NADPH oxidase gp91^phox 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 (O₂^•-). Gp91^phox, 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^phox (gp91^phox-/-) 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^phox-/- mice resulted from impaired osteoclast differentiation. In addition, gp91^phox-/- mice were defective for RANKL-induced expression of nuclear factor of activated T cells c1 (NFATc1). However, H₂O₂ treatment compensated for gp91^phox 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^phox-/- BMMs. Therefore, our results demonstrate that gp91^phox-derived superoxide is important for promoting efficient osteoclast differentiation by inducing NFATc1 as a downstream signaling mediator of RANK.