In vivo and in vitro action mechanism of treatment of glucocorticoid-induced osteoporosis by regulation of osteoprotegerin/receptor activator of nuclear factor-κB pathways by denshensu.

The research aimed to discuss the action mechanism of the treatment of glucocorticoid-induced osteoporosis (GIOP) by denshensu. In the research, 60 rats were purchased and divided into a control group, model group, estradiol group, and denshensu treatment group. Except for the control group, GIOP models were established for all other groups, and then the structural changes of osseous tissues as well as osteoprotegerin (OPG), expression of receptor activator of nuclear factor-κB ligands (RANKL) were detected. Besides, the changes in osteoclasts were observed by bone marrow-derived mononuclear phagocytes in vitro. The results showed that the micro-structure of bone trabeculae, bone mineral density (BMD), and bone metabolic markers of rats in the denshensu treatment group were enhanced significantly, while trabecular separation and structural model index were reduced (P<0.05). OPG messenger ribonucleic acid (mRNA) and protein levels in the hypothalamus and femur tissues were increased, while RANKL content was remarkably decreased (P<0.05). In addition, in vitro experiments revealed that denshensu inhibited the differentiation of positive osteoclasts, and osteoclast-related genes were reduced (P<0.05). To conclude, denshensu might inhibit the expressions of OPG and RANKL and further play a role in treating GIOP.

Isoimperatorin attenuates bone loss by inhibiting the binding of RANKL to RANK.

Osteoporosis, an immune disease characterized by bone mass loss and microstructure destruction, is often seen in postmenopausal women. Isoimperatorin (ISO), a bioactive, natural furanocoumarin isolated from many traditional Chinese herbal medicines, has therapeutic effects against various diseases; however, its effect on bone homeostasis remains unclear. In this study, we investigated the effect of ISO on the differentiation and activation of osteoclast and its molecular mechanism in vitro, and evaluated the effect of ISO on bone metabolism by ovariectomized (OVX) rat model. In vitro experiments showed that ISO affected RANKL-induced MAPK, NFAT, NFATc1 trafficking and expression, osteoclast F-actin banding, osteoclast-characteristic gene expression, ROS inhibitory activity, and calcium oscillations, NF-κB signaling pathway. In vivo experiments showed that oral administration of ISO effectively reduced bone loss caused by ovariectomy and retained bone mass.Collectively, ISO inhibits RANK/RANKL binding, thereby reducing the activity of NFATc1, calcium, and ROS and inhibiting osteoclast generation. In addition, ISO protects bone mass by slowing osteoclast production and downregulating NFATc1 gene and protein expression in the bone tissue microenvironment and inhibits OVX-induced bone loss in vivo.

Soy Isoflavones and Bone Health: Focus on the RANKL/RANK/OPG Pathway.

Bone remodels via resorption and formation, two phenomena that continuously occur in bone turnover. The RANKL/RANK/OPG pathway is one of the several mechanisms that affect bone turnover. The RANKL/OPG ratio has a substantial role in bone resorption. An imbalance between formation and resorption is related to an increased RANKL/OPG balance. OPG, a member of this system, can bind to RANKL and suppress RANK-RANKL interaction, and subsequently, inhibit further osteoclastogenesis. The serum levels of RANKL and OPG in the bone microenvironment are vital for osteoclasts formation. The RANK/RANKL/OPG system plays a role in the pathogenesis of bone disorders. This system can be considered a new treatment target for bone disorders. Soy isoflavones affect the RANK/RANKL/OPG system through numerous mechanisms. Soy isoflavones decrease RANKL levels and increase OPG levels. Therefore, isoflavones improve bone metabolism and decrease bone resorption. Soy isoflavones decrease serum markers of bone resorption and improve bone metabolism. However, while the available data are promising, the results of several studies reported no change in RANKL and OPG levels with isoflavones supplementation. In this regard, current evidence is insufficient for conclusive approval of the efficacy of isoflavones on RANKL/RANK/OPG and further research, including animal and human studies, are needed to confirm the effect of soy isoflavones on the RANKL/RANK/OPG pathway. This study was a review of available evidence to determine the role of isoflavones in bone hemostasis and the RANK/RANKL/OPG pathway. The identification of the effects of isoflavones on the RANKL/RANK/OPG pathway directs future studies and leads to the development of effective treatment strategies for bone disorders.

