摘要
该文旨在探讨带核定位信号的维甲酸受体α(nuclear localization signal retinoic acid receptor alpha,NLS-RARα)对人急性早幼粒白血病(acute promyelocytic leukemia,APL)细胞株NB4分化的影响及其机制。免疫印迹实验检测全反式维甲酸(all-trans retinoic acid,ATRA)诱导的NB4细胞分化标志物C/EBPβ、CD11b和p38α蛋白质水平;利用慢病毒介导的NLS-RARα基因过表达,进一步用免疫印迹实验验证过表达效率并检测NLS-RARα对NB4细胞分化标志物C/EBPβ、CD11b和p38α蛋白质水平的影响;间接免疫荧光实验分析NLS-RARα与p38α的空间共定位;免疫共沉淀实验分析NLS-RARα与p38α的相互作用。结果显示,生理浓度和药理浓度的ATRA促进NB4细胞分化的同时也激活了p38α,且p38α的活性变化与髓系分化标志物C/EBPβ变化一致;髓系分化表面标志物CD11b表达量在药理浓度ATRA(1μmol/L)处理下达到最高;NLS-RARα抑制NB4细胞的分化,且只有在ATRA存在的条件下,NLS-RARα抑制NB4细胞的分化与下调p38α活性相关;NLSRARα与p38α存在空间共定位且NLS-RARα与p38α直接相互作用。该研究结果提示,当存在ATRA诱导时,NLS-RARα与p38α直接相互作用后下调p38α的活性进而抑制NB4细胞的分化。
This paper is to investigate the effects of NLS-RARα on differentiation of human promyelocytic leukemia cell line NB4 and its potential mechanisms. The expressions of myeloid differentiation markers, C/EBPβ and CD1 lb, p38α induced by ATRA in NB4 cells were detected by Western blot. NLS-RARα was overexpressed mediating by lentivirus and its efficiency of NLS-RARα overexpress, the expressions of myeloid differentiation markers, C/EBPβ and CD1 1b, p38α were detected by Westem blot. The localization of NLS-RARα and its interaction with p38α was analysed by indirect immunofluorescence assay and co-immunopreci-pitation assay, respectively. Our results demonstrated that the two ATRA concentrations (physiological [10 nmol/L] and pharmacological [ 1 μmol/L] concentrations) that we investigated promoted differentiation and activated p38α in NB4 cells. Further, ATRA-induced expression of the myeloid differentiation marker, C/EBPβ, was proportional to phosphorylated p38α (p-p38α). We also observed a higher peak in the surface of myeloid differentiation marker, CDllb, following treatment with pharmacological concentrations of ATRA (1 μmol/L). NLS-RARα also inhibited NB4 cell differentiation, more importantly, this effect was related to downregulation of the active form of p38α in the presence of ATRA. Finally, co-localization of NLS-RARα and p38α in three-dimensional space was confirmed, and we found that NLS-RARa could interact with p38α directly. Taken together, these data indicated that NLS-RARa inhibited ATRA-induced differentiation of NB4 cells by downregulating the active form of p38α via a direct interaction with p38α.
出处
《中国细胞生物学学报》
CAS
CSCD
2016年第7期794-801,共8页
Chinese Journal of Cell Biology
基金
国家自然科学基金(批准号:81171658)
重庆市自然科学基金计划重点项目(批准号:2011BA5037)资助的课题~~