MEK5$\alpha $ 和 MEK5$\beta $ 调控 ${\bf Beclin}$ 1 启动子

Beclin 1是哺乳动物自噬相关基因, 调控自噬起始和自噬体成熟. 在肌肉分化过程中, Beclin 1 基因表达上调, 自噬增加; 此外 MEK5-ERK5 信号活化并调控成肌细胞分化. 因此, 在肌肉分化过程中, MEK5-ERK5 信号通路可能调控 Beclin 1 基因表达. 目的是阐明 MEK5 对成肌细胞 Beclin 1 基因启动子活性的调控. 将不同长度 Beclin 1 启动子片段克隆至荧光素酶报告基因载体pGL3-Basic并转染成肌细胞C2C12. 双荧光素酶报告基因检测实验结果显示, 含 Beclin 1 基因起始密码子上游 586 碱基对 DNA 片段的载体(p-354)具有强荧光素酶活性. MEK5$\alpha $显著增加 p-354 荧光素酶活性, 并有剂量依赖性; 而 MEK5$\beta $ 显著降低 p-354 荧光素酶活性. MEK5$\beta $能够拮抗 MEK5$\alpha $对 p-354 荧光素酶活性的调控. 与 MEK5 对 Beclin 1 基因启动子调控结果一致, MEK5$\alpha $CA 上调细胞Beclin 1 mRNA 表达, MEK5$\beta $DD 下调 Beclin 1 mRNA 表达, 并且 MEK5$\beta $DD 抑制 MEK5$\alpha $CA 对 Beclin 1 mRNA 表达的促进作用. 此外, 转录因子 CREB 家族成员 CREB3, CREBP 和 CREBL1 能够显著上调 p-354 荧光素酶活性. CREB3 呈剂量依赖性显著上调 p-354 荧光素酶活性, 并与 MEK5$\alpha $ 具有协同效应. MEK5$\alpha $ 和 MEK5$\beta $ 对 Beclin 1 启动子具有不同调控作用, CREB 可能是其下游效应因子.

MEK5$\alpha $ and MEK5$\beta $ differentially regulate Beclin 1 promoter

Beclin 1, a mammalian autophagy-related gene, regulates autophagy initiation and autophagosome maturation. During muscle differentiation, Beclin 1 is upregulated. Additionally, MEK5-ERK5 is activated to regulate muscle differentiation. Thus, MEK5-ERK5 may regulate Beclin 1 gene expression during muscle differentiation. The aim of this study was to determine MEK5 regulation of Beclin 1 promoter in myoblast cells. A series of promoter-luciferase constructs harbouring different lengths of Beclin 1 promoter were created and transfected into myoblast C2C12 cells. In the luciferase assay, the construct containing 586 base pairs upstream of the start codon (p-354) exhibited the most potent luciferase activity. MEK5$\alpha $ and MEK5$\beta $ enhanced and suppressed p-354 luciferase activity, respectively. MEK5$\beta $ is capable of antagonizing the effect of MEK5$\alpha $ on p-354 luciferase activity. Consistent with the results on the regulatory effects of MEK5 on Beclin 1 promoter, MEK5$\alpha $CA and MEK5$\beta $DD upregulated and downregulated Beclin 1 mRNA expression, respectively. Moreover, MEK5$\beta $DD antagonised the stimulatory effects of MEK5$\alpha $CA on Beclin 1 mRNA expression. Members of the CREB family, including CREB3, CREBP, and CREBL1, promoted p-354 luciferase activity. Furthermore, CREB3 dose-dependently increased p-354 luciferase activity and exhibited a synergistic effect with MEK5$\alpha $ on p-354 luciferase activity. Collectively, these findings indicate that MEK5$\alpha $ and MEK5$\beta $ differentially regulate Beclin 1 promoter activity and that CREB family members may be downstream effectors.