The cell cycle factor E2F-1 activates bnip3 and the intrinsic death pathway in ventricular myocytes
The cell cycle factor E2F-1 activates bnip3 and the intrinsic death pathway in ventricular myocytes
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DOI：
10.1161/circresaha.107.164731
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发表时间：
2008-02-29
影响因子：
20.1
通讯作者：
Kirshenbaum, Lorrie A.
中科院分区：
文献类型：
--
作者：
Yurkova, Natalia;Shaw, James;Kirshenbaum, Lorrie A.
已知细胞周期因子E2 F-1调节包括凋亡在内的多种细胞过程。以前，我们表明，Rb-E2 F-1复合物的中断引起有丝分裂后的成人和新生儿心室肌细胞的凋亡，然而，其潜在的机制是不确定的。在这份报告中，我们表明，E2 F-1引起细胞死亡的心室肌细胞通过一种机制，直接影响内在的死亡途径。此外，我们机械地表明，缺氧诱导的死亡因子Bnip 3是E2 F-1的直接转录靶点，这对于E2 F-1诱导的细胞死亡是必要的和足够的。通过Hoechst 33258染料和活体染色，E2 F-1的表达导致核小体DNA断裂和细胞死亡增加4.9倍（P < 0.001）。E2 F-1引起的线粒体扰动与渗透性转换孔开放一致。如通过定量实时PCR分析所确定的，在表达野生型E2 F-1的细胞中观察到内源性Bnip 3基因转录的6.2倍增加（P < 0.001），但在表达DNA结合缺陷的E2 F-1突变的细胞中未观察到。Rb是细胞E2 F-1活性的主要调节因子，在缺氧期间在心室肌细胞中被蛋白水解裂解并失活。与Rb的蛋白水解裂解一致，染色质免疫沉淀分析显示在缺氧期间E2 F-1与Bnip 3启动子的结合增加，这一发现与Bnip 3基因转录的诱导一致。Bnip 3同系物Nix/Bnip 3L在心室肌细胞中不受E2 F-1或缺氧的影响。基因敲低E2 F-1或表达抗半胱天冬酶形式的Rb抑制基础和缺氧诱导的Bnip 3基因转录。线粒体膜插入缺陷的Bnip 3功能缺失突变或针对Bnip 3的小干扰RNA抑制E2 F-1引起的细胞死亡信号。据我们所知，这些数据提供了第一个直接证据，即E2 F-1对固有线粒体死亡途径的激活与Bnip 3的转录激活相互依赖并必然相关。
The cell cycle factor E2F-1 is known to regulate a variety of cellular processes including apoptosis. Previously we showed that disruption of Rb-E2F-1 complexes provoked apoptosis of postmitotic adult and neonatal ventricular myocytes; however, the underlying mechanism was undetermined. In this report, we show that E2F-1 provokes cell death of ventricular myocytes through a mechanism that directly impinges on the intrinsic death pathway. Furthermore, we show mechanistically that the hypoxia-inducible death factor Bnip3 is a direct transcriptional target of E2F-1 that is necessary and sufficient for E2F-1-induced cell death. Expression of E2F-1 resulted in a 4.9-fold increase (P < 0.001) in nucleosomal DNA fragmentation and cell death by Hoechst 33258 dye and vital staining. E2F-1 provoked mitochondrial perturbations that were consistent with permeability transition pore opening. As determined by quantitative real-time PCR analysis, a 6.2-fold increase (P < 0.001) in endogenous Bnip3 gene transcription was observed in cells expressing wild-type E2F-1 but not in cells expressing a mutation of E2F-1 defective for DNA binding. Rb, the principle regulator of cellular E2F-1 activity, was proteolytically cleaved and inactivated in ventricular myocytes during hypoxia. Consistent with the proteolytic cleavage of Rb, chromatin immunoprecipitation analysis revealed increased binding of E2F-1 to the Bnip3 promoter during hypoxia, a finding concordant with the induction of Bnip3 gene transcription. The Bnip3 homolog Nix/Bnip3L was unaffected in ventricular myocytes by either E2F-1 or hypoxia. Genetic knockdown of E2F-1 or expression of a caspase-resistant form of Rb suppressed basal and hypoxia-inducible Bnip3 gene transcription. Loss-of-function mutations of Bnip3 defective for mitochondrial membrane insertion or small interference RNA directed against Bnip3 suppressed cell death signals elicited by E2F-1. To our knowledge, the data provide the first direct evidence that activation of the intrinsic mitochondrial death pathway by E2F-1 is mutually dependent on and obligatorily linked to the transcriptional activation of Bnip3.