Association Between Serum Selenium Concentration and OPG/RANKL/RANK Axis in Patients with Arterial Hypertension.

The aim of the study was to determine the relationship between the serum selenium concentration (Se-S) and the blood concentrations of osteoprotegerin (OPG), receptor activator of nuclear factor kappa-Β ligand (RANKL) and the OPG/RANKL ratio in patients with arterial hypertension. The study group comprised 138 patients with arterial hypertension (age: 56.04 ± 11.59 years). Se-S was determined in all the subjects. Based on the Se-S, the following subgroups were distinguished: a subgroup of patients with a lower Se-S ("low-Se", Se-S < median) and a subgroup of patients with a higher Se-S ("high-Se", Se-S ≥ median). Moreover, the blood concentrations of the parameters of bone metabolism and extraskeletal calcification were assessed: OPG and RANKL. The OPG/RANKL ratio was calculated. In the "low-Se" subgroup, the RANKL concentration was statistically significantly lower, and the OPG/RANKL ratio was statistically significantly higher than in the patients in the "high-Se" subgroup. The correlation analysis showed the negative linear relationships between Se-S and OPG (r = - 0.25, p < 0.05) and between Se-S and OPG/RANKL (r = - 0.47, p < 0.05). Moreover, Se-S positively correlated with RANKL (r = 0.33, p < 0.05). In regression analysis, higher body mass index (BMI), smoking and lower Se-S were independently associated with a higher OPG/RANKL ratio, while lower BMI, use of diuretics, ß-blockers and ACE inhibitors and lower OPG/RANKL ratio with effective blood pressure control. In summary, in the group of patients with arterial hypertension, lower Se-S is associated with an unfavourable prognostic panel of parameters of bone metabolism and extraskeletal calcification. Lower Se-S is an independent risk factor for a higher OPG/RANKL ratio, which is an independent prediction factor of ineffective blood pressure control in patients with hypertension.

Magnesium Picolinate Improves Bone Formation by Regulation of RANK/RANKL/OPG and BMP-2/Runx2 Signaling Pathways in High-Fat Fed Rats.

Magnesium (Mg) deficiency may affect bone metabolism by increasing osteoclasts, decreasing osteoblasts, promoting inflammation/oxidative stress, and result in subsequent bone loss. The objective of the present study was to identify the molecular mechanism underlying the bone protective effect of different forms of Mg (inorganic magnesium oxide (MgO) versus organic magnesium picolinate (MgPic) compound) in rats fed with a high-fat diet (HFD). Forty-two Wistar albino male rats were divided into six group (n = 7): (i) control, (ii) MgO, (iii) MgPic, (iv) HFD, (v) HFD + MgO, and (vi) HFD + MgPic. Bone mineral density (BMD) increased in the Mg supplemented groups, especially MgPic, as compared with the HFD group (p < 0.001). As compared with the HFD + MgO group, the HFD + MgPic group had higher bone P (p < 0.05) and Mg levels (p < 0.001). In addition, as compared to MgO, MgPic improved bone formation by increasing the levels of osteogenetic proteins (COL1A1 (p < 0.001), BMP2 (p < 0.001), Runx2 (p < 0.001), OPG (p < 0.05), and OCN (p < 0.001), IGF-1 (p < 0.001)), while prevented bone resorption by reducing the levels of RANK and RANKL (p < 0.001). In conclusion, the present data showed that the MgPic could increase osteogenic protein levels in bone more effectively than MgO, prevent bone loss, and contribute to bone formation in HFD rats.

Marantodes pumilum Var Alata (Kacip Fatimah) ameliorates derangement in RANK/RANKL/OPG pathway and reduces inflammation and oxidative stress in the bone of estrogen-deficient female rats with type-2 diabetes.

