Receptor Activator Of NF-kappaB Research Articles

Xanthoxyletin blocks the RANK/RANKL signaling pathway to suppress the growth of human pancreatic cancer cells.

Xanthoxyletin is a vital plant-derived bioactive coumarin. It has been shown to exhibit anticancer effects against different human cancers. Nonetheless, the anticancer effects of xanthoxyletin against human pancreatic cancer cells have not been evaluated. Against this backdrop, the present study was designed to evaluate the anticancer effects of xanthoxyletin in human pancreatic cancer cells and to decipher the underlying molecular mechanisms. The results revealed a significant (p < 0.05) upregulation of receptor activator of NF-kappaB (RANK), receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) in human pancreatic tissues and cell lines at both transcriptional and translational levels. The administration of pancreatic cancer cells with xanthoxyletin diminished the viability of Capan-2 cells in a concentration-dependent manner and led to a significant decline in RANK, RANKL, and OPG expression. Silencing of RANK and xanthoxyletin treatment declined the viability of Capan-2 pancreatic cancer cells via induction of apoptosis. However, pancreatic cancer cells overexpressing RANK could rescue the growth inhibitory effects. Collectively, xanthoxyletin targets the RANK/RANKL signaling pathway in pancreatic cancer cells to induce cell apoptosis and may prove to be an important lead molecule.

Effect of different access cavity designs on fracture toughness of endodontically treated teeth: a systematic review and network meta-analysis.

Objective To compare the effect of different endodontic access cavities on fracture toughness of extracted endodontically treated human teeth.Data/sources An electronic literature search was performed in seven databases as well as hand search until September 2020. Risk-of-bias tool was used to evaluate the quality of included studies. Random effects frequentist network meta-analysis was performed, with mean difference (MD) and 95% confidence interval (CI) as the effect measure. Confidence in the documented evidence was assessed through the newly fuelled Confidence in Network Meta-analysis (CINeMA) framework based on the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach.Study selection A total of 844 articles were obtained in the electronic and hand search. After the application of the eligibility criteria and duplicate removal, 14 studies were included in this systematic review. All included studies were in vitro that evaluated the influence of conservative endodontic cavities (CECs) on fracture toughness in extracted endodontically treated human teeth and compared to traditional endodontic cavities (TECs). In total, ten studies (n = 456) were included in the network meta-analysis for molars. The overall risk of bias was moderate.Results The results showed that when compared to intact teeth, the greatest reduction in fracture resistance was reported for TEC (MD: -927.52; 95% CI [-1304.80; -550.24]) and CEC showed least reduction in fracture resistance (MD: -365.59; 95% CI [-759.02; 27.83]). The surface under the cumulative ranking curve (SUCRA) value for intact teeth was highest (85.4% probability of being ranked as first), followed by CEC (51.4% probability of being ranked as second), with CEC presenting the highest probabilities to be the most effective access cavity design, according to the RANK (receptor activator of NF-kappaB) table. Level of confidence varied from low to moderate across all formulated comparisons.Conclusion Overall, based on the included in vitro studies in this systematic review, CEC was the most favourable access cavity design when compared to other (truss access cavities, TEC and ninja cavities) and TEC was the least favourable for fracture resistance; however, the level of evidence was moderate.

Molecular Triad RANK/ RANKL/ OPG in Mandible and Femur of Wistar Rats (Rattus norvegicus) With Type 2 Diabetes Mellitus

A successful treatment of dental implant needs a good jaw bone support, which depends on healthy bone metabolism. Bone metabolism can be affected by Diabetes Mellitus (DM). It may trigger various complications, including osteoporosis. Molecular triads consisting of Receptor Activator of NF-kappaB (RANK), Activator of nF-κB Ligand (RANKL), and osteoprotegerin (OPG), have an important role in the formation, function, and osteoclast survival. In this study, molecular triads were observed on mandible and femur bones in type 2 DM Wistar rats. The aim of this study was to observe the molecular triad RANK / RANKL / OPG expressions in type 2 DM Wistar rats. This laboratory research used 18 male Wistar rats divided into three groups: nondiabetic group (control), uncontrolled DM injected with single dose of Streptozotocin (STZ), and controlled DM treated with Metformin. On day 20, the mandible and femur were collected and specimen processing was carried out. The results of RANK / RANKL / OPG expressions were obtained from immunohistochemical staining. In both mandible and femur groups, RANK, RANKL, OPG expressions showed no difference between the control and uncontrolled DM groups. RANKL / OPG ratio in uncontrolled DM was higher than that in the control group. RANK expression was lower in uncontrolled DM group compared with controlled DM, and the RANKL expression in uncontrolled DM group was higher than that in the controlled DM group. RANKL / OPG ratio was lower in the controlled DM group. The study suggested that DM affects resorptive activity in mandible and femur bones which can be observed via RANK/RANKL/OPG.

