Receptor Activator Of Nuclear Kappa Research Articles

Effects of 8-week increment aerobic exercise program on bone metabolism and body composition in young non-athletes.

The effects of aerobic exercise on bone metabolism are still unclear. Thus, the main goal of this study was to explore if there was an effect of the short-term aerobic exercise program on the bone remodeling process and if there were sex differences in the effect of the training program on bone metabolism. Twenty-one participants (men and women) aged 20-23 performed an 8-week aerobic exercise program three times per week in 1-h sessions with increases in the exercise load every 2 weeks. Bone density, bone mineral content and concentration of markers of bone metabolism: osteocalcin, C-terminal procollagen type I peptide, pyridinoline, parathyroid hormone, osteoprotegerin, and the receptor activator of nuclear kappa B ligand by ELISA were measured at the start and at the end of the study, while changes in body composition were assessed by a bioelectric impedance analysis method 6 times during the study. The aerobic exercise program increased the concentration of osteocalcin (11.34 vs 14.24ng/ml), pyridinoline (67.51 vs 73.99nmol/l), and the receptor activator of nuclear kappa B ligand (95.122 vs 158.15pg/ml). A statistically significant increase in bone density at neck mean (1.122 vs 1.176g/cm3) and in bone mineral content at dual femur (33.485 vs 33.700g) was found in women, while there was no statistically significant change at any site in men. 8 weeks of the aerobic exercise program with increment in intensity increased some of bone remodeling biomarkers and showed different effects for men and women.

LAMP-2 Is Involved in Surface Expression of RANKL of Osteoblasts In Vitro.

Lysosome associated membrane proteins (LAMPs) are involved in several processes, among which is fusion of lysosomes with phagosomes. For the formation of multinucleated osteoclasts, the interaction between receptor activator of nuclear kappa β (RANK) and its ligand RANKL is essential. Osteoclast precursors express RANK on their membrane and RANKL is expressed by cells of the osteoblast lineage. Recently it has been suggested that the transport of RANKL to the plasma membrane is mediated by lysosomal organelles. We wondered whether LAMP-2 might play a role in transportation of RANKL to the plasma membrane of osteoblasts. To elucidate the possible function of LAMP-2 herein and in the formation of osteoclasts, we analyzed these processes in vivo and in vitro using LAMP-2-deficient mice. We found that, in the presence of macrophage colony stimulating factor (M-CSF) and RANKL, active osteoclasts were formed using bone marrow cells from calvaria and long bone mouse bone marrow. Surprisingly, an almost complete absence of osteoclast formation was found when osteoclast precursors were co-cultured with LAMP-2 deficient osteoblasts. Fluorescence-activated cell sorting FACS analysis revealed that plasma membrane-bound RANKL was strongly decreased on LAMP-2 deficient osteoblasts. These results suggest that osteoblastic LAMP-2 is required for osteoblast-induced osteoclast formation in vitro.

Vav1 inhibits RANKL-induced osteoclast differentiation and bone resorption

Vav1 is a Rho/Rac guanine nucleotide exchange factor primarily expressed in hematopoietic cells. In this study, we investigated the potential role of Vav1 in osteoclast (OC) differentiation by comparing the ability of bone marrow mononuclear cells (BMMCs) obtained from Vav1-deficient (Vav1−/−) and wild-type (WT) mice to differentiate into mature OCs upon stimulation with macrophage colony stimulating factor and receptor activator of nuclear kappa B ligand in vitro. Our results suggested that Vav1 deficiency promoted the differentiation of BMMCs into OCs, as indicated by the increased expression of tartrate-resistant acid phosphatase, cathepsin K, and calcitonin receptor. Therefore, Vav1 may play a negative role in OC differentiation. This hypothesis was supported by the observation of more OCs in the femurs of Vav1−/− mice than in WT mice. Furthermore, the bone status of Vav1−/− mice was analyzed in situ and the femurs of Vav1−/− mice appeared abnormal, with poor bone density and fewer number of trabeculae. In addition, Vav1-deficient OCs showed stronger adhesion to vitronectin, an αvβ3 integrin ligand important in bone resorption. Thus, Vav1 may inhibit OC differentiation and protect against bone resorption.