BACKGROUND: M. pumilum has been claimed to protect the bone against the adverse effect of estrogen deficiency. Additionally, it also exhibits anti-diabetic activity. In view of these, this study aims to identify the mechanisms underlying the bone protective effect of M. pumilum in the presence of both estrogen deficiency and diabetes mellitus (DM). METHODS: Ovariectomized, diabetic female rats were given M. pumilum leave aqueous extract (MPLA) (50 and 100 mg/kg/day), estrogen, glibenclamide and estrogen plus glibenclamide for 28 consecutive days. At the end of the treatment, fasting blood glucose (FBG), serum insulin, Ca2+, PO43- and bone alkaline phosphatase (BALP) levels were measured. Rats were sacrificed and femur bones were harvested for determination of expression level and distribution of RANK, RANKL, OPG and oxidative stress and inflammatory proteins by molecular biological techniques. RESULTS: 100 mg/kg/day MPLA treatment decreased the FBG and BALP levels but increased the serum insulin, Ca2+ and PO43- levels in estrogen deficient, diabetic rats. Expression and distribution of RANKL, NF-κB p65, IKKß, IL-6, IL-1ß and Keap-1 decreased however expression and distribution of RANK, OPG, BMP-2, Type-1 collagen, Runx2, TRAF6, Nrf2, NQO-1, HO-1, SOD and CAT increased in the bone of estrogen deficient, diabetic rats which received 100 mg/kg/day MPLA with greater effects than estrogen-only, glibenclamide-only and estrogen plus glibenclamide treatments. CONCLUSION: MPLA helps to overcome the adverse effect of estrogen deficiency and DM on the bone and thus this herb could potentially be used for the treatment and prevention of osteoporosis in postmenopausal women with diabetes.

Pinus roxburghii alleviates bone porosity and loss in postmenopausal osteoporosis by regulating estrogen, calcium homeostasis and receptor activator of nuclear factor-κB, osteoprotegerin, cathepsin bone markers.

OBJECTIVES: The study was aimed to evaluate the potential of hydroalcoholic extract of Pinus roxburghii (PRE) stem bark in post-menopausal osteoporosis and its underlying mechanisms. METHODS: In silico docking of the markers was done using AutoDock version 4.2. for molecular targets: receptor activator of nuclear factor-κB (RANK), osteoprotegerin (OPG) and Cathepsin. Female Wistar rats of bodyweight 200-250 g were employed and surgical ovariectomy (OVX) was performed. PRE was administered at a dose of 100 and 200 mg/kg whereas standard drug, raloxifene given at 1 mg/kg orally for eight weeks. KEY FINDINGS: PRE (20 and 40 µg/mL) significantly increased the cellular proliferation in osteoblastic UMR cell lines 11.58 and 15.09% respectively. Eight weeks after surgical removal of ovaries, a significant bone porosity was confirmed by modulation in bone breaking strength of tibia, lumber, and femur; bone mineral density (BMD), calcium, phosphorus, hydroxyproline levels in OVX group. Treatment with PRE 100 and 200 mg/kg significantly restored the bone loss. Real-time polymerase chain reaction (RT-PCR) analysis of molecular markers RANK, OPG and cathepsin and histology also confirmed the attenuation of bone loss. The quantification of quercetin, gallic acid, caffeic acid, catechin, tannic acid and ascorbic acid was done by high-performance liquid chromatography (HPLC) and high performance thin layer chromatography. CONCLUSIONS: P. roxburghii produced anti-osteoporotic effect possibly due to estrogenic modulation, and improved bone remodeling.

The receptor activator of nuclear factor κΒ ligand receptor leucine-rich repeat-containing G-protein-coupled receptor 4 contributes to parathyroid hormone-induced vascular calcification.

BACKGROUND: In chronic kidney disease, serum phosphorus (P) elevations stimulate parathyroid hormone (PTH) production, causing severe alterations in the bone-vasculature axis. PTH is the main regulator of the receptor activator of nuclear factor κB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system, which is essential for bone maintenance and also plays an important role in vascular smooth muscle cell (VSMC) calcification. The discovery of a new RANKL receptor, leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4), which is important for osteoblast differentiation but with an unknown role in vascular calcification (VC), led us to examine the contribution of LGR4 in high P/high PTH-driven VC. METHODS: In vivo studies were conducted in subtotally nephrectomized rats fed a normal or high P diet, with and without parathyroidectomy (PTX). PTX rats were supplemented with PTH(1-34) to achieve physiological serum PTH levels. In vitro studies were performed in rat aortic VSMCs cultured in control medium, calcifying medium (CM) or CM plus 10-7 versus 10-9 M PTH. RESULTS: Rats fed a high P diet had a significantly increased aortic calcium (Ca) content. Similarly, Ca deposition was higher in VSMCs exposed to CM. Both conditions were associated with increased RANKL and LGR4 and decreased OPG aorta expression and were exacerbated by high PTH. Silencing of LGR4 or parathyroid hormone receptor 1 (PTH1R) attenuated the high PTH-driven increases in Ca deposition. Furthermore, PTH1R silencing and pharmacological inhibition of protein kinase A (PKA), but not protein kinase C, prevented the increases in RANKL and LGR4 and decreased OPG. Treatment with PKA agonist corroborated that LGR4 regulation is a PTH/PKA-driven process. CONCLUSIONS: High PTH increases LGR4 and RANKL and decreases OPG expression in the aorta, thereby favouring VC. The hormone's direct pro-calcifying actions involve PTH1R binding and PKA activation.