ID: 81: Therapeutic efficacy of RANKL blockade in non-small cell lung cancer

With over 1.5 million deaths yearly, lung cancer has become the leading cause of cancer-related mortality worldwide. Although targeted therapies are emerging to combat non-small cell lung cancer (NSCLC), the principal type of lung cancer, they are currently limited to a small fraction of tumors with defined oncogenic alterations, and no specific treatment exists against tumors carrying activating mutations in the most frequently mutated proto-oncogene, KRAS. Here we show an association between expression of the osteoclast differentiation factor, receptor activator of NF-kappaB (RANK) ligand (RANKL) and overall survival in NSCLC, and an impaired progression of Kras-mutant tumors upon pharmacological blockade of RANKL in several genetically-engineered mouse models of NSCLC. RANKL blockade used as monotherapy led to a rapidly diminished tumor growth rate, with shrinkage in a large proportion of tumors, as monitored by in vivo micro-computed tomography. The anti-tumor response, which was characterized by apoptosis induction and NF-kappaB pathway attenuation in tumor cells, as well as increased dendritic cell maturation with proliferation of CD8+ T cells in the tumor microenvironment, lasted for a prolonged period of time without noticeable resistance. Combined administration of the standard chemotherapy cisplatin with the RANKL inhibitor to mirror an ongoing clinical trial revealed increased efficacy compared to each treatment alone. Finally, we report the case of a patient whose primary lung tumor responded to a RANKL inhibitory drug. Altogether, our results indicate that RANKL blocking agents used as monotherapies or in combination treatment impair the growth of primary lung tumors.

Urinary messenger RNA of the receptor activator of NF-kappaB could be used to differentiate between minimal change disease and membranous nephropathy

Podocyte damage and loss together have an important role in the pathogenesis and progression of glomerulonephritis. Glomerulonephritis patients and healthy controls were enrolled in this study. Biochemical, clinical and experimental procedures included measurement of total urinary protein, renal biopsy and gene expression analysis of the receptor activator of NF-kappaB (RANK). The urinary mRNA levels of RANK were significantly higher in the glomerulonephritis group compared to the controls. The urinary RANK level of glomerular subtypes was correlated significantly with proteinuria. The calculated area of RANK mRNA levels under the curve was 0.61 for minimal change disease (MCD), 0.97 for membranous nephropathy (MN), 0.65 for IgA nephropathy (IgAN), 0.70 for lupus nephritis (LN) and 0.70 for focal segmental glomerulosclerosis (FSGS). The urinary mRNA of RANK might be used to differentiate histologic subtypes of glomerulonephritis, particularly between MCD and MN.

Myeloid Dendritic Cells Contribute to Hematopoietic Progenitor Mobilization by G-CSF.

