NADPH Oxidase Gp91 Contributes to RANKL-induced Osteoclast Differentiation by Upregulating NFATc1

Bone-marrow derived monocyte-macrophages (BMMs) differentiate into osteoclasts by M-CSF along subsequent RANKL stimulation possibly in collaboration with many other unknown cytokines released by pre- or mature osteoblasts. The differentiation process requires receptor activator of nuclear factor kappa-B ligand (RANKL)/RANK signaling and reactive oxygen species (ROS) such as superoxide anion (O). Gp91, a plasma membrane subunit of NADPH oxidase (Nox), is constitutively expressed in BMMs and plays a major role in superoxide anion production. In this study, we found that mice deficient in gp91 (gp91) showed defects in osteoclast differentiation. Femurs of these mice produced osteoclasts at about 70% of the levels seen in femurs from wild-type mice, and accordingly exhibited excessive bone density. This abnormal bone growth in the femurs of gp91 mice resulted from impaired osteoclast differentiation. In addition, gp91 mice were defective for RANKL-induced expression of nuclear factor of activated T cells c1 (NFATc1). However, HO treatment compensated for gp91 deficiency in BMMs, almost completely rescuing osteoclast differentiation. Treating wild-type BMMs with antioxidants and superoxide inhibitors resulted in a differentiation defect resembling the phenotype of gp91 BMMs. Therefore, our results demonstrate that gp91-derived superoxide is important for promoting efficient osteoclast differentiation by inducing NFATc1 as a downstream signaling mediator of RANK.