Oral Exposure to ZnO Nanoparticles Disrupt the Structure of Bone in Young Rats via the OPG/RANK/RANKL/IGF-1 Pathway.

PURPOSE: To evaluate the effects of ZnO NPs on bone growth in rats and explore the possible mechanisms of action. MATERIALS AND METHODS: Three-week-old male rats received ultrapure water or 68, 203, and 610 mg/kg zinc oxide nanoparticles (ZnO NPs) for 28 days, orally. RESULTS: The high-dosage groups caused significant differences in weight growth rate, body length, and tibia length (P<0.05), all decreasing with increased ZnO NP dosage. There were no significant differences in body mass index (BMI) (P>0.05). The zinc concentration in liver and bone tissue increased significantly with increased ZnO NP dosage (P<0.05). Clearly increased aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were observed in the 610 mg/kg ZnO NP group (P>0.05), whereas alkaline phosphatase (ALP) increased in the 610 mg/kg ZnO NP group (P<0.05). Significant differences in insulin-like growth factor type 1 (IGF-1) levels and a decrease in calcium (Ca) levels were observed in 203 and 610 mg/kg ZnO NP groups (P<0.05). Phosphorus (P) levels increased and the Ca/P ratio decreased in the 610 mg/kg ZnO NP group (P<0.05). Micro-computed tomography (micro-CT) of the tibia demonstrated signs of osteoporosis, such as decreased bone density, little trabecular bone structure and reduced cortical bone thickness. Micro-CT data further demonstrated significantly decreased bone mineral density (BMD), trabecular number (Tb.N), and relative bone volume (BV/TV) with increasing dosage of ZnO NPs. Osteoprotegerin (OPG) expression and the ratio of OPG to receptor activator of nuclear factor-κB ligand (RANKL) were statistically lower in the 610 mg/kg ZnO NP group (P<0.05), whereas RANKL expression did not change significantly (P>0.05). CONCLUSION: We infer that ZnO NPs affect bone growth in young rats directly or indirectly by altering IGF-1 levels. Overall, the results indicate that ZnO NPs promote osteoclast activity and increase bone loss through the OPG/RANK/RANKL/IGF-1 pathway.

Network pharmacology approach to elucidate possible action mechanisms of Sinomenii Caulis for treating osteoporosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Sinomenii Caulis (SC) is a well-konwn traditional Chinese medicine used for treatment of rheumatoid arthritis (RA), dermatophytosis and paralysis. Patients with RA are usually secondary to osteoporosis, but the potential protective effect of SC on osteoporosis (OP) is seldom reported and its possible action mechanism is little known. AIM: The purpose of this study was to demonstrate the anti-osteoporosis effects of SC extract and alkaloids in prednisolone (Pre)-induced OP of zebrafish, and then to explore the potential mechanism of SC on system level by network pharmacology. METHODS: Firstly, zebrafish OP model was established to investigate the anti-osteoporosis effect of SC. Secondly, the targets of SC and OP from multiple databases were collected, and Compound-Target-Pathway network based on protein-protein interaction (PPI) was constructed. Moreover, gene enrichment and annotation were performed via the DAVID server. Finally, the reliability of the network pharmacology prediction results in Pre-induced OP of zebrafish was verified by qRT-PCR. RESULTS: The results indicated that SC extract and alkaloids have remarkable ability to promote bone formation of cranial bones and reduce TRAP contents in Pre-induced OP of zebrafish. 32 OP-related ingredients in SC and 77 OP-related targets were screened from multiple databases, and 15 OP-related pathways were enriched by the KEGG database. Further experimental validation indicated that SC extract and alkaloids could regulate the expression of MAPK14, CASP3, CXCL8, IL-1ß, IL6, PTGS2, TNF-α, ESR1, and MMP9 for treatment of OP. CONCLUSION: In summary, we conducted an integrative analysis to provide convincing evidence that SC may partially alleviate OP by inhibiting pro-inflammatory cytokines and regulating of RANK/RANKL/OPG system.

