Immunohistochemical localization of Nox1, Nox4 and Mn-SOD in mouse femur during endochondral ossification

• We investigated expression of the Nox family in endochondral ossification.
• Nox1 may be necessary for hypertrophic process in chondrocyte.
• Nox1 and Nox4 involve in the bone matrix formation.
• Mn-SOD is related to the inhibition of chondrocyte differentiation.

Enzymes synthesizing reactive oxygen (Nox family) have recently been identified. Elucidation of the production mechanism has been initiated, and the involvement of reactive oxygen in metabolism, intracellular transport, signal transmission and apoptosis has been reported. We immunohistochemically investigated expression and localization of the Nox family in endochondral ossification using a normal mouse femur. Weakly positive reactions with Nox1, Noxa1, and Noxo1 were observed in the zones of proliferative and prehypertrophic chondrocytes at 3 weeks of age. Nox4 was widely positive from the resting over the hypertrophic cell zone. At 18 weeks of age, none of the Nox types was expressed in chondrocytes as the zones disappeared. On the other hand, positive reactions with Nox1, Noxa1, Noxo1, and Nox4 were observed in osteoblasts in the zone of ossification at 3 weeks of age, and each Nox was also positive in osteoblasts arranged on the bone marrow side in the epiphyseal cartilage at 18 weeks of age. In addition, a reactive oxygen-eliminating enzyme, Mn-SOD, was observed only in prehypertrophic chondrocytes at 3 weeks of age, and not detected in osteoblasts. It was suggested that the Nox family is closely associated with endochondral ossification of the mouse femur, and Nox1 and Nox4 are closely involved in the chondrocyte maturation process and bone matrix formation.