AbstractAbstract 2319Granulocyte-colony stimulating factor (G-CSF) is the prototypic agent used to mobilize hematopoietic stem and progenitor cells (HSPCs) into the blood where they can then be harvested for stem cell transplantation. G-CSF acts in a non-cell-intrinsic fashion to induce HSPC mobilization. We recently showed that G-CSF signaling in a CD68+ monocyte/macrophage lineage cell within the bone marrow initiates the HSPC mobilization cascade (Christopher et al., 2011). Consistent with this finding, two other groups showed that ablation of monocytes/macrophages induces HSPC mobilization (Winkler et al., 2010; Chow et al., 2011). CD68 marks a heterogeneous cell population that includes monocytes, macrophages, myeloid dendritic cells, and osteoclasts. To further define the relevant cell population(s) for HSPC mobilization by G-CSF, we first examined the role of osteoclasts. Receptor activator of NF-kappaB (RANK) signaling is required for osteoclast development. Osteoprotegerin (OPG) is a decoy receptor for RANK ligand, and treatment with OPG-Fc (a stabilized form of OPG) results in osteoclast ablation in mice. We treated mice with 100 μg of OPG-Fc and documented complete osteoclast ablation by histomorphometry. Osteoclast ablation did not result in constitutive HSPC mobilization, nor did it affect G-CSF-induced HSPC mobilization. To further assess the role of osteoclasts, we transplanted RANK−/− fetal liver cells into irradiated Csf3r−/− (G-CSF receptor deficient) recipients. Since RANK is required for osteoclast development, the osteoclasts in these bone marrow chimeras lack the G-CSFR, while other hematopoietic cells (including monocytes/macrophages) are G-CSFR sufficient. Again, G-CSF-induced HSPC mobilization in these mice was normal. Based on these data, we conclude that osteoclasts are dispensable for HSPC mobilization by G-CSF.We next quantified changes in monocytic/macrophage cell populations in the bone marrow after G-CSF treatment (250 μg/kg per day for 5 days) using a novel multi-color flow cytometry assay that includes CD115, F4/80, MHC class II, Gr-1, B220, and CD11c. Using this assay, we observed a significant decrease in macrophages (11.8 ± 3.6-fold) and, surprisingly, myeloid dendritic cells (MDCs; 5.5 ± 1.2-fold) in the bone marrow with G-CSF treatment. To further assess the role of MDCs, we used transgenic mice expressing the diphtheria toxin receptor under the control of the CD11c promoter (CD11c-DTR) to conditionally ablate MDCs. To avoid systemic toxicity, we transplanted CD11c-DTR bone marrow into congenic wild type recipients prior to MDC ablation. The resulting bone marrow chimeras were treated with diphtheria toxin (DT; 400 ng per day for 6 days), which resulted in a 92% reduction in MDCs. Ablation of MDCs resulted in a significant increase in colony-forming cells in the blood and spleen (figure 1A). Moreover, MDC ablation significantly increased mobilization of colony-forming cells and c-Kit+lineage−Sca-1+ (KLS) cells by G-CSF (figures 1B and 1C). Taken together, these data suggest that myeloid dendritic cells, but not osteoclasts, contribute to HSPC mobilization by G-CSF. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Genetic polymorphisms of the OPG gene associated with breast cancer.

1523 Background: The receptor activator of NfkappaB (RANK) and osteoprotegerin (OPG) cascade system have been reported to play a role in the pathogenesis of breast cancer (BC). Genetic variations in the genes coding for RANK, RANK ligand (RANKL), and OPG are supposed to play a role in the susceptibility of breast cancer. Methods: In the present case-control study genomic DNA was obtained from 307 BC patients (age: median 56) and 396 healthy female controls (healthy blood donors, HC; age median: 45). We studied six single nucleotide polymorphisms (SNPs) in the genes coding for RANK (2 SNPs, rs1805034, rs35211496), OPG (2 SNPs, rs3102735, rs2073618), and RANKL (2 SNPs, rs9533156, rs2277438) using TaqMan assay-guided PCR for the respective SNPs. The genotype and allelic frequencies comparing BC with HC were analyzed with χ2 test for 2x3 and 2x2 tables, respectively. Results: The genotype distributions as well as the allelic frequencies of the SNP rs3102735 in the OPG gene differed significantly (p=0.006 and p=0.019, respectively) between BC patients and HC; the genotypes containing the minor allele were more frequent in BC patients (table). In the OPG SNP rs2073618 the minor allele C was significantly less frequent in BC patients compared to HC (43.8 vs. 49.7%; OR: 0.788; p=0.031). In addition, BC patients carried less frequently the homozygous genotype of the minor allele compared to major allele (18.6 vs. 23.9%, p=0.083). No significant risk was detected for the other SNPs investigated in this study. Conclusions: This is the first study reporting a significant association of the SNP rs3102735 in the OPG gene with the susceptibility to develop BC in the Caucasian population. The minor allele C of OPG SNP rs2073618, however, seems to be protective against BC disease. The impact of the OPG SNP rs3102735 (location: 5`near region, chromosome 8q24) and of the OPG SNP rs2073618 (a missense SNP, leading to amino acid exchanges Leu3Asn) on the pathogenesis of BC are unclear. [Table: see text]

