PICK1 Regulates Osteoclastogenesis by Binding to Calcineurin B

Novel molecular mechanisms and molecular targets for osteoclastogenesis could form the basis for effective therapeutic strategies against alveolar bone resorption.  We previously identified PICK1 (protein interacting with C kinase) as a novel calcineurin B-binding protein that modulates NFAT activity in neuron-like PC12 cells (Biochem Biophys Res Commun, 2008).  We hypothesized that PICK1 may also positively regulate calcineurin B in osteoclasts to activate NFAT, which is critical for osteoclastogenesis.  Objective: To investigate the involvement of PICK1 in calcineurin-NFAT-regulated osteoclastogenesis.  Methods: Mouse primary osteoclast progenitor bone marrow macrophages (BMMs) and RAW264.7 osteoclast progenitor cells (RAWs) were stimulated with RANKL to induce osteoclast differentiation.  PICK1 gene and protein expression in BMMs during osteoclastogenesis was examined by real-time RT-PCR and Western blotting, respectively.  The interaction of PICK1 with calcineurin B in osteoclastogenic BMMs was determined by co-immunoprecipitation.  RAWs were transduced with lentiviral PICK1 or GFP (control) expression vectors, and the number of TRAP-positive multinucleated cells during osteoclastogenesis was evaluated.  RAWs were transfected with an NFAT response element/luciferase reporter vector, and tetracycline-regulated PICK1 expression RAWs (Tet-PICK1/NFAT/Luc-RAWs) were then lentivirally engineered.  The NFAT activity in these cells during the forced expression of PICK1 by tetracycline (doxycycline) was measured by the luciferase reporter assay.  Results: PICK1 gene and protein expression in BMMs was significantly increased during osteoclast differentiation.  The interaction of PICK1 with calcineurin B in BMMs was confirmed by co-immunoprecipitation.  PICK1 overexpression in RAWs significantly increased the number of TRAP-positive mature osteoclasts (ANOVA; P<0.01).  In addition, Tet-PICK1/NFAT/Luc-RAWs showed significantly increased NFAT activation in the presence of doxcycline during osteoclastogenesis (ANOVA; P<0.01).  Conclusion: These results suggest that PICK1 binds to calcineurin B in osteoclast progenitor cells and promotes osteoclast differentiation via the activation of calcineurin-NFAT signaling.  The novel molecular target PICK1 may be useful for therapeutic strategies against alveolar bone resorption in prosthetic treatments.