Yurkova Natalia, Shaw James, Blackie Karen, Weidman Danielle, Jayas Ravi, Flynn Bryan, Kirshenbaum Lorrie A
Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Taché Ave, Winnipeg, Manitoba, Canada R2H 2A6.
Circ Res. 2008 Feb 29;102(4):472-9. doi: 10.1161/CIRCRESAHA.107.164731. Epub 2007 Dec 20.
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.
细胞周期因子E2F-1已知可调节包括凋亡在内的多种细胞过程。此前我们发现，Rb-E2F-1复合物的破坏会引发有丝分裂后成年和新生心室肌细胞的凋亡；然而，其潜在机制尚不清楚。在本报告中，我们表明E2F-1通过直接影响内在死亡途径的机制引发心室肌细胞死亡。此外，我们从机制上表明，缺氧诱导死亡因子Bnip3是E2F-1的直接转录靶点，对于E2F-1诱导的细胞死亡是必需且充分的。E2F-1的表达导致核小体DNA片段化增加4.9倍（P<0.001），并通过Hoechst 33258染色和活体染色导致细胞死亡。E2F-1引发的线粒体扰动与通透性转换孔开放一致。通过定量实时PCR分析确定，在表达野生型E2F-1的细胞中观察到内源性Bnip3基因转录增加6.2倍（P<0.001），而在表达DNA结合缺陷型E2F-1突变体的细胞中未观察到。Rb是细胞E2F-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切割并失活。与Rb的蛋白水解切割一致，染色质免疫沉淀分析显示缺氧期间E2F-1与Bnip3启动子的结合增加，这一发现与Bnip3基因转录的诱导一致。Bnip3同源物Nix/Bnip3L在心室肌细胞中不受E2F-1或缺氧的影响。E2F-1的基因敲低或抗半胱天冬酶形式的Rb的表达抑制了基础和缺氧诱导的Bnip3基因转录。线粒体膜插入缺陷的Bnip3功能丧失突变或针对Bnip3的小干扰RNA抑制了E2F-1引发的细胞死亡信号。据我们所知，这些数据提供了首个直接证据，表明E2F-1激活内在线粒体死亡途径与Bnip3的转录激活相互依赖且必然相关。
