唑来膦酸对破骨细胞分化中Ca2+/钙调蛋白赖性蛋白激酶Ⅱ与钙调蛋白结合及下游基因表达的影响

摘要

目的 探讨唑来膦酸对破骨细胞分化中Ca2+/钙调蛋白依赖性蛋白激酶Ⅱ(Ca2+/calmodulin-dependent protein kinaseⅡ,CaMKⅡ)与钙调蛋结合及下游活化T细胞核因子c1(nuclear factor of activation of T cells-1,NFATc1)、抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)基因表达的影响,探索其抑制破骨细胞分化的分子机制.方法 将小鼠RAW264.7细胞分为A、B两组,每组均以5x103个/孔接种于直径为30 mm的培养皿中培养.A组(对照组)在NF-κB受体激活蛋白配体(receptor activator of NF-κB ligand,RANKL)诱导下向破骨细胞形成,B组(唑来膦酸组)在RANKL诱导培养1d后加用1×10-6mol/L唑来膦酸处理2d.应用免疫共沉淀(co-immunoprecipitation,Co-IP)及反向Co-IP对CaMKⅡ与钙调蛋白的结合进行分析;应用蛋白质印迹法及免疫荧光细胞化学检测两组NFATc1、TRAP蛋白表达,并对破骨细胞生成进行评价.结果 B组新生多核破骨细胞数、牙本质吸陷窝数和面积分别为(11.3±1.5)个、(8.7±2.1)个和(5 034.4±775.4) μm2,均显著低于A组[分别为(37.7±5.7)个、(23.0±4.0)个和(15 042.7±1 906.0) μm2](P＜0.01).Co-IP及反向Co-IP检测显示,B组CaMKⅡ与钙调蛋白的结合较A组分别显著下降了59.8％和50.9％(P＜0.01);在总蛋白中,B组钙调蛋白与CaMKⅡ蛋白较A组,分别显著降低了52.1％和51.5％(P＜0.01).NFATc1、TRAP蛋白水平B组较A组显著下降了52.4％和38.9％(P＜0.01);免疫荧光化学检测也显示B组蛋白表达强度较A组弱.结论 唑来膦酸可以显著抑制破骨细胞分化中CaMKⅡ与钙调蛋白的结合,并下调下游NFATc1、TRAP的蛋白表达.%Objective To investigate the effect of zoledronate on protein interaction between Ca2+/ calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) 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 × l0-6 zoledronate for two days from day 2.Co-immunoprcipitation (Co-IP) and reverse Co-IP were used to detect the protein-binding between CaMK Ⅱ and calmodulin.Western-blotting and immunofluorescent cytochemistry were also used to detect the protein level of NFATc 1 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) μm2respevtively,which were significantly lower than those (37.7±5.7,23.0±4.0 and [15 042.7± 1 906.0] μm2) in group A (P＜0.01).Co-IP and reverse Co-IP examination indicated that protein-binding between CaMK Ⅱ 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 CaMK lⅡ 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 CaMK Ⅱ and calmodulin and down-regulate protein level of NFATc1 and TRAP.