Genetic Polymorphisms of the RANKL and OPG Genes Associated with Myeloma

Abstract 2911The bone disease in myeloma is the result of the complex interactions between myeloma cells and the bone marrow osteoclasts plus other accessory cells and microenvironmental components. The receptor activator of NfkappaB (RANK) and osteoprotegerin (OPG) cascade system have been reported to be an essential pathway in the osteoclastogenesis. Genetic variations in the genes coding for RANK, RANK ligand (RANKL), and OPG are supposed to play a role in the susceptibility of myeloma.In the present case control study genomic DNA was obtained in 217 myeloma patients including smoldering myeloma (age: mean 62, 33–88) and 516 healthy controls (healthy blood donors, HC; age mean: 44, 18–65). We studied six single-nucleotide-polymorphisms (SNPs) in the genes of RANK (2 SNPs, rs1805034, rs35211496), OPG (2 SNPs, rs3102735, rs2073618), and of RANKL (2 SNPs, rs9533156, rs2277438) using TaqMan assay guided polymerase chain reaction for the respective SNPs. The genotype and allelic frequencies comparing myeloma patients with HC were analyzed with χ2 test for 2×3 and 2×2 tables, respectively.The genotype distributions of the SNP rs2277438 in the RANKL gene and SNP rs3102735 in the OPG gene differed highly significantly (p=0.0001 and p=0.005, respectively) between myeloma patients and HC (table 1). No significantly increased risk was detected in the other SNP analyzed in the study. A subgroup analysis of the myeloma patients stratified into beta2 microglobulin levels lower or higher than 3.5 mg/l showed lower values to be significantly associated with the minor allele of the SNP rs3102735 in the OPG gene (major vs. minor allele in myeloma vs. HC: 77/23% vs. 89/11%; OR 2.49; CI 1.38–4.49). Further stratification into gender, serum calcium levels, hemoglobin values at diagnosis showed no differences.Table 1Myeloma patientsHealthy controls (HC)OR (CI)pRANKL rs2277438Allelen = 434 (%)n = 1024 (%)1324 (75)860 (84)1.7790.00012110 (25)164 (16)(1.373–2.341)Genotypen = 217 (%)n = 512 (%)11132 (61)360 (70)0.00011260 (28)140 (27)2225 (12)12 (2)OPG rs 3102735Allelen = 420 (%)n = 1028 (%)167 (16)109 (11)1.6000.0052353 (84)919 (89)(1.153–2.222)Genotypen = 210 (%)n = 514 (%)119 (4)5 (1)0.0051249 (23)99 (19)22152 (72)410 (80)RANK = receptor activator of NfkappaB, RANKL = receptor activator of NfkappaB ligand, OPG = osteoprotegerin, OR = odds ratio, CI = confidence intervals 95%To the best of our knowledge, this is the first study reporting a highly significant association of the SNP rs2277438 of the RANKL gene and the SNP rs3102735 in the OPG gene, respectively, with the susceptibility to develop myeloma in the Caucasian population. Both SNPs are associated with an increased risk for myeloma. However, myeloma patients carrying the minor allele of OPG SNP rs3102735 developed lower levels of the proliferation marker beta2 microglobulin. The impact of the OPG SNP rs3102735 (location: promoter region, chromosome 8q24) or of the SNP rs2277438 located in intron 1 (chromosome 13q14.11), a potentially regulatory region of the RANKL gene, on the regulation of RANKL expression is unclear. Disclosures:No relevant conflicts of interest to declare.