Baicalin Ameliorates Dexamethasone-Induced Osteoporosis by Regulation of the RANK/RANKL/OPG Signaling Pathway.

BACKGROUND: Osteoporosis is a chronic bone metabolism disorder affecting millions of the world population. The RANKL/RANK/OPG signaling pathway has been confirmed to be the main regulator of osteoporosis. It is of great interest to identify appropriate therapeutic agents that can regulate the RANKL/RANK/OPG pathway. Baicalin (BA) is a well-known traditional Chinese medicine formula against various inflammatory diseases with a proven role of the RANKL/RANK/OPG pathway regulation. However, the potential effect of BA on osteoporosis and the mechanisms underlying this remain unclear. In the present study, we aimed to evaluate the efficacy of BA in the prevention of dexamethasone (DEX)-induced osteoporosis in zebrafish. METHODS: In this study, growth and development changes of zebrafish and calcein staining were assessed with a micrograph. The expression levels of RANKL and OPG and transcription factors in response to DEX induction and BA administration were evaluated by Western blotting and qRT-PCR. In addition, the intermolecular interactions of BA and RANKL were investigated by molecular docking. RESULTS: Results show that BA enhances the growth and development of dexamethasone (DEX)-induced osteoporosis in zebrafish larvae. Calcein staining and calcium and phosphorus determination revealed that BA ameliorates mineralization of DEX-induced osteoporosis zebrafish larvae. BA also regulates the expression of RANKL and OPG and hampers the changes in gene expression related to bone formation and resorption under the induction of DEX in zebrafish. It can be inferred by molecular docking that BA may interact directly with the extracellular domain of RANKL. CONCLUSION: The findings, herein, reveal that BA ameliorates DEX-induced osteoporosis by regulation of the RANK/RANKL/OPG signaling pathway.

Zinc supplements and bone health: The role of the RANKL-RANK axis as a therapeutic target.

BACKGROUND: To this day, empirical data suggests that zinc has important roles in matrix synthesis, bone turnover, and mineralization and its beneficial effects on bone could be mediated through different mechanisms. The influence of zinc on bone turnover could be facilitated via regulating RANKL/RANK/OPG pathway in bone tissue. Therefore, the aim of the study was to conduct a review to investigate the possible effect of the zinc mediated bone remodeling via RANKL/RANK/OPG pathway. METHODS: A comprehensive systematic search was performed in MEDLINE/PubMed, Cochrane Library, SCOPUS, and Google Scholar to explore the studies investigating the effect of zinc as a bone remodeling factor via RANKL/RANK/OPG pathway regulation. Subsequently, the details of the pathway and the impact of zinc supplements on RANKL/RANK/OPG pathway regulation were discussed. RESULTS: The pathway could play an important role in bone remodeling and any imbalance between RANKL/RANK/OPG components could lead to extreme bone resorption. Although the outcomes of some studies are equivocal, it is evident that zinc possesses protective properties against bone loss by regulating the RANKL/RANK/OPG pathway. There are several experiments where zinc supplementation resulted in upregulation of OPG expression or decreases RANKL level. However, the results of some studies oppose this. CONCLUSION: It is likely that sufficient zinc intake will elicit positive effects on bone health by RANKL/RANK/OPG regulation. Although the outcomes of a few studies are equivocal, it seems that zinc can exert the protective properties against bone loss by suppressing osteoclastogenesis via downregulation of RANKL/RANK. Additionally, there are several experiments where zinc supplementation resulted in upregulation of OPG expression. However, the results of limited studies oppose this. Therefore, aside from the positive role zinc possesses in preserving bone mass, further effects of zinc in RANKL/RANK/OPG system requires further animal/human studies.

