Acetylated myocyte enhancer factor-2D can promote T cell apoptosis by regulating the expression of Nur77 / 中国组织工程研究

ABSTRACT

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. ①Escherichia coli DH5?and plasmid pcDNA3 were held by the Research Center in the First Hospital of Jilin University. Plasmids pET32M, Flag-p300 and pSilencerTM-p300 RNAi were gifts from Professor Zhengguo Wu from Hong Kong University of Science and Technology. Plasmid NFATp was contributed by Yun Chen from Harvard University. Jurkat cells were provided by Department of Hematology- Oncology in the First Hospital of Jilin University. ②The 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. ③The 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: ①Although 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.②MEF2D 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. ③The 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. ④The 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%. ⑤When 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.