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...

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BackgroundA shift from oxygen phosphorylation to aerobic glycolysis was known as the Warburg effect and a characteristic of cancer cell metabolism facilitating metastasis. Mitochondrial calcium uniporter (MCU), a key ion channel that mediates Ca2+ uptake into mitochondria, was found to promote cancer progression and metastasis. However, its explicit role in shifting metabolism of breast cancer cells has not been defined.MethodsWe evaluated MCU overexpression or knock-down on migration, invasion and glucose metabolismin breast cancer cells. Mitochondrial Ca2+ dynamics were monitored with Rhod-2 fluorescence imaging. Luciferase reporter assay was used to confirm the interaction between miR-340 and 3’-untranslated region (3’-UTR) of MCU gene. Mouse models of lung metastasis were used to determine whether gain-/loss-of-MCU impacts metastasis. MCU expression was assessed in 60 tumor samples from breast cancer patients by immunohistochemistry (IHC).ResultsKnockdown of MCU in MDA-MB-231 cells significantly reduced cell migration and invasion in vitro and lung metastasis in vivo; whereas overexpression of MCU in MCF-7 cells significantly increased migration and invasion in vitro and lung metastasis in vivo. Overexpression of MCU promoted lung metastasis by enhancing glycolysis, whereas suppression of MCU abolished this effect. Moreover, a novel mechanism was identified that MCU was a direct target of microRNA-340, which suppressed breast cancer cell motility by inhibiting glycolysis. Consistently, significantly increased MCU protein was found in metastatic breast cancer patients.ConclusionsWe identified a novel mechanism that upregulated MCU promotes breast cancer metastasis via enhancing glycolysis, and that this process is posttranscriptionally and negatively regulated by microRNA-340.

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