Chronic treatment with zoledronic acid alters the expression levels of inflammatory, bone, and apoptotic markers and Toll-like receptors 2 and 4 in rat dental pulp

The aim of this study was to evaluate the immunostaining of inflammatory, apoptotic, and bone markers, as well as Toll-like-receptors (TLRs) 2 and 4 in the dental pulp in rats treated with zoledronic acid (ZA). We administered 4 intravascular infusions of saline (control group) or 0.20 mg.kg-1 ZA in Wistar rats (n = 6/group). After 70 days, the 3 rights molars (n = 18/group) were microscopically evaluated (presence of ectasic/dilated blood vessels and inflammatory cells). Immunohistochemistry was performed for tartrate resistant acid phosphatase 5 (TRAP; cell counting), cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), TLR2, TLR4, receptor activator of nuclear kappa B ligand (RANKL), osteoprotegerin (OPG), and caspase-3 (scored 0-3 in odontoblast and nonodontoblast dental pulp cells). Mann-Whitney and Fisher's exact tests and Spearman's correlation were used (GraphPad Prism 5.0). There was no alteration in ectasic/dilated blood vessels (P = .101) or inflammatory cells (P = .500), but the number of TRAP-positive cells was reduced in the ZA-group (P = .027). In ZA-group odontoblasts, immunostaining for COX-2 (P = .044), TLR4 (P = .003), OPG (P = .035) and caspase-3 (P = .039) increased, and that for RANKL (P = 0.045) decreased. In nonodontoblast dental pulp cells, RANKL immunostaining decreased (P = .009). In the ZA group, the RANKL/OPG ratio decreased in odontoblast (P = .022) and nonodontoblast dental pulp cells (P = .007). IL-6 did not differ between the groups. ZA increases the expression levels of inflammatory, apoptotic markers, and TLR4 and alters bone makers in the dental pulp of rats.

A comparative evaluation of osteoprotegerin and receptor activator of nuclear kappa B ligand in gingival crevicular fluid during canine retraction with elastomeric chain and nickel titanium coil spring: Anin vivostudy

Objective: The objective of this study is to evaluate and compare the level of receptor activator of nuclear kappa B ligand (RANKL) and osteoprotegerin (OPG) in gingival crevicular fluid (GCF) during canine retraction with nickel-titanium (NiTi) coil spring and elastomeric chain. Materials and Methods: Ten patients between the ages of 13–17 years requiring canine distalization after first premolar extraction were selected. NiTi coil spring was placed on one side and elastomeric chain on the contralateral side. GCF sampling was done at baseline, 1 h, 24 h, 168 h, 1 month from the distal site of the test teeth after the application of compressive force. RANKL and OPG concentration were detected by enzyme-linked immunosorbent assay. Results: In the NiTi coil spring side, the RANKL levels in GCF increased and OPG levels decreased in a time-dependent manner. Increase in RANKL and decrease in OPG were significant when compared with the baseline. In the elastomeric chain side, the RANKL levels were significantly higher and the OPG levels significantly lower at 24 h, the RANKL levels showed a significant decrease and OPG levels showed a significant increase from 24 h to 1 month time intervals. The RANKL and OPG levels in GCF when compared individually to the NiTi coil spring and elastomeric chain showed a significant difference at 24 h, 168 h, and 1 month after initiation of compressive force. Conclusion: The result suggests that RANKL and OPG expression during orthodontic tooth movement varies at different time intervals and according to the type of force delivery system.

