The induction of RANKL molecule clustering could stimulate early osteoblast differentiation

Abstract We recently found that the membrane-bound receptor activator of NF-κB ligand ( RANKL) on osteoblastsworks as a receptor to stimulate osteoblastdifferentiation, however, the reason why the RANKL-binding molecules stimulate osteoblastdifferentiation has not been well clarified. Since the induction of cell-surface receptorclustering is known to lead to cell activation, we hypothesized that the induction of membrane- RANKLclustering on osteoblastsmight stimulate osteoblastdifferentiation. Immunoblotting showed that the amount of RANKLon the membrane was increased by the RANKL-binding peptide OP3-4, but not by osteoprotegerin(OPG), the other RANKL-binding molecule, in Gfp- Rankl-transfected ST2 cells. Observation under a high-speed atomic force microscope (HS-AFM) revealed that RANKLmolecules have the ability to form clusters. The induction of membrane- RANKL-OPG-Fc complex clustering by the addition of IgM in Gfp- Rankl-transfected ST2 cells could enhance the expression of early markers of osteoblastdifferentiation to the same extent as OP3-4, while OPG-Fc alone could not. These results suggest that the clustering-formation of membrane- RANKLon osteoblastscould stimulate early osteoblastdifferentiation.