The Effect Of Zoledronate On Gene Expression Of Calmodulin And Calcineurin During Osteoclast Differentiation

Objectives The study is aimed to examine the effects of zoledronate acid on gene expression of calmodulin and calcineurin, two key molecules for transmittion of Ca2+signal during differentiation of osteoclasts and to explore their function in zoledronic acid-induced inhibition of osteoclastogenesis.Methods Mouse leukemia macrophage monocyte cells, RAW264.7,were used in this study for osteoclast differentiation induced with 100ng/ml Ligand of Receptor Activator of Nuclear FactorκB(RANKL). The cells were divided into two groups, group A and goup B. Cells in both groups were induced with RANKL for 4 days, while cells in group B were also treated with 1×10-6M zoledronate from day 2 for 48 h. Four days later, the cells were harvested. Following examinations were conduced to study the effect of zoledronate on osteoclastogenesis and gene expression of calmodulin and calcineurin during osteoclast differentiation. 1 Tartrate-resistant acid phosphatase(TRAP) staining and dentin resorption lacunae examination were performed to explore the difference of formation of multinuclear osteoclasts and their bone resorption function between group A and group B. 2 Real-time PCR, Western-blot, as well as immunofluorescent cytochemistry were also used to detect the m RNA and protein level of calmodulin and calcineurin during osteoclast differentiation in group A and B.(3) Protein phosphotase activity of calcineurin was analysized using a commercial analysis kit to determine the effect of zoledronate on calcineurin activity in osteoclasts.Results 1 RAW264.7 cells were successfully differentiated into multinuclear osteoclasts after 4 days` induction with RANKL. 2 TRAP positive multinuclear osteoclasts were formed in both group A and B, whereas osteoclastogenesis were inhibited by zoledronate in group B. The number of osteoclasts in group B was 26.7±3.5,which was significantly decreased when compared with that(49.0±2.6) in group A(P<0.01) and the decrease is about 44.9%. The number and areas of absorption lacunae were 6.7±.1.2 and 997.1±14.8μm2 respectively in group B, which were also significantly decreased when compared with those(13.3±1.5 and 1676.9±24.9μm2respectively) in group A, and the decrease were 49.6% and 40.5%, respectively. 3. Zoledronate also inhibited gene expression of calmodulin and calcineurin and it was confirmed by Real-time PCR, which showed that m RNA level of two genes decreased about 51% and 37%respectively in group B when compared with group A(P<0.05). Western-blot also demonstrated significant decrease of protein level of calmodulin and calcineurin in group B when compared with group A(P<0.05), and the decrease were 78.7% and 69.5%,respectively. Immunofluorescent cytochemistry showed that fluorescence expression of Calmodulin and Calcineurin in group B was weaker than the control group A.4.Phosphotase activity analysis further confirmed signficant inhibition of calcineurin acivity by zoledronate in group B at 24 h, 48 h,72h after zoledronate treatment(P<0.05 or P<0.01), although no significant difference was found at 0h between the two groups.Conclusion The results of this study suggested that zoledronate could inhibit osteoclastogenesis and downregulate m RNA and protein level of calmodulin and calcineurin during osteoclast differentiation. As the two genes are both key molecules for Ca2+ signal transmission in Ca2+/Calmodulin/NFATc1 axis, the signaling pathway may play a certain role in zoledronate-induced inhibition of osteoclastogenesis.