CaMKi>Ⅱδ基因沉默对破骨细胞分化功能及c-fosi>/c-juni>/CREBi>基因的影响

doi:10.7518/gjkq.2019048

摘要/Abstract

摘要：

目的探索编码钙调蛋白依赖性激酶（CaMK）Ⅱδ的基因沉默对破骨细胞分化功能的影响以及c-fos、c-jun、CREB基因在CaMKⅡδ调控破骨细胞分化中的作用,以揭示破骨细胞分化调控的分子机制。方法构建CaMKⅡδ RNA干扰载体,转染至小鼠RAW264.7细胞,确定干扰效率。病毒转染细胞后,用50 ng·mL ^-1核因子-κB受体活化因子配体（RANKL）诱导,5 d后收获,通过耐酒石酸酸性磷酸酶（TRAP）检测、牛骨吸收陷窝检测沉默CaMKⅡδ基因对破骨细胞分化、骨吸收功能的影响;48 h后收获,利用实时聚合酶链式反应（PCR）、Western-blot等方法检测c-fos、c-jun、CREB的表达情况。结果 CaMKⅡδ RNA干扰在mRNA和蛋白质水平的干扰效率分别为70.6%和72.2%。 CaMKⅡδ RNA干扰可显著降低 c-fos、c-jun和CREB的mRNA水平,下调幅度分别为74%、49% 和24%,CaMKⅡδ RNA干扰可显著降低c-Fos、c-Jun和CREB的水平,下调幅度分别为74.3%、61.3%和59.2%。结论 CaMKⅡδ RNA干扰可在mRNA和蛋白质水平显著抑制c-fos、c-jun、CREB的表达,其共同参与CaMKⅡδ对破骨细胞分化的调控。

关键词: 破骨细胞, 钙调蛋白依赖性激酶Ⅱδ, c-Fos, c-Jun, 环腺苷酸反应原件结合蛋白

Abstract:

Objective This study aims to explore the effect of calmodulin-dependent kinase (CaMK) Ⅱδ gene silencing on osteoclast differentiation, investigate the roles of the c-fos, c-jun and CREB genes in CaMKⅡδ regulation of osteoclast differentiation, and reveal the molecular mechanism underlying osteoclast differentiation.Methods A CaMKⅡδ RNA interference vector was constructed and transfected into mouse RAW264.7 cells to determine interference efficiency. The cells were transfected with the virus, induced with 50 ng·mL ^-1 nuclear factor-κB receptor activator ligand and then harvested after 5 days. The effect of silencing the CaMKⅡδ gene on osteoclast differentiation was detected by tartrate-resistant acid phosphatase detection and bovine bone resorption lacuna. After 48 h, the expression levels of the c-fos, c-jun and CREB genes were detected by real-time polymerase chain reaction and Western blot.Results The interference efficiencies of the CaMKⅡδ RNA interference vector at the mRNA and protein levels were 70.6% and 72.2%, respectively. CaMKⅡδ RNA interference significantly reduced the mRNA levels of c-fos, c-jun and CREB by 74%, 49% and 24%, respectively. CaMKⅡδ RNA interference significantly reduced the protein levels of c-Fos, c-Jun and CREB by 74.3%, 61.3% and 59.2%, respectively.Conclusion CaMKⅡδ RNA interference can significantly inhibit the expression of c-fos, c-jun and CREB at the mRNA and protein levels. These results indicate that c-Fos, c-Jun and CREB are involved in the regulatory effects of CaMKⅡδ on osteoclast differentiation.

Key words: osteoclast, calmodulin-dependent kinase Ⅱδ, c-Fos, c-Jun, cyclic adenosine monophosphate responsive element-binding protein

中图分类号:

Q786

张誉泓,戚孟春,董伟,孙红. CaMKi>Ⅱδ基因沉默对破骨细胞分化功能及c-fosi>/c-juni>/CREBi>基因的影响[J]. 国际口腔医学杂志, 2019, 46(4): 420-425.

Zhang Yuhong,Qi Mengchun,Dong Wei,Sun Hong. Effect of CaMKⅡδ gene silencing on osteoclast differentiation and c-fos/c-jun/CREB gene[J]. Int J Stomatol, 2019, 46(4): 420-425.

