Detection of RANKL-producing cells and osteoclastic activation by the addition of exogenous RANKL in the regenerating scales of goldfish

We have previously reported that microgravity promotes the activation of osteoclasts in cultured regenerating scales. This osteoclastic activation was induced by increased levels of receptor activator of the nuclear factor-κB ligand (RANKL). Therefore, we determined that RANKL is an important factor in evaluating osteoclastogenesis in bone tissue. However, the role of RANKL in fish scales is poorly understood. In the present study, we prepared antiserum against goldfish RANKL in rabbits and detected RANKL-producing cells in regenerating goldfish scales. Furthermore, we studied osteoclastic activation by the addition of RANKL to examine exogenous RANKL on osteoclastogenesis in regenerating goldfish scales. As a result, RANKL immune-positive cells were detected in grooves of regenerating scales. In addition, treating the regenerating scales with mammalian RANKL for 3 h significantly increased the expression of the nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), which is essential for osteoclast differentiation. After 6 h of incubation with RANKL, the expression of cathepsin K, a functional osteoclastic gene, significantly increased. Furthermore, the molecules for osteoclast multinucleation and differentiation significantly increased following treatment with mammalian RANKL. Therefore, in fish scales as well as mammalian bone, we concluded that RANKL plays an important role in osteoclastogenesis.