TRPV-5 Mediates a Receptor Activator of NF-κB (RANK) Ligand-induced Increase in Cytosolic Ca2+ in Human Osteoclasts and Down-regulates Bone Resorption

Most of the signaling effectors located downstream of receptor activator of NF-kappaB (RANK) activation are calcium-sensitive. However, the early signaling events that lead to the mobilization of intracellular calcium in human osteoclasts are still poorly understood. The Ca(2+)-sensitive fluorescent probe Fura2 was used to detect changes in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) in a model of human osteoclasts. Stimulating these cells with receptor activator of NF-kappaB ligand (RANKL) induced a rapid and significant increase in [Ca(2+)](i). Adding extracellular Ca(2+) chelators, depleting intracellular stores, and the use of a phospholipase C inhibitor all indicated that the Ca(2+) was of extracellular origin, suggesting the involvement of a Ca(2+) channel. We showed that none of the classical Ca(2+) channels (L-, T-, or R-type) were involved in the RANKL-induced Ca(2+) spike. However, the effect of high doses of Gd(3+) did suggest that TRP family channels were present in human osteoclasts. The TRPV-5 channel was expressed in osteoclasts and was mainly located in the cellular area in contact with the bone surface. Furthermore, the RNA inactivation of TRPV-5 channel completely inhibited the RANKL-induced increase in [Ca(2+)](i), which was accompanied in the long term by marked activation of bone resorption. Overall, our results show that RANKL induced a significant increase in [Ca(2+)](i) of extracellular origin, probably as a result of the opening of TRPV-5 calcium channels on the surface of human osteoclasts. Our findings suggest that TRPV-5 contributes to maintaining the homeostasis of the human skeleton via a negative feedback loop in RANKL-induced bone resorption.

IKKβ activation is sufficient for RANK-independent osteoclast differentiation and osteolysis

Monocytes differentiate into osteoclasts through stimulation of receptor activator of NF-κB (RANK). Many downstream effectors of RANK play a positive role in osteoclastogenesis, but their relative importance in osteoclast differentiation is unclear. We report the discovery that activation of a single pathway downstream of RANK is sufficient for osteoclast differentiation. In this regard, introduction of constitutively activated IKKβ (IKKβSSEE) but not wild-type IKKβ into monocytes stimulates differentiation of bona fide osteoclasts in the absence of RANK ligand (RANKL). This phenomenon is independent of upstream signals because IKKβSSEE induced the development of bone-resorbing osteoclasts from RANK and IKKα knockout monocytes and in conditions in which NEMO-IKKβ association was inhibited. NF-κB p100 and p105, but not RelB, were critical mediators of this effect. Inflammatory autocrine signaling by tumor necrosis factor α (TNF-α) and interleukin 1 (IL-1) were dispensable for the spontaneous osteoclastogenesis driven by IKKβSSEE. More important, adenoviral gene transfer of IKKβSSEE induced osteoclasts and osteolysis in calvariae and knees of mice. Our data establish the sufficiency of IKKβ activation for osteolysis and suggest that IKKβ hyperactivation may play a role in conditions of pathologic bone destruction refractory to RANK/RANKL proximal therapeutic interventions. © 2010 American Society for Bone and Mineral Research.

Gu Ling Pian, a traditional Chinese medicine, regulates function and OPG/RANKL synthesis of osteoblasts via the p38 MAPK pathway

Osteoporosis is a common disease that makes bones prone to fracture and can affect both men and women. Many traditional Chinese medicine formulations have the potential effect of preventing osteoporosis. Gu Ling Pian (GLP), a traditional Chinese medicine formulation, comprised of tonifying kidney herbal medicines, has been demonstrated to prevent osteoporosis by increasing bone mineral density, however the exact mechanism has not yet been elucidated. Osteoprotegerin (OPG), a receptor activator of NF-kappaB (RANK), and RANK ligand (RANKL) play critical roles in bone remodelling by regulating the function of osteoclasts. In this study, we investigated the effect of GLP on osteoblasts, namely MG-63 cells. The cell proliferation and differentiation, synthesis of OPG/RANKL and p38 expression were tested on MG-63 cells exposed to serum from rats fed with GLP or not. The results showed that GLP significantly promoted MG-63 cell proliferation and differentiation. Upregulation of OPG and down-regulation of RANKL at the protein and mRNA level were observed in GLP serum treated MG-63 cells using an enzyme-linked immunosorbent assay and real-time polymerase chain reaction. Further, treatment with GLP serum increased the level of p38 phosphorylation but did not affect the total p38 expression. These effects can be blocked by the p38 specific inhibitor SB203580. The results indicate that GLP can effectively promote the proliferation and differentiation of osteoblasts and regulate their OPG/RANKL expression, while the effects may be mediated via the p38 MAPK pathway. The findings suggest that GLP induces bone formation and may be beneficial for patients with osteoporosis.