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链接本文: https://www.gjkqyxzz.cn/CN/10.7518/gjkq.2019048

https://www.gjkqyxzz.cn/CN/Y2019/V46/I4/420

图/表 5

图 1

实时PCR检测CaMKⅡδ mRNA水平 ▼与对照组比较,P>0.05;●与对照组及阴性载体组比较,P<0.01。n=3。"

图 1

图 2

Western-blotting检测CaMKⅡδ表达水平"

图 2

图 3

TRAP、牛骨吸收陷窝检测破骨细胞的形成 A、D：对照组;B、E：阴性载体组;C、F：干扰载体组。A~C：TRAP染色;D~F：SEM。"

图 3

图 4

实时PCR检测c-fos mRNA、c-jun mRNA和CREB mRNA水平阴性载体组与对照组比较,P>0.05;**干扰载体组c-fos和CREB mRNA水平与对照组及阴性载体组比较,P<0.01;*干扰载体组c-jun mRNA水平与对照组及阴性载体组比较,P<0.05。n=3。"

图 4

图 5

Western-blotting检测c-Fos、c-Jun和CREB表达"

图 5

参考文献 15

[1] Pereira M, Petretto E, Gordon S , et alCommon signalling pathways in macrophage and osteoclast multinucleation[J]. J Cell Sci, 2018,131:11. pii:jcs216267.

[2] Yuan FL, Wu QY, Miao ZN , et al. Osteoclast-derived extracellular vesicles: novel regulators of osteoclastogenesis and osteoclast-osteoblasts communication in bone remodeling[J]. Front Physiol, 2018,9:628.

[3] Yao CH, Zhang P, Zhang L . Differential protein and mRNA expression of CaMKs during osteoclastogenesis and its functional implications[J]. Biochem Cell Biol, 2012,90(4):532-539.

[4] Kim JY, Cheon YH, Yoon KH , et al. Parthenolide inhibits osteoclast differentiation and bone resorbing activity by down-regulation of NFATc1 induction and c-Fos stability, during RANKL-mediated osteoclastogenesis[J]. BMB Rep, 2014,47(8):451-456.

[5] Mohamed SG, Sugiyama E, Shinoda K , et al. Interleukin-10 inhibits RANKL-mediated expression of NFATc1 in part via suppression of c-Fos and c-Jun in RAW264.7 cells and mouse bone marrow cells[J]. Bone, 2007,41(4):592-602.
doi: 10.1016/j.bone.2007.05.016

[6] Park KH, Gu DR, Jin SH , et al. Pueraria lobate inhibits RANKL-mediated osteoclastogenesis via dow-nregulation of CREB/PGC1β/c-Fos/NFATc1 signaling[J]. Am J Chin Med, 2017,45(8):1725-1744.

[7] 刘娟娟, 董伟, 戚孟春 , 等. 钙离子/钙调蛋白依赖性蛋白激酶Ⅱδ RNA干扰对下游基因表达及破骨细胞分化的影响[J]. 第三军医大学学报, 2017,39(1):22-27.

Liu JJ, Dong W, Qi MC , et al. Influence of CaMK Ⅱ δ RNA interference on expression of its downstream molecules and osteoclast differentiation[J]. J Third Mil Med Univ, 2017,39(1):22-27.

[8] Teitelbaum SL . Bone resorption by osteoclasts[J]. Science, 2000,289(5484):1504-1508.

[9] Matsuo K, Galson DL, Zhao C , et al. Nuclear factor of activated T-cells (NFAT) rescues osteoclastogenesis in precursors lacking c-Fos[J]. J Biol Chem, 2004,279(25):26475-26480.

[10] Matsuo K, Ray N . Osteoclasts, mononuclear phagocytes, and c-Fos: new insight into osteoimmunology[J]. Keio J Med, 2004,53(2):78-84.

[11] Nie S, Xu J, Zhang C , et al. Salicortin inhibits osteoclast differentiation and bone resorption by down-regulating JNK and NF-κB/NFATc1 signaling pathways[J]. Biochem Biophys Res Commun, 2016,470(1):61-67.

[12] 王想福, 孙凤歧, 石瑞芳 , 等. 靶向抑制PI3K对人体外周血破骨细胞分化p38/c-Fos信号通路调控的研究[J]. 中国骨质疏松杂志, 2016,22(1):49-52.

Wang XF, Sun FQ, Shi RF , et al. The regulation of p38/c-Fos signal pathway by targeting inhibition of PI3K in human peripheral osteoclasts[J]. Chin J Osteopor, 2016,22(1):49-52.

[13] Izawa T, Arakaki R, Mori H , et al. The nuclear receptor AhR controls bone homeostasis by regulating osteoclast differentiation via the RANK/c-Fos signaling Axis[J]. J Immunol, 2016,197(12):4639-4650.

[14] 王红美, 董伟, 戚孟春 , 等. CREB和ERK1/2在CaMKⅡδ调控破骨细胞分化中的作用[J]. 解放军医学杂志, 2018,43(2):91-95.

Wang HM, Dong W, Qi MC , et al. Regulation of osteoclast differentiation by CaMKⅡδ mediated through CREB and ERK1/2 signal pathways[J]. Med J Chin PLA, 2018,43(2):91-95.

