Reactive Oxygen Species Stimulates Receptor Activator of NF-κB Ligand Expression in Osteoblast

It has been established that reactive oxygen species (ROS) such as H2O2 or superoxide anion is involved in bone loss-related diseases by stimulating osteoclast differentiation and bone resorption and that receptor activator of NF-kappaB ligand (RANKL) is a critical osteoclastogenic factor expressed on stromal/osteoblastic cells. However, the roles of ROS in RANKL expression and signaling mechanisms through which ROS regulates RANKL genes are not known. Here we report that increased intracellular ROS levels by H2O2 or xanthine/xanthine oxidase-generated superoxide anion stimulated RANKL mRNA and protein expression in human osteoblast-like MG63 cell line and primary mouse bone marrow stromal cells and calvarial osteoblasts. Further analysis revealed that ROS promoted phosphorylation of cAMP response element-binding protein (CREB)/ATF2 and its binding to CRE-domain in the murine RANKL promoter region. Moreover, the results of protein kinase A (PKA) inhibitor KT5720 and CREB1 RNA interference transfection clearly showed that PKA-CREB signaling pathway was necessary for ROS stimulation of RANKL in mouse osteoblasts. In human MG63 cells, however, we found that ROS promoted heat shock factor 2 (HSF2) binding to heat shock element in human RANKL promoter region and that HSF2, but not PKA, was required for ROS up-regulation of RANKL as revealed by KT5720 and HSF2 RNA interference transfection. We also found that ROS stimulated phosphorylation of extracellular signal-regulated kinases (ERKs) and that PD98059, the inhibitor for ERKs suppressed ROS-induced RANKL expression either in mouse osteoblasts or in MG63 cells. These results demonstrate that ROS stimulates RANKL expression via ERKs and PKA-CREB pathway in mouse osteoblasts and via ERKs and HSF2 in human MG63 cells.