DOES ATORVASTATIN ALONE AMELIORATE CHRONIC PERIODONTITIS IN HYPERLIPIDEMIC PATIENTS? (A CROSS-SECTIONAL STUDY)

The present study was conducted to assess whether atorvastatin (ATV) alone could improve periodontal parameters and reduce inflammation and bone resorption in generalized chronic periodontitis (gCP) patients with hyperlipidemia. One hundred and two subjects were classified into three groups: Group I included 34 gCP patients with hyperlipidemia and maintained on a daily dose of 20 mg atorvastatin at least for 6 months. Group II comprised 34 gCP patients with hyperlipidemia and not taking any hypolipidemic drugs. Group III consisted of 34 normolipidemic individuals with gCP. The periodontal parameters including plaque index (PI), bleeding on probing (BOP), gingival index (GI), probing depth (PD) and clinical attachment loss (CAL) were recorded and compared. Unstimulated whole saliva and blood samples were harvested from all subjects. Salivary levels of interleukin-1beta (IL-1β) and receptor activator of nuclear kappa B ligand (RANKL) were evaluated by enzyme-linked immunosorbent assay (ELISA) technique. The blood samples were used to assess the lipid profile for all patients including total cholesterol (TC), triglycerides (TG), low-density lipoprotein-cholesterol (LDL-C) and high-density lipoprotein-cholesterol (HDL-C). The results demonstrated significantly greater decrease of periodontal parameters and salivary IL-1β and RANKL levels in group I compared to other groups. BOP, PD and CAL were positively correlated with the levels of salivary IL-1β, TC, TG and LDL-C whereas HDL-C measurements were negatively associated with BOP, PD and CAL. In conclusion, ATV could be a viable therapeutic modality in gCP management, due to its anti-inflammatory and antiosteopathic effects. Salivary IL-1β and RANKL could serve as reliable biomarkers in periodontal disease.

Lrp4 in osteoblasts suppresses bone formation and promotes osteoclastogenesis and bone resorption

Bone mass is maintained by balanced activity of osteoblasts and osteoclasts. Lrp4 (low-density lipoprotein receptor related protein 4) is a member of the LDL receptor family, whose mutations have been identified in patients with high-bone-mass disorders, such as sclerosteosis and van Buchem diseases. However, it remains unknown whether and how Lrp4 regulates bone-mass homeostasis in vivo. Here we provide evidence that Lrp4-null mutation or specific mutation in osteoblast-lineage cells increased cortical and trabecular bone mass, which was associated with elevated bone formation and impaired bone resorption. This phenotype was not observed in osteoclast-selective Lrp4 knockout mice. Mechanistic studies indicate that loss of Lrp4 function in osteoblast-lineage cells increased serum levels of sclerostin, a key factor for bone-mass homeostasis that interacts with Lrp4, but abolished the inhibition of Wnt/β-catenin signaling and osteoblastic differentiation by sclerostin. Concomitantly, sclerostin induction of RANKL (receptor activator of nuclear kappa B ligand) was impaired, leading to a lower ratio of RANKL over OPG (osteoprotegerin) (a key factor for osteoclastogenesis). Taken together, these results support the view for Lrp4 as a receptor of sclerostin to inhibit Wnt/β-catenin signaling and bone formation and identify Lrp4 as a critical player in bone-mass homeostasis.

The Roles of Epithelial-to-Mesenchymal Transition (EMT) and Mesenchymal-to-Epithelial Transition (MET) in Breast Cancer Bone Metastasis: Potential Targets for Prevention and Treatment.

Many studies have revealed molecular connections between breast and bone. Genes, important in the control of bone remodeling, such as receptor activator of nuclear kappa (RANK), receptor activator of nuclear kappa ligand (RANKL), vitamin D, bone sialoprotein (BSP), osteopontin (OPN), and calcitonin, are expressed in breast cancer and lactating breast. Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) effectors play critical roles during embryonic development, postnatal growth, and epithelial homeostasis, but also are involved in a number of pathological conditions, including wound repair, fibrosis, inflammation, as well as cancer progression and bone metastasis. Transforming growth factor β (TGFβ), insulin-like growth factor I & II (IGF I & II), platelet-derived growth factor (PDGF), parathyroid hormone-related protein (PTH(rP)), vascular endothelial growth factor (VEGF), epithelial growth factors II/I (ErbB/EGF), interleukin 6 (IL-6), IL-8, IL-11, IL-1, integrin αvβ3, matrix metalloproteinases (MMPs), catepsin K, hypoxia, notch, Wnt, bone morphogenetic proteins (BMP), and hedgehog signaling pathways are important EMT and MET effectors identified in the bone microenviroment facilitating bone metastasis formation. Recently, Runx2, an essential transcription factor in the regulation of mesenchymal cell differentiation into the osteoblast lineage and proper bone development, is also well-recognized for its expression in breast cancer cells promoting osteolytic bone metastasis. Understanding the precise mechanisms of EMT and MET in the pathogenesis of breast cancer bone metastasis can inform the direction of therapeutic intervention and possibly prevention.

