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RANKL mRNA Expression (rankl + mrna_expression)
Selected AbstractsBumetanide, the Specific Inhibitor of Na+ -K+ -2Cl, Cotransport, Inhibits 1,,25-Dihydroxyvitamin D3 -Induced Osteoclastogenesis in a Mouse co-culture SystemEXPERIMENTAL PHYSIOLOGY, Issue 5 2003Hyun-A Lee The Na+ -K+ -2Cl, cotransporter (NKCC1) is responsible for ion transport across the secretory and absorptive epithelia, the regulation of cell volume, and possibly the modulation of cell growth and development. It has been reported that a variety of cells, including osteoblasts, contain this cotransporter. In this study, the physiological role of NKCC1 in osteoclastogenesis was exploited in a co-culture system. Bumetanide, a specific inhibitor of NKCC1, reduced the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. In order to investigate the mechanism by which bumetanide inhibits osteoclastogenesis, the mRNA expressions of the receptor activator of nuclear factor (NF)-,B ligand (RANKL) and osteoprotegerin (OPG) were analysed by RT-PCR. Exposure of osteoblastic cells to a medium containing 1 µM bumetanide reduced RANKL mRNA expression induced by 10 nM 1,,25-dihydroxyvitamin D3 (1,,25(OH)2D3, in a dose-dependent manner. In addition, RANKL expression was also analysed with enzyme-linked immunosorbant assay (ELISA) using anti-RANKL antibody. The expression of RANKL was decreased with the increase of bumetanide concentration. In contrast, the expression of OPG mRNA, a novel tumour necrosis factor (TNF) receptor family member was increased in the presence of bumetanide. These results imply that bumetanide inhibits osteoclast differentiation by reducing the RANKL/OPG ratio in osteoblastic cells. However, no significant difference in M-CSF mRNA expression was observed when bumetanide was added. Also, we found that the phosphorylation of c-Jun NH2 -terminal kinase (JNK), which regulates the activity of various transcriptional factors, was reduced by bumetanide treatment. Conclusively, these findings suggest that NKCC1 in osteoblasts has a pivotal role in 1,,25(OH)2D3 -induced osteoclastogenesis partly via the phosphorylation of JNK. [source] Selective Blockade of Voltage-Gated Potassium Channels Reduces Inflammatory Bone Resorption in Experimental Periodontal Disease,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2004Paloma Valverde Abstract The effects of the potassium channel (Kv1.3) blocker kaliotoxin on T-cell-mediated periodontal bone resorption were examined in rats. Systemic administration of kaliotoxin abrogated the bone resorption in conjunction with decreased RANKL mRNA expression by T-cells in gingival tissue. This study suggests a plausible therapeutic approach for inflammatory bone resorption by targeting Kv1.3. Introduction: Kv1.3 is a critical potassium channel to counterbalance calcium influx at T-cell receptor activation. It is not known if Kv1.3 also regulates RANKL expression by antigen-activated T-cells, and consequently affects in vivo bone resorption mediated by activated T-cells. Materials and Methods:Actinobacillus actinomycetemcomitans 29-kDa outer membrane protein-specific Th1-clone cells were used to evaluate the expression of Kv1.3 (using reverse transcriptase-polymerase chain reaction [RT-PCR] and Western blot analyses) and the effects of the potassium channel blocker kaliotoxin (0,100 nM) on T-cell activation parameters ([3H]thymidine incorporation assays and ELISA) and expression of RANKL and osteoprotegerin (OPG; flow cytometry, Western blot, and RT-PCR analyses). A rat periodontal disease model based on the adoptive transfer of activated 29-kDa outer membrane protein-specific Th1 clone cells was used to analyze the effects of kaliotoxin in T-cell-mediated alveolar bone resorption and RANKL and OPG mRNA expression by gingival T-cells. Stimulated 29-kDa outer membrane protein-specific Th1 clone cells were transferred intravenously on day 0 to all animals used in the study (n = 7 animals per group). Ten micrograms of kaliotoxin were injected subcutaneously twice per day on days 0, 1, 2, and 3, after adoptive transfer of the T-cells. The control group of rats was injected with saline as placebo on the same days as injections for the kaliotoxin-treated group. The MOCP-5 osteoclast precursor cell line was used in co-culture studies with fixed 29-kDa outer membrane protein-specific Th1-clone cells to measure T-cell-derived RANKL-mediated effects on osteoclastogenesis and resorption pit formation assays in vitro. Statistical significance was evaluated by Student's t -test. Results: Kaliotoxin decreased T-cell activation parameters of 29-kDa outer membrane protein-specific Th1 clone cells in vitro and in vivo. Most importantly, kaliotoxin administration resulted in an 84% decrease of the bone resorption induced in the saline-treated control group. T-cells recovered from the gingival tissue