Involvement of Calcineurin and PICK1 in RANKL-induced Osteoclast Differentiation

Calcineurin is a Ca²⁺/calmodulin-dependent protein phosphatase that plays a role in the activation of NFATc1 during RANKL-induced osteoclast differentiation. However, little is known about the mechanisms that govern calcineurin signaling during osteoclast differentiation. Objectives: To investigate the role of calcineurin in the regulation of osteoclast differentiation and to isolate new calcineurin B-interacting proteins using a yeast two-hybrid screen assay. Methods: M-CSF-dependent osteoclast precursors isolated from mouse bone marrow cells were stimulated with M-CSF and RANKL to induce osteoclast differentiation. The calcineurin inhibitor tacrolimus was added to the culture medium and its effect on osteoclast differentiation was evaluated by TRAP-positive multinucleated cell counts and real-time RT-PCR analysis for NFATc1 mRNA expression. In the yeast two-hybrid screen assay, calcineurin B was used as the bait and its interaction with the identified protein was confirmed using a pull-down assay in HEK293 cells as well as lysate immunoprecipitation in mouse osteoclasts. A deletion mutant of the consensus C-terminal peptide “VDV” sequence in calcineurin B was also constructed and involvement of this sequence in specific interactions with the identified protein was investigated by co-immunoprecipitation in HEK293 cells. Results: Addition of 1 µM tacrolimus reduced the number of TRAP-positive multinucleated cells and inhibited the expression of NFATc1 mRNA. The yeast two-hybrid screen specifically identified an interaction between calcineurin B and protein interacting with C kinase (PICK1). Real-time RT-PCR demonstrated that the expression of PICK1 mRNA increased during osteoclast differentiation. Sequence analysis showed that PICK1 protein contains a “PDZ” domain, which is known to interact with the VDV sequence. Co-immunoprecipitation revealed that deletion of the VDV sequence in calcineurin B was sufficient to abolish its interaction with PICK1. Conclusions: These results suggest that PICK1 interacts with calcineurin B by targeting its VDV sequence. PICK1 may regulate calcineurin and thereby modulate the calcineurin-NFAT pathway during RANKL-induced osteoclast differentiation.