IntroductionReceptor activator of NF-B (nuclear factor-B) ligand (RANKL; also called TRANCE [tumor necrosis factor (TNF) activationinduced cytokine], ODF [osteoclast differentiation factor], and OPGL [osteoprotegerin ligand]) 1-4 is a key factor stimulating the differentiation and activation of osteoclasts and, therefore, is essential for bone remodeling. 5 The binding of RANKL to its receptor RANK leads to recruitment of TNF receptor-associated factor 6 (TRAF6) to the cytoplasmic domain of RANK, thereby resulting in the activation of distinct signaling cascades mediated by mitogen-activated protein (MAP) kinases, including c-Jun N-terminal kinase (JNK), p38 MAP kinase (p38), and extracellular signal-regulated kinase (ERK). 6 It has been shown that JNK1-activated c-Jun signaling in cooperation with nuclear factor of activated T cells (NFAT) is key to RANKL-regulated osteoclast differentiation. 7 In addition, stimulation of p38 results in the downstream activation of the mi/Mitf (microphthalmia/microphthalmia transcription factor), which controls the expression of the genes encoding tartrate-resistant acid phosphatase (TRAP) and cathepsin K, indicating the importance of p38 signaling cascades. 6 Although our understanding of signaling pathways associated with osteoclast differentiation has advanced considerably recently, the mechanism of RANKL-mediated osteoclastogenesis, specifically the molecular linkage between TRAF6 and MAP kinases, is still unknown.At high concentrations, reactive oxygen species (ROSs) cause oxidative stress that has been viewed as deleterious phenomena, including inflammatory response, apoptosis, or ischemia. 8 Recent studies, however, indicate that small nontoxic amounts of ROS may play a role as a second messenger in the various receptor signaling pathways. [9][10][11][12][13] Osteoclasts have shown to be activated by ROSs to enhance bone resorption, 14 but little attention has been given to the role of ROSs in differentiation of macrophages and monocytes into osteoclasts. Signaling molecules such as JNK and p38, which are known to be essential for osteoclast differentiation, 6,7 are sensitive to activation by ROSs. 11,12 Thus, we hypothesized that signaling cascade(s) can be modulated by ROSs in bone marrow monocyte-macrophage lineage (BMM) cells.Here, we show that RANKL generates ROSs in BMM cells. Examination of the mechanism by which RANKL generates ROSs revealed the involvement of TRAF6, Rac1, and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 1 (Nox1). These data suggest that RANKL-mediated ROS production serves to regulate RANKL signaling pathways, including JNK and p38 activation required for osteoclast differentiation. Materials and methods Reagents and plasmids2Ј,7Ј-dichlorofluorescein diacetate (DCFH-DA) was purchased from Molecular Probes (Leiden, The Netherlands); all other chemicals and FLAG (five NH2-terminally deleted epitope-tagged) epitope (M2) were from Expression constructs encoding FLAG-tagged wild-type RANK, HAtagged TRAF6 (amino acids [aa] 289-530), and...