Development of Small-Molecules Targeting Receptor Activator of Nuclear Factor-κB Ligand (RANKL)-Receptor Activator of Nuclear Factor-κB (RANK) Protein-Protein Interaction by Structure-Based Virtual Screening and Hit Optimization.

Targeting RANKL/RANK offers the possibility of developing novel therapeutic approaches to treat bone metabolic diseases. Multiple efforts have been made to inhibit RANKL. For example, marketed monoclonal antibody drug Denosumab could inhibit the maturation of osteoclasts by binding to RANKL. This study is an original approach aimed at discovering small-molecule inhibitors impeding RANKL/RANK protein interaction. We identified compound 34 as a potent and selective RANKL/RANK inhibitor by performing structure-based virtual screening and hit optimization. Disruption of the RANKL/RANK interaction by 34 effectively inhibits RANKL-induced osteoclastogenesis and bone resorption. The expression of osteoclast marker genes was also suppressed by treatment of 34. Furthermore, 34 markedly blocked the NFATc1/c-fos pathway. Thus, our current work demonstrates that the chemical tractability of the difficult PPI (RANKL/RANK) target by a small-molecule compound 34 offers a potential lead compound to facilitate the development of new medications for bone-related diseases.

2-O-digalloyl-1,3,4,6-tetra-O-galloyl-ß-D-glucose isolated from Galla Rhois suppresses osteoclast differentiation and function by inhibiting NF-κB signaling.

Natural compounds isolated from medicinal herbs and plants have immense significance in maintaining bone health. Hydrolysable tannins have been shown to possess a variety of medicinal properties including antiviral, anticancer, and anti-osteoclastogenic activities. As a part of a study on the discovery of alternative agent against skeletal diseases, we isolated a hydrolysable tannin, 2-O-digalloyl-1,3,4,6-tetra-Ogalloyl- ß-D-glucose (DTOGG), from Galla Rhois and examined the effect on osteoclast formation and function. We found that DTOGG significantly inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation by downregulating the expression of the key regulator in osteoclastogenesis as well as osteoclast-related genes. Analysis of RANKL/RANK signaling revealed that DTOGG impaired activation of IκBα and p65 in the nuclear factor kappa-lightchain- enhancer of activated B cells (NF-κB) signaling pathway. Furthermore, DTOGG reduced bone resorbing activity of osteoclasts, compared to the vehicle-treated control. These results suggest that DTOGG could be a useful natural compound to manage osteoclast-mediated skeletal diseases. [BMB Reports 2019; 52(6): 409-414].

Protective effects of Dipsacus asper polysaccharide on osteoporosis in vivo by regulating RANKL/RANK/OPG/VEGF and PI3K/Akt/eNOS pathway.

A homogenous polysaccharide (DAP), with a molecular weight of 2.61 × 104 Da, was isolated from the roots of Dipsacus asper Wall. Gas chromatography (GC) indicated that DAP was composed of galactose and mannose with a molar ratio of 1:1. The purpose of this study was to evaluate the effect of DAP on the progress of bone loss in the ovariectomized (OVX) rat model of osteoporosis. Administration of DAP (50 and 200 mg/kg/body wt. day) for 12 weeks significantly prevented OVX-induced bone loss, biomechanical reduction, the body weight gain, the loss of the uterus weight, as well as increased U-Ca/Cr, U-P/Cr, ALP, TRAP, OC and DPD/Cr levels in rats, which was further supported by the histopathological examinations. Furthermore, we found that the mechanism by which DAP elicited anti-osteoporotic effects was mediated by up-regulation of VEGF and OPG, but down-regulation of RANK and RANKL in both protein and mRNA expression in OVX rats, as well as the activation of PI3K/Akt/eNOS signaling pathway, indicating that DAP can be clinically used as a potential alternative medicine for the prevention and treatment of postmenopausal osteoporosis.

Is vitamin D deficiency behind the scenes for high incidence of Giant cell tumor amongst the Indian population? Unraveling the vitamin D - RANKL association.