of kaliotoxin-treated rats displayed lower ratios of RANKL and OPG mRNA expression than those recovered from the control group. The ratio of RANKL and osteoprotegerin protein expression and induction of RANKL-dependent osteoclastogenesis by the activated T-cells were also markedly decreased after kaliotoxin treatments in vitro. Conclusion: The use of kaliotoxin or other means to block Kv1.3 may constitute a potential intervention therapy to prevent alveolar bone loss in periodontal disease. [source] In vivo real-time imaging of TGF-,-induced transcriptional activation of the RANK ligand gene promoter in intraosseous prostate cancerTHE PROSTATE, Issue 4 2004Jian Zhang Abstract BACKGROUND Current animal models of prostate cancer (CaP) bone metastasis do not allow measurement of either tumor growth in bone over time or activation of gene promoters in intraosseous tumors. To develop these methods, we used bioluminescent imaging (BLI) to determine if expression of receptor activator of NF-,B ligand (RANKL), a pro-osteoclastogenic factor that promotes CaP bone metastases, is modulated by the bone matrix protein transforming growth factor-, (TGF-,) in vivo. METHODS C4-2B human CaP cells were treated with TGF-, in vitro and RANKL mRNA and protein production were measured by polymerase chain reaction (PCR) and ELISA, respectively. Then C4-2B cells stably transfected with the RANKL promoter driving luciferase (lux) were injected intra-tibially into severe combined immundeficient (SCID) mice. Tumors were subjected to BLI every 2 weeks for 6 weeks and serum prostate specific antigen (PSA) was measured using ELISA. Vehicle (V), 1,25 dihydroxyvitamin D (VitD), or TGF-, was administered to mice with established tumors and BLI to measure RANKL promoter activity was performed. Tumors were then subjected to immunohistochemistry for lux and assayed for RANKL mRNA levels. RESULTS TGF-, induced RANKL protein and mRNA expression and activated the RANKL promoter activity in a dose-dependent manner in vitro. BLI demonstrated an increase in intraosseous tumor size over time, which correlated with serum PSA levels. Administration of TGF-, and VitD to mice with established intraosseous tumors increased lux activity compared to V. Intratibial tumor RANKL mRNA expression paralleled the increased promoter activity. Immunohistochemistry confirmed the presence of lux in the intraosseous tumors. CONCLUSIONS These results demonstrate the ability to measure intraosseous tumor growth over time and gene promoter activation in an established intraosseous tumor in vivo and also demonstrate that TGF-, induces activates the RANKL promoter. These results provide a novel method to explore the biology of CaP bone metastases. © 2004 Wiley-Liss, Inc. [source] Effect of interleukin-32, on differentiation of osteoclasts from CD14+ monocytesARTHRITIS & RHEUMATISM, Issue 2 2010Yong-Gil Kim Objective Interleukin-32 (IL-32) induces various inflammatory molecules in human monocytes and differentiation of monocytes into macrophage-like cells. This study was undertaken to evaluate the effects of IL-32,, the most biologically active isoform, on the differentiation and activation of osteoclasts. Methods CD14+ monocytes were obtained from healthy volunteers, and samples of synovial tissue and synovial fluid were obtained from patients with rheumatoid arthritis (RA) and patients with osteoarthritis (OA). The concentration and expression levels of IL-32, in RA and OA samples were evaluated by enzyme-linked immunosorbent assay and immunoblotting, respectively. To examine the osteoclastogenic effects and functional activities, isolated monocytes were treated with either IL-32, or IL-17 in the presence or absence of soluble RANKL (sRANKL) on a culture system and on Osteologic disks. The expression of RANKL and osteoprotegerin (OPG) messenger RNA (mRNA) in RA fibroblast-like synoviocytes (FLS) was measured using reverse transcription,polymerase chain reaction (PCR) and real-time PCR. Results The concentration and expression levels of IL-32, were higher in the RA samples than in the OA samples. Upon costimulation with sRANKL, the osteoclast count and resorbed area increased more significantly in the IL-32,,stimulated cultures than in those stimulated with IL-17. In the IL-32,,treated group without sRANKL stimulation, osteoclasts were differentiated, but the cells displayed low resorption activity. In RA FLS, RANKL mRNA expression increased in the presence of both IL-32, and IL-17. However, transcription of OPG decreased following IL-32, stimulation, resulting in a significant increase in the RANKL:OPG ratio. Conclusion Our results suggest that IL-32, is a potent mediator of active osteoclast generation in the presence of sRANKL. Moreover, this novel cytokine creates more favorable conditions for osteoclastogenesis in the RA joint by increasing the RANKL:OPG ratio in FLS. [source] | ||||||||||