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

Abstract

It has been established that reactive oxygen species (ROS) such as H 2 O 2 or superoxide anion is involved in bone loss-related diseases by stimulating osteoclast differentiation and bone resorption and that receptor activator of NF-B 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 H 2 O 2 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.Bone remodeling depends on a delicate balance between bone formation and bone resorption, wherein bone-forming osteoblast and bone-resorbing osteoclasts play central roles (1, 2). Indeed, tipping this balance in the favor of osteoclasts leads to pathological bone resorption, as seen in bone diseases such as osteoporosis and rheumatoid arthritis. The differentiation or lifespan of osteoblast and osteoclast is believed to be particularly important in pathogenesis of these bone diseases (3-5). Osteoclast formation from hematopoietic prcecursors requires factors that promote their differentiation and survival. Such factors are produced by stromal/osteoblastic cells that originate from mesenchymal progenitors residing in the bone marrow. Two such factors appear to be essential. One is macrophage colony stimulating factor (M-CSF), 1 which is necessary but not sufficient for osteoclast formation. The other is a membrane cytokine, receptor activator of NF-B ligand (RANKL). RANKL is essential and, together with M-CSF, is sufficient for osteoclast differentiation. RANKL acts by binding to its receptor, RANK, on the surface of hematopoietic precursors, stimulating their differentiation into mature osteoclasts. The action of RANKL is prevented by osteoprotegerin (OP...

High d(+)glucose concentration inhibits RANKL-induced osteoclastogenesis

“…ROS are important for stimulating osteoclast differentiation and function [21][22][23][24]. The effect of high glucose on intracellular ROS production in RAW264.7 cultures was analyzed using the peroxide-sensitive DCF-DA probe.…”

“…Binding of RANKL to its cognant receptor on osteoclast precursors leads to activation of NF-κB that is required for osteoclast differentiation [19,20]. Reactive oxygen species (ROS) are also crucial for RANKL-induced osteoclastogenesis [21][22][23][24] and NF-κB is among the redoxsensitive cell signaling pathways [25][26][27]. Recent studies indicate that ROS such as hydrogen peroxide produced in BMM cells stimulate osteoclast differentiation, whereas decreasing the level of ROS reverses RANKL responses [28].…”

Inhibitory Effect of Ginsenoside-Rp1, a Novel Ginsenoside Derivative, on the Functional Activation of Macrophage-like Cells

“…Cells (5×10 6 cells/ml) were lysed in lysis buffer (in mM: 20 TRIS-HCl, pH 7.4, 2 EDTA, 2 EGTA, 50 β-glycerophosphate, 1 sodium orthovanadate, 1 dithiothreitol, 1% Triton X-100, 10% glycerol, 10 µg/ml leupeptin, 10 µg/ml aprotinin and 10 µg/ml pepstatin, 1 benzimidine and 2 hydrogen peroxide) for 30 min rotating at 4 o C. Lysates were clarified by centrifugation at 16,000 × g for 10 min at 4 o C. Soluble cell lysates were immunoblotted and total levels of iNOS and β-actin were visualized as previously reported (Bai et al, 2005).…”

GATA-3 transduces survival signals in osteoblasts through upregulation of bcl-x L gene expression

“…(5) In bone loss-related diseases, ROS-induced oxidative stress can stimulate expression of key osteoclastogenic factors such as the receptor activator of nuclear factor-kB ligand to regulate osteoblast activities. (6) Nitric oxide (NO), one of the ROS, has biphasic effects on osteoblast metabolism. (7,8) Under stimulation of inflammatory cytokines or mechanical stress, NO can be overproduced and lead to osteoblast injuries.…”