Regulatory mechanisms of proton extrusion in osteoclasts via RANKL-TRAF signal.

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Although RANKL (receptor activator of NF-kB ligand) is well known to be the essential factorfor osteoclastgenesis, it remains unknown how RANKL regulates osteoclastic H^+ transporter in bone resorption. In this study, we examined effects of RANKL on H^+ extrusion activity in resorbing rat osteoclasts using intracellular pH (pHi) indicator, BCECF (2'7'-bis-(2-carboxyethyl)-5-carboxyfluorescein). Both basal pHi and H^+ extrusion activity in cultured resorbing osteoclasts, which were identified by forming the resorbing pits on calcium phosphate-coated quartz coverslips, were significantly both higher than those of non-resorbing osteoclasts. Pharmacological classification showed that resorbing osteoclasts expressed two types of H^+ -extruding systems : bafilomycin A_1- and amiloride-sensitive H^+ extrusion systems (vacuolar type proton pump (V-ATPase)-and Na^+ /H^+ exchanger (NHE)-mediated H^+ extrusion, respectively). RANKL increased both H^+ extrusion activities in mouse osteoclasts. On the other hand, pretreatment with osteoprotegerin (OPG : decoy receptor of RANKL) suppressed the RANKL-induced action.These results indicate that RANKL up-regulates the intracellular H^+ production and activates the H^+ extrusion generated by V-ATPase and NHE. These mechanisms of H^+ transport by RANKL are likely to contribute to its regulation of bone resorption. Furthermore, we investigated the effects of RNKL in human odontoclasts (osteoclast-like cells) collected from human deciduous tooth. RANKL also promoted the bone resorption in human osteoclast-like cells, suggesting that human osteoclasts be useful in the following experiment system.