[15] 陆大壮, 刘娟娟, 戚孟春 , 等. CaMKⅡδ在破骨细胞分化不同阶段表达规律的研究[J]. 中国病理生理杂志, 2016,32(10):1870-1874.

Lu DZ, Liu JJ, Qi MC , et al. Expression profiles of CaMKⅡδ at different stages of osteoclast differen-tiation[J]. Chin J Pathophysiol, 2016,32(10):1870-1874.

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[9] 胡巍,王译凡,袁一方,李影,郭斌. 节律基因调控成骨和破骨活动机制的研究进展[J]. 国际口腔医学杂志, 2019, 46(3): 302-307.

[10] 高鑫,曾融生. 骨保护素在口腔领域的研究进展[J]. 国际口腔医学杂志, 2019, 46(3): 316-319.

[11] 崔跃, 姜欢, 胡敏. 破骨细胞蛋白酪氨酸磷酸酶与正畸移动牙牙根吸收的关系[J]. 国际口腔医学杂志, 2017, 44(1): 87-91.

[12] 侯玉帛1 刘歆婵2 于海燕1 崔磊华3 于维先4. 牙龈蛋白及其对破骨和成骨细胞功能的影响[J]. 国际口腔医学杂志, 2016, 43(5): 609-613.

[13] 谭秀君，叶玲，汪成林. c-Jun N-末端激酶信号转导通路调节细胞迁移的机制[J]. 国际口腔医学杂志, 2015, 42(5): 586-591.

[14] 周正赵长铭焦凯王美青. 交感神经系统-肾上腺素能受体对骨改建的调节作用[J]. 国际口腔医学杂志, 2015, 42(3): 348-351.

[15] 孟庆阳，郑嵘，朱阳，王丹丹，吴立鹏，孙宏晨. 破骨细胞分化因子及其信号转导通路[J]. 国际口腔医学杂志, 2015, 42(2): 189-193.

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CaMKi>Ⅱδ基因沉默对破骨细胞分化功能及c-fosi>/c-juni>/CREBi>基因的影响

Effect of CaMKⅡδ gene silencing on osteoclast differentiation and c-fos/c-jun/CREB gene

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[1]	Pereira M, Petretto E, Gordon S , et alCommon signalling pathways in macrophage and osteoclast multinucleation[J]. J Cell Sci, 2018,131:11. pii:jcs216267.
[2]	Yuan FL, Wu QY, Miao ZN , et al. Osteoclast-derived extracellular vesicles: novel regulators of osteoclastogenesis and osteoclast-osteoblasts communication in bone remodeling[J]. Front Physiol, 2018,9:628.
[3]	Yao CH, Zhang P, Zhang L . Differential protein and mRNA expression of CaMKs during osteoclastogenesis and its functional implications[J]. Biochem Cell Biol, 2012,90(4):532-539.
[4]	Kim JY, Cheon YH, Yoon KH , et al. Parthenolide inhibits osteoclast differentiation and bone resorbing activity by down-regulation of NFATc1 induction and c-Fos stability, during RANKL-mediated osteoclastogenesis[J]. BMB Rep, 2014,47(8):451-456.
[5]	Mohamed SG, Sugiyama E, Shinoda K , et al. Interleukin-10 inhibits RANKL-mediated expression of NFATc1 in part via suppression of c-Fos and c-Jun in RAW264.7 cells and mouse bone marrow cells[J]. Bone, 2007,41(4):592-602. doi: 10.1016/j.bone.2007.05.016
[6]	Park KH, Gu DR, Jin SH , et al. Pueraria lobate inhibits RANKL-mediated osteoclastogenesis via dow-nregulation of CREB/PGC1β/c-Fos/NFATc1 signaling[J]. Am J Chin Med, 2017,45(8):1725-1744.
[7]	刘娟娟, 董伟, 戚孟春 , 等. 钙离子/钙调蛋白依赖性蛋白激酶Ⅱδ RNA干扰对下游基因表达及破骨细胞分化的影响[J]. 第三军医大学学报, 2017,39(1):22-27.
	Liu JJ, Dong W, Qi MC , et al. Influence of CaMK Ⅱ δ RNA interference on expression of its downstream molecules and osteoclast differentiation[J]. J Third Mil Med Univ, 2017,39(1):22-27.
[8]	Teitelbaum SL . Bone resorption by osteoclasts[J]. Science, 2000,289(5484):1504-1508.
[9]	Matsuo K, Galson DL, Zhao C , et al. Nuclear factor of activated T-cells (NFAT) rescues osteoclastogenesis in precursors lacking c-Fos[J]. J Biol Chem, 2004,279(25):26475-26480.
[10]	Matsuo K, Ray N . Osteoclasts, mononuclear phagocytes, and c-Fos: new insight into osteoimmunology[J]. Keio J Med, 2004,53(2):78-84.
[11]	Nie S, Xu J, Zhang C , et al. Salicortin inhibits osteoclast differentiation and bone resorption by down-regulating JNK and NF-κB/NFATc1 signaling pathways[J]. Biochem Biophys Res Commun, 2016,470(1):61-67.
[12]	王想福, 孙凤歧, 石瑞芳 , 等. 靶向抑制PI3K对人体外周血破骨细胞分化p38/c-Fos信号通路调控的研究[J]. 中国骨质疏松杂志, 2016,22(1):49-52.
	Wang XF, Sun FQ, Shi RF , et al. The regulation of p38/c-Fos signal pathway by targeting inhibition of PI3K in human peripheral osteoclasts[J]. Chin J Osteopor, 2016,22(1):49-52.
[13]	Izawa T, Arakaki R, Mori H , et al. The nuclear receptor AhR controls bone homeostasis by regulating osteoclast differentiation via the RANK/c-Fos signaling Axis[J]. J Immunol, 2016,197(12):4639-4650.
[14]	王红美, 董伟, 戚孟春 , 等. CREB和ERK1/2在CaMKⅡδ调控破骨细胞分化中的作用[J]. 解放军医学杂志, 2018,43(2):91-95.
	Wang HM, Dong W, Qi MC , et al. Regulation of osteoclast differentiation by CaMKⅡδ mediated through CREB and ERK1/2 signal pathways[J]. Med J Chin PLA, 2018,43(2):91-95.
[15]	陆大壮, 刘娟娟, 戚孟春 , 等. CaMKⅡδ在破骨细胞分化不同阶段表达规律的研究[J]. 中国病理生理杂志, 2016,32(10):1870-1874.
	Lu DZ, Liu JJ, Qi MC , et al. Expression profiles of CaMKⅡδ at different stages of osteoclast differen-tiation[J]. Chin J Pathophysiol, 2016,32(10):1870-1874.

