mRNA Expression Levels Of RANKL Research Articles

Effect of diesel exhaust particles on RANK/RANKL expression in in vivo and in vitro models of middle ear inflammation

ObjectiveIncreasing evidence suggests a link between middle ear inflammation and the development of diesel exhaust particles (DEPs). Chronic middle ear inflammation can lead to bone damage and remodeling. This study aimed to explore the impact of DEPs on the expression of interleukin (IL)-6 and RANKL under conditions of middle ear inflammation. MethodsDEPs were collected by burning fuel in a diesel engine at the Gwangju Institute of Science and Technology. Human middle ear epithelial cells were cultured to 70–80% confluence in culture plates and then treated with DEPs at concentrations of 0, 5, 10, 20, 40, and 80 μg/mL for 24 h. Cell viability was assessed manually. B6.SJL mice, aged 9 weeks, were exposed to DEPs at a concentration of 200 μg/m3 for 1 h daily over a period of 28 days. The expression levels of IL-6, tumor necrosis factor α, RANKL, and RANK were evaluated using hematoxylin and eosin staining and western blot analysis of the harvested middle ear samples. ResultsThe viability of human middle ear epithelial cells was found to decrease in a dose-dependent manner after 24 h. The mRNA expression level of IL-6 exhibited the most significant increase at the 48-h mark. In contrast, the mRNA expression levels of RANKL and RANK showed a marked increase as early as 6 h post-exposure, with both genes subsequently displaying a time-dependent decrease. Histological analysis revealed that the middle ear mucosa was thicker in the group exposed to DEPs compared to the control group. Additionally, the protein expression levels of IL-6 and RANKL were elevated in the DEP-exposed group relative to the normal control group. ConclusionsWe confirmed the expression of osteoclast-related proteins in the mouse middle ear. These results imply that air pollutants might affect RANKL/RANK signaling, which is associated with bone remodeling.

The Interaction of Heat and Lipopolysaccharide on the Expression Levels of Receptor Activator of NF-κB Ligand and Osteoprotegerin in Human Periodontal Ligament Cells

Aim: To investigate the expression levels of receptor activator of NF-κB ligand (RANKL) and oste-oprotegerin (OPG) in human periodontal ligament fibroblasts when stimulated with heat in infective conditions. Methods: Periodontal ligament fibroblasts were subjected to various temperature increases for 5 min with or without 10 ng/mL lipopolysaccharide (LPS) and then maintained at 37℃ for 6 h. After that, the expression levels of RANKL and OPG were investigated using real-time RT-PCR and ELISA. As a positive or negative control, the cells were cultured at 37℃ with or without 10 ng/mL LPS. Data were analyzed using one-way ANOVA at a significant level of p = 0.05. Results: The mRNA expression levels of RANKL and OPG were both down-regulated when the cells were heated in infective conditions. The release of sRANKL was increased at low temperatures in such infection; while at high temperatures heat treatment down-regulated the release of sRANKL induced by LPS. The relative RANKL/OPG expression ratios were increased at low temperatures in infective conditions. Conclusions: The interactions between heat and LPS would affect the balance between RANKL and OPG in periodontal ligament fibroblasts when they were heated in infective conditions. Such infection may be the reason why bone resorption occurs in the local area after warm vertical compaction.

THE EFFECTS OF COMBINED AMLODIPINE AND ATORVASTATIN ON THE BALANCE OF ACTIVATED RANKL/RANK/OPG SYSTEM IN SHR

Objectives IntroductionTo explore the effect of amlodipine, atorvastatin, and their combination on the activated RANKL/RANK/OPG system, by investigating the changes of RANKL, RANK and OPG expression in SHR.Methods36-week-old SHR were...

Effect of heat stress on the expression levels of receptor activator of NF‐κB ligand and osteoprotegerin in human periodontal ligament cells

To investigate the expression levels of the receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) in human periodontal ligament fibroblasts (PDLs) when stimulated with heat. Periodontal ligament fibroblasts were subjected to various temperature increases for 5 min and then maintained at 37 °C. After that, cell viability was determined using an MTT assay. The expression levels of RANKL and OPG were investigated using real-time RT-PCR and ELISA. As a control, the cells were cultured at 37 °C. Data were analysed using one-way anova at a significant level of P = 0.05. Results Cell viability was reduced significantly in the heated groups (P < 0.05). Heat stress downregulated the mRNA expression levels of RANKL and OPG (P < 0.05). When the cells were heated at 39 °C, the protein release of OPG was increased (P < 0.05). There were no significant differences between the heated groups and the control in the release of soluble RANKL (P > 0.05). The relative RANKL/OPG expression ratios were decreased at 39, 43 and 50 °C (P < 0.05), but increased at 47 °C (P < 0.05). Heat influenced the balance between RANKL and OPG in PDLs. Low heat downregulated their relative ratio, whilst high heat upregulated it.

Heparin enhances osteoclastic bone resorption by inhibiting osteoprotegerin activity

Heparin is a highly sulfated glycosaminoglycan and has been shown to activate osteoclastic bone resorption though how is not yet clear. Here we investigate the molecule involved in heparin-induced activation of osteoclasts using an in vitro osteoclast culture assay. The formation and activation of osteoclasts are induced by receptor activator of NFκB ligand (RANKL) on osteoblasts, and inhibited by osteoprotegerin (OPG), a decoy receptor of RANKL, which is secreted from osteoblasts. In a coculture of mouse bone marrow cells and osteoblasts treated with 1,25-dihydroxyvitamin D 3 and prostaglandin E 2 on dentin slices, the bone marrow cells differentiate into osteoclasts, and resorption pits are formed on the dentin slices. Addition of heparin, various glycosaminoglycans, and chemically modified heparins to the coculture reveals that heparin enhances the pit-forming activity of osteoclasts, and this effect of heparin on the activation of osteoclasts is dependent on its sugar chain structure. By contrast, mRNA expression levels of RANKL, RANK, and OPG in the coculture are not altered by heparin treatment. Furthermore, neither RANK nor RANKL binds to heparin, suggesting that heparin does not directly interact with these proteins. Instead, heparin specifically binds to OPG and prevents OPG-mediated inhibition of osteoclastic bone resorption in the coculture. Heparin treatment does not enhance osteoclastic bone resorption in a monoculture of osteoclasts derived from bone marrow cells, and in the coculture using osteoblasts from OPG-deficient mice. A 125I-OPG binding assay showed that OPG binds to osteoblasts and that this binding is inhibited by the addition of heparin, suggesting that OPG binds to RANKL on the osteoblast membrane and that heparin blocks this interaction. These results demonstrate that heparin enhances osteoclastic bone resorption by inhibiting OPG activity.