Dual Oxidase Maturation Factor 1 Positively Regulates RANKL-Induced Osteoclastogenesis via Activating Reactive Oxygen Species and TRAF6-Mediated Signaling.

Receptor activator of NF-κB ligand (RANKL) induces generation of intracellular reactive oxygen species (ROS), which act as second messengers in RANKL-mediated osteoclastogenesis. Dual oxidase maturation factor 1 (Duoxa1) has been associated with the maturation of ROS-generating enzymes including dual oxidases (Duox1 and Duox2). In the progression of osteoclast differentiation, we identified that only Duoxa1 showed an effective change upon RANKL stimulation, but not Duox1, Duox2, and Duoxa2. Therefore, we hypothesized that Duoxa1 could independently act as a second messenger for RANKL stimulation and regulate ROS production during osteoclastogenesis. Duoxa1 gradually increased during RANKL-induced osteoclastogenesis. Using siRNA or retrovirus transduction, we found that Duoxa1 regulated RANKL-stimulated osteoclast formation and bone resorption positively. Furthermore, knockdown of Duoxa1 decreased the RANKL-induced ROS production. During Duoxa1-related control of osteoclastogenesis, activation of tumor necrosis factor receptor-associated factor 6 (TRAF6)-mediated early signaling molecules including MAPKs, Akt, IκB, Btk, Src and PLCγ2 was affected, which sequentially modified the mRNA or protein expression levels of key transcription factors in osteoclast differentiation, such as c-Fos and NFATc1, as well as mRNA expression of osteoclast-specific markers. Overall, our data indicate that Duoxa1 plays a crucial role in osteoclastogenesis via regulating RANKL-induced intracellular ROS production and activating TRAF6-mediated signaling.