Astronauts experience osteoporosis‐like loss of bone mass because of microgravity conditions during space flight. To prevent bone loss, they need a riskless and antiresorptive drug. Melatonin is reported to suppress osteoclast function. However, no studies have examined the effects of melatonin on bone metabolism under microgravity conditions. We used goldfish scales as a bone model of coexisting osteoclasts and osteoblasts and demonstrated that mRNA expression level of acetylserotonin O‐methyltransferase, an enzyme essential for melatonin synthesis, decreased significantly under microgravity. During space flight, microgravity stimulated osteoclastic activity and significantly increased gene expression for osteoclast differentiation and activation. Melatonin treatment significantly stimulated Calcitonin (an osteoclast‐inhibiting hormone) mRNA expression and decreased the mRNA expression of receptor activator of nuclear factor κB ligand (a promoter of osteoclastogenesis), which coincided with suppressed gene expression levels for osteoclast functions. This is the first study to report the inhibitory effect of melatonin on osteoclastic activation by microgravity. We also observed a novel action pathway of melatonin on osteoclasts via an increase in CALCITONIN secretion. Melatonin could be the source of a potential novel drug to prevent bone loss during space flight.

Using fish scales in which osteoclasts and osteoblasts coexist on the calcified bone matrix, we examined the effects of low-intensity pulsed ultrasound (LIPUS) on both osteoclasts and osteoblasts. At 3 hours of incubation after LIPUS treatment, osteoclastic markers such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K mRNA expressions decreased significantly while mRNA expressions of osteoblastic markers, osteocalcin, distal-less homeobox 5, runt-related transcription factor 2a, and runt-related transcription factor 2b, increased significantly. At 6 and 18 hours of incubation, however, both osteoclastic and osteoblastic marker mRNA expression did not change at least present conditions. Using GeneChip analysis of zebrafish scales treated with LIPUS, we found that cell death-related genes were upregulated with LIPUS treatment. Real-time PCR analysis indicated that the expression of apoptosis-related genes also increased significantly. To confirm the involvement of apoptosis in osteoclasts with LIPUS, osteoclasts were induced by autotransplanting scales in goldfish. Thereafter, the DNA fragmentation associated with apoptosis was detected in the osteoclasts using the TUNEL (TdTmediated dUTP nick-end labeling) method. The multi-nuclei of TRAP-stained osteoclasts in the scales were labeled with TUNEL. TUNEL staining showed that the number of apoptotic osteoclasts in goldfish scales was significantly elevated by treatment with LIPUS at 3 hours of incubation. Thus, we are the first to demonstrate that LIPUS directly functions to osteoclasts and to conclude that LIPUS directly causes apoptosis in osteoclasts shortly after exposure.Key words: LIPUS, osteoclasts, osteoblasts, fish scales, apoptosis, GeneChip analysis Abbreviations: ALP, alkaline phosphatase; Bcl-2, B-cell lymphoma 2; CTSK, Cathepsin K; Dlx5, distal less homeobox5; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; LIPUS, low-intensity pulsed ultrasound; OCN, osteocalcin; PMAIP, phorbol-12-myristate-13-acetateinduced protein 1; SAFHS, sonic accelerated fracture healing system; pNP, para-nitrophenol; TRAP, tartrate-resistant acid phosphatase; TUNEL, TdT-mediated dUTP nick end labelling; TNFRSF10A, tumor necrosis factor receptor superfamily, member a; Runx2a, runt related transcription factor 2a; Runx2b, runt related transcription factor 2b 3

