Effect of zoledronate on protein interaction between Ca(2+)/calmodulin-dependent protein kinaseⅡ and calmodulin and expression of downstream genes during osteoclast differentiation

Abstract

Objective: To investigate the effect of zoledronate on protein interaction between Ca(2+)/calmodulin-dependent protein kinaseⅡ(CaMKⅡ) and calmodulin and protein expression of nuclear factor of activation of T cells-1 (NFATc1) and tartrate resistant acid phosphatase (TRAP) during osteoclast differentiation. Methods: Mouse RAW264.7 cells were divided into group A and B and were cultured. Group A was induced with 50 mg/L receptor activator of NF-κB ligand (RANKL) for osteoclastogenesis, and group B was treated with 1×10(-6) zoledronate for two days from day 2. Co-immunoprcipitation (Co-IP) and reverse Co-IP were used to detect the protein-binding between CaMKⅡ and calmodulin. Western-blotting and immunofluorescent cytochemistry were also used to detect the protein level of NFATc1 and TRAP in both groups. Osteoclast formation was also analyzed. Results: In group B, the number of osteoclasts, number and size of dentin resorption lacunaes were 11.3±1.5, 8.7±2.1 and (5 034.4±775.4) μm(2) respevtively, which were significantly lower than those (37.7±5.7, 23.0±4.0 and [15 042.7±1 906.0] μm(2)) in group A (P<0.01). Co-IP and reverse Co-IP examination indicated that protein-binding between CaMKⅡ and calmodulin significantly decreased by 59.8% and 50.9% in group B compared with group A (P<0.01). The protein level of calmodulin and CaMKⅡ in total cellular proteins also significantly decreased by 52.1% and 51.5% in group B compared with group A (P<0.01). NFATc1 and TRAP protein decreased by 52.4% and 38.9% in group B than in group A (P<0.01), respectively. Conclusions: Zoledronate could significantly inhibit protein-binding between CaMKⅡ and calmodulin and down-regulate protein level of NFATc1 and TRAP.