The cell cycle factor E2F-1 activates Bnip3 and the intrinsic death pathway in ventricular myocytes.
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.
Yurkova
Natalia
N
Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Taché Ave, Winnipeg, Manitoba, Canada R2H 2A6.
Shaw
James
J
Blackie
Karen
K
Weidman
Danielle
D
Jayas
Ravi
R
Flynn
Bryan
B
Kirshenbaum
Lorrie A
LA
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002453:Cell Cycle / Q000502:physiology; D002478:Cells, Cultured; D050687:E2F1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology; D000860:Hypoxia / Q000378:metabolism / Q000503:physiopathology; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D008928:Mitochondria / Q000502:physiology; D024101:Mitochondrial Proteins; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000502:physiology; D015533:Transcriptional Activation / Q000502:physiology; D014162:Transfection
C505650:BNIP3 protein, rat; D050687:E2F1 Transcription Factor; C493755:E2f1 protein, rat; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011518:Proto-Oncogene Proteins