Abstract 1330: Divergent Control of the Mitochondrial Death Gene BNIP3 by Opposing Actions of the Cellular Factors Nuclear Factor kappaB and E2F-1 in Ventricular Myocytes.

The hypoxia-inducible death factor Bnip3 is known to provoke mitochondrial perturbations and cell death of ventricular myocytes. The transcriptional control processes that govern Bnip3 gene expression under basal and inducible conditions remain cryptic. Sequence analysis of the Bnip3 promoter revealed the presence of distinct but overlapping DNA binding elements for the cell cycle factor E2F-1 and cellular factor NF-κB. Previously, we reported a survival role for NF-κB in ventricular myocytes. As a step toward elucidating the regulation of Bnip3 gene expression in ventricular myocytes, we tested the impact of E2F-1 and NF-κB on basal and inducible expression of Bnip3. A 2.0 fold increase in Bnip3 gene transcription was observed in cells expression wild type E2F-1 but not in cells expressing an E2F-1 mutant defective for DNA binding. Interestingly, basal Bnip3 gene transcription was increased by 2.5 fold in myocytes rendered defective for NF-κB activation with a non-phosphorylatable form of IκBα. Importantly, genetic ablation of E2F-1 inhibited basal and inducible Bnip3 transcription in NF-κB defective cells. Expression of the p65 subunit of NF-κB in NF-κB defective cells inhibited E2F-1 mediated Bnip3 transcription. Western blot analysis of cardiac cell lysate revealed that p65 NF-κB immunoprecipitated with E2F-1. ChIP analysis of the Bnip3 promoter indicated that the p65 NF-κB bound DNA under normoxic conditions. During hypoxia E2F-1 activity increased where as p65 NF-κB protein levels were decreased. ChIP analysis revealed increased binding of E2F-1 to the Bnip3 promoter during hypoxia which coincided with a 3.5 fold increase in Bnip3 gene transcription. IKKβ mediated activation of NF-κB activation abrogated hypoxia-induced E2F-1 binding to the Bnip3 promoter and Bnip3 gene transcription. To our knowledge our data provide the first direct evidence that a novel relationship exists between p65 NF-κB and E2F-1 for basal and hypoxia-inducible regulation of the Bnip3 promoter. Furthermore, our data highlight a novel survival pathway by which NF-κB averts hypoxia - induced cell death by antagonizing the E2F-1 dependent transcription of Bnip3 in ventricular myocytes.

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