Hypertonicity down-regulates 1a,25-dihydroxyvitamin D3-induced osteoclastogenesis via TonEBP

Objectives:Our unique finding was the fact that hypertonic stress surprisingly down-regulates 1a,25-dihydroxyvitamin D3-induced osteoclastogenesis in co-culture system. We tested the hypothesis that the effect of hypertonic stress on the osteoclastogenesis is induced via TonEBP (tonicity-responsive enhancer binding protein), which plays a central role in protecting renal medullary cells from the stress of high osmolality. Methods:The cellular responses by the hypertonicity in the process of osteoclastogenesis were exploited in co-culture system as well as osteoblastic and RAW 264.7 cell (osteoclast cell line). Results:Hypertonicity (sucrose 25, 50 mM) were applied to co-culture system, inhibits the number of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells induced by 10 nM 1a,25(OH)2D3. In order to investigate the mechanism by which hypertonicity inhibits osteoclastogenesis, the mRNA expression of receptor activator of nuclear factor (NF)-kB ligand (RANKL) and osteoprotegerin (OPG) were analyzed by RT-PCR. An exposure of osteoblastic cells to a medium containing sucrose (25, 50 mM) reduced RANKL mRNA expression induced by 10 nM 1a,25(OH)2D3, in a dose-dependent manner while the changes in OPG mRNA were not detected. Similar to the changes in mRNA, the addition of sucrose(25, 50 mM) on osteoblastic cells decreased the RANKL protein expression, but not in the case of OPG protein. Furthermore, hypertonicity inhibited the osteoclastogenesis of RAW 264.7 cell treated with 50 ng/ml RANKL. On the other hand, hypertonicity increased the TonEBP expression in osteoblastic cell. Conclusions:Hypertonic stress inhibits osteoclast differentiation by reducing RANKL/OPG ratio in osteoblastic cells. Our findings suggest that the TonEBP might be involved in the process of 1a,25(OH)2D3 induced osteoclastogenesis in terms of signal transduction pathway.