual role in RA, and that the resistance of RA FLS to TRAIL-induced apoptosis is associated with a disease-promoting activity of TRAIL in RA.

Mechanisms and clinical relevance of TRAIL-triggered responses in synovial fibroblasts of rheumatoid arthritis patients

ObjectiveWe have shown that TRAIL induces apoptosis only in a portion of RA fibroblast-like synoviocytes (RAFLS) and that in the surviving cells, TRAIL induced proliferation. In the present study, we...

Porphyromonas gingivalis Induces RANKL in T-cells

Porphyromonas gingivalis is an oral pathogen highly implicated in chronic periodontitis, a disease characterized by inflammatory destruction of the tooth-supporting alveolar bone and eventually, tooth loss. T-cell innate immune responses are actively involved in this pathological process. Receptor activator of NF-κB Ligand (RANKL) is a cytokine that stimulates bone resorption, while its soluble decoy receptor osteoprotegerin (OPG) blocks its action. This study aimed to investigate in Jurkat T-cells the effects of P. gingivalis on the RANKL-OPG system and the major inflammatory mediator of bone resorption prostaglandin E(2) (PGE(2)). P. gingivalis caused concentration-dependent up-regulation of RANKL gene expression and protein production, assessed by quantitative PCR and ELISA, respectively. PGE(2) production was also enhanced. However, OPG was not detected. In conclusion, P. gingivalis induces RANKL and PGE(2) in T-cells, potentially favoring bone resorption. These T-cell responses to P. gingivalis may contribute to the pathogenesis of inflammatory alveolar bone destruction occurring in chronic periodontitis.

Inhibition of RANKL increases the anti-tumor effect of the EGFR inhibitor panitumumab in a murine model of bone metastasis

Bone metastases cause severe skeletal complications and are associated with osteoclast-mediated bone destruction. RANKL is essential for osteoclast formation, function, and survival, and is the primary effector of tumor-induced osteoclastogenesis and osteolysis. RANKL inhibition by its soluble decoy receptor osteoprotegerin (OPG) prevents tumor-induced osteolysis and decreases skeletal tumor burden. Because osteoclast-mediated bone resorption releases growth factors from the bone matrix, the host bone microenvironment induces a vicious cycle of bone destruction and tumor proliferation and survival. A prediction of this vicious cycle hypothesis is that targeting the host bone microenvironment by osteoclast inhibition would reduce tumor growth and survival and may enhance the anti-tumor effects of targeted therapies. The epidermal growth factor receptor (EGFR) pathway regulates critical processes such as cell growth and survival, and anti-EGFR therapies can cause tumor cell arrest and apoptosis. We evaluated whether reduction of osteolysis by RANKL inhibition could enhance the anti-tumor effects of an anti-EGFR antibody (panitumumab) in a novel murine model of human A431 epidermoid carcinoma bone metastasis. Skeletal tumor progression was assessed longitudinally by bioluminescence imaging. RANKL inhibition by OPG-Fc treatment resulted in a reduction in tumor progression in bony sites. OPG-Fc treatment also caused a dose-dependent reduction in tumor-induced osteolysis, supporting the essential role of RANKL in this process. In combination, RANKL inhibition increased the anti-tumor efficacy of an anti-EGFR antibody, and completely blocked tumor-induced bone breakdown, demonstrating that addition of the indirect anti-tumor effect of RANKL inhibition increases the anti-tumor efficacy of panitumumab, a targeted anti-EGFR antibody.

Markers of bone remodeling and skeletal morbidity in patients with solid tumors metastatic to the skeleton receiving the biphosphonate zoledronic acid

The molecular triad, which includes the receptor activator of nuclear factor kappa-B ligand (RANKL), its receptor RANK, and the endogenous soluble RANKL decoy receptor osteoprotegerin (OPG), has emerged as an important determinant of bone metabolism. We aimed to evaluate the effect of treatment with the biphosphonate zoledronic acid (ZA) on biochemical markers of bone remodeling and to detect possible correlations of markerlevel changes with skeletal morbidity and clinical outcomes in patients with solid tumors and osseous metastases. The following serum markers were measured at the onset of skeletal metastases and after 6 months of treatment with ZA (4 mg intravenously monthly) in 70 patients with breast (n = 30), lung (n = 18), or prostate (n = 22) cancer: RANKL, OPG, C-terminal cross-linking telopeptide of type I collagen (CTX), tartrate-resistant acid phosphatase isoform 5b (TRACP-5b), bone-specific alkaline phosphatase (bALP), and osteopontin (OPN). Logistic regression models were applied to assess the correlation between marker-level changes and skeletal related events (SRE, primary endpoint), recurrence or progression, and death. Within a median follow-up of 32 months, 34 patients (48.6%) presented with at least 1 SRE and 48 patients (68.6%) relapsed. The RANKL/OPG ratio was upregulated in patients with breast and lung cancer, and it tended to decline after treatment with ZA, whereas prostate cancer patients presented with profound elevation of OPG only that persisted after treatment. CTX levels were significantly reduced after treatment in the whole study population (P = 0.003). None of the markers was able to predict skeletal morbidity or clinical outcomes independently of well-established prognostic clinical parameters.

Silibinin inhibits osteoclast differentiation mediated by TNF family members.

Silibinin is a polyphenolic flavonoid compound isolated from milk thistle (Silybum marianum), with known hepatoprotective, anticarcinogenic, and antioxidant effects. Herein, we show that silibinin inhibits receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis from RAW264.7 cells as well as from bone marrow-derived monocyte/macrophage cells in a dose-dependent manner. Silibinin has no effect on the expression of RANKL or the soluble RANKL decoy receptor osteoprotegerin (OPG) in osteoblasts. However, we demonstrate that silibinin can block the activation of NF-κB, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein (MAP) kinase, and extracellular signal-regulated kinase (ERK) in osteoclast precursors in response to RANKL. Furthermore, silibinin attenuates the induction of nuclear factor of activated T cells (NFAT) c1 and osteoclast-associated receptor (OSCAR) expression during RANKL-induced osteoclastogenesis. We demonstrate that silibinin can inhibit TNF-α-induced osteoclastogenesis as well as the expression of NFATc1 and OSCAR. Taken together, our results indicate that silibinin has the potential to inhibit osteoclast formation by attenuating the downstream signaling cascades associated with RANKL and TNF-α.