INTRODUCTION: The major neoplastic and proliferative component of GCTB is the stromal tumor cells; that they have shown no evidence of bone destruction, instead the massive tissue destruction appears to be a result of tumor induced osteoclastogenesis. The discovery of receptor activator of nuclear factor kB (RANK) and RANK binding ligand (RANKL) uncovered the bone homeostasis and molecular mechanism by which multiple compounds (including vitamin D) regulated osteoclast differentiation; a function mediated by osteoblastic cells and osteoclast-precursor cells. HYPOTHESIS: In a country burdened by vitamin D deficiency, causal relation between hypovitaminosis D and GCTB was hypothesized based on the vitamin D mediated RANKL expression and osteoclastogenesis, as India is also a population with higher incidence of GCTB as compared to Western populations described in the literature. The possibility of vitamin D regulated osteoclastogenesis in GCTB is postulated on the evidence from molecular research linking it to the RANK/RANKL/OPG pathway. The aim of this study was to analyse the prevalence of Vitamin D deficiency in patients with primary GCTB and to elucidate any difference in serum Vitamin 25(OD)D3 levels amongst the matched control population data. MATERIALS AND RESULTS: 130 patients of primary GCTBs were matched to 310 controls from the general health check population and serum levels of 25(OH)D3 were analyzed. Statistical analysis performed on the non-parametric data and Mann Whitney U Test used to derive inference with significance set at p < 0.05. 56 females and 76 males with median Vitamin D level in the GCTB group was 15.9 ng/ml (Mean 19.41; Range 1.03 to 92) as compared to the control population with median level of 22.2 ng/ml (Mean 25.1; Range 2.6 to 87.9). The results were significant (p value < 0.05) as compared to the control population in all decades except the third decade (p value 0.0548). DISCUSSION: The differential expression of RANKL and OPG in response to levels of vitamin D has been established. The stromal cells of osteolytic GCTB express high levels of RANKL, which is a key signal regulator in development of this disease and bone destruction typical of GCTBs. This has resulted in research targeting this pathway for therapeutic approach in GCTBs. As vitamin D supplementation is simple and safe, increased awareness to assess and if necessary correct vitamin D status of patients is warranted, however the question as to whether patients with low vitamin D levels are more prone to develop GCTB and thus would profit from vitamin D supplementation remains unanswered. To conclude, it is essential to assess vitamin D levels in patients with GCTB as deficiency is pronounced. Future research on this hypothesis might lead to an association between Vitamin D deficiency and the onset/natural history of GCTB that may in the future help us cure or prevent GCTBs.

RANKL/RANK/OPG system beyond bone remodeling: involvement in breast cancer and clinical perspectives.

RANKL/RANK/OPG system consists of three essential signaling molecules: i) the receptor activator of nuclear factor (NF)-kB-ligand (RANKL), ii) the receptor activator of NF-kB (RANK), and iii) the soluble decoy receptor osteoprotegerin (OPG). Although this system is critical for the regulation of osteoclast differentiation/activation and calcium release from the skeleton, different studies have elucidated its specific role in mammary gland physiology and hormone-driven epithelial proliferation during pregnancy. Of note, several data suggest that progesterone induces mammary RANKL expression in mice and humans. In turn, RANKL controls cell proliferation in breast epithelium under physiological conditions typically associated with higher serum progesterone levels, such as luteal phase of the menstrual cycle and pregnancy. Hence, RANKL/RANK system can be regarded as a major downstream mediator of progesterone-driven mammary epithelial cells proliferation, potentially contributing to breast cancer initiation and progression. Expression of RANKL, RANK, and OPG has been detected in breast cancer cell lines and in human primary breast cancers. To date, dysregulation of RANKL/RANK/OPG system at the skeletal level has been widely documented in the context of metastatic bone disease. In fact, RANKL inhibition through the RANKL-blocking human monoclonal antibody denosumab represents a well-established therapeutic option to prevent skeletal-related events in metastatic bone disease and adjuvant therapy-induced bone loss in breast cancer. On the other hand, the exact role of OPG in breast tumorigenesis is still unclear. This review focuses on molecular mechanisms linking RANKL/RANK/OPG system to mammary tumorigenesis, highlighting pre-clinical and clinical evidence for the potential efficacy of RANKL inhibition as a prevention strategy and adjuvant therapy in breast cancer settings.

Cynomorium songaricum prevents bone resorption in ovariectomized rats through RANKL/RANK/TRAF6 mediated suppression of PI3K/AKT and NF-κB pathways.

