摘要
目的:Nur77是介导T细胞凋亡的重要基因,它的激活需要第二信使钙离子,而Nur77基因启动子的钙反应元件是转录因子肌细胞增强因子2D的结合位点。探讨在Nur77诱导T细胞凋亡过程中肌细胞增强因子2D是否可以被乙酰化,以及通过调节Nur77基因乙酰化对T细胞凋亡的影响。方法:实验于2006-11/2007-09在吉林大学第一医院血液肿瘤科完成。①材料:大肠杆菌DH5α、pcDNA3质粒由吉林大学第一医院中心研究室保存。pET32M质粒、Flag-p300质粒、pSilencerTM-p300RNAi质粒由香港科技大学Zhengguo Wu博士馈赠。NFATp质粒由哈佛大学Yun Chen博士馈赠。Jurkat细胞由吉林大学第一医院血液肿瘤科提供。②实验方法:PCR法产生全长的Nur77和依赖Nur77的荧光报告基因被亚克隆到pcDNA质粒中,产生的肌细胞增强因子2D(1-514)、(1-121)、(1-300)、(301-514)各片段以及含4个赖氨酸(K245/K250/K267/K279)位点突变为精氨酸位点的肌细胞增强因子2D(4KR)突变体亚克隆到pcDNA后在氨基端含有Flag抗原簇,于细菌表达载体上进行质粒构建,验证正确的质粒使用脂质体DMRIE-C进行转染。③实验评估:将Nur77荧光报告基因与肌细胞增强因子2D、p300、NFATp共转染到Jurkat细胞中,检测p300对肌细胞增强因子2D促进Nur77转录的影响。免疫沉淀法检测内源性肌细胞增强因子2D是否能够被乙酰化,以及阻滞p300表达后是否影响其乙酰化。体外乙酰化法检测肌细胞增强因子2D的乙酰化位点。荧光素报告分析法检测肌细胞增强因子2D的乙酰化功能缺失对Nur77转录活性的影响。采用流式细胞仪检测其功能缺陷对Nur77诱导T细胞凋亡的影响。结果:①肌细胞增强因子2D与NFATp的二聚体虽然不能促进Nur77的转录,但p300、NFAT、肌细胞增强因子2D的三聚体却能明显促进Nur77的转录,且p300与肌细胞增强因子2D的二聚体也明显促进Nur77的转录。②PMA/Iono活化钙信号传导通路后,肌细胞增强因子2D被乙酰化。当内源性p300RNAi后,p300的表达被阻滞时,肌细胞增强因子2D的乙酰化水平显著下降。③K245,K250,K267,K279氨基酸同时突变可以使肌细胞增强因子2D乙酰化程度大部分丧失,说明在体外这4个赖氨酸位点是肌细胞增强因子2D的主要乙酰化位点。④与空载体比较,转染乙酰化缺陷的肌细胞增强因子2D可使Nur77转录功能提高2.48倍;与转染野生型的肌细胞增强因子2D比较,转染乙酰化缺陷的肌细胞增强因子2D能降低Nur77基因70.4%的转录功能。⑤与空载体比较,Nur77表达能够促进T细胞凋亡,早晚期凋亡之和从4.3%提高至16.3%;共表达野生型肌细胞增强因子2D与Nur77能够进一步促进T细胞的凋亡,早晚期凋亡之和从16.3%提高到31.0%;但肌细胞增强因子2D乙酰化功能缺失的突变体肌细胞增强因子2D明显降低Nur77介导的T细胞凋亡,早晚期凋亡之和从16.3%降低到9.2%。结论:p300对肌细胞增强因子2D促进Nur77的转录起主要作用。肌细胞增强因子2D在Nur77诱导的T细胞凋亡过程中可以被乙酰化,乙酰化的肌细胞增强因子2D通过上调Nur77基因的转录来促进T细胞凋亡。
AIM： Nur77 expression can lead to T cell apoptosis, and it is activated by the second messenger calcium, The calcium-responsive elements in the Nur77 promoter are two putative binding sites for the transcription factor, myocyte enhancer factor-2D （MEF2D）. In this study, we explored whether MEF2D could be acetylated in Nur77-induced T cell apoptosis and whether MEF2D acetylation was affected by the regulation of Nur77 expression.
METHODS： The experiment was performed at Department of Hematology-Oncology in the First Hospital of Jilin University from November 2006 to September 2007. （1）Escherichia coil DH5 a and plasmid pcDNA3 were held by the Research Center in the First Hospital of Jilin University. Plasmids pET32M, Flag-p300 and pSilencerr^TM-p300 RNAi were gifts from Professor Zhengguo Wu from Hong Kong University of Science and Technology. Plasmid NFATp was contributed by Yun then from Harvard University. Jurkat cells were provided by Department of Hematology- Oncology in the First Hospital of Jilin University. （2）The full-length Nur77 gene, generated by PCR, and the Nur77-dependent luciferase reporter gene were subcloned into plasmid pcDNA. The variant sequences of MEF2D （1-514, 1-121, 1-300, 301-514） and the MEF2D （4KR） lysine-to-arginine mutants, at lysine sites K245/K250/K267/K279, had the Flag epitope at their amino termini. All of the above plasmids were constructed in bacterial expression vectors and the constructed clones were verified by sequencing and then transfected with liposome DMRIE-C. （3）The impact of p300 on the trans-activation function of Nur77, mediated by MEF2D, was detected by luciferase reporter assays after Nur77-dependent reporter gene was transfected together with MEF2D, p300 or NFATp into Jurkat cells. In vivo acetylation of endogenous MEF2D and whether acetylation of MEF2D was affected by blocking the expression of p300 were detected by immunoprecipitation. The acetylated sites of MEF2D were detected by acetylation assays in vitro. The impacts of both MEF2D acetylation defects on trans-activation of Nur77 and on apoptosis of T cells induced by Nur77 were detected by luciferase reporter assays and flow cytometry, respectively.
RESULTS： （1）Although-the dimeric-complex of MEF2D and NFATp could not induce the transcription of Nur77, significant transcription was achieved with the ternary complex of p300, NFAT and MEF2D. The dimeric complex of p300 and MEF2D could also significantly induce Nur77transcription.（2）MEF2D was acetylated after the calcium signaling pathway was activated by PMA/Iono. The acetylation level of MEF2D was markedly reduced when p300 expression was blocked by p300 RNAi. （3）The simultaneous mutation of K245/K250/K267/K279 lysine sites largely prevented the acetylation of MEF2D, which demonstrated that K245/K250/K267/K279 lysine sites in vitro were the major sites of MEF2D acetylation, （4）The MEF2D acetylation defect increased by 2.48 fold the transcription f of Nur77, when compared with vacant vector, But compared with wild type MEF2D, transcription of Nur77 was decreased by 70.4%. （5）When compared with vacant vector, Nur77 expression could increase total T cell apoptosis of early and late periods from 4.3% up to 16.3%; the co-expression of wild type MEF2D and Nur77 could further increase total T cells apoptosis of early and late periods from 16.3% to 31.0%. However, the co-expression of acetylation defective MEF2D mutant and Nur77 significantly decreased total T cells apoptosis of early and late periods from 16.3% to 9.2%.
CONCLUSION： p300 has a major impact on the transactivation function of Nur77 mediated by MEF2D, MEF2D could be acetylated in Nur77-induced T cell apoptosis and acetylated MEF2D could promote T cell apoptosis by upregulating the transcription function of Nur77.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第16期3127-3132,共6页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金(30671091)~~