The influence of sodium fluoride (NaF) on calcium metabolism was examined in goldfish (fresh water teleost). At 2 days after administration of NaF (500 ng/g body weight; 5µg/g body weight)(around 10 -5 to 10 -4 M in goldfish), we indicated that plasma calcium levels upregulated in both doses of NaF-treated goldfish. To examine the mechanism of hypercalcemia by NaF treatments, therefore, direct effects of NaF on osteoblasts and osteoclasts in goldfish were investigated by an original assay system using teleost scale which has osteoblasts, osteoclasts and bone matrix. Alkaline phosphatase activity in the scales increased with the treatment of NaF (10 -6 and 10 -5 M) during 6 hours of incubation. Also, tartrate-resistant acid phosphatase activity increased after exposure to NaF (10 -5 M) at the 6 hours of incubation. To investigate the osteoclastic activation, the mRNA expression of osteoclastogenesis related factors were examined. The receptor activator of the nuclear factor-κB ligand (RANKL) which is known as a factor for osteoclastogenesis, increased in the NaF-treated scales after 6 hours of incubation.The ratio of RANKL/osteoprotegerin (osteoclastogenesis inhibitory factor) significantly increased after 6 hours of incubation. Resulting from the increase of RANKL mRNA level, the expression of transcription-regulating factors was significantly increased. Furthermore, the expression of functional genes, cathepsin K and matrix metalloproteinase-9 mRNA, was significantly increased. In our knowledge, this is the first report concerning the effects of NaF on osteoblasts and osteoclasts in teleosts. We concluded that NaF influences calcium metabolism via osteoclastic activation in goldfish.3 Keywords: sodium fluoride; osteoblasts; osteoclasts; scales; calcium metabolism; RANKL;OPG; goldfish Abbreviations: ALP, alkaline phosphatase; CTSK, cathepsin K; MMP-9, matrix metalloproteinase-9; NFATc1, nuclear factor of activated T-cells and cytoplasmic 1; TRAF6, TNF receptor-associated factor 6; TRAP, tartrate-resistant acid phosphatase; OPG, osteoprotegerin; RANK, receptor activator of the nuclear factor-κB ; RANKL, receptor activator of the nuclear factor-κB ligand 4

The effects of low-intensity pulsed ultrasound (LIPUS) on osteoclastogenesis were examined using fish scales that had both osteoclasts and osteoblasts. The binding of the receptor activator of NF-κB ligand (RANKL) in osteoblasts to the receptor activator of NF-κB (RANK) in osteoclasts induced osteoclastogenesis. Therefore, we focused on RANK/RANKL signaling. After 6 h of incubation following LIPUS treatment, mRNA expression of RANKL increased significantly. Resulting from the increased RANKL mRNA level, the expression of transcription-regulating factors significantly increased after 6 h of incubation, and then the mRNA expression of functional genes was significantly up-regulated after 12 h of incubation. However, the mRNA expression of osteoprotegerin (OPG), which is known as an osteoclastogenesis inhibitory factor, also significantly increased after 6 h of incubation and tended to further increase after 12 h of incubation. At 24 h of incubation, osteoclastic functional genes' mRNA expression decreased to the level of the control. Furthermore, we performed an in vivo experiment with goldfish. Two weeks after daily LIPUS exposure, osteoclastic marker enzymes tended to decrease while osteoblastic marker enzymes were activated. The regeneration rate of the LIPUS-treated scales was significantly higher than that of the control scales. Thus, LIPUS moderately activates osteoclasts and induces bone formation.Low-intensity pulsed ultrasound (LIPUS), a noninvasive remedial measure, promotes the repair of bone fracture and distraction osteogenesis (see a review, 25). Until now, the study of LIPUS has focused on osteoblastic growth and differentiation (3,8,10). However, the details of the direct effect of

At 3 hours of incubation after LIPUS treatment, osteoclastic marker expression decreased while osteoblastic markers increased. Using GeneChip analysis of zebrafish scales treated by LIPUS, we found that cell death-related genes were up-regulated by LIPUS treatment. TUNEL staining showed that the number of apoptotic osteoclasts in goldfish scales was elevated by treatment with LIPUS at 3 hours of incubation. Thus, we conclude that LIPUS promotes apoptosis in osteoclasts shortly after exposure.