Receptor activator of nuclear factor-κB ligand-induced mouse osteoclast differentiation is associated with switching between NADPH oxidase homologues

Reactive oxygen species (ROS) have been suggested to regulate receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclast differentiation. Stimulation of wild-type mouse bone marrow monocyte/macrophage lineage (BMM) cells by RANKL down-regulated NADPH oxidase 2 (Nox2) mRNA expression by half. RANKL reciprocally increased Nox1 mRNA levels and newly induced Nox4 transcript expression. BMM cells from Nox1 knockout (Nox1−/−) as well as Nox2−/− mice generated ROS in response to RANKL and differentiated into osteoclasts in the same way as wild-type BMM cells, which was assessed by the appearance of tartrate-resistant acid phosphatase-positive, multinucleated cells having the ability to form resorption pits and by the expression of osteoclast marker genes. A small interfering RNA (siRNA) targeting Nox1 or Nox2 failed to inhibit the RANKL-stimulated ROS generation and osteoclast formation in wild-type cells, whereas Nox1 and Nox2 siRNAs significantly suppressed the ROS generation and osteoclast formation in Nox2−/− and Nox1−/− cells, respectively. We also confirmed that Nox4 siRNA did not affect the RANKL-dependent events in Nox2−/− cells, whereas p22phox siRNA suppressed the events in both wild-type and Nox1−/− cells. Collectively, our results suggest that there may be a flexible compensatory mechanism between Nox1 and Nox2 for RANKL-stimulated ROS generation to facilitate osteoclast differentiation.