AIM: Cynomorium songaricum Rupr., an edible and important Traditional Chinese medicine has long been used in folk for treatment of kidney deficiency, was chosen to estimate the antiosteoporotic activity and underlying molecular mechanism on rats induced by ovariectomy (OVX). MAIN METHODS: 9 of 45 rats were underwent bilateral laparotomy without removing the ovaries as sham group, remains were underwent bilateral ovariectomy and equally randomized into four groups: with vehicle (0.5% CMC-Na) as model group, estradiol valerate (1 mg/kg body weight/day) as positive control, with 100 and 300 mg/kg body weight/day of ethanol extracts of C. songaricum extract (CSE) as low and high dosage groups, respectively. KEY FINDINGS: After 12 weeks of continues orally intervention, the decreases of bone mineral density, bone mineral content, tissue mineral content, as well as the increases of bone trabecular separation and bone resorption markers were significantly reversed by CSE in the OVX rats, and in particular, a contradictory phenomenon on calcium and phosphorus contents was observed and elucidated. Mechanistically, the expressions of tumor-necrosis factor receptor-associated factor 6 (TRAF 6), nuclear factor kappa B (RANK) and its ligand (RANKL), as well as the nuclear factor kappa B (NF-κB), phosphoinositide 3­kinase (PI3K) and protein kinase B (AKT) levels were significantly down-regulated by CSE intervention, whereas the osteoprotegerin (OPG) was significantly up-regulated by CSE as compared to the control. SIGNIFICANCE: Concisely, C. songaricum exhibited potential therapeutic effect on bone metabolism of ovariectomized rats, and this effect was possibly exerted by RANKL/RANK/TRAF6 mediated down-regulation of NF-κB and PI3K/AKT pathways.

Radix Paeoniae Rubra stimulates osteoclast differentiation by activation of the NF-κB and mitogen-activated protein kinase pathways.

BACKGROUND: Radix Paeoniae Rubra (RPR), a traditional Chinese herb, has anti-inflammatory and immuno-regulatory properties. This study explored the effects of RPR on stimulation of osteoclast differentiation in RAW264.7 cells and peripheral blood mononuclear cells (PBMC)s. METHODS: The mature osteoclasts were measured by bone resorption assays and TRAP staining. JNK, ERK, p38 and NF-κB inhibitors were used applied in order to verify their contribution in RPR-induced osteoclast differentiation. The NF-κB and MAPK pathways were evaluated by western blotting, RT-PCR and luciferase assay. RESULTS: RPR induced osteoclast differentiation in a dose-dependent manner and induced the resorption activity of osteoclasts differentiation of RAW264.7 cells and PBMCs. Western blotting showed that RPR treatment induced phosphorylation of JNK, ERK, and p38 in RAW 264.7 cells. Treatment of JNK, ERK, and p38 MAP kinase inhibitors verified the contribution of JNK, ERK and p38. RPR treatment induced c-Fos and NFATc1 protein expression; NF-κB inhibitor treatment and luciferase assay verified the contribution of the NF-κB pathway. CONCLUSIONS: This study demonstrated the interesting effect, in which RPR stimulated osteoclast differentiation in murine RAW264.7 cells and human monocytes.

Central RANK signalling in NPY neurons alters bone mass in male mice.

RANKL signalling known to be important for the control of bone mass, has recently also been implicated in the brain to control thermoregulation, however, it is not known which neuronal pathways are involved and whether other aspects of energy homeostasis are also affected. Here we show that selective deletion of RANK from NPY neurons down-regulated NPY mRNA expression in the hypothalamus. While comprehensive phenotyping of germline-induced NPY neuron specific RANK deficient mice revealed no significant changes in physical or metabolic parameters, adult onset deletion of RANK from NPY neurons led to a significant increase in fat mass and a decrease in whole body bone mineral content and bone mineral density. Intriguingly, when these conditional knockout mice were placed on a high fat diet, body weight and fat mass did not differ to control mice. However, they were able to significantly increase their bone mass to match their increased body weight, an ability that was lacking in control mice. Taken together, results from this study demonstrate that RANK signalling in NPY neurons is involved in modulating NPY levels and through that matching bone mass to body weight.