The induction of RANKL molecule clustering could stimulate early osteoblast differentiation

We recently found that the membrane-bound receptor activator of NF-κB ligand (RANKL) on osteoblasts works as a receptor to stimulate osteoblast differentiation, however, the reason why the RANKL-binding molecules stimulate osteoblast differentiation has not been well clarified. Since the induction of cell-surface receptor clustering is known to lead to cell activation, we hypothesized that the induction of membrane-RANKL clustering on osteoblasts might stimulate osteoblast differentiation. Immunoblotting showed that the amount of RANKL on 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 RANKL molecules 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 osteoblast differentiation to the same extent as OP3-4, while OPG-Fc alone could not. These results suggest that the clustering-formation of membrane-RANKL on osteoblasts could stimulate early osteoblast differentiation. [Display omitted] •The RANKL-binding peptide OP3-4 enhanced ALP activity, but OPG did not.•OP3-4 increased membrane-RANKL, but OPG did not in osteoblastic cell line ST2.•HS-AFM imaging revealed the ability of RANKL molecules to form clusters.•HS-AFM imaging also revealed highly flexible OPG clinging to RANKL.•Induction of heteromer of membrane-RANKL-OPG-Fc by IgM stimulated ALP activity.