[1]	余岳霖,孔卫东. 甲状旁腺激素受体1基因相关与原发性牙齿萌出障碍的研究进展[J]. 国际口腔医学杂志, 2023, 50(5): 573-580.
[2]	安宁,李姣,梅志丹. 骨保护素/核因子-κB受体活化因子/核因子κB-受体活化因子配体信号分子调控牙萌出的研究进展[J]. 国际口腔医学杂志, 2022, 49(1): 116-120.
[3]	吕辉,王华,孙雯. 辅助性T细胞17与牙周炎骨免疫[J]. 国际口腔医学杂志, 2020, 47(6): 661-668.
[4]	付世锦,曾刊,李鑫,杨静,汪成林,叶玲. 骨保护素/核因子κB受体活化因子配体影响肺癌细胞下颌骨与股骨转移差异的初步研究[J]. 国际口腔医学杂志, 2020, 47(5): 538-546.
[5]	孙坚炜,雷利红,谭静怡,陈莉丽. 微小RNA 155对骨免疫的调控及其在牙周炎中作用的研究进展[J]. 国际口腔医学杂志, 2020, 47(5): 607-615.
[6]	杨佩佩,杨羽晨,张强. 尼古丁对牙槽骨破骨细胞的作用及其机制的研究进展[J]. 国际口腔医学杂志, 2020, 47(5): 616-620.
[7]	朱俊瑾,周佳琦,伍颖颖. 哺乳动物雷帕霉素靶蛋白复合物1介导的自噬对骨代谢的调控[J]. 国际口腔医学杂志, 2020, 47(1): 84-89.
[8]	卢可心,张迪亚,吴燕岷. 蛋白酶激活受体在牙周组织细胞中相关作用的研究进展[J]. 国际口腔医学杂志, 2019, 46(6): 657-662.
[9]	胡巍,王译凡,袁一方,李影,郭斌. 节律基因调控成骨和破骨活动机制的研究进展[J]. 国际口腔医学杂志, 2019, 46(3): 302-307.
[10]	高鑫,曾融生. 骨保护素在口腔领域的研究进展[J]. 国际口腔医学杂志, 2019, 46(3): 316-319.
[11]	崔跃, 姜欢, 胡敏. 破骨细胞蛋白酪氨酸磷酸酶与正畸移动牙牙根吸收的关系[J]. 国际口腔医学杂志, 2017, 44(1): 87-91.
[12]	侯玉帛1 刘歆婵2 于海燕1 崔磊华3 于维先4. 牙龈蛋白及其对破骨和成骨细胞功能的影响[J]. 国际口腔医学杂志, 2016, 43(5): 609-613.
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