Immunohistochemical expression of RANKL, RANK, and OPG in human oral squamous cell carcinoma

The mechanism of oral squamous cell carcinoma (SCC) invading jawbone remains controversial. Interactions between receptor activator of NF-kappaB (RANK) and its ligand (RANKL) are required for osteoclastogenesis. The binding of RANK and RANKL induces differentiation of osteoclasts, leading to bony destruction. Osteoprotegerin (OPG), a decoy receptor for RANKL, also binds to RANKL by competing with RANK, and this could protect against osseous destruction. Immunoexpression of RANKL, RANK, and OPG in 25 cases of human buccal SCCs without bony invasion and 15 cases of gingival SCCs with mandibular bony invasion was investigated. Normal oral mucosa from five individuals without betel-quid chewing or cigarette smoking was used as a control. The scores are designated as percentage of positive staining x intensity of staining for each section. Strong cytoplasmic staining of RANKL proteins is detected in cancer cells of both buccal and gingival SCCs. The same protein is identified in cytoplasm of osteoclasts for all cases involving bony invasion. Strong cytoplasmic staining of RANKL is confined to basal layer for all normal mucosa. A similar staining pattern is noted for RANK protein in all buccal and gingival SCCs. An absence of staining of RANK protein is noted for all normal tissues. Weak to negative cytoplasmic stained OPG protein is present in all buccal and gingival SCCs, but is absent in all normal tissues. These findings suggest the potential value of the RANK/RANKL/OPG pathway as biomarkers in human oral SCCs.

Water-soluble fullerene (C60) inhibits the osteoclast differentiation and bone destruction in arthritis

Recently, it has been demonstrated that oxygen free radicals have an important role as a signaling messenger in the receptor activator NFkappaB (RANK) signal pathway required for osteoclast differentiation. The aim of this study was to examine the potential of a strong free-radical scavenger, water-soluble fullerene (C60), as a protective agent against the RANK-induced osteoclastogenesis and osteoclastic bone destruction in arthritis, both in vitro and in vivo. The effects of C60 on the RANK-induced osteoclastogenesis and osteoclastic bone resorption were examined in vitro. Adjuvant-induced arthritic rats were used as an animal model of arthritis. Rats were divided into two subgroups: control and treatment with C60 at 1.0 microM. The left ankle joint was injected intra-articularly with water-soluble C60 (20 microl) in the C60-treated group, while, as a control, the left ankle joint in the control rats received phosphate-buffered saline (20 microl) once weekly for eight weeks. Ankle joint tissues were prepared for histologic analysis. C60 significantly inhibited the responses of osteoclast precursor cells to RANK ligand, including osteoclast differentiation and osteoclastic bone resorption in vitro. In adjuvant-induced arthritic rats, intra-articular treatment with C60 in vivo reduced the number of osteoclasts and alleviated bone resorption and destruction in the joints, while control ankle joints showed progression of joint destruction with time. These findings indicate that C60 downregulates the RANK-induced osteoclast differentiation and is a potential therapeutic agent for inhibition of osteoclastic bone destruction in arthritis.

Caffeic acid phenethyl ester, an active component of honeybee propolis attenuates osteoclastogenesis and bone resorption via the suppression of RANKL‐induced NF‐κB and NFAT activity

Receptor activator NF-kappaB ligand (RANKL)-activated signaling is essential for osteoclast differentiation, activation and survival. Caffeic acid phenethyl ester (CAPE), a natural NF-kappaB inhibitor from honeybee propolis has been shown to have anti-tumor and anti-inflammatory properties. In this study, we investigated the effect of CAPE on the regulation of RANKL-induced osteoclastogenesis, bone resorption and signaling pathways. Low concentrations of CAPE (<1 microM) dose dependently inhibited RANKL-induced osteoclastogenesis in RAW264.7 cell and bone marrow macrophage (BMM) cultures, as well as decreasing the capacity of human osteoclasts to resorb bone. CAPE inhibited both constitutive and RANKL-induced NF-kappaB and NFAT activation, concomitant with delayed IkappaBalpha degradation and inhibition of p65 nuclear translocation. At higher concentrations, CAPE induced apoptosis and caspase 3 activities of RAW264.7 and disrupts the microtubule network in osteoclast like (OCL) cells. Taken together, our findings demonstrate that inhibition of NF-kappaB and NFAT activation by CAPE results in the attenuation of osteoclastogenesis and bone resorption, implying that CAPE is a potential treatment for osteolytic bone diseases.