Abstract 1426: Functional analysis of membrane bound versus soluble RANKL: implications for cancer-induced osteolysis.

Abstract The primary site of prostate cancer metastasis is the skeleton with the resultant metastases containing areas of extensive osteogenesis and osteolysis. The latter is a process controlled by multinucleated osteoclasts and receptor activator of nuclear kappa B ligand (RANKL) is critical for osteoclastogenesis. RANKL is a type II transmembrane molecule that forms a trimer on the cell surface and mediates its effects via RANK. We have shown that matrix metalloproteinases-3 and -7 (MMP-3, MMP-7) can process RANKL to a soluble form, sRANKL. The objective of this study is to determine the functionality of membrane bound RANKL versus sRANKL in promoting osteoclastogenesis. Full-length murine RANKL cDNA was isolated and inserted into the pcDNA 3.1(+) expression construct. In vitro translation experiments demonstrated that RANKL was being generated and could be processed by MMP-3 and MMP-7. N-terminal amino acid sequencing revealed that the site of cleavage occurs in the juxtamembrane region of RANKL between amino acids 145 and 146. We hypothesized that cleavage in the juxtamembrane region could generate an active soluble form of sRANKL. Osteoclastogenesis and resorption assays with myeloid precursors demonstrated the functionality of sRANKL. To determine whether RANKL or sRANKL is the major mediator of osteoclastogenesis in vivo we have generated RANKL mutants that are resistant to cleavage by MMPs and other proteases. Using a site directed mutagenesis kit we have designed a number of mutants and found that Δ158, (deletion of amino acids 71-157) and, Δ148, deletion of amino acids 72-147), were resistant to proteolytic cleavage. We anticipate that the while Δ158 and Δ148 are resistant to cleavage, they will be functional. We will therefore test the functionality of these two deletion mutants in-vitro by co-culturing myeloid precursor cells with Cos-7/Cos-1 cells expressing wild type and mutant RANKL proteins. In conclusion, we have demonstrated that RANKL is proteolytically cleaved to generate a soluble active form. We have generated non-cleavable mutant forms of RANKL that will allow us to assess the role of membrane bound versus soluble RANKL in-vitro and in-vivo. The results of these experiments will further elucidate the roles of RANKL and sRANKL in prostate to bone metastases in addition to being informative for the engineering of future RANKL based therapies. Citation Format: Jeremy J. McGuire, Mitsuru Futakuchi, Conor Lynch. Functional analysis of membrane bound versus soluble RANKL: implications for cancer-induced osteolysis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1426. doi:10.1158/1538-7445.AM2013-1426

Osteoprotegerin expression in bone marrow by treatment with tocilizumab in rheumatoid arthritis

The aim of this study was to investigate histological changes of bone marrow in response to tocilizumab for rheumatoid arthritis (RA). After tocilizumab therapy, bone marrow tissues were extracted from ten RA patients at the time of total knee arthroplasty (TKA). Control samples were obtained from ten RA patients who underwent MTX mono-therapy. Histological examination of structural differences between the tocilizumab and control groups in bone marrow was performed using hematoxylin and eosin (H&E) to evaluate differences. In immunohistochemical examination, the expression of seven molecules including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), CD68, osteoprotegerin (OPG), receptor activator of nuclear kappa B ligand (RANKL), CD4 and osteopontin (OPN) were compared between two groups. NTx was significantly low at 44.5 ± 2 nM BCE/mM Cr compared with control at 73.2 ± 8 nM BCE/mM Cr. Immunohistochemical examination revealed that the bone marrow tissues of the RA patients who underwent tocilizumab therapy demonstrated significant positive OPG as compared with the control. However, immunohistochemical examinations after tocilizumab revealed that TNF-α, IL-6, CD68, CD4, OPN and RANKL were not significantly different with control of MTX in bone marrow. Therefore, treatment with tocilizumab increased the expression of OPG as the histological changes with respect to inhibit RANKL-related bone resorption of bone marrow in RA.

