RANKL subcellular trafficking and regulatory mechanisms in osteocytes

The receptor activator of the NF-κB ligand (RANKL) is the central player in the regulation of osteoclastogenesis, and the quantity of RANKL presented to osteoclast precursors is an important factor determining the magnitude of osteoclast formation. Because osteoblastic cells are thought to be a major source of RANKL, the regulatory mechanisms of RANKL subcellular trafficking have been studied in osteoblastic cells. However, recent reports showed that osteocytes are a major source of RANKL presentation to osteoclast precursors, prompting a need to reinvestigate RANKL subcellular trafficking in osteocytes. Investigation of molecular mechanisms in detail needs well-designed in vitro experimental systems. Thus, we developed a novel co-culture system of osteoclast precursors and osteocytes embedded in collagen gel. Experiments using this model revealed that osteocytic RANKL is provided as a membrane-bound form to osteoclast precursors through osteocyte dendritic processes and that the contribution of soluble RANKL to the osteoclastogenesis supported by osteocytes is minor. Moreover, the regulation of RANKL subcellular trafficking, such as OPG-mediated transport of newly synthesized RANKL molecules to lysosomal storage compartments, and the release of RANKL to the cell surface upon stimulation with RANK are confirmed to be functional in osteocytes. These results provide a novel understanding of the regulation of osteoclastogenesis.