VEGF165 Promotes the Osteolytic Bone Destruction of Ewing's Sarcoma Tumors by Upregulating RANKL

The purpose of this study was to determine whether vascular endothelial growth factor-165 (VEGF165) contributed to the osteolytic process in Ewing's sarcoma. VEGF165 induced osteoclast formation from murine bone marrow cells. Tartrate-resistant acid phosphatase (TRAP) staining demonstrated significantly fewer osteoclasts in VEGF-inhibited TC/siVEGF7-1 tumors compared to TC71 parental or TC/si-control tumors. Receptor activator NF-kappaB (RANKL), a critical osteoclastogenic factor, was decreased in TC/siVEGF7-1 cells. Incubation of these cells with recombinant VEGF165 upregulated RANKL in a dose- and time-dependent manner. The induction of (RANKL) by VEGF165 was also demonstrated in MC3T3-E1 mouse osteoblast cells and bone marrow stromal cells. This upregulation was transcriptionally mediated by an effect on the RANKL promoter. Both VEGF and EWS/FLI-1 increased RANKL promoter activity. Taken together, these data suggest that modulation of RANKL by VEGF165 may be one of the mechanisms responsible for the osteolytic process induced by Ewing's sarcoma cells. VEGF165 may, therefore, play an important role in modulating RANKL gene expression in the bone marrow microenvironment during the metastatic process, thereby contribution to tumor induced bone lysis.

The role of T-cell leukemia translocation-associated gene (TCTA) protein in human osteoclastogenesis

Synovial tissues of patients with rheumatoid arthritis (RA) include factors regulating bone resorption, such as receptor activator NF-kappaB ligand (RANKL), TNF-alpha, IL-6, IL-17, and IFN-gamma. However, in addition to these cytokines, other factors expressed in synovial tissues may play a role in regulating bone resorption. In 2009, we demonstrated that novel peptides from T-cell leukemia translocation-associated gene (TCTA) protein expressed in synovial tissues from patients with RA inhibit human osteoclastogenesis, preventing cellular fusion via the interaction between TCTA protein and a putative counterpart molecule. Only a few studies on the role of TCTA protein have been reported, including our report published in 2009. In the current review paper, we summarized papers on TCTA protein before 2009 and our recent findings.

INOS -derived Nitric Oxide Modulates Infection-stimulated Bone Loss

Nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) plays an important role in host defense, as well as in inflammation-induced tissue lesions. Here we evaluated the role of NO in bone loss in bacterial infection-induced apical periodontitis by using iNOS-deficient mice (iNOS(-/-)). The iNOS(-/-) mice developed greater inflammatory cell recruitment and osteolytic lesions than WT mice. Moreover, tartrate-resistant acid-phosphatase-positive (TRAP(+)) osteoclasts were significantly more numerous in iNOS(-/-) mice. Furthermore, the increased bone resorption in iNOS(-/-) mice also correlated with the increased expression of receptor activator NF-kappaB (RANK), stromal-cell-derived factor-1 alpha (SDF-1 alpha/CXCL12), and reduced expression of osteoprotegerin (OPG). These results show that NO deficiency was associated with an imbalance of bone-resorption-modulating factors, leading to severe infection-stimulated bone loss.

