Activation of RANKL-producing cells under simulated microgravity with a three-dimensional clinostat in regenerating goldfish scales

Exposure to microgravity during space flight has caused astronauts to experience decreased bone density, resulting from bone resorption by osteoclastic activation. Activation of the nuclear factor-κB ligand (RANKL) of the RANKL-producing cells, a known osteoclastogenesis-promoting factor, seems to be induced under microgravity. However, the role of the RANKL-producing cells under microgravity has not yet been demonstrated due to the lack of suitable in vitro organ culture of osteoblasts, osteoclasts, and osteocytes with the intact bone matrix under microgravity. Previous reports demonstrated that RANKL-producing cells were detected with a specific antiserum for goldfish RANKL in the regenerating goldfish scales. In this study, the response of RANKL-producing cells to simulated microgravity with a three-dimensional clinostat was examined using in vitro organ culture system with regenerating scales. In addition, mRNA expression analysis of osteoclastic and osteoblastic markers via quantitative real-time PCR was conducted together with histological analysis of RANKL-producing cells. After 4 days of exposure to simulated microgravity, the number of RANKL immune-positive cells increased compared with the RANKL immune-positive cells in control regenerating scales. The Rankl mRNA expression in the regenerating scales after simulated microgravity treatments was significantly higher than that in the control (1G) scales. The mRNA expression of osteoprotegerin (Opg), an osteoclastogenesis inhibitory factor, significantly decreased under simulated microgravity conditions. The ratio of Rankl/Opg in the simulated microgravity-treated scales was significantly higher than that in the scales of 1G control. The change in the ratio of Rankl/Opg implied that osteoclastic activation is promoted after simulated microgravity treatments. Actually, in the regenerating scales exposed to simulated microgravity, the mRNA expression of osteoclastic markers, such as receptor activator of NFκB, cathepsin K, integrin beta-3, and cellular-Src, significantly increased. On the other hand, the mRNA expression of the osteoblastic markers (collagen type I alpha 1 and osteocalcin) significantly decreased. The changes in the osteoclastic and osteoblastic markers were consistent with our previously reported experiment on the International Space Station. To the best of our knowledge, we are the first to demonstrate the activation of RANKL-producing cells under simulated microgravity conditions by in vitro organ culture using regenerating goldfish scales. We strongly emphasize that fish scales serve as an excellent bone model for the analysis of gravitational responses while maintaining conditions similar to in vivo.