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CREB和ERK1/2在CaMKⅡδ调控破骨细胞分化中的作用
作者:王红美 董伟 戚孟春 冯晓洁 陆大壮 李任 孙红 
单位:063000  河北唐山  华北理工大学口腔医学院口腔颌面外科教研室(王红美、董伟、戚孟春、冯晓洁、陆大壮、李任)  基础医学院病理学教研室(孙红) 
关键词:破骨细胞 钙调蛋白依赖性激酶Ⅱδ RNA干扰 cAMP反应原件结合蛋白 细胞外信号调节激酶1/2 活化T细胞核因子c1 
分类号:R743.31
出版年,卷(期):页码:2018,43(2):91-95
摘要:

[摘要] 目的 探索cAMP反应原件结合蛋白(CREB)、细胞外信号调节激酶1/2(ERK1/2)在钙调蛋白依赖性激酶Ⅱ (CaMK调控破骨细胞分化中的作用及分子机制。方法 构建CaMKδ RNA干扰载体并转染小鼠RAW264.7胞,确定其干扰效率。病毒转染细胞后,用50ng/ml核因子-κB受体活化因子配体(RANKL)诱导,于不同时间点检测 CREBERK1/2蛋白磷酸化水平。应用ERK1/2信号阻断剂PD98059处理细胞,检测ERK1/2信号阻断对活化T细胞核因c1(NFATc1)基因表达及破骨细胞分化的影响。结果 CaMKδ干扰载体在mRNA和蛋白水平的干扰效率分别为77.2% 70.2%CaMKδ干扰可明显降低CREBERK1/2蛋白磷酸化水平,下调幅度分别为21%~55%55%~64%。此外, ERK1/2信号阻断明显下调了NFATc1蛋白表达,使破骨细胞数目、骨吸收陷窝数目及面积分别下降了39.3%50.0% 52.3%结论 CaMKδ RNA干扰可明显抑制CREBERK1/2蛋白磷酸化;CREBERK1/2参与了CaMKδ对破骨细胞分化的调控。

[Abstract] Objective To investigate the function of cAMP response element binding protein (CREB) and extracellular signal-regulated kinase (ERK1/2) in osteoclast differentiation mediated by Ca2+/calmodulin-dependent kinase (CaMK)δ, and elucidate the molecular mechanism thereof. Methods CaMKδ RNA interference lentivirus vector was constructed and mouse RAW264.7 cells were transfected with the virus to determine the interference efficiency. After virus transfection, RAW264.7 cells were treated with 50ng/ml receptor activator of nuclear factor κB ligand (RANKL) and the phosphorylation levels of CREB and ERK1/2 were detected at different time points. The cells were also treated with PD98059, an ERK1/2 inhibitor, to determine the effect of ERK1/2 signal blocking on the expression of nuclear factor activated T-cells cytoplasmic 1 (NFATc1) and osteoclast differentiation. Results Interference efficiency of recombinant CaMKδ virus vector was 77.2% at mRNA level and 70.2% at protein level. CaMKδ RNA interference significantly suppressed phosphorylation of CREB and ERK1/2, and the levels of p-CREB and p-ERK1/2 were down-regulated by 21%-55% and 55%-64%, respectively. ERK1/2 inhibitor significantly down-regulated the protein expression of NFATc1, and the number of osteoclast, the number and size of bone resorption lacunae decreased by 39.3%, 50.0% and 52.3%, respectively. Conclusion CaMKδ RNA interference may significantly suppress the phosphorylation of CREB and ERK1/2, and CREB and ERK1/2 have mediated the CaMKδ-induced osteoclast differentiation.

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