The Tumor Necrosis Factor Family Receptors RANK and CD40 Cooperatively Establish the Thymic Medullary Microenvironment and Self-Tolerance

Medullary thymic epithelial cells (mTECs) establish T cell self-tolerance through the expression of autoimmune regulator (Aire) and peripheral tissue-specific self-antigens. However, signals underlying mTEC development remain largely unclear. Here, we demonstrate crucial regulation of mTEC development by receptor activator of NF-kappaB (RANK) and CD40 signals. Whereas only RANK signaling was essential for mTEC development during embryogenesis, in postnatal mice, cooperation between CD40 and RANK signals was required for mTEC development to successfully establish the medullary microenvironment. Ligation of RANK or CD40 on fetal thymic stroma in vitro induced mTEC development in a tumor necrosis factor-associated factor 6 (TRAF6)-, NF-kappaB inducing kinase (NIK)-, and IkappaB kinase beta (IKKbeta)-dependent manner. These results show that developmental-stage-dependent cooperation between RANK and CD40 promotes mTEC development, thereby establishing self-tolerance.

Lipopolysaccharide suppresses RANK gene expression in macrophages by down-regulating PU.1 and MITF

Receptor activator of NF-kappaB (RANK) is a receptor for RANK ligand (RANKL), and signals transduced by RANK-RANKL interaction are prerequisite for the differentiation and activation of osteoclasts. We cloned and characterized a 6-kb fragment containing the 5'-flanking region of the mouse RANK gene. A fragment of 1-kb from the transcription start sites containing four Sp-1 sites and putative binding sites for MITF, CRE/AP-1, and PU.1 was ligated to the pGL3-basic vector, and the promoter activity was confirmed by transfection studies. By electrophoretic gel motility shift assay, both PU.1 and proximal MITF binding site showed specific DNA-protein binding. Co-transfection studies with MITF- and PU.1-expression vectors revealed that MITF and PU.1 increased RANK promoter activity three- and twofold, respectively, and sixfold synergistically. Taken together, these results show that RANK transcription is positively regulated by both PU.1 and MITF. The effect of lipopolysaccharide (LPS) on RANK gene expression, analyzed by in situ hybridization using mouse bone tissue, showed that LPS decreased RANK transcripts of both precursor and mature osteoclasts. Furthermore, LPS treatment of RAW.264.7 cells decreased their RANK mRNA expression by 70%, mirroring the decrease of PU.1 and MITF mRNA. Short-term treatment with LPS decreased the promoter activity of pGL3-WT by 70%. Although LPS has been reported to promote osteoclastogenesis in chronic and local pyogenic inflammation, we speculate that LPS per se may directly suppress RANK expression in the osteoclastic cell lineage by down-regulating the expression of PU.1 and MITF genes in acute and systemic severe endotoxemia, such as in septic shock.

Dried Plum Polyphenols Inhibit Osteoclastogenesis by Downregulating NFATc1 and Inflammatory Mediators

Dried plums and their polyphenols have been shown to suppress bone resorption by downregulating receptor activator NF-kappaB ligand (RANKL). Due to the anti-inflammatory and antioxidant properties of these compounds, this study was designed to investigate whether dried plum polyphenols exert additional, more direct effects on osteoclasts and their precursors. RAW 264.7 macrophages were used as a model to study osteoclast precursors and osteoclast differentiation and activity. Under inflammatory conditions induced by lipopolysaccharide (LPS), polyphenols extracted from dried plum (10, 20, and 30 microg/mL) downregulated osteoclast precursor cyclooxygenase expression and nitric oxide (NO) by inhibiting inducible NO synthase. NO and tumor necrosis factor (TNF)-alpha were also suppressed in the presence of RANKL during osteoclastogenesis by the polyphenols. Increased TNF-alpha production in response to oxidative stress, but not LPS, was decreased over time. As expected, LPS and H2O2 significantly increased the number of tartrate-resistant acid phosphatase-positive cells by 127% and 30%, respectively. Dried plum polyphenols decreased osteoclast differentiation under normal as well as inflammatory and oxidative stress conditions, coincident with the suppression of the transcription factor, nuclear factor for activated T cells (NFATcl). These inhibitory effects on osteoclastogenesis were confirmed in primary bone marrow cultures. Resorption pit formation was decreased to a similar extent as osteoclast differentiation, suggesting that dried plum polyphenols primarily affect osteoclast differentiation as opposed to activity. Our data demonstrate that dried plum polyphenols directly inhibit osteoclastogenesis, leading to a decrease in osteoclast activity, by downregulating NFATc1 and inflammatory mediators.