Human cytomegalovirus, Epstein‐Barr virus and bone resorption‐inducing cytokines in periapical lesions of deciduous teeth

A connection of herpesvirus periapical infection with symptomatic and large-size periapical lesions has been recognized in adult patients, but no data exist about a possible involvement of herpesviruses in severe periapical pathosis in children. Herpesviruses have the potential to elicit potent bone resorption-inducing cytokines in mammalian cells. This study aimed to determine the occurrence of human cytomegalovirus and Epstein-Barr virus DNA, and mRNA transcripts of receptor activator of nuclear kappa B ligand (RANKL), osteoprotegerin, core binding factor alpha-1, colony stimulating factor-1, transforming growth factor-beta, and monocyte chemoattractant protein-1 in periapical symptomatic pathosis of deciduous teeth. Twelve deciduous molar teeth from patients aged 2-8 years were extracted due to severe periapical infection, and granulomatous tissue adherent to the root tip of the extracted teeth was collected using a surgical knife. Non-diseased pulpal tissue, obtained from 12 teeth extracted for orthodontic reasons, served as negative control. Polymerase chain reaction assays were employed to identify herpesvirus DNA and cytokine gene expression, using established polymerase chain reaction primers and procedures. Seven (58%) of the periapical lesions yielded human cytomegalovirus and eight (67%) Epstein-Barr virus. Only one (8%) periapical lesion showed neither human cytomegalovirus nor Epstein-Barr virus. In healthy pulpal tissue, one (8%) specimen demonstrated human cytomegalovirus and another (8%) specimen revealed Epstein-Barr virus. Of the cytokines examined, RANKL expression showed significantly higher occurrence in periapical pathosis than in healthy pulpal tissue (P < 0.040). No relationship was identified between the type of herpesvirus and cytokine expression in the periapical lesions studied. The present findings provide evidence of a putative role of human cytomegalovirus and Epstein-Barr virus in the pathogenesis of symptomatic periapical pathosis in deciduous teeth. Increased RANKL expression in periapical lesions may be of pathogenetic significance.

Mechanical Strain Differentially Regulates Endothelial Nitric-oxide Synthase and Receptor Activator of Nuclear κB Ligand Expression via ERK1/2 MAPK

Exercise promotes positive bone remodeling through controlling cellular processes in bone. Nitric oxide (NO), generated from endothelial nitric-oxide synthase (eNOS), prevents resorption, whereas receptor activator of nuclear kappa B ligand (RANKL) promotes resorption through regulating osteoclast activity. Here we show that mechanical strain differentially regulates eNOS and RANKL expression from osteoprogenitor stromal cells in a magnitude-dependent fashion. Strain (0.25-2%) induction of eNOS expression was magnitude-dependent, reaching a plateau at 218 +/- 36% of control eNOS. This was accompanied by increases in eNOS protein and a doubling of NO production. Concurrently, 0.25% strain inhibited RANKL expression with increasing response up to 1% strain (44 +/- 3% of control RANKL). These differential responses to mechanical input were blocked when an ERK1/2 inhibitor was present during strain application. Inhibition of NO generation did not prevent strain-activated ERK1/2. To confirm the role of ERK1/2, cells were treated with an adenovirus encoding a constitutively activated MEK; Ad.caMEK significantly increased eNOS expression and NO production by more than 4-fold and decreased RANKL expression by half. In contrast, inhibition of strain-activated c-Jun kinase failed to prevent strain effects on either eNOS or RANKL. Our data suggest that physiologic levels of mechanical strain utilize ERK1/2 kinase to coordinately regulate eNOS and RANKL